COMPOSITIONS AND METHODS FOR BIO-BUTADIENE

PRODUCTION SCREENING

RELATED APPLICATIONS

This application is a Patent Convention Treaty (PCT) International Application which claims benefit of priority to U.S. Provisional Patent Application Serial No. (USSN) 61/747,806, filed December 31, 2012, which is expressly incorporated by reference herein in its entirety for all purposes.

TECHNICAL FIELD

This invention generally relates to detection methods of products of biosynthetic processes and sustainable chemical production processes. In alternative embodiments, the invention provides compositions and methods for the detection of organisms having butadiene biosynthetic capability. In alternative embodiments, the invention provides compositions and methods for screening or assaying a population of cells for: the production of a butadiene; or, for the rate of production of a butadiene; or, for the improved production of a butadiene.

BACKGROUND

Over 25 billion pounds of butadiene (1,3 -butadiene, BD, BDE) are produced annually and are applied in the manufacture of polymers such as synthetic rubbers and ABS resins, and chemicals such as hexamethylenediamine and 1 ,4-butanediol. Butadiene is typically produced as a by-product of the steam cracking process for conversion of petroleum feedstocks such as naphtha, liquefied petroleum gas, ethane or natural gas to ethylene and other olefins. The ability to manufacture butadiene from alternative and/or renewable feedstocks represents a major advance in the quest for more sustainable chemical production processes. Butadiene can be produced renewably by fermentation of sugars or other feedstocks to produce diols, such as 1 ,4- butanediol or 1,3-butanediol, which are separated, purified, and then dehydrated to butadiene in a second step involving metal-based catalysis.

However, direct fermentative production of butadiene from renewable feedstocks obviates the need for dehydration steps since butadiene gas (boiling point, or bp, is -4.4°C) could be continuously emitted from the fermenter and readily collected, e.g. by condensation. The direct fermentative production process eliminates the need for fossil-based butadiene and would allow substantial savings in cost, energy, and harmful waste and emissions relative to petrochemically-derived butadiene.

Improved microbial organisms and methods for effectively producing butadiene from cheap renewable feedstocks such as molasses, sugar cane juice, and sugars derived from biomass sources, including agricultural and wood waste, as well as CI feedstocks such as syngas and carbon dioxide, are needed.

SUMMARY

In alternative embodiments, the invention provides compositions and methods that can efficiently detect butadiene (1,3-butadiene, BD, BDE), including butadiene gas, produced as a product of a biosynthetic process, e.g., as a product of a microbial organism biosynthetic process.

In alternative embodiments, the invention provides methods for screening or assaying a population of cells for production of a butadiene, or rate of production of a butadiene, or improved production of a butadiene (BD), the method comprising the steps of:

(a) arranging the cells in a spatial array such that each position in the spatial array is occupied by identical cells, or a single cell, or a plurality of cells derived from a single cell,

wherein optionally the array comprises a cylindrical array, a square array, a cubical array, or a rectangular array, or a plate or a microtiter plate, or a tape or a membrane, or a solid or a semisolid surface, or a textile surface, or a combination thereof;

(b) cultivating, growing or culturing the cell or cells under or in a media, a culture or under growth conditions suitable for production or synthesis of a butadiene (BD), optionally under culture or growth conditions suitable for screening or assaying for BD;

(c) screening or assaying each spatial array position for production of a butadiene (BD), or for the amount or rate of production of BD, or for the presence of BD, or for improved production of a butadiene (BD),

wherein optionally screening or assaying for improved production of a BD comprises screening or assaying for and/or determining: an increased yield of BD; an increased ratio of BD production to feedstock carbon; an increased total amount of BD produced; and/or, an increased ratio of BD to an impurity, wherein optionally the screening or assaying is or comprises a medium or a high throughput screening (HTS) method or system, and optionally each spatial array position is assayed or screened simultaneously,

and optionally the screening or assaying is or comprises detection of a butadiene gas, and optionally the butadiene gas is detected, measured and/or quantified: by a sensing device, or directly or indirectly by a chemical or an enzymatic reaction, optionally using a chromo-reactant to form a detectable product or a spectrophotometrically, colorimetrically or magnetically detectable product; and

(d) identifying the cells or cells that produce the butadiene, or identifying an array position that produces the butadiene, or identifying, quantifying or measuring the amount or rate of butadiene from each array position,

thereby screening or assaying the population of cells for production of a butadiene or butadiene gas, or rate of production of a butadiene or butadiene gas, or for improved production of a butadiene or butadiene gas.

In alternative embodiments, methods of the invention comprise detecting a cell or a population of cells having an improved, faster and/or increased or higher production of butadiene or butadiene gas under a particular or a changed set of media, growth or culture conditions, wherein the method comprises screening or assaying each spatial array position for a changed rate or amount of production of a butadiene or butadiene gas under a particular or a changed set of conditions by screening or assaying each spatial array position before and at least once or multiple times after changing the culture or growth conditions,

wherein optionally the change in culture or growth conditions comprises one or more of: a change in culture or media pH or salinity; a change in amount or rate of oxygen or carbon dioxide provision or exchange to the culture or media; a change in temperature in the culture or media; a change in concentration of, or the presence or absence of, one or more a chemical, molecule, substrate, feedstock or feedstock composition, or chemical or molecule that triggers a response from the cell or cells, wherein optionally the chemical or molecule modifies, begins or stops expression of a protein, lipid, polysaccharide or nucleic acid in the cell by influencing or inducing an inducible transcriptional or translational modifier, activator or repressor.

In alternative embodiments, the spatial array is or comprises a microtiter array, a tape, a membrane, a plate or a platform or equivalent, a solid or a semisolid surface, a textile surface or a combination thereof, wherein optionally the microtiter array, tape, plate or platform or equivalent comprises about 96, 384, 1536, 3456 or 9600 wells, optionally in an automated sampling format.

In alternative embodiments, after step (b) or step (c) a sample is transferred from each position in the spatial array to a position in a second spatial array, and optionally the culture or growth conditions are the same (duplicated) or changed, and the positions in the spatial arrays are screened or assayed for production of a butadiene, or for the amount or rate of production of a butadiene, and/or cells or cells that produce butadiene are identified, or the amount or rate of production of a butadiene is determined, quantified or measured.

In alternative embodiments, each position in the spatial array is occupied by one or more beads comprising one or the same cell, or a plurality of the same cells, and optionally the bead is or comprises an agarose bead.

In alternative embodiments, the butadiene is measured, quantified or assayed directly or indirectly (e.g., by detecting or measuring the amount of a BD-reactive compound that remains after its reaction with the BD) in a whole broth, a cell supernatant or a cell lysate, or the butadiene is measured, quantified or assayed directly or indirectly intracellularly.

In alternative embodiments, cell or cells used to practice the invention are: of natural origin; of mammalian origin; transfected, infected or recombinant cells; of microbial origin; bacterial or fungal cells; from the genus Escherichia, Bacillus, Aspergillus or Saccharomyces; or are cells selected from the group consisting of Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Bacillus clausii, Aspergillus niger, Aspergillus oryzae, Aspergillus nidulans, and Saccharomyces cerevisiae,

and optionally the cell or cells are recombinant or non-naturally occurring organisms, and optionally comprise a 1,3 -butadiene pathway,

or optionally the cell or cells are recombinant or non-naturally occurring organisms, and optionally comprise a 1,3 -butadiene pathway which includes at least one nucleic acid encoding a 1,3-butadiene pathway enzyme over-expressed compared to the unmodified organisms in a sufficient amount to produce 1,3-butadiene,

or optionally the cell or cells are recombinant or non-naturally occurring organisms, and optionally comprise a 1,3-butadiene pathway which includes at least one exogenous nucleic acid encoding a 1,3 -butadiene pathway enzyme expressed in a sufficient amount to produce 1,3- butadiene,

or optionally the cell or cells are recombinant or non-naturally occurring organisms, and optionally comprise a 1,3 -butadiene pathway, which optionally includes at least one exogenous nucleic acid encoding a 1,3-butadiene pathway enzyme expressed in a sufficient amount to produce 1,3-butadiene, and optionally a 1,3-butadiene pathway is selected from the group consisting of: (A) 1) trans, trans-muconate decarboxylase and 2) tra/?s-2,4-pentadienoate decarboxylase; (B) 1) cis, trans-muconate cz ^' s-decarboxylase and 2) tra/?s-2,4-pentadienoate decarboxylase; (C) 1) cis, trans-muconate trans-decarboxylase 2) cz ^' s-2,4-pentadienoate decarboxylase; and (D) 1) cis, cz ^' s -muconate decarboxylase and 2) cis -2,4-pentadienoate decarboxylase;

or optionally the 1,3-butadiene pathway comprises two exogenous nucleic acids each encoding a 1,3-butadiene pathway enzyme, or optionally the two exogenous nucleic acids can encode a set selected from the group consisting of: (A) 1) trans, trans-muconate decarboxylase and 2) trans -2,4-pentadienoate decarboxylase; (B) 1) cis, trans -muconate cz ^' s-decarboxylase and 2) trans-2,4-pentadienoate decarboxylase; (C) 1) cis, trans-muconate trans-decarboxylase 2) cis- 2,4-pentadienoate decarboxylase; and (D) 1) cis, cz ^' s-muconate decarboxylase and 2) cis-2,4- pentadienoate decarboxylase.

In alternative embodiments, the chemical, molecule, substrate is selected from the group consisting of a polypeptide, a peptide, a small molecule, a small peptide, a lipopeptide, a polysaccharide or a sugar, an antimicrobial, a lipid, an ion, a metal ion, a co-factor, an alcohol, methanol, ¾, formate, and a pharmaceutical molecule.

In alternative embodiments, the media, culture or growth conditions suitable for production of a butadiene comprise a feedstock, a renewable feedstock, a molasses, a cane sugar, a sugar cane juice, a sugar derived from a biomass source, an agricultural or a wood source or waste, a CI (one carbon) compound, a syngas or a carbon dioxide.

In alternative embodiments, the presence or amount butadiene (BD) gas is analyzed or measured in an air sample by drawing known volume of air through a sampling tube containing charcoal adsorbent which has been coated with 4-tert-butylcatechol, and the samples are desorbed with carbon disulfide and then analyzed by gas chromatography using a flame ionization detector. In alternative embodiments, the presence or amount butadiene (BD) gas is analyzed or measured by a chemical or an enzymatic reaction, or the butadiene (BD) gas is analyzed or measured in its soluble form in the cell culture medium, or in its gas form in the cell culture headspace, or in its soluble form in a liquid which trapped the BDE gas produced by the cell culture, and/or in its gaseous form in the headspace of that liquid.

In alternative embodiments, the presence or amount of butadiene (BD) gas is analyzed or measured by a chemical or an enzymatic reaction which results in modification or creation of a detectable substrate, wherein optionally the substrate is modified by isomerization, ligation or other modification; and optionally the substrate is a polymer, or a polypeptide, a cellulose, a polysaccharides or a starch, and optionally the substrate is immobilized, e.g., to the surface of a solid material, or an array, a metal, a textile, a cellulose, or a ceramic, or a solid surface, or a cellulose swatch, and optionally the substrate is labeled with a detectable probe, and optionally the probe is fluorescent or a fluorescein 5-isothiocyanate (FITC) or a dichlorotriazino-5- aminofluorescein (DTAF).

In alternative embodiments, the presence or amount of butadiene (BD) gas is analyzed or measured by detection of a signal or molecule whose presence or amount is proportional to the presence of amount of butadiene (BD) gas in a sample, wherein optionally the signal comprises a visible signal, ultraviolet signal, fluorescence, a fluorescence polarization, an absorbance, a radioactivity, a nuclear magnetic resonance (NMR) and/or a luminescence signal.

In alternative embodiments, the growth conditions are optimized to give minimal variation in the amount produced of butadiene (BD), BD gas, or a detectable molecule, in each position of the spatial array, and optionally the growth conditions are chosen so as to mimic the conditions of a full-scale fermentation, or the growth conditions comprise a highly dilute media.

In alternative embodiments, methods of the invention further comprise screening for a nucleic acid, a gene or a DNA, or a DNA library, for a nucleic acid, a gene or a DNA of interest, or a nucleic acid, a gene or a DNA capable of increasing the amount or rate of butadiene (BD) or BD gas production; wherein optionally the DNA library is generated from a natural DNA sequence by mutagenesis, error prone PCR, rationale mutagenesis, GSSM, SSM, DNA shuffling or directed evolution.

In alternative embodiments, the presence or amount of butadiene (BD) gas is analyzed or measured using a non-intrusive sensor, wherein optionally the non-intrusive sensor is a electrochemical sensor , a spectrophotometric sensor, or the presence or amount of butadiene (BD) gas is analyzed or measured by: an off-axis integrated cavity output spectroscopy (OA ICOS), a laser absorption spectroscopy (LAS), an infrared spectroscopy (IR), a near-infrared spectroscopy (NIR), a Fourier transform infrared spectroscopy (FTIR); a Gas Chromatography - Flame Ionization Detector or GC-FID and/or a Gas chromatography-mass spectrometry (GC- MS), and optionally the butadiene is directly detected in the headspace of each well of a plate, or a culture plate using the non-intrusive sensor, or the sensor is sequentially connected to multiple wells to read BD in each well, and optionally the sensor is connected to a headspace in a way that allows BD diffusion from the headspace to the sensor, and optionally the sensor is connected and measurements taken at any period of time during culturing or during growth or production phase or both, and optionally a manifold comprising an array of plugs or caps, one for each well of the culture plate, and each plug or cap connected to a tube connecting to the BD sensor, which is put in place at a desired time period, and optionally the plug or cap comprises a tube or passageway connecting the well's headspace to BD sensor, allowing diffusion of the BD gas directly to the sensor, and optionally the sensor is equipped with a means to flush air thru its detector without disturbing the well headspace.

In alternative embodiments, the presence or amount of butadiene (BD) gas is analyzed or measured using a two-plate or a two-microtiter plate system having a lower plate as a cell culture plate and an upper plate as a screening or assay plate, the lower and upper plates separated by a gas-permeable liquid-impermeable membrane or equivalent, and the cells are grown or cultured in the lower plate,

and optionally the upper plate comprises: a liquid composition that traps the butadiene (BD) gas for direct measurement of BD; a liquid composition comprising a reactive compound that reacts with the butadiene (BD) gas to produce a detectable compound; a liquid composition comprising an enzyme or enzymes and, optionally one or more co-factors or additional substrates, that catalyze a reaction with the butadiene (BD) gas to produce a detectable compound; or, a microbe that utilizes the butadiene (BD) gas for growth and whose growth can be detected or converts the butadiene (BD) gas to a detectable compound;

or optionally to each of the above the amount or presence of BD is determined by the detecting or measuring the amount of the reactive compound that remains after its reaction with the BD, for example, in the exemplary embodiment wherein BD reacts with a maleimide and then the remaining maleimide is measured by reacting with a thiol-compound.

In alternative embodiments, the presence or amount of butadiene (BD) gas is analyzed or measured by: detection of trapped butadiene (BD) off-gas or soluble butadiene (BD) gas in cell culture, optionally using a chromo-reactant to form spectrophotometric detectable products; detection of trapped butadiene (BD) off-gas or soluble butadiene (BD) gas in cell culture via enzymatic reaction to generate spectrophotometric detectable products; very fast sequential direct detection of BD liquid and/or headspace off-gas, optionally using Fourier transform infrared spectroscopy (FTIR), near-infrared spectroscopy (N IRS), or equivalent; or simultaneous direct detection of headspace BD by one or an array of commercial BD sensors.

In alternative embodiments, the presence or amount of butadiene (BD) gas is analyzed or measured using a gas-trapping chromo-reactant filter membrane or an upper BD-trapping liquid plate, wherein optionally the filter membrane or BD-trapping liquid plate is present for the entire culture or growth and production period, or it can be added at any time point, for example, to measure BD during production phase only; or optionally to each of the above the amount or presence of BD is determined by the detecting or measuring the amount of the reactive compound that remains after its reaction with the BD,

In alternative embodiments, the presence or amount of butadiene (BD) gas is analyzed or measured using an apparatus comprising two plates: a lower plate containing media and cells to be tested for butadiene production and an upper plate for containing a liquid composition comprising a chromo-reactant, and a gas permeable, liquid impermeable membrane separating the two plates that allows a headspace gas of the lower plate in fluid contact with the liquid composition of the upper plate; and optionally the solutions in the upper plate can further comprise: a BD-reactive agent; a BD-reactive dye or BD-reactive co-substrate; a BD-reactive co- substrate for an enzyme reaction; a catalyst; a buffer; a co-factor; and/or, an enzyme for a BD detection method, or optionally to each of the above the amount or presence of BD is determined by the detecting or measuring the amount of the reactive compound that remains after its reaction with the BD. In alternative embodiments the upper plate has a microbe for detection of BD, and optionally the upper plate comprises a minimal media with no carbon source with a microbe that grows on BD, and optionally the microbial growth is detected by an increase in solution turbidity. In alternative embodiments, the presence or amount of butadiene (BD) gas is analyzed or measured using a single plate apparatus, optionally with a BD-reactant membrane, wherein optionally the single plate apparatus comprises a single microtiter plate apparatus comprising a single plate and a membrane or a filter paper impregnated with a BD-reactive compound capable of reacting with the BD gas diffusing from the cell culture headspace, where the membrane is in contact with the cell culture headspace, and where host cells are grown in the single plate,

wherein optionally the microtiter plate is designed such that the butadiene (BD) gas that escapes from the cell medium into the cell culture headspace diffuses into the membrane where the BD reacts with a BD-reactive compound to produce a detectable compound,

and optionally the microtiter plate is a 96-well microtiter plate, or a plate or microtiter plate comprising an array of cells in a culture media that are subjected to conditions that allow BD production, an optionally the plate is a colony growth plate with a solid growth media, or is an agar plate or a Petri dish where individual colonies are grown;

and optionally the single plate apparatus comprises a solvent phase in which BD is highly soluble and optionally the solvent further comprises a BD-reactive compound or a BD-reactive enzyme reaction compound, and optionally the BD-reactive compounds or enzyme reaction reagents can be added at any desired time point, and optionally the solvent can be more dense than water, where the plate is such that it allows spectrophotometric reading through the bottom of the plate, or the solvent can be less dense than water and the detection is made through the top of the plate; and optionally BD-soluble organic solvents are used, optionally comprising an ethanol, a diethyl ether, an acetone, a benzene, or a polar or a nonpolar organic solvent compatible with microbial cells;

and optionally the single culture plate is used to grow an array of cells to be assayed for BD production, and the cell culture medium is isolated and tested for BD, either directly or indirectly, and optionally the microtiter culture plate is used to grow the cells and the cell culture medium is clarified by centrifugation of the plates to create a clear supernatant, the clear supernatant isolated, and the BD in the supernatant is detected directly or indirectly.

and optionally the single culture plate is used to grow an array of cells to be assayed for BD production, and the cell culture medium is clarified by addition of a cell lysing agent, and optionally also a viscosity reducing agent is added, and the clarified cell culture is tested for BD, either directly or indirectly; and optionally to reduce microbial-produced turbidity, a microbe- lysing agent, a lysozyme, a chelators and/or an organic solvent is added to each well; or optionally to each of the above the amount or presence of BD is determined by the detecting or measuring the amount of the BD-reactive compound that remains after its reaction with the BD.

In alternative embodiments, the cells and/or an enzyme convert butadiene to a more soluble product, resulting in a butadiene-derived product in liquid phase, and the butadiene- derived product is detected or measured to determine the amount or rate of production of BD, and optionally the enzyme or enzymes are added to the cell culture or are recombinant enzymes generated by the cells,

wherein optionally the cells and/or enzymes convert the butadiene to a 3-butene-l,2-diol, a l-hydroxy-2-butanone, a l-hydroxy-2-butenone and/or a 2-hydroxybut-3-eneone, and optionally the BD to 3-butene-l,2-diol, a l-hydroxy-2-butanone, a l-hydroxy-2-butenone and/or a 2-hydroxybut-3-eneone conversion is enzymatic, and optionally the 3-butene-l,2-diol, a 1- hydroxy-2-butanone, a l-hydroxy-2-butenone and/or a 2-hydroxybut-3-eneone is detected or measured by Gas chromatography-mass spectrometry (GC-MS) or by Fourier transform infrared spectroscopy (FTIR), or equivalents,

and optionally after the cell or enzyme produces BD and converts it to the 3-butene-l,2- diol, a l-hydroxy-2-butanone, a l-hydroxy-2-butenone and/or a 2-hydroxybut-3-eneone, a cell free system or clarified cell culture with the BD soluble in a liquid is generated and the soluble BD is detected by adding an alcohol dehydrogenase (ADH) enzyme or a 3-butene-l,2-diol dehydrogenase and a co-factor NAD, and measuring the amount of NADH formation by change in absorbance,

and optionally the enzyme or enzymes that convert the butadiene to a secondary product, or a 3-butene-l,2-diol, a l-hydroxy-2-butanone, a l-hydroxy-2-butenone and/or a 2-hydroxybut- 3-eneone, are added to the cell culture or are recombinant enzymes generated by the cells,

and optionally the cells or enzymes convert the butadiene to a butadiene-derived product by oxidation, and optionally the butadiene-derived product comprises a butadiene monoxide (BMO, also called l,2-epoxy-3-butene), and optionally BMO is subsequently converted to a diepoxybutane, a 3 -butane- 1,2-diol, a crotonaldehyde and/or a glutathione conjugate,

and optionally the cells and/or enzymes that convert the butadiene to BMO comprise a toluene-4-monooxygenase, a toluene monooxygenase, a cytochrome P450 enzyme, a butadiene monooxygenase, a monooxygenase, a chloroperoxidase and/or a myeloperoxidase, which can be recombinantly added to the cell or added an enzyme to the cell culture or the media; and optionally the butadiene monooxygenase converts butadiene, 0 ₂ and an electron donor to BMO and an electron acceptor; and optionally the expression of the enzyme or the butadiene monooxygenase is under control of a promoter inducible by a toluene or other inducer,

and optionally the BMO is further converted to 3-butene-l,2-diol (BDD) by an enzyme with a BMO hydrolase activity, and optionally the BMO hydrolase activity enzyme is an epoxide hydrolase; and optionally BDD is further oxidized to a l-hydroxy-2-butanone, a 1- hydroxybuteneone or a 2-hydroxy-l-ketobutenone by a secondary alcohol dehydrogenase, and optionally the secondary alcohol dehydrogenase is an alcohol dehydrogenase, a malate dehydrogenase or a lactate dehydrogenase;

and optionally the cells or enzymes convert the butadiene to an epoxide, and optionally the enzyme is a cytochrome P450 or a cytochrome P450 BM3, and optionally the cell culture is clarified by centrifugation or cell lysis and the supernatant is used in a cell-free assay to detect an epoxide formed colorimetrically, or the epoxide is detected using pNTP (para-nitrothiolphenol).

In alternative embodiments, the presence or amount of butadiene (BD) is analyzed or measured using an Diels-Alder reaction, which optionally can be carried out in an aqueous phase, and optionally the Diels-Alder the reaction is carried out in water at neutral conditions, and optionally the Diels-Alder the reaction generates a maleimide, which optionally can be directly assayed spectrophotometrically at 302 nm, and optionally a maleimide group that did not react with BD can be quantified by first reacting a maleimide sample with a known amount of a thiol present in excess and then assaying the remaining unreacted thiol, optionally using a 4,4'- DTDP; and optionally a spectrophotometric assay for the determination of maleimide groups is a reverse glutathione (GSH) assay.

In alternative embodiments, the presence or amount of butadiene (BD) or 1,3 -Butadiene is by a reaction with an acetylene-dicarboxylic acid or its di-methyl ester to form a cyclodiene- dicarboxylic acid or its dimethyl ester, and optionally the cyclodiene-dicarboxylic acid or methyl ester is aromatized to form a phthalic acid, which optionally can be detected using an UV absorption assay; and optionally an aromatization step to phthalic acid is implemented in an aqueous solution in the presence of a hydrogen peroxide, a sodium bromide and a hydrochloric acid, In alternative embodiments, the presence or amount of butadiene (BD) or 1,3 -Butadiene gas is by use of a BD-sensitive membrane, paper or filter paper, impregnated with BD-reactive compound (a reactive membrane, or sensor layer) over a plate, a microtiter plate, or over a colony dish; and optionally the BD-sensitive membrane in which the BD reaction has taken place is detected spectrophotometrically,

and optionally the dishes or plates or culture or growth conditions are anaerobic to order to induce BD production, and the reactive membrane or sensor layer is applied, and at the end of a desired BD production period the reactive membrane or sensor layer is analyzed for location of spots or "dots" where BD reacted with a chemical reagent on the reactive membrane or sensor layer, and optionally the reactive membrane or sensor layer is removed and the BD-sensing reaction developed prior to detection of and/or quantitation of the reactive spots,

and optionally the BD-sensitive membrane, paper or filter paper comprises a

polytetrafluoroethylene (PTFE), nylon, nitrocellulose, parafilm or other BD-resistant porous plastic membrane.

In alternative embodiments, the presence or amount of butadiene (BD) or 1,3 -Butadiene gas comprises: detection in the cell media of a plate or a microtiter plate, optionally with or without reducing turbidity by optionally centrifugation or cell lysing; direct detection in the headspace of microtiter plate; using an apparatus allowing BD gas trapping,

wherein optionally the apparatuses comprises: (i) a chromo-reactant impregnated membrane or a paper or a filter paper over a plate or a microtiter plate or over an agar colony plate; or, (ii) a two-plate system comprising a lower plate or microtiter culture plate and an upper BD-trapping plate or microtiter plate, and optionally the lower plate or microtiter culture plate is separated via a gas-permeable liquid-impermeable membrane from the upper BD-trapping liquid-containing plate or microtiter plate, and optionally the membrane is a separate component from either plate, and optionally the membrane comprises a bottom surface of the upper plate, optionally a headspace gas comprises a butadiene gas, and optionally the membrane comprises a chromo-reactant and a headspace gas of the container is in fluid contact with chromo-reactant.

In alternative embodiments, the presence or amount of butadiene (BD) or 1,3 -Butadiene gas comprises use of a sensing device, and optionally the device comprises: a membrane having at least one nanochannel, and optionally the membrane comprises a polycarbonate, glass, polytetrafluoroethylene, polyethylene terephthalate, acrylonitrile-butadiene-styrene, acrylonitrile-methyl acetate copolymer, cellophane, ethyl cellulose, cellulose acetate, cellulose acetate butyrate, cellulose propionate, cellulose triacetate, polyethylene, polyethylene-vinyl acetate copolymers, ethylene polymers, polyethylene-nylon copolymers, polypropylene, methyl pentene polymers, polyvinyl fluoride, polypyrrole, polyaniline, polythiophene, or an aromatic polysulfone.

In alternative embodiments, the presence or amount of butadiene (BD) or 1,3 -Butadiene gas comprises use of a magnetic field or applying a magnetic field to the spatial array, and performing magnetic resonance imaging spectroscopy (MRI) on the one or more samples of the spatial array, thereby identifying the array position for each of the one or more samples having one or more MRI detectable chemical shifts, which one or more chemical shifts corresponds to the production of butadiene (BD) gas.

In alternative embodiments, the BD-reactive compound is a maleimide or iodoacetamide, and optionally the amount or presence of BD is determined by the amount of conjugated BD or optionally the amount or presence of BD is determined by the amount of unreacted maleimide or iodoacetamide that is detected by reaction with a thiol. In alternative embodiments, the BD- reactive compound is a maleimide dye or iodoacetamide dye, and optionally where the maleimide or iodoacetamide dye is pyrene maleimide or pyrene iodoacetamide, and optionally the amount or presence of BD is determined by the amount of pyrene-conjugated BD or optionally the amount or presence of BD is determined by the amount of unreacted pyrene maleimide or pyrene iodoacetamide that is detected by reaction with a thiol.

In alternative embodiments, the presence or amount of butadiene (BD) or 1,3 -Butadiene gas in an aqueous solution comprises detection of BD in a reaction with a p-nitro- benzendiazonium ion or its chloride salt, the reaction optionally in an acid medium or an acetic acid-based acid medium, which optionally yields a yellow product, and optionally a product's optical density is determined photocolorimetrically.

In alternative embodiments, the presence or amount of butadiene (BD) or 1,3 -Butadiene gas is determined using a BD-gas detector tube, or array of tubes, or by measuring or quantifying a BD-reactive reagent used in the BD-gas detector tube, or array of tubes, and optionally the tube or tubes can be used to sample the BD gas in a headspace of a plate or culture plate, or the upper plate in a dual plate system, and optionally an open tube or tubes are inserted in a manifold of the same a spatial array pattern as the culture plate or upper plate, and the manifold placed over the plate at the desired time point such that each tube in the array is in contact with its corresponding well in the plate array, wherein optionally the tubes are read by color development along the length of the tube, or the intensity and/or hue of the developed color indicates the presence and/or amount of BD gas detected in the given volume of headspace, and optionally a vacuum is applied to the distal end of each tube to draw up a precise amount of headspace air into each tube to enhance accuracy of a reading;

and optionally each tube comprises a reagent to impregnate a membrane, a paper or a filter paper, to create a BD-reactive membrane,

and optionally dry reagents are placed in an upper plate of a two plate system without any liquid, and optionally the reagents can be placed directly in the upper plate wells adsorbed to a solid support or a silica gel or a silica glass, and optionally the reagents can be placed in the wells without their solid support,

and optionally the presence or amount of BD is determined colorimetrically, by a plate reader or by a CCD image reader, and optionally color amount, intensity and hue are measured, and optionally a BD-trapping color reaction comprises: (a) a reduction of chromate or dichromate to chromous ion, which produces a pale yellow to pale blue color change, (b) a reduction of ammonium molybdate plus palladium sulfate to molybdenum blue which produces a pale yellow to white color change, and/or (c) a reduction of potassium permanganate to produce a pink to white color change.

In alternative embodiments, the invention provides a composition or method according to any embodiment of the invention, substantially as hereinbefore described, or described herein, with reference to any one of the examples.

The details of one or more embodiments of the invention are set forth in the

accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

All publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes. BRIEF DESCRIPTION OF THE DRAWINGS

The drawings set forth herein are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.

Figure 1 illustrates exemplary pathways for production of butadiene from acetyl-CoA, glutaconyl-CoA, glutaryl-CoA, 3-aminobutyryl-CoA or 4-hydroxybutyryl-CoA via crotyl alcohol. Enzymes for transformation of the identified substrates to products include: A. acetyl- CoA:acetyl-CoA acyltransferase, B. acetoacetyl-CoA reductase, C. 3-hydroxybutyryl-CoA dehydratase, D. crotonyl-CoA reductase (aldehyde forming), E. crotonaldehyde reductase (alcohol forming), F. crotyl alcohol kinase, G. 2-butenyl-4-phosphate kinase, H. butadiene synthase, I. crotonyl-CoA hydrolase, synthetase, transferase, J. crotonate reductase, K. crotonyl- CoA reductase (alcohol forming), L. glutaconyl-CoA decarboxylase, M., glutaryl-CoA dehydrogenase, N. 3-aminobutyryl-CoA deaminase, O. 4-hydroxybutyryl-CoA dehydratase, P. crotyl alcohol diphosphokinase.

Figure 2 illustrates exemplary pathways for production of butadiene from erythrose-4- phosphate. Enzymes for transformation of the identified substrates to products include: A.

Erythrose-4-phosphate reductase, B. Erythritol-4-phospate cytidylyltransferase, C. 4-(cytidine 5'- diphospho)-erythritol kinase, D. Erythritol 2,4-cyclodiphosphate synthase, E. l-Hydroxy-2- butenyl 4-diphosphate synthase, F. l-Hydroxy-2-butenyl 4-diphosphate reductase, G. Butenyl 4- diphosphate isomerase, H. Butadiene synthase I. Erythrose-4-phosphate kinase, J. Erythrose reductase, K. Erythritol kinase.

Figure 3 illustrates an exemplary pathway for production of butadiene from malonyl-CoA plus acetyl-CoA. Enzymes for transformation of the identified substrates to products include: A. malonyl-CoA:acetyl-CoA acyltransferase, B. 3-oxoglutaryl-CoA reductase (ketone-reducing), C. 3-hydroxyglutaryl-CoA reductase (aldehyde forming), D. 3-hydroxy-5-oxopentanoate reductase, E. 3,5-dihydroxypentanoate kinase, F. 3H5PP kinase, G. 3H5PDP decarboxylase, H. butenyl 4- diphosphate isomerase, I. butadiene synthase, J. 3-hydroxyglutaryl-CoA reductase (alcohol forming), K. 3-oxoglutaryl-CoA reductase (aldehyde forming), L. 3,5-dioxopentanoate reductase (ketone reducing), M. 3,5-dioxopentanoate reductase (aldehyde reducing), N. 5-hydroxy-3- oxopentanoate reductase, O. 3-oxo-glutaryl-CoA reductase (CoA reducing and alcohol forming). Compound abbreviations include: 3H5PP = 3-Hydroxy-5-phosphonatooxypentanoate and 3H5PDP = 3-Hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate. Figure 4 illustrates exemplary pathways for the conversion of muconate stereoisomers to

1.3 - butadiene. Enzymes are A. trans, trans-muconate decarboxylase, B. cis, trans-muconate cis- decarboxylase, C. cis, trans-muconate trans-decarboxylase, D. cis, cis-muconate decarboxylase, E. trans-2,4-pentadienoate decarboxylase, F. cis-2,4-pentadienoate decarboxylase.

Figure 5 illustrates an exemplary pathway for the formation of butadiene from 3- hydroxypent-4-enoate (3HP4) by 3-hydroxypent-4-enoate decarboxylase.

Figure 6 illustrates exemplary pathways for the production of butadiene, 3-hydroxypent- 4-enoate (3HP4), 2,4-pentadienoate and 3-butene-l-ol from 3-HP-CoA and/or acrylyl-CoA. Enzymes are A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA dehydratase, D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase, F. 3-oxo-5- hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, G. 3,5-dihydroxypentanoyl- CoA synthetase, transferase and/or hydrolase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5- dihydroxypentanoate dehydratase, K. 3-hydroxypropanoyl-CoA dehydratase, L. 3-oxo-5- hydroxypentanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, N. 3-oxopent-4-enoyl- CoA reductase, O. 3-oxopent-4-enoyl-CoA synthetase, transferase and/or hydrolase, P. 3- oxopent-4-enoate reductase, Q. 5-hydroxypent-2-enoate dehydratase, R. 3-hydroxypent-4-enoyl- CoA dehydratase, S. 3-hydroxypent-4-enoate dehydratase, T. 3-hydroxypent-4-enoyl-CoA transferase, synthetase or hydrolase, U. 3,5-dihydroxypentanoate decarboxylase, V. 5- hydroxypent-2-enoate decarboxylase, W. 3-butene-l-ol dehydratase (or chemical conversion), X.

2.4- pentadiene decarboxylase, Y. 3-hydroxypent-4-enoate decarboxylase. 3-HP-CoA is 3- hydroxypropanoyl-CoA.

Figure 7, or Figure A, illustrates exemplary reactions for detection of butadiene in the exemplary high through-put screening (HTS) methods of the invention, e.g., as described herein.

Figures 8A, 8B and 8C; or Figures Bl, B2 and B3, respectively: illustrate exemplary reactions for detection of BD in exemplary HTS methods of the invention, e.g., as described herein.

Figure 9, or Figure C, illustrates exemplary reactions for detection of BD in exemplary HTS methods of the invention, e.g., as described herein. Figure 10, or Figure D, illustrates exemplary enzyme pathways for use in the detection of BD in exemplary HTS methods of the invention, e.g., as described herein. Exemplary enzyme reactions for converting butadiene to 3-butene-l,2-diol or l-hydroxy-2-butanone are shown. Enzymes are: A. butadiene monooxygenase, B. butadiene monoxide hydrolase, C. 3-butene-l,2- diol dehydrogenase, D. butadiene monoxide monooxygenase.

Figure 11, or Figure E, illustrates an exemplary system of the invention comprising a single microtiter plate, e.g. 96-well plate, with a chromo-reactant impregnated membrane for detection of BDE off-gas when the membrane is placed over the plate.

Figure 12, or Figure F, illustrates an exemplary system of the invention comprising a colony growth dish, e.g. agar plate, Petri dish, solid support growth media, with a chromo- reactant impregnated membrane for detection of BDE off-gas emitted by each colony when the membrane is placed over the dish. In alternative embodiments, the colonies are spotted in a grid pattern with the location of a reactant spot on the sensor membrane correlated to a specific colony that produced BD.

Figure 13A and 13B, or Figures Gl and G2, respectively, illustrate exemplary two-plate systems of the invention. Figure Gl shows an exemplary two plate apparatus of the invention comprising an upper plate with an integrated gas-permeable, liquid-impermeable membrane. Figure G2 shows an exemplary two plate apparatus of the invention comprising a separate gas- permeable, liquid-impermeable membrane.

Figure 14, or Figure H, illustrates an exemplary two plate apparatus of the invention comprising an upper plate with an integrated membrane bottom.

Like reference symbols in the various drawings indicate like elements, unless otherwise stated.

Reference will now be made in detail to various exemplary embodiments of the invention, examples of which are illustrated in the accompanying drawings. The following detailed description is provided to give the reader a better understanding of certain details of aspects and embodiments of the invention, and should not be interpreted as a limitation on the scope of the invention. DETAILED DESCRIPTION

In alternative embodiments, the invention provides compositions and methods that can efficiently detect butadiene (1,3-butadiene, BD, BDE), including butadiene gas, produced as a product of a biosynthetic process, e.g., as a product of a microbial organism biosynthetic process. In alternative embodiments, the invention provides compositions and methods for making and detecting butadiene, including butadiene gas.

"Butadiene" has the molecular formula C4H6 and a molecular mass of 54.09 g/mol (see e.g., Figures 2 to 4; IUPAC name Buta-l,3-diene), and is used interchangeably throughout with 1,3-butadiene, biethylene, erythrene, divinyl, vinylethylene and the acronyms "BD" or "BDE". Butadiene is a volatile, colorless, toxic, carcinogenic, flammable, and highly reactive gas.

Butadiene is both explosive and flammable because of its low flash point. Butadiene as a liquefied gas is colorless, non-corrosive with a mild aromatic or gasoline-like odor.

In alternative embodiments, the invention provides compositions and methods used in the development of a bio-based butadiene production process; and in alternative embodiments, compositions and methods of the invention can rapidly screen enzymes and host organisms for butadiene production, and for incremental improvements in butadiene production. In alternative embodiments, the compositions and methods of the invention address the challenges the chemical properties of butadiene present to traditional high-throughput screening (HTS) approaches. Butadiene is colorless, eliminating the possibility of direct detection via

calorimetry. Its toxicity, flammability and reactivity present a safety hazard. The gaseous form and low solubility also present challenges to screening.

In alternative embodiments, compositions and methods of the invention provide screening methods, e.g., medium- and high-throughput screening (HTS) methods, that take advantage of the chemical and/or enzymatic reactivity of butadiene and its physical properties. In alternative embodiments, the invention provides methods for performing assays that can efficiently and accurately screen large numbers of cell populations (e.g., bacterial cells cultures), e.g., libraries, for producing butadiene. The library can be a homogenous or heterologous population of cells (e.g., microbial cells) that differ in their ability to produce butadiene during cell culture. Such a library can be a population of cells producing variants of a protein or proteins or enzyme or enzymes of interest, e.g. enzymes in a butadiene biosynthetic pathway in that microbe, that affect BDE production in each cell. The cell or cells can be genetically engineered or can be obtained from nature. In alternative embodiments, compositions and methods of the invention also can be used to assay cells (e.g., microbial cells, such as bacterial cells) subjected to varying culture conditions that affect BDE production by each cell. In alternative embodiments, methods of the invention are or comprise high throughput screening (HTS) of the above populations of cells.

In alternative embodiments, screening methods comprise assaying a large population of host cells for production of butadiene, the method comprising the steps of: a) arranging the host cells in a spatial array so each position in the spatial array is occupied by one cell, b) cultivating the host cells under growth conditions suitable for HTS, c) assaying each array position for production of butadiene, and d) selecting the cells from those positions where butadiene was produced, as determined in step c). In alternative embodiments, the assay detects improved, e.g., faster and/or more (e.g., increased or higher), production of butadiene, e.g., by comparing BD production of the "parent" (or initial) host cell to a modified or "evolved" host cell, or by comparing the BD production of the host cell cultured on a starting culture condition to a test culture condition; e.g., where a test culture condition comprises one or more of: a change in culture pH; a change in rate of oxygen provision to the culture; a change in temperature; or, presence or absence of or a change in concentration of substrates or feedstocks or feedstock composition and the like, as would be known to one of ordinary skill in the cell fermentation arts.

In alternative embodiments, compositions and methods of the invention can efficiently obtain improved microbial organisms, e.g., bacteria, for producing butadiene, including producing butadiene from renewable feedstocks, e.g., cheap renewable feedstocks such as molasses, sugar cane juice, sugars derived from biomass sources, including agricultural and wood waste, as well as CI (one carbon compounds) feedstocks such as syngas and carbon dioxide.

In alternative embodiments, compositions and methods of the invention overcome problems associated with detecting and handling butadiene (BD), a volatile, colorless, toxic, carcinogenic, flammable, highly reactive gas, produced during microbial fermentation. In alternative embodiments, compositions and methods of the invention use qualitative and/or quantitative screening, e.g., a high-throughput screening, to identify an organism, e.g., a microbial organism such as a bacteria, having an improved or a desired ability to produce a BD. In alternative embodiments, compositions and methods of the invention comprise use of any method or apparatus to detect an organic volatile, e.g., BD or BD gas, or a microbially- produced organic volatile (e.g., BD gas), by e.g., employing invasive sampling of either fermentation medium or headspace followed by subjecting the sample to gas chromatography or liquid chromatography often coupled with mass spectroscopy. In alternative embodiments, any"state-of-the-art" apparatus can be used, e.g., for high throughput" screening, e.g., an Agilent 7697A HEADSPACE SAMPLER™ (Agilent Technologies, Santa Clara CA, USA) having a 111-vial capacity (10 mL, 20 mL, or 22 mL vials) and three 36-vial racks that can be exchanged while the headspace sampler is operating, or equivalent, can be used. In addition to limited sample configurations and numbers, the apparatus when coupled with GC or GC/MS would typically require 10-30 minutes to analyze each sample.

In alternative embodiments, compositions and methods of the invention test or assay large numbers of variations, e.g., libraries, using a high throughput screening (HTS) process to screen for individual variations in cells or cell culture conditions that produce or result in a desired effect, e.g., to produce more BD, or produce BD at a faster rate. In alternative embodiments, test procedures are automated HTS, and can be designed to be as simple and as fast as possible and, optionally, only require a minimum of simple automatable process steps which do not require manual steps in between. In alternative embodiments time-consuming manipulations such as removal of aliquots, further incubations using coupled (enzyme) reactions, centrifugation steps which cannot be automated etc., are avoided by the HTS methods and materials, e.g., as described herein. High throughput methods and materials can provide reliable results, which can be either qualitative or quantitative. In alternative embodiments HTS systems can provide superior speed, simplicity, efficiency, quantity and safety compared to the fermentation vial system and sampling and detection systems. In alternative embodiments HTS systems allow screening of more samples in a shorter period of time, allowing a wider variety of sample configurations and fermentation conditions and microbial variants, with less operator involvement, providing a safer, more effective, and higher throughput screening.

In alternative embodiments HTS systems microtiter plates (MTP), arrays or tapes, or equivalent, are used in high throughput screening (HTS) methods, and can be an efficient and accurate assay that can screen large numbers of variants and/or variant conditions for production of a butadiene (BD) or a butadiene gas. In alternative embodiments HTS systems can identify and be used to develop functional biosynthetic pathways, improved enzymes in a pathway, and growth and fermentation conditions enhancing butadiene production. The methods are compatible for use with microbial engineering that creates libraries using recombinant DNA technologies including Gene Site Saturation Mutagenesis, DNA shuffling, error prone PCR mutagenesis, random DNA

mutagenesis and in vivo recombination, that allow the generation of enormous populations of variant cells that produce variants of a certain protein, RNA, or small molecule, or with large libraries of natural enzymes cloned from other organisms.

In alternative embodiments, methods of the invention are used to screen a nucleic acid (e.g., a DNA) library for nucleic acid, gene or DNA of interest, the method comprising: creating host cells comprising (having contained within) the nucleic acid, gene or DNA library, and using a method of this invention to assay for a host cell producing a molecule of interest (e.g., nucleic acid, a polypeptide, peptide, an enzyme) that results in a desired amount of BD production, e.g., a greater rate of BD gas production. Recombinant vectors comprising the nucleic acid, gene or DNA of interest can be used to infect or transfect the cells to be screened using a method of this invention.

Detection Strategies for HTS of Butadiene Production

In alternative embodiments, the invention provides high throughput screening (HTS) of a large population of cells for butadiene production that takes advantage of butadiene's high reactivity. In alternative embodiments, methods and apparatus of the invention are designed or configured for HTS of cell, e.g., microbial, e.g., bacterial, butadiene production by detecting and/or measuring BDE either directly or indirectly, e.g., by chemical or enzymatic reaction, e.g., in its soluble form in the cell culture medium, in its gas form in the cell culture headspace, in its soluble form in a liquid which trapped the BDE gas produced by the cell culture, and/or in its gaseous form in the headspace of that liquid.

In alternative embodiments, methods are automatable and suitable for use with laboratory robotic systems, eliminating or reducing operator involvement, while proving high-throughput screening. In some embodiments the apparatus and methods exploit the volatile nature of BDE either by its direct detection in cell culture headspace or by trapping the off-gas BD followed by its detection in the trapped state. In alternative embodiments, compared to current state-of-the- art apparatus and methods, the present methods provide the advantage of faster screening that allows screening of larger numbers of host cells and cell culture conditions in a shorter period of time.

Apparatus for HTS of Butadiene Production

Two-plate apparatus. In alternative embodiments, a two-microtiter plate system having a lower plate (the cell culture plate) and an upper plate (the screening or assay plate) separated by a gas-permeable liquid-impermeable membrane, where host cells grown in the lower plate is used. For example, in one embodiment, a microtiter plate produces a butadiene gas that escapes from the cell medium into the cell culture headspace and diffuses through the gas-permeable membrane to an upper plate comprising a solution where the butadiene (BDE) gas is trapped, and then either detected directly or indirectly through chemical or enzymatic reaction of the trapped BDE. See for example Figures Gl, G2 and H, wherein in alternative embodiments an upper plate can comprise: a liquid composition that traps the BDE for direct measurement of BDE; a liquid composition comprising a reactive compound that reacts with the BDE to produce a detectable compound; a liquid composition comprising an enzyme or enzymes and, optionally one or more co-factors or additional substrates, that catalyze a reaction with the BDE to produce a detectable compound; or, a microbe that utilizes the BDE for growth and whose growth can be detected or converts the BDE to a detectable compound.

In alternative embodiments, the membrane can be a separate component, or it can be integrated into the upper plate, e.g., as a "filter-bottom" plate. Oxygen available to the upper plate oxygen can pass thru the separating membrane to the culture medium. Optionally the lower plate can be a deep well to increase starting oxygen or can be a special plate allowing sparging of or access to oxygen or other gases to the culture medium, e.g. a "filter-bottom" plate. In alternative embodiments BD-resistant seals, e.g. sealing mats of silicone or rubber, are used between each plate and/or membrane to isolate each well from adjacent wells.

In alternative embodiments, methods and apparatus of the invention allow fully robotic simultaneous reactions with or detection of BD from multiple colonies or fermentation conditions, followed by simultaneous measurement, thus HTS. In alternative embodiments, as when Fourier transform infrared spectroscopy (FTIR) is used, the results is a fast sequential measurement when compared to conventional GC/MS. In alternative embodiments,

conventional Gas Chromatography - Flame Ionization Detector or GC-FID, or Gas chromatography-mass spectrometry (GC-MS), of headspace or cell culture sampling also, or alternatively, can be used.

In alternative embodiments, methods and apparatus of the invention are fully robotic, requiring minimal to no operator intervention. In alternative embodiments, non-robotic plate handling is used, e.g., for microtiter centrifugation when low solution turbidity is desired.

In alternative embodiments, a gas-trapping chromo-reactant filter membrane or an upper BDE-trapping liquid plate is present for the entire culture/ growth and production period, or it can be added at any time point, for example, to measure BDE during production phase only. An the assay apparatus comprises two plates, a lower plate for containing media and cells to be tested for butadiene production and an upper plate for containing a liquid composition comprising a chromo-reactant, and a gas permeable, liquid impermeable membrane separating the two plates that allows a headspace gas of the lower plate in fluid contact with the liquid composition of the upper plate. In alternative embodiments, the solutions in the upper plate can further comprise in addition to the BD-reactive agent (e.g. dye) or co-substrate (e.g. for some enzyme reactions) a catalyst, a buffer, a co-factor, and/or an enzyme as appropriate for the detection method selected and as would be known in the art of butadiene chemistry and enzymology.

In alternative embodiments, the lower plate is a microtiter plate, e.g., a 96-well microtiter plate, and can comprise an array of cells in a culture media that are subjected to conditions that allow BD production.

In alternative embodiments, the liquid impermeable membrane is inert to BD; and optionally the membrane is PTFE, nylon, nitrocellulose, or other BD-resistant plastic.

An exemplary upper plate has a microbe for detection of BD; e.g., an upper plate can comprise a minimal media with no carbon source with a microbe that grows on BD, for example a Nocardia strain BT1; e.g., as described by: van Ginkel et al, Oxidation of Gaseous and Volatile Hydrocarbons by Selected Alkene-Utilizing Bacteria. Applied and Environmental Microbiology. 1987; 53:2903-2907. Microbial growth can be detected as for example by an increase in solution turbidity.

In alternative embodiments, the systems are used in a high-throughput manner allowing a large number of cells and /or conditions to be tested in a short period of time, as the detectable compound can be spectrophotometrically detected allowing simultaneous detection and assay of each well on a plate, e.g. with a plate reader.

In alternative embodiments, the sealing mats or gaskets (see for example Figures Gl, G2 and H) can be transparent, allowing isolation between wells if desired, but allowing

spectrophotometric or other detection of a well's contents. A suitable transparent sealing material is silicone rubber. The sealing mats or gaskets can be perforated, with the perforations or holes over each well, allowing isolation between wells if desired, but allowing

spectrophotometric or other detection of a well's contents.

For example, with reference to Figures Gl, G2 and H, a sealing mat when used as part of the lid or top plate can be transparent to allow a direct reading of the reaction in the screening plate (upper plate) wells without disassembly of the apparatus. Alternatively, a top sealing mat may be obviated when trapping of the BD gas and detection reaction are highly efficient such that insignificant amounts of BD gas are released from the upper screening plate. In another embodiment, the top sealing mat is perforated above each well such when used with a clear hard top plate over the mat the escape of BDE gas from each well of the upper plate is prevented. An alternative to the sealing mat are multiple separate gaskets or O-rings around each well to isolate each well from its adjacent wells.

Construction of the apparatus can be simple and all parts are commercially available. The color change can be stable and, due to the physical separation of the indicator solution from the microbe cells, is not affected by cellular interactions or nonvolatile metabolites. In alternative embodiments the assays are not significantly affected by oxygen, carbon dioxide, or volatile fatty acids at levels that could be experienced in biological systems.

In alternative embodiments, a single plate apparatus with a BD-reactant membrane is used. Another alternative embodiment is an apparatus comprising a single microtiter plate apparatus comprising a single plate and a membrane (e.g. filter paper) impregnated with a BD- reactive compound capable of reacting with the BD gas diffusing from the cell culture headspace, where the membrane in contact with the cell culture headspace, and where host cells grown in the single plate. For example a microtiter plate, produce butadiene that escapes from the cell medium into the cell culture headspace and diffuses into the membrane where the BD reacts with the BD-reactive compound to produce a detectable compound. In alternative embodiments, a single plate, for example a microtiter plate such as a 96-well microtiter plate, can comprise an array of host cells in culture media that are subjected to conditions that allow BD production. The plate can also be a colony growth plate with a solid growth media, such as an agar plate, Petri dish or the like, where individual colonies are grown, e.g., in an array, and produce BDE. See for example Figures E and F.

In alternative embodiments, the systems are used in a high-throughput manner since a large number of cells and /or colonies and/or conditions can be tested in a short period of time as the detectable compound can be spectrophotometrically detected allowing simultaneous detection and assay of each well or colony on a plate. For example, a detectable compound in the membrane can be detected without further operator involvement. In some embodiments, the reacted membrane is removed from the single plate and subjected to means to detect the detectable compound such as by subjecting the membranes to further conditions that develop the color or other detectable property, e.g. fluorescence, of the detectable compound, e.g. with a plate reader.

In another embodiment, a single culture plate is used to grow the array of cells to be assayed for butadiene production and can further comprise a solvent phase in which BD is highly soluble. The solvent can further contain a BDE-reactive compound or enzyme reaction compounds, or the BD-reactive compounds or enzyme reaction reagents can be added at any desired time point. The solvent can be solvent can be more dense that water where the plate is such that it allows spectrophotometric reading through the bottom of the plate, or the solvent can be less dense than water and the detection is made through the top of the plate. BDE is soluble in a number of organic solvents including ethanol, diethyl ether, acetone, benzene, and polar and nonpolar organic solvents compatible with microbial cells.

In alternative embodiments, on contact with air, butadiene can form peroxides, and an inhibitor of butadiene peroxidation, e.g. 4-tertiary-butyl catechol, can be added as needed to reduce peroxide formation.

In another embodiment, a single culture plate is used to grow the array of cells to be assayed for butadiene production, and the cell culture medium is isolated and tested for BD, either directly or indirectly. In one such embodiment, the microtiter culture plate is used to grow the host cells, the cell culture medium is clarified by centrifugation of the plates to create a clear supernatant, the clear supernatant isolated, and the BDE in the supernatant is detected directly or indirectly. In another embodiment, a single culture plate is used to grow the array of cells to be assayed for butadiene production, and the cell culture medium is clarified by addition of a cell lysing agent, and if necessary also a viscosity reducing agent is added, and the clarified cell culture is tested for BD, either directly or indirectly. During fermentation the presence of microbes in the well will typically provide a turbid solution that can interfere with

spectrophotometric methods for detection of produced BD or the detectable compound. A typical fermentation can produce microbial densities from 0.1 to 1.0 UV absorbance units or higher. In one embodiment, to reduce microbial-produced turbidity, a microbe-lysing agent, e.g. a lysozyme, a chelator, an organic solvent, can be added to each well. Suitable lysozymes include hen egg white lysozyme or T4 lysozyme or a combination of lysozymes. Chelators include EDTA or EGTA. Cell lysis and lysis time will be improved if the wells undergo shaking and/or are incubated at room temperature or, more preferably at least at 35-40 degrees. The turbidity decreases upon lysis, and the solution will become sufficiently clear to allow detection methods directly in each well. Should the viscosity increase unacceptably upon lysis due to DNA release from the lysed cells, one or more nucleases can be added, e.g. DNase, Benzonase. To further increase efficiency of lysis, more than one lysis agent can be added. The wells can be heated at even higher temperatures to lyse the cells, where the temperature and time are limited so as not to significantly drive off the soluble BD. For example, 65 degrees for 30 minutes can be used to lyse the microbes.

BDE-Reaction Strategies

The solubility of butadiene in water is 735 mg/L. Thus the cell culture media can contain sufficient BD either for direct detection or for indirect detection, and in view of its partitioning between the liquid and gas phases, the soluble BD will proportionately reflect total BD produced. Alternatively, as BDE is produced it can be immediately reacted with BD-reagent or enzymatic reaction.

Enzymatic Conversion of Butadiene to Detectable Compounds

Enzymatic conversion of butadiene to an intermediate with increased solubility, stability and reduced toxicity offers several advantages. One advantage is that the butadiene-producing organism can more safely be cultured in an oxygen-rich environment. Converting butadiene to a more soluble product will result in increased concentration of the butadiene-derived product in the liquid phase. The use of enzymes (biosynthesized by a microorganism or supplemented to the media) rather than chemical reagents may also reduce cost of reagents, waste disposal and even equipment, if the equipment used in the assay can be simplified or reduced (one plate vs. two, no need for an expensive BD-reactive filter membrane).

Exemplary enzymatic based BD detection reactions include those in which the microbe produces BD and converts it to 3-butene-l,2-diol (Figure D Steps A/B), where the diol is detected directly, for example by Gas chromatography-mass spectrometry (GC-MS), or alternatively, by faster methods using Fourier transform infrared spectroscopy (FTIR) for example. Exemplary enzymatic based BD detection reactions include those in which the microbe produces BD and in a cell free system (e.g. the clarified cell culture or BD trapped in a liquid), the soluble BDE is detected by adding a cofactor and (see Figure D) Enzyme A

(butadiene monooxygenase) and/or D (butadiene monoxide monooxygenase) and a PNTP/MBP to yield a detectable color in plate reader. Exemplary enzymatic based BD detection reactions include those in which the cell or microbe produces BD converts it to 3-butene-l,2-diol, and in a cell free system (e.g. the clarified cell culture or BD trapped in a liquid), the soluble BD detected by adding alcohol dehydrogenase (ADH) enzyme, or 3-butene-l,2-diol dehydrogenase (see Figure D, Step C) and co-factor NAD and measuring NADH formation by change in absorbance using a plate reader.

In one embodiment, a screening organism is developed that contains the butadiene pathway, and expresses enzymes that convert butadiene to 3-butene-l,2-diol (BDD).

Alternatively such enzymes can be added to the cell culture to convert BDE outside the cell. See Figure D. BDD, being more soluble and less volatile than BD, accumulates in the media and can be detected by GCMS or preferably by other faster direct methods described herein such as FTIR. One approach to screening for BDD production is to obtain clarified cell culture, e.g. by centrifugation of the culture plate or by cell lysis, adding an alcohol dehydrogenase that converts 3-butene-l,2-diol to l-hydroxy-2-butanone, l-hydroxy-2-butenone, or 2-hydroxybut-3-eneone. See Figure D.

The following describes approaches for enzymatic transformation of butadiene to 3- butene-l,2-diol or l-hydroxy-2-butanone. These metabolites are less toxic and more easily assayable: Figure D provides an exemplary pathway for converting butadiene to 3-butene-l,2- diol or l-hydroxy-2-butanone. Enzymes are: A. butadiene monooxygenase, B. butadiene monoxide hydrolase, C. 3-butene-l,2-diol dehydrogenase. Since butadiene is readily metabolized by some organisms such as mammals enzymes for BD conversion are available. Metabolism of butadiene has been characterized in mice and other model organisms (Melnick and Huff, Rev Environ Contam Toxicol 124: 111-44 (1992)). The first step of butadiene metabolism in mammals entails oxidation of butadiene to butadiene monoxide (BMO), also called l,2-epoxy-3-butene (Step A of Figure D). BMO is subsequently converted to

intermediates such as diepoxybutane, 3 -butane- 1,2-diol, crotonaldehyde and glutathione conjugates (Kemper et al, Drug Met Disp 29, 830-6 (1991); Sharer et al, Chem Res Toxicol 4:430-6 (1991); Cheng and Ruth, Drug Metab Dispos 21 : 121-4 (1993)).

Butadiene monooxygenase: Butadiene is converted to BMO by an enzyme with butadiene monooxygenase activity (Step A of Figure D). Several classes of enzymes convert butadiene to BMO including cytochrome P450 enzymes, monooxygenases, chloroperoxidase and myeloperoxidase. Enzymes are described in further detail below.

Cytochrome P450 enzymes convert butadiene, 0 ₂ and NAD(P)H to BMO. Enzymes with this activity include CYP2E1 and CYP2A6, found in human liver (Malvoisin et al, J Chromatogr 178:419-25 (1979); Csanady et al, Carcinogenesis 13: 1143-53 (1992), Duescher and Elfarra, Arch Biochem Biophys 311 :342-9 (1994)). Both CYP2E1 and CYP2A6 can further convert BMO to its diepoxide, diepoxybutane (Seaton et al, Carcinogenesis 16:2287-93 (1995)). Further exemplary enzymes are provided in the following table:

Butadiene monooxygenase converts butadiene, 0 ₂ and an electron donor to BMO and an electron acceptor. Enzymes with this activity include methane monooxygenase and toluene monooxygenase. The membrane-bound particulate methane monooxygenase (pMMO) from Methylosinus trichosporum utilizes quinol as an electron donor (Mijaji et al, Methods Enzymol 495: Ch 14 (2011)). Although the enzyme has been crystallized, it has not been functionally expressed in E. coli to date; it has been expressed in Rhodococcus erythropolis . A similar pMMO enzyme is found in Methylococcus capsulatus (Semrau et al, J Bacteriol 177: 3071-9 (1995)). M. trichosporium also has a soluble form of methane monooxygenase, sMMO, which catalyzes the epoxidation of 1,3 -butadiene to BMO (Ono et al, J Mol Catal, 61 : 113-122 (1990)). Further exemplary enzymes are provided in the following table:

Toluene monooxygenase and other non-heme di-iron monooxygenase enzymes also catalyze the conversion of butadiene to BMO. The toluene-4-monooxygenase (T4MO) of Pseudomonas mendocina K 1, when expressed in E. coli, formed epoxides from butadiene and other linear alkenes (Steffan et al, PCT Int Appl 2000073425). The toluene-4-monooxygenase of P. mendocina is encoded by six genes, tmoABCDEF (Yen and Karl, J Bacteriol 174:7253-61 (1992)). Additional monooxygenase enzymes with activity on butadiene are found in Ralstonia pickettii PKOl, Burkholderia cepacia G4 (ATCC 53617), Pseudomonas sp. strain ENVBFl, and Pseudomonas sp. strain ENVPC5, Pseudomonas sp. strain JS150 and Pseudomonas stutzeri OX1 (Steffan et al, PCT Int Appl 2000073425). Several of the enzymes are induced in the presence of toluene. Maintaining control of butadiene monooxygenase under a promoter inducible by toluene or some other inducer would be useful for screening purposes as the BMO forming enzyme could only be expressed during high-throughput screens and not during larger scale fermentations. Further exemplary enzymes are provided in the following table:

BMO hydrolase: BMO is further converted to 3-butene-l,2-diol (BDD) by an enzyme with BMO hydrolase activity. Enzymes with this activity are found in the EC class 3.3.2.

Microsomal and soluble epoxide hydrolase enzymes (EC 3.3.2.9; EC 3.3.2.10) in rat, mouse and human liver catalyze this activity (Cheng and Ruth, Drug Metab Dispos 21 : 121-4 (1993);

Guengerich et al, Rev Biochem Toxicol 4, 5-30 (1982)). The cytosolic epoxide hydrolase from rat liver, cEH, was functionally expressed in E. coli (Knehr et al, J Biol Chem 268: 17623-7 (1993). The microsomal rat liver epoxide hydrolase is encoded yb Ephxl (Qamar et al, Toxicol 291 :25-31 (2012)). Human epoxide hydrolase enzymes are EPHX2 and (Beetham et al, Arch Biochem Biophys 305: 197-201 (1993); Skoda et al, J Biol Chem 263: 1549-54 (1988)).

Another epoxide hydrolase that converts BMO to BDD is hyl 1 of Aspergillus niger (US Patent App 2009/0061494). Further exemplary enzymes are provided in the following table:

3 -Butene-l,2-diol dehydrogenase: BDD is further oxidized to l-hydroxy-2-butanone, 1- hydroxybuteneone or 2-hydroxy-l-ketobutenone by secondary alcohol dehydrogenase enzymes. Alcohol dehydrogenase enzymes from horse liver, and rat, mouse and human cytosols catalyze the oxidation of BDD to l-hydroxy-2-butanone (Kemper et al, Chem Res Toxicol 9, 1127-34 (1996)). Other suitable secondary alcohol dehydrogenase enzymes are found in microorganisms. Secondary alcohol dehydrogenase enzymes from E. coli are malate dehydrogenase (mdh: EC 1.1.1.37, 1.1.1.82, 1.1.1.299) and lactate dehydrogenase (ldhA). S. cerevisiae encodes three copies of malate dehydrogenase, MDH1 (McAlister-Henn and Thompson, J. Bacteriol.

169:5157-5166 (1987), MDH2 (Minard and McAlister-Henn, Mol. Cell. Biol. 11 :370-380 (1991); Gibson and McAlister-Henn, J. Biol. Chem. 278:25628-25636 (2003)), and MDH3 (Steffan and McAlister-Henn, J. Biol. Chem. 267:24708-24715 (1992)), which localize to the mitochondrion, cytosol, and peroxisome, respectively. Close homologs to the cytosolic malate dehydrogenase, MDH2, from S. cerevisiae are found in several organisms including

Kluyveromyces lactis and Candida tropicalis. Alcohol dehydrogenase enzymes of C. beijerinckii (Ismaiel et al, J.Bacteriol. 175:5097-5105 (1993)) and T. brockii (Lamed et al, BiochemJ. 195: 183-190 (1981); Peretz et al, Biochemistry. 28:6549-6555 (1989)) convert acetone to isopropanol. Methyl ethyl ketone reductase catalyzes the reduction of MEK to 2- butanol. Exemplary MEK reductase enzymes can be found in Rhodococcus ruber (Kosjek et al., Biotechnol Bioeng. 86:55-62 (2004)) and Pyrococcus furiosus (van der Oost et al,

Eur.J.Biochem. 268:3062-3068 (2001)). Further exemplary enzymes are provided in the following table:

An example of the enzymatic conversions, from Ono et al. (J Mol Catal, 61 : 113-122 (1990)) cited above, suitable for use herein are shown in the following table showing that methane monooxygenase (pMMO) from Methylosinus trichosporum can convert 1,3-butadiene to 1,2- epoxybut-3-ene, which is readily detected:

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Cytochrome P450 BM3 can epoxidize terminal alkenes (see Kubo et al., 2006). A CytP450 BM3 can be expressed in the cell to convert butadiene to an epoxide. Then, the cell culture is clarified, by centrifugation or cell lysis, and the supernatant is used in a cell-free assay to detect epoxides formed colorimetrically in 96 well plate (see Kubo et al., 2006), for example using 4-(p-nitrobenzyl)pyridine (pNBP) as described in Kubo et al, 2006. Epoxides can also be detected using pNTP (para-nitrothiolphenol). CytP450 uses NADPH and 0 ₂ to epoxidize alkenes and butadiene and crotyl alcohol are both potential substrates. In alternative embodiments, Cyt P450 is used to detect formation of a crotyl alcohol by converting it to an epoxide and then detecting the epoxide colorimetrically using pNTP.

Cyt P450 enzymes have been highly amenable to laboratory evolution. Mutagenesis methods can be used to identify an enzyme variant that can distinguish between butadiene and crotyl alcohol and only epoxidize butadiene. For further enzymes and reaction conditions see also Tee et al. "A p-nitrothiophenolate screening system for the directed evolution of a two- component epoxygenase (StyAB)" Journal of Molecular Catalysis B: Enzymatic . 50 (2008) 121-127; Kubo et al. "Enantioselective Epoxidation of Terminal Alkenes to (R)- and (S)- Epoxides by Engineered Cytochromes P450 BM-3" Chem. Eur. J. (2006), 12, 1216 - 1220; and Schrader PS, Burrows EH, Ely RL. "High-throughput screening assay for biological hydrogen production." Anal Chem (2008) 80(11):4014-9.

Detection of Butadiene by Diels-Alder Reaction or other Reactions

In alternative embodiments, compositions and methods of the invention comprise use of Diels-Alder reactions to detect butadiene (BD). In alternative embodiments, these Diels-Alder reactions can be carried out in an aqueous phase; and the reaction can be tremendously accelerated in water at neutral conditions. Extinction coefficient of maleimides is conducive to the assays described herein. In alternative embodiments, the invention provides screening methods that entail rapid trapping of gaseous butadiene and/or liquid butadiene (which take advantage of butadiene's high reactivity) that allow high throughput screening of cells for BD production. Exemplary chemical methods are described in Figure A and elsewhere herein. The upper reaction in Figure A demonstrates trapping of BD by a Diels-Alder reaction with a maleimide to form a directly detectable compound, as further described herein. One alternative reaction is with an iodoacetamide compound. The middle reaction of Figure A demonstrates trapping of BD by reaction with a maleimide to form a compound that can be further reacted or modified to a readily detectable compound, as further described herein. One alternative is a reaction with an iodoacetamide compound. The lower reaction of Figure A demonstrates trapping of BD by use and "A-B" compound that is an N-heterocyclic carbene or alternatively an inorganic compound, e.g. sulfur. An alternative to each of the above reactions is to detect or measure the amount of BD by detecting or measuring the amount or presence of unreacted BD- reactive or -trapping compound either directly or by its reaction with a second reagent to generate a readily detectable compound.

Maleimides can be directly assayed spectrophotometrically, for example at 302 nm. The extinction coefficient of 620 M-lcm-1 for maleimide is suitable for detection. However, maleimide dyes with higher extinction coefficients are preferred. Alternatively, a colorimetric maleimide assay measures unreacted maleimide can be used. For example, maleimide groups that did not react with BD can be quantified by first reacting a maleimide sample with a known amount of thiol present in excess and then assaying the remaining unreacted thiol, for example using 4,4'-DTDP that has a molar extinction coefficient of 19,800 M-lcm-1. The amount of maleimide is calculated as the difference between the initial amount of thiol and the amount of unreacted thiol after the complete reaction of all maleimide groups. This spectrophotometric assay for the determination of maleimide groups is a reverse glutathione (GSH) assay. It takes advantage of the high reactivity of thiols of GSH (reduced glutathione) with the maleimide moiety. Maleimide of the sample is allowed to form a stable thiosuccinimidyl linkage with GSH. After the reaction of the sample is complete, the excess GSH, i.e., the remaining thiols of GSH in the reaction mixture, is estimated by using 4,4'-DTDP. The amount of GSH reacted with the sample is titrated to determine the extent of maleimide. For more sensitive maleimide quantitation, fluorimetric based spectrophotometric assays are available.

In alternative embodiments, the invention comprises use of a BD-reactive compound that is non-colored or non- fluorescent until reacted with BD or a thiol, or alternatively, a BD-reactive whose conjugate with a thiol is even more colored or fluorescent than its conjugate with BD. An exemplary reactant is a maleimide dye.

In alternative embodiments, a BD-reactive compound can be a maleimide or

iodoacetamide. Optionally the amount or presence of BD is determined by the amount of conjugated BD or optionally the amount or presence of BD is determined by the amount of unreacted maleimide or iodoacetamide that is detected by reaction with a thiol.

In alternative embodiments, a BD-reactive compound can be a maleimide dye or iodoacetamide dye, and optionally the amount or presence of BD is determined by the amount of dye-conjugated BD or optionally the amount or presence of BD is determined by the amount of unreacted maleimide dye or iodoacetamide dye that is detected by reaction with a thiol.

Pyrene Derivatives

In alternative embodiments, an exemplary BD-reactant used to practice the invention is a pyrene maleimide or N-(l-pyrene)maleimide. It is essentially nonfluorescent until it has reacted with a thiol or BD, but once excited, pyrene-thiol or pyrene-BD conjugates can interact to form excited-state dimers (excimers) that emit at longer wavelengths than the excited monomeric fluorophore. Pyrene maleimide conjugates often have very long fluorescence lifetimes (>100 nanoseconds), giving proximal pyrene rings within 6 to 10 A of each other ample time to form the spectrally altered excimer. The amount of BD can be determined either by the amount of pyrene-conjugated BD detected spectrophotometrically or by the amount of unreacted pyrene maleimide that is detected by reaction of the unreacted pyrene maleimide with a thiol. The pyrene maleimide is conducive to any of the HTS apparatuses described herein.

In alternative embodiments, another BD-reactive compound used to practice the invention is non-colored or non- fluorescent until reacted with BD, or with a thiol such as pyrene iodoacetamide, N-(l-pyrenemethyl)iodoacetamide. The BD-reactive compound is essentially nonfluorescent until it has reacted with a thiol or a BD, but once excited it conjugates can interact to form excited-state dimers (excimers) that emit at longer wavelengths than the excited monomeric fluorophore. The amount of BD can be determined either by the amount of pyrene- conjugated BD detected spectrophotometrically or by the amount of unreacted pyrene iodoacetamide that is detected by reaction with a thiol. This compound is conducive to any of the HTS apparatuses described herein.

1,3 -Butadiene reaction with acetylene-dicarboxylic acid (or its di-methyl ester) forming cyclodiene-dicarboxylic acid (or its dimethyl ester)

1,3 -Butadiene can be reacted with acetylene-dicarboxylic acid (or its di-methyl ester) to form cyclodiene-dicarboxylic acid (or its dimethyl ester). The reactions in Figures Bl, B2 and B3 are described in literature and can be adapted to BD screening using the methods and apparatuses described herein. See for example Chandrasekhar et al., Green Chemistry, 2010, Vol 3, No. 1, p 39-47, Letters and Reviews, Reaction of bromine with 4,5-dimethyl-l,4- cyclohexadiene-l,2-dicarboxylic acid: a green chemistry puzzle for organic chemistry students; Grieco et al, JACS 1990, V. 112, No. 11, p. 4595. Dramatic rate accelerations of Diels-Alder reactions in 5 M lithium perchlorate-diethyl ether: the cantharidin problem reexamined; Havis et al. Journal of Agricultural and Food Chemistry, Vol. 45, No. 6, p. 2341-2344, Synthesis, fungicidal activity, and effects on fungal polyamine metabolism of novel cyclic diamines;

Cataldo, Croatica Chemica Acta 2000, 73(2), 435-450, A New Approach to the Anodic

Decarboxylation of Unsaturated Dicarboxylic Acids. Part 1 : Fumaric, Maleic and

Acetylenedicarboxylic Acids; and Emerman and Meinwald, J. Org. Chem., 1956, 21 (3), pp 375-375, The Ultraviolet Absorption Spectrum of 1,4-Cyclohexadien-l-carboxylic Acid, an Intermediate in the Preparation of 1 ,4-Cyclohexadienyl Methyl Ketone.

BD can react with acetylene-dicarboxylic acid to form cyclodiene-dicarboxylic acid or corresponding dimethyl ester. See Figure C. Cyclodiene-dicarboxylic acid (or methyl ester) can be further aromatized to form phthalic acid for more sensitive detection based on UV absorption. All the compounds in the reaction below are available commercially, and their properties and spectra are known. Additional last aromatization step to phthalic acid can be implemented in aqueous solution in the presence of hydrogen peroxide, sodium bromide and hydrochloric acid. Details on UV absorption maxima and extinction coefficients of the compounds can be derived or predicted based on the literature as needed. The BD assay according to the reactions above can be set up using plate screening assay as described herein, for example, in a two-plate apparatus or used in an impregnated-membrane.

Detection of BD using p-Nitro-Benzendiazonium Ion

The presence of butadiene in aqueous solution can be determined by reaction, in an acid medium, e.g., acetic acid, of butadiene with a p-nitro-benzendiazonium ion, e.g., the chloride salt, which yields a yellow product whose optical density is determined photocolorimetrically. The sensitivity of the method for butadiene is 2 micrograms per milliliter. The method is usable with the 2-plate BD trapping apparatus described herein. Alternatively, when used with a single plate system, the cell culture can be acidified and the cells removed or lysed prior to plate reading. Detection of BP using Gas Detector Tubes and Reagents

The presence of butadiene gas can be determined using BD-gas detector tubes or the reagents used in BD-gas detector tubes, for example those from Kitagawa (Pompton Lakes, New Jersey USA) or Gastec (Kanagawa, Japan). Butadiene Gas Detector Tubes that can be adapted directly to a single plate or dual plate system include Gastec tube product numbers 174, 174L and 174LL and Kitagawa tubes 168A, 168B, 168C, 168SA, 168SB, 168SC and 168SE, commercially available. The tubes can be used to sample the BD gas in the headspace of the culture plate or the upper plate in a dual plate system. For example, active, open tubes can be inserted in a manifold of the same spatial array pattern as the culture plate or upper plate, and the manifold placed over the plate at the desired time point such that each tube in the array is in contact with its corresponding well in the plate array. The tubes are read according to manufactures instructions; typically either the position of color development along the length of the tube or the intensity and/or hue of the developed color will indicate the presence and/or amount of BD gas detected in the given volume of headspace. If needed, a vacuum can be applied to the distal end of each tube to draw up a precise amount of headspace air into each tube to further enhance accuracy of the readings.

One alternative is to use the reagents contained within each tube to impregnate a membrane, e.g. filter paper, to create a BD-reactive membrane as described herein, or to place the dry reagents in an upper plate of a two plate system without any liquid. In the latter case, the reagents can be placed directly in the upper plate wells as found in the gas detector tubes- adsorbed to a solid support such as silica gel or silica glass--, or the reagents can be placed in the wells without their solid support. The presence or amount of BD is determined calorimetrically, preferably by a plate reader or CCD image reader. The color amount, intensity and hue can be measured. IN addition, in a further embodiment, the area of the color spot can be measured, preferably with a plate reader or CCD image reader, since the amount of diffusing BD gas can determine the size of the reacted, and thus colored, area, e.g. more gas diffusing to and reacting with reagent further from the point of contact of the membrane with the headspace. In a further embodiment the surface area contact of the membrane with the headspace is physically limited by a window or aperture having an opening less than the area of the plate well and oriented so that its opening is essentially in the center of the window or aperture. This restricts the headspace BD gas to contacting the membrane at a relatively small point that will allow the BD-gas to diffuse from that point to the surrounding reactive-area of the membrane above that well; the extent of the diffusion and thus size of the colored spot, reflecting the amount of BD present in the headspace. This is similar to the theory used in gas detector tubes but employing a radial rather than linear gas diffusion and color development.

The BD-trapping color reactions include (a) reduction of chromate or dichromate to chromous ion, which produces a pale yellow to pale blue color change, (b) reduction of ammonium molybdate plus palladium sulfate to molybdenum blue which produces a pale yellow to white color change, and (c) reduction of potassium permanganate to produce a pink to white color change.

High-throughput assays using a membrane

Figures E and F illustrate alternative embodiments of high-throughput assays of the invention using a membrane, e.g. filter paper, impregnated with BD-reactive compound over a microtiter plate (Figure E) or over a colony dish (Figure F). The BD-sensitive membrane in which the BD reaction can be detected spectrophotometrically allows screening of large numbers of colonies on solid media for BD production. Individual colonies surviving mutagenesis and selection or screening, or engineered colonies, are transferred to microtiter plates or to dish, e.g. agar based Petri dish, that can easily accommodate an a colony matrix and a chemo-chromic sensor membrane. If desired during or following an appropriate growth period, the plates can be made to go anaerobic to order to induce BD production. The reactive membrane (sensor layer) can be applied and the colonies produce BD. At the end of the desired production period, the sensor layers are analyzed for the location of spots or "dots" where BD reacted with the chemical reagent on the sensor layer. If necessary, the sensors layers can be removed and the BD-sensing reaction developed prior to detection of, and quantitation of, the reactive spots. The spots correspond to the microbial colony that evolved or improved BD-production under the conditions tested. The identified clones can be transferred from the original plate to liquid media for further cultivation and additional characterization.

Preparing the Membrane Sensors

A wide variety of inert solid porous supports can be employed for the BD-reactant impregnated membrane (i.e. sensor layer, detector layer, filter paper). The supports may be in the form of sheets or strips or any shape compatible with the cell culture plate or colony dish or plate. For example, filter papers formed from cellulose fibers or glass fibers can be used, as well as woven cloth formed from cotton or synthetic fibers. In alternative embodiments, the membrane is inert to BD. The membrane can be a po!ytetrafluoroethy!ene (PTFE), nylon, nitrocellulose, parafilm or other BD-resistant porous plastic membrane. In alternative embodiments, the support is readily wettable by aqueous solutions. The porosity can be such that the membrane retains the reactant solution over the period of use.

In alternative embodiments the membrane is impregnated with a BD-reactant, and optionally a buffer, catalyst or co-factor, and where an enzymatic reaction is employed, one or more enzymes. The membranes may be used immediately as prepared or stored for later use.

Direct Butadiene Detection Approaches

Direct Detection by GC and GCMS (gas chromatography mass spectrometry)

BD can be monitored by conventional gas chromatography-flarne ionization detector (GC-FID) or GCMS by either sampling the headspace or the culture medium of the cell culture vials/wells. Various GC columns are available for that: PLOT (porous layer open tubular, particle-based stationary phase) type columns are typically interfaced with FID (flame ionization detectors), and mass spec compatible proprietary phase capillary columns, e.g. GasPro (Agilent). Using high selectivity MS detector is preferred. Cryo-cooling of GC oven using liquid N2 is typically required for gas measurements to ensure adequate retention on the column and separation efficiency. Headspace GC analysis has advantage of cleaner samples and low background interference resulting into higher signal to noise ratio and better detection limits. BD solutions can be analyzed as well, preferably, following by liquid- liquid extraction of BD into organic solvent (e.g. toluene). In HTP plate format LLE can be done by using the same sandwich plate system, described by Cara and references therein, where organic solvent is in the upper filter plate trapping BD escaping from the cell cultures. It might be of importance to use glass microtiter plates (e.g. in the form of inserts) for BD cell culture experiments, considering possible reactivity of BD and diffusion into plastic materials.

GCMS approaches to monitor gas fermentation products (e.g. BD) are described in a number of patents and journal articles. See U.S. Patent Number 5,849,970 entitled "Materials and methods for the bacterial production of BD" and see WO2009132220 entitled "BD synthase variants for improved microbial production of BD." While such methods are useful, they are not conducive to high-throughput screens in large part due to the general requirement for sequential sampling of wells and the amount of time needed for each separation and detection.

Direct Detection by FTIR and FTNIR

1,3-Butadiene molecule possesses conjugated double bonds which exhibit a quite unique absorption band pattern in IR and NIR spectra. There are examples in the literature describing direct online or offline quantitative monitoring of BD or similar molecules, either their formation or consumption, in chemical reactions, polymerization or depolymerization processes, in solution and/or gas phase, using transmission and/or ATR (attenuated total reflection) probes. A number of vibrational C-H and C=C bond stretches give a characteristic signature of IR and NIR spectra of BD. NIR spectra are based on combinations of overtone transitions corresponding to IR vibrational bands. NIR is likely to be a method of choice for analyzing aqueous solutions due to high interference of water signal in IR. Modern chemometrics approaches and data processing packages based on Fourier transformation, PLS (partial least square regression) and PCA (principal components) analysis, allow quantitative and sensitive measurements in complex matrices, such as fermentation broth or cell cultures. Commercial FTIR and FTNIR systems are available from Bruker, Thermofisher and other vendors. Bruker offers a HTP version model, HTS-XT 96-well plate reader coupled to a Vertex 70 FTIR/NIR light source and detector. BD monitoring in transmission mode in a 96 well plate, interrogating both solution (cell culture) and headspace, can be envisioned. In one embodiment the system uses a plate design enabling handling growing cultures followed by FTNIR detection without interruption and/or sample transfer procedures.

Descriptions of real-time FTIR monitoring include: Shikh et al, Journal of polymer science A, Polymer chemistry, 2004, 42, 4084, entitled "A new high-throughput approach to measure copolymerization reactivity ratios using real-time FTIR monitoring."; Denzer et al., Analyst, 2011, 136(4), 801-806, entitled "Trace species detection in the near infrared using Fourier transform broadband cavity enhanced absorption spectroscopy: initial studies on potential breath analytes." For example, see Figure 2 of Denzer et al. reporting measurement of neat BDE using FTIR. See also Denzer et al, Analyst (Cambridge, United Kingdom) (2009), 134(11), 2220-2223, Near-infrared broad-band cavity enhanced absorption spectroscopy using a superluminescent light emitting diode.; Mendes et al. Analytica Chimica Acta (2003), 493(2), 219-231, Determination of ethanol in fuel ethanol and beverages by Fourier transform (FT)-near infrared and FT-Raman spectrometries; and Lanzendoerfer et al., 221st ACS National Meeting], San Diego, CA, United States, April 1-5, 2001 (2003), 67-81, In Situ Spectroscopy of Monomer and Polymer Synthesis Application of FT-NIR spectroscopy for monitoring the kinetics of living polymerizations.

Direct Detection by LAS (laser absorption spectroscopy) and OA ICOS (off-axis integrated cavity output spectroscopy)

LAS and its enhanced version OA ICOS techniques are similar to IR / NIR spectroscopy but utilize tunable near infrared lasers to excite molecules in gas phase and generate absorption spectra. LAS devised have advantages of high sensitivity due to monochromaticity and high energy of laser light, high selectivity and speed of measurements due to high spectral resolution

(l-3MHz) of modern lasers. Off-axis integrated cavity designed can greatly enhance sensitivity by increasing optical path-length up to several kilometers. See for example,

www.gasesmag.com, May/June 2012, p. 23, M. Gupta, Cavity-enhanced laser absorption spectroscopy for industrial applications, and in particular Figures 2 and 3 therein. See also

Industrial & Engineering Chemistry Research (2012), 51(39), 12674-12684. Knighton, W. Berk;

Herndon, Scott C; Franklin, Jon F.; Wood, Ezra C; Wormhoudt, Jody; Brooks, William;

Former, Edward C; Allen, David T. Direct measurement of volatile organic compound emissions from industrial flares using real-time online techniques: Proton Transfer Reaction

Mass Spectrometry and Tunable Infrared Laser Differential Absorption Spectroscopy; Faming

Zhuanli Shenqing Gongkai Shuomingshu (2009), CN 101545857 A 20090. Xing, Longchun;

Liu, Haisheng; Liu, Pei; Li, Ting; Fan, Xiaowei; Du, Wenxuan; Wu, Kefeng; Zhao, Xiuqin; Li,

Bin; Xu, Cuihong. Method for determining oxygen content in butadiene gas by diode laser absorption spectrometry; Icarus (1995), 116(2), 415-22; and Fahr, Askar; Monks, Paul S.; Stief,

Louis J.; Laufer, Allan H. Experimental determination of the rate constant for the reaction of

C2H3 with H2 and implications for the partitioning of hydrocarbons in atmospheres of the outer planets.

Direct Detection by BDE Sensors

Butadiene can be directly detected in the headspace of each well of the culture plate using a non-intrusive sensor designed to detect and monitor butadiene in air using electrochemical sensor technology. A sensor is fluidly and removably connected to the headspace of a well. In one embodiment a sensor is dedicated to each well in a microtiter plate such that multiple sensors would be used, for example, a 96-well plate could have up to 96 sensors. In another

embodiment a sensor is sequentially connected to multiple wells to read BD in each well. The plug-in, field replaceable detection cell provides automatic recognition of gas type and range, and features over-sized gold-plated connections that help prevent corrosion. Sensors sensitivity in the range of 0-100 ppm can be used, but the sensitivity will depend on rate of BDE

production. For example, the Detcon Model DM-700-C4H6 (Detcon, Inc. The Woodlands, Texas USA) is an electro-polished 316 stainless steel housing with fully encapsulated electronics and dual layer surge protection. The Model 700 is equipped with standard analog 4-20mA, and Modbus™ RS-485 outputs and has a wireless option that can be used to transmit measurement data over time. The Detcon Model DM-100-C4H6 is a gas detection sensor designed to detect and monitor Butadiene in air over the range of 0-100 ppm using electrochemical sensor technology whose method of detection is by diffusion adsorption. Air and gas molecules diffuse through a porous membrane contacting an electrolyte solution which creates a change in electrical conductance between a reference and measure electrode. This change in conductance is conditioned by internal electronic circuitry to provide a linear 4-20 milliamp signal proportional to the gas concentration.

In alternative embodiments, the sensor is connected to the headspace in a way that allows BD diffusion from the headspace to the sensor. As in any of the methods herein, the sensor can be connected and measurements taken at any period of time during culturing, e.g. during growth or production phase or both.

In one embodiment is a manifold containing an array of plugs or caps, one for each well of the culture plate, and each plug or cap connected to a tube connecting to the BD sensor, and that is put in place at the desire time period. The plug or cap contains a tube or passageway connecting the well's headspace to BD sensor, allowing diffusion of the BDE gas directly to the sensor. At the desired time point, the array of plugs or caps can be placed in functional contact with the culture plate, for example by replacing a lid present on the cell culture plate.

Alternatively, the lid of each culture plate can have a flexible, puncturable seal, e.g. a rubber or silicone seal or a pre-split rubber or silicone seal, which when in contact with the array of plugs flexes to open sufficiently to allow headspace gas to diffuse into the plug and to the BDE sensor. Alternatively, each plug of the array can contain a needle that punctures the flexible seal to allow contact between the headspace and the BDE sensor. Removal of the needle allows closure of the seal for continued fermentation of the culture if desired. In a further embodiment the plug can contain a passageway or gas permeable membrane allow passage of oxygen or air into the cell culture as needed.

The use of an array of BD sensors, coupled with a corresponding array of plugs or caps or needles, essentially one for each well of a plate, enables simultaneous detection and

measurement of BDE in each well of that plate enabling high-throughput analysis.

In a further embodiment, the sensor is equipped with a means to flush air thru its detector without disturbing the well headspace.

While any microtiter plate is suitable for the HTS methods and apparatuses described herein, alternatively are microtiter plates suitable for HTS with at least 96, 384, 1536, and even 3456 wells per plate. Microtiter plates for cell culture can be those modified for allowing sparging of gases, e.g. oxygen, at desired rates, aerobic, microaerobic, non-aerobic, during the fermentation. Microtiter plates modified to monitor or control process information are suitable, such as those that where process information can be acquired in microtiter plates, such as pH, dissolved oxygen tension (DOT), dissolved carbon dioxide tension (DCT) and temperature (T). Commercially, microtiter plates with integrated pH- or DOT-optodes are available including those for 96-well microtiter plates (Precision Sensing GmbH, Regensburg, Germany).

The apparatuses and methods described herein are suitable for use with common microplate readers or the BioScreen C reader (Oy Growth Curves Ab Ltd., Finland). In alternative embodiments, the plate reader is adapted for automated reading of microtiter plates. Fluorescence measurements using microplate readers are very common and widely applied in biotechnology and are suitable depending on the selected BD-reactant as described herein.

In accordance with the subject invention, characteristic changes can include, but are not limited to: optical changes; i.e., optical changes observed by means of light source such as fluorescence, chemiluminescence, or electrogenerated chemiluminescence changes; electrical changes (i.e., change in electrical current through at least one nanochannel in a membrane); changes in fluid flow (either gas or liquid) through at least one nanochannel; change in signal agent (i.e., target analyte-induces an increase or decrease in the concentration of a particular signal agent such as hydronium ion (see change in pH)). The assay means for detecting such characteristic changes include, but are not limited to, fluorescence spectroscopy; UV-VIS absorption spectroscopy; Raman spectroscopy; Fourier transform infrared spectroscopy (FTIR); nuclear magnetic resonance (NMR); electrochemical methods such as amperometry or cyclic voltammetry (if the signaling agent is redox active), potentiometry (if the signaling agent is an ion), and radiometric methods (if the signaling agent is radioactive); and the like.

In further embodiments, the assay means involves various known techniques and/or

instrumentation that enable the detection of signaling agents that are released from at least one nanochannel when exposed to a target analyte. For example, assay means of the invention for detecting signaling agents include, but are not limited to, gross examination (e.g., detection with the human eye/by observation); radiographic systems; and microscopic systems (e.g., light microscopy, transmission electron microscopy, and laser capture microscopy).

Exemplary Cell Culture Media

In alternative embodiments, the invention comprises use of a cell or a population of cells in a culture to produce butadiene (1,3 -butadiene, BD). In alternative embodiments, "cells in culture" is meant two or more cells in a solution (e.g., a cell medium) that allows the cells to undergo one or more cell divisions. Alternatively, the cells can be not dividing or growing, but vivable, and producing BD.

Any carbon source can be used to cultivate the host cells. In alternative embodiments the term "carbon source" refers to one or more carbon-containing compounds capable of being metabolized by a host cell or organism. For example, the cell medium used to cultivate the host cells may include any carbon source suitable for maintaining the viability or growing the host cells. In some embodiments, the carbon source is a carbohydrate (such as monosaccharide, disaccharide, oligosaccharide, or polysaccharides), invert sugar (e.g., enzymatically treated sucrose syrup), glycerol, glycerine (e.g., a glycerine byproduct of a biodiesel or soap-making process), dihydroxyacetone, one-carbon source, fatty acid (e.g., a saturated fatty acid, unsaturated fatty acid, or polyunsaturated fatty acid), lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, polypeptide (e.g., a microbial or plant protein or peptide), renewable carbon source (e.g., a biomass carbon source such as a hydrolyzed biomass carbon source; beet sugar or cane sugar molasses), yeast extract, component from a yeast extract, polymer, acid, alcohol, aldehyde, ketone, amino acid, succinate, lactate, acetate, ethanol, or any combination of two or more of the foregoing. In some embodiments, the carbon source is a product of photosynthesis, including, but not limited to, glucose. Exemplary monosaccharides include glucose and fructose; exemplary oligosaccharides include lactose and sucrose, and exemplary polysaccharides include starch and cellulose. Exemplary carbohydrates include C6 sugars (e.g., fructose, mannose, galactose, or glucose) and C5 sugars (e.g., xylose or arabinose). In some embodiments, the cell medium includes a carbohydrate as well as a carbon source other than a carbohydrate (e.g., glycerol, glycerine, dihydroxyacetone, one-carbon source, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, or a component from a yeast extract). In some embodiments, the cell medium includes a carbohydrate as well as a polypeptide (e.g., a microbial or plant protein or peptide). In some embodiments, the microbial polypeptide is a polypeptide from yeast or bacteria. In some embodiments, the plant polypeptide is a polypeptide from soy, corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower, sesame, or linseed.

In alternative embodiments the concentration of the carbohydrate is at least or about 5 grams per liter of broth (g/L, wherein the volume of broth includes both the volume of the cell medium and the volume of the cells), such as at least or about 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, 300, 400, or more g/L. In some embodiments, the concentration of the carbohydrate is between about 50 and about 400 g/L, such as between about 100 and about 360 g/L, between about 120 and about 360 g/L, or between about 200 and about 300 g/L. In some embodiments, this concentration of carbohydrate includes the total amount of carbohydrate that is added before and/or during the culturing of the host cells.

In alternative embodiments, exemplary lipids are any substance containing one or more fatty acids that are C4 and above fatty acids that are saturated, unsaturated, or branched.

Exemplary fatty acids include compounds of the formula R-COOH, where "R" is a hydrocarbon. Exemplary unsaturated fatty acids include compounds where "R" includes at least one carbon- carbon double bond. Exemplary unsaturated fatty acids include, but are not limited to, oleic acid, vaccenic acid, linoleic acid, palmitelaidic acid, and arachidonic acid. Exemplary polyunsaturated fatty acids include compounds where "R" includes a plurality of carbon-carbon double bonds. Exemplary saturated fatty acids include compounds where "R" is a saturated aliphatic group. In some embodiments, the carbon source includes one or more C12-C22 fatty acids, such as a Cu saturated fatty acid, a C _M saturated fatty acid, a Ci ₆ saturated fatty acid, a C ₁₈ saturated fatty acid, a C20 saturated fatty acid, or a C22 saturated fatty acid. In an exemplary embodiment, the fatty acid is palmitic acid. In some embodiments, the carbon source is a salt of a fatty acid (e.g., an unsaturated fatty acid), a derivative of a fatty acid (e.g., an unsaturated fatty acid), or a salt of a derivative of fatty acid (e.g., an unsaturated fatty acid). Suitable salts include, but are not limited to, lithium salts, potassium salts, sodium salts, and the like. Di- and triglycerols are fatty acid esters of glycerol. In alternative embodiments, the concentration of the lipid, fatty acid, monoglyceride, diglyceride, or triglyceride is at least or about 1 gram per liter of broth (g/L, wherein the volume of broth includes both the volume of the cell medium and the volume of the cells), such as at least or about 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, 300, 400, or more g/L. In some embodiments, the concentration of the lipid, fatty acid, monoglyceride, diglyceride, or triglyceride is between about 10 and about 400 g/L, such as between about 25 and about 300 g/L, between about 60 and about 180 g/L, or between about 75 and about 150 g/L. In some embodiments, the concentration includes the total amount of the lipid, fatty acid, monoglyceride, diglyceride, or triglyceride that is added before and/or during the culturing of the host cells. In some embodiments, the carbon source includes both (i) a lipid, fatty acid, monoglyceride, diglyceride, or triglyceride and (ii) a carbohydrate, such as glucose. In some embodiments, the ratio of the lipid, fatty acid, monoglyceride, diglyceride, or triglyceride to the carbohydrate is about 1 : 1 on a carbon basis (i.e., one carbon in the lipid, fatty acid, monoglyceride, diglyceride, or triglyceride per carbohydrate carbon). In particular embodiments, the amount of the lipid, fatty acid, monoglyceride, diglyceride, or triglyceride is between about 60 and 180 g/L, and the amount of the carbohydrate is between about 120 and 360 g/L.

In alternative embodiments, exemplary microbial polypeptide carbon sources include one or more polypeptides from yeast or bacteria. Exemplary plant polypeptide carbon sources include one or more polypeptides from soy, corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower, sesame, or linseed.

In alternative embodiments, renewable carbon sources are used, including e.g., cheese whey permeate, cornsteep liquor, sugar beet molasses, barley malt, and components from any of the foregoing. Exemplary renewable carbon sources also include glucose, hexose, pentose and xylose present in biomass, such as corn, switchgrass, sugar cane, cell waste of fermentation processes, and protein by-product from the milling of soy, corn, or wheat. In some embodiments, the biomass carbon source is a lignocellulosic, hemicellulosic, or cellulosic material such as, but are not limited to, a grass, wheat, wheat straw, bagasse, sugar cane bagasse, soft wood pulp, corn, corn cob or husk, corn kernel, fiber from corn kernels, corn stover, switch grass, rice hull product, or a by-product from wet or dry milling of grains (e.g., corn, sorghum, rye, triticate, barley, wheat, and/or distillers grains). Exemplary cellulosic materials include wood, paper and pulp waste, herbaceous plants, and fruit pulp. In some embodiments, the carbon source includes any plant part, such as stems, grains, roots, or tubers. In some embodiments, all or part of any of the following plants are used as a carbon source: corn, wheat, rye, sorghum, triticate, rice, millet, barley, cassava, legumes, such as beans and peas, potatoes, sweet potatoes, bananas, sugarcane, and/or tapioca. In some embodiments, the carbon source is a biomass hydrolysate, such as a biomass hydrolysate that includes both xylose and glucose or that includes both sucrose and glucose. In alternative embodiments, the renewable carbon source (such as biomass) is pretreated before it is added to the cell culture medium. In some embodiments, the pretreatment includes enzymatic pretreatment, chemical pretreatment, or a combination of both enzymatic and chemical pretreatment (see, for example, Farzaneh et al, Bioresource Technology 96 (18): 2014- 2018, 2005; U.S. Patent No. 6,176,176; U.S. Patent No. 6,106,888; which are each hereby incorporated by reference in their entireties, particularly with respect to the pretreatment of renewable carbon sources). In some embodiments, the renewable carbon source is partially or completely hydrolyzed before it is added to the cell culture medium. In some embodiments, the renewable carbon source (such as corn stover) undergoes ammonia fiber expansion (AFEX) pretreatment before it is added to the cell culture medium (see, for example, Farzaneh et ah, Bioresource Technology 96 (18): 2014-2018, 2005). During AFEX pretreatment, a renewable carbon source is treated with liquid anhydrous ammonia at moderate temperatures (such as about 60 to about 100 °C) and high pressure (such as about 250 to about 300 psi) for about 5 minutes. Then, the pressure is rapidly released. In this process, the combined chemical and physical effects of lignin solubilization, hemicellulose hydrolysis, cellulose decrystallization, and increased surface area enables near complete enzymatic conversion of cellulose and

hemicellulose to fermentable sugars. AFEX pretreatment has the advantage that nearly all of the ammonia can be recovered and reused, while the remaining serves as nitrogen source for microbes in downstream processes. Also, a wash stream is not required for AFEX pretreatment. Thus, dry matter recovery following the AFEX treatment is essentially 100%. AFEX is basically a dry-to-dry process. The treated renewable carbon source is stable for long periods and can be fed at very high solid loadings in enzymatic hydrolysis or fermentation processes. Cellulose and hemicellulose are well preserved in the AFEX process, with little or no degradation. There is no need for neutralization prior to the enzymatic hydrolysis of a renewable carbon source that has undergone AFEX pretreatment. Enzymatic hydrolysis of AFEX-treated carbon sources produces clean sugar streams for subsequent fermentation use.

In alternative embodiments, the concentration of the carbon source (e.g., a renewable carbon source) is equivalent to at least or about 0.1, 0.5, 1, 1.5 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50% glucose (w/v). The equivalent amount of glucose can be determined by using standard HPLC methods with glucose as a reference to measure the amount of glucose generated from the carbon source. In some embodiments, the concentration of the carbon source (e.g., a renewable carbon source) is equivalent to between about 0.1 and about 20% glucose, such as between about 0.1 and about 10%> glucose, between about 0.5 and about 10%> glucose, between about 1 and about 10%) glucose, between about 1 and about 5%> glucose, or between about 1 and about 2%> glucose. In some embodiments, the carbon source includes yeast extract or one or more components of yeast extract. In some embodiments, the concentration of yeast extract is at least 1 gram of yeast extract per liter of broth (g/L, wherein the volume of broth includes both the volume of the cell medium and the volume of the cells), such at least or about 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, 300, or more g/L. In some embodiments, the concentration of yeast extract is between about 1 and about 300 g/L, such as between about 1 and about 200 g/L, between about 5 and about 200 g/L, between about 5 and about 100 g/L, or between about 5 and about 60 g/L. In some embodiments, the concentration includes the total amount of yeast extract that is added before and/or during the culturing of the host cells. In some embodiments, the carbon source includes both yeast extract (or one or more components thereof) and another carbon source, such as glucose. In some embodiments, the ratio of yeast extract to the other carbon source is about 1 :5, about 1 : 10, or about 1 :20 (w/w).

In alternative embodiments the carbon source is a one-carbon substrate such as carbon dioxide, or methanol. Glycerol production from single carbon sources (e.g., methanol, formaldehyde, or formate) has been reported in methylotrophic yeasts (Yamada et al., Agric. Biol. Chem., 53(2) 541-543, 1989, which is hereby incorporated by reference in its entirety, particularly with respect to carbon sources) and in bacteria (Hunter et. al, Biochemistry, 24, 4148-4155, 1985, which is hereby incorporated by reference in its entirety, particularly with respect to carbon sources). These organisms can assimilate single carbon compounds, ranging in oxidation state from methane to formate, and produce glycerol. The pathway of carbon assimilation can be through ribulose monophosphate, through serine, or through xylulose- monophosphate (Gottschalk, Bacterial Metabolism, Second Edition, Springer- Verlag: New York, 1986, which is hereby incorporated by reference in its entirety, particularly with respect to carbon sources). The ribulose monophosphate pathway involves the condensation of formate with ribulose-5 -phosphate to form a six carbon sugar that becomes fructose and eventually the three carbon product glyceraldehyde- 3 -phosphate. Likewise, the serine pathway assimilates the one-carbon compound into the glycolytic pathway via methylenetetrahydrof olate.

In alternative embodiments, in addition to one and two carbon substrates, methylotrophic organisms are also known to utilize a number of other carbon containing compounds such as methylamine, glucosamine and a variety of amino acids for metabolic activity. For example, methylotrophic yeast are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion et al, Microb. Growth CI Compd., Int. Symp., 7 ^th ed., 415-32. Editors: Murrell et al, Publisher: Intercept, Andover, UK, 1993, which is hereby incorporated by reference in its entirety, particularly with respect to carbon sources). Similarly, various species of Candida metabolize alanine or oleic acid (Suiter et al., Arch. Microbiol. 153(5), 485-9, 1990, which is hereby incorporated by reference in its entirety, particularly with respect to carbon sources).

[0271] In some embodiments, cells are cultured in a standard medium containing physiological salts and nutrients (see, e.g., Pourquie, J. et al, Biochemistry and Genetics of Cellulose

Degradation, eds. Aubert et al, Academic Press, pp. 71-86, 1988; and Ilmen et al, Appl.

Environ. Microbiol. 63: 1298-1306, 1997, hereby incorporated by reference, particularly with respect to cell media). Exemplary growth media are common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, or Yeast medium (YM) broth. Other defined or synthetic growth media may also be used, and the appropriate medium for growth of particular host cells are known by someone skilled in the art of microbiology or fermentation science.

In alternative embodiments, in addition to an appropriate carbon source, the cell medium may comprise suitable minerals, salts, cofactors, buffers, and other components known to those skilled in the art suitable for the growth of the cultures or the enhancement of BD production (see, for example, WO 2004/033646; WO 96/35796). Microbes that produce Butadiene

In alternative embodiments, methods and apparatuses of the invention are suitable for use in screening naturally occurring microorganisms or non-naturally occurring microbial organisms containing butadiene biosynthetic pathways. The pathways can comprise endogenous genes, and further can be combined with modifications of the organism, such as deletions or additions of genetic elements, such as promoters and genes that encode enzymes or proteins that enhance butadiene production. The pathway can comprise at least one exogenous nucleic acid encoding a butadiene pathway enzyme expressed in a sufficient amount to produce butadiene. The methods and apparatuses for screening are suitable for use with such microbial organisms that produce butadiene by culturing the natural or non-naturally occurring microbial organism containing butadiene pathways as described herein under conditions and for a sufficient period of time to produce butadiene.

In alternative embodiments, any microbe or bio-synthetic pathway that produces butadiene can be used to practice the invention, and microbes that produce butadiene, biosynthetic pathways for producing butadiene, methods to engineer microbes to produce butadiene, target enzymes and proteins for improving butadiene production, and methods to create libraries of butadiene producing microbes are described in the literature, for example see WIPO Patent Application publication WO/2011/140171, published 10 November 2011, entitled

Microorganisms And Methods For The Biosynthesis Of Butadiene, and see WIPO Patent Application publication WO/2012/106516 published 8 September 2012 entitled Microorganisms And Methods for the Biosynthesis of Butadiene.

In some embodiments, the invention provides a non-naturally occurring microbial organism, including a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding a butadiene pathway enzyme expressed in a sufficient amount to produce butadiene, the butadiene pathway including an acetyl-CoA:acetyl-CoA

acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonyl- CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase, or transferase, a crotonate reductase, a crotonyl-CoA reductase (alcohol forming), a glutaconyl-CoA decarboxylase, a glutaryl-CoA dehydrogenase, an 3-aminobutyryl-CoA deaminase, a 4-hydroxybutyryl-CoA dehydratase or a crotyl alcohol diphosphokinase (Figure 1). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3- hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a

crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene synthase (Figure 1, steps A-H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an acetyl-CoA:acetyl- CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA reductase (alcohol forming) (Figure 1, steps A-C, K, F, G, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an acetyl-CoA:acetyl- CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a butadiene synthase, a crotonyl-CoA reductase (alcohol forming) and a crotyl alcohol diphosphokinase (Figure 1, steps A-C, K, P, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase, or transferase and a crotonate reductase, (Figure 1, steps A-C, I, J, E, F, G, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an acetyl-CoA:acetyl- CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonaldehyde reductase (alcohol forming), a butadiene synthase, a crotonyl-CoA hydrolase, synthetase or transferase, a crotonate reductase and a crotyl alcohol diphosphokinase (Figure 1, steps A-C, I, J, E, P, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a butadiene synthase and a crotyl alcohol diphosphokinase (Figure 1, steps A-E, P, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaconyl-CoA decarboxylase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene synthase (Figure 1 , steps L, D-H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaconyl-CoA decarboxylase, a crotyl alcohol kinase, a 2-butenyl-4- phosphate kinase, a butadiene synthase and crotonyl-CoA reductase (alcohol forming) (Figure 1 , steps L, K, F, G, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaconyl-CoA decarboxylase, a butadiene synthase, a crotonyl- CoA reductase (alcohol forming) and a crotyl alcohol diphosphokinase (Figure 1, steps L, K, P, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaconyl-CoA decarboxylase, a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase, or transferase and a crotonate reductase (Figure 1, steps L, I, J, E, F, G, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaconyl-CoA decarboxylase, a crotonaldehyde reductase (alcohol forming), a butadiene synthase, a crotonyl-CoA hydrolase, synthetase or transferase, a crotonate reductase and a crotyl alcohol diphosphokinase (Figure 1, steps L, I, J, E, P, H). In one aspect, the non- naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 3- hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a

crotonaldehyde reductase (alcohol forming), a butadiene a glutaconyl-CoA decarboxylase and a crotyl alcohol diphosphokinase (Figure 1, steps L, C, D, E, P, H). In one aspect, the non- naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaryl-CoA dehydrogenase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene synthase (Figure 1, steps M, D-H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaryl-CoA dehydrogenase, a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA reductase (alcohol forming) (Figure 1, steps M, K, F, G, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaryl-CoA dehydrogenase, a butadiene synthase, a crotonyl-CoA reductase (alcohol forming) and a crotyl alcohol diphosphokinase (Figure 1, steps M, K, P, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaryl-CoA dehydrogenase, a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase, or transferase and a crotonate reductase (Figure 1, steps M, I, J, E, F, G, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a glutaryl-CoA dehydrogenase, a crotonaldehyde reductase (alcohol forming), a butadiene synthase, a crotonyl- CoA hydrolase, synthetase or transferase, a crotonate reductase and a crotyl alcohol

diphosphokinase (Figure 1, steps M, I, J, E, P, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a butadiene synthase, a glutaryl-CoA dehydrogenase and a crotyl alcohol

diphosphokinase (Figure 1, steps M, C, D, E, P, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an 3-aminobutyryl-CoA deaminase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene synthase (Figure 1, steps N, D-H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an 3-aminobutyryl-CoA deaminase, a crotyl alcohol kinase, a 2- butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA reductase (alcohol forming) (Figure 1, steps N, K, F, G, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an 3-aminobutyryl-CoA deaminase, a butadiene synthase, a crotonyl-CoA reductase (alcohol forming) and a crotyl alcohol diphosphokinase (Figure 1, steps N, K, P, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an 3-aminobutyryl-CoA deaminase, a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase, or transferase and a crotonate reductase (Figure 1, steps N, I, J, E, F, G, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an 3-aminobutyryl- CoA deaminase, a crotonaldehyde reductase (alcohol forming), a butadiene synthase, a crotonyl- CoA hydrolase, synthetase or transferase, a crotonate reductase and a crotyl alcohol

diphosphokinase (Figure 1, steps N, I, J, E, P, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a butadiene synthase, a 3-aminobutyryl-CoA deaminase and a crotyl alcohol diphosphokinase (Figure 1, steps N, C, D, E, P, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 4-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase and a butadiene synthase (Figure 1, steps O, D-H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 4-hydroxybutyryl-CoA dehydratase, a crotyl alcohol kinase, a 2- butenyl-4-phosphate kinase, a butadiene synthase and crotonyl-CoA reductase (alcohol forming) (Figure 1, steps O, K, F, G, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 4-hydroxybutyryl-CoA dehydratase, a butadiene synthase, a crotonyl-CoA reductase (alcohol forming) and a crotyl alcohol diphosphokinase (Figure 1, steps O, K, P, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 4-hydroxybutyryl-CoA dehydratase, a

crotonaldehyde reductase (alcohol forming), a crotyl alcohol kinase, a 2-butenyl-4-phosphate kinase, a butadiene synthase, a crotonyl-CoA hydrolase, synthetase, or transferase and a crotonate reductase (Figure 1, steps O, I, J, E, F, G, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 4-hydroxybutyryl- CoA dehydratase, a crotonaldehyde reductase (alcohol forming), a butadiene synthase, a crotonyl-CoA hydrolase, synthetase or transferase, a crotonate reductase and a crotyl alcohol diphosphokinase (Figure 1, steps O, I, J, E, P, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a butadiene synthase, a 4-hydroxybutyryl-CoA dehydratase and a crotyl alcohol diphosphokinase (Figure 1, steps L, C, D, E, P, H).

In some embodiments, the invention provides a non-naturally occurring microbial organism, including a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding a butadiene pathway enzyme expressed in a sufficient amount to produce butadiene, the butadiene pathway including an erythrose-4-phosphate reductase, an erythritol-4-phospate cytidylyltransferase, a 4-(cytidine 5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate synthase, a l-hydroxy-2-butenyl 4-diphosphate synthase, a 1- hydroxy-2-butenyl 4-diphosphate reductase, a butenyl 4-diphosphate isomerase, a butadiene synthase, an erythrose-4-phosphate kinase, an erythrose reductase or an erythritol kinase (Figure 2). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an erythrose-4-phosphate reductase, an erythritol-4-phospate cytidylyltransferase, a 4- (cytidine 5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate synthase, a 1- hydroxy-2-butenyl 4-diphosphate synthase, a l-hydroxy-2-butenyl 4-diphosphate reductase and a butadiene synthase (Figure 2, steps A-F, and H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an erythrose-4-phosphate reductase, an erythritol-4-phospate cytidylyltransferase, a 4-(cytidine 5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate synthase, a l-hydroxy-2-butenyl 4-diphosphate synthase, a l-hydroxy-2-butenyl 4-diphosphate reductase, a butenyl 4-diphosphate isomerase and butadiene synthase (Figure 2, steps A-H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an erythritol-4-phospate

cytidylyltransferase, a 4-(cytidine 5'-diphospho)-erythritol kinase, an erythritol 2,4- cyclodiphosphate synthase, a l-hydroxy-2-butenyl 4-diphosphate synthase, a l-hydroxy-2- butenyl 4-diphosphate reductase, a butadiene synthase, an erythrose-4-phosphate kinase, an erythrose reductase and a erythritol kinase (Figure 2, steps I, J, K, B-F, H). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including an erythritol-4-phospate cytidylyltransferase, a 4-(cytidine 5'-diphospho)-erythritol kinase, an erythritol 2,4-cyclodiphosphate synthase, a l-hydroxy-2-butenyl 4-diphosphate synthase, a 1- hydroxy-2 -butenyl 4-diphosphate reductase, a butenyl 4-diphosphate isomerase, a butadiene synthase, an erythrose-4-phosphate kinase, an erythrose reductase and an erythritol kinase (Figure 2, steps I, J, K, B-H).

In some embodiments, the invention provides a non-naturally occurring microbial organism, including a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding a butadiene pathway enzyme expressed in a sufficient amount to produce butadiene, the butadiene pathway including a malonyl-CoA:acetyl-CoA

acyltransferase, an 3-oxoglutaryl-CoA reductase (ketone-reducing), a 3-hydroxyglutaryl-CoA reductase (aldehyde forming), a 3-hydroxy-5-oxopentanoate reductase, a 3,5- dihydroxypentanoate kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a 3-hydroxy-5- [hydroxy(phosphonooxy)phosphoryl]oxy pentanoate decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene synthase, a 3-hydroxyglutaryl-CoA reductase (alcohol forming), an 3- oxoglutaryl-CoA reductase (aldehyde forming), a 3,5-dioxopentanoate reductase (ketone reducing), a 3,5-dioxopentanoate reductase (aldehyde reducing), a 5-hydroxy-3-oxopentanoate reductase or an 3-oxo-glutaryl-CoA reductase (CoA reducing and alcohol forming) (Figure 3). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a malonyl-CoA:acetyl-CoA acyltransferase, an 3-oxoglutaryl-CoA reductase (ketone- reducing), a 3-hydroxyglutaryl-CoA reductase (aldehyde forming), a 3-hydroxy-5-oxopentanoate reductase, a 3,5-dihydroxypentanoate kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a 3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate decarboxylase, a butenyl 4- diphosphate isomerase and a butadiene synthase (Figure 3, steps A-I). In one aspect, the non- naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a malonyl-CoA:acetyl-CoA acyltransferase, a 3,5-dihydroxypentanoate kinase, a 3-hydroxy-5- phosphonatooxypentanoate kinase, a 3-hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene synthase, an 3- oxoglutaryl-CoA reductase (aldehyde forming), a 3,5-dioxopentanoate reductase (aldehyde reducing) and a 5-hydroxy-3-oxopentanoate reductase. (Figure 3, steps A, K, M, N, E, F, G, H, I). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a malonyl-CoA:acetyl-CoA acyltransferase, a 3-hydroxy-5-oxopentanoate reductase, a 3,5-dihydroxypentanoate kinase, a 3-Hydroxy-5-phosphonatooxypentanoate kinase, a 3- Hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate decarboxylase, a butenyl 4- diphosphate isomerase, a butadiene synthase, an 3-oxoglutaryl-CoA reductase (aldehyde forming) and a 3,5-dioxopentanoate reductase (ketone reducing). (Figure 3, steps A, K, L, D, E, F, G, H, I). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a malonyl-CoA:acetyl-CoA acyltransferase, a 3,5-dihydroxypentanoate kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a 3-hydroxy-5- [hydroxy(phosphonooxy)phosphoryl]oxy pentanoate decarboxylase, a butenyl 4-diphosphate isomerase, a butadiene synthase, a 5-hydroxy-3-oxopentanoate reductase and a 3-oxo-glutaryl- CoA reductase (CoA reducing and alcohol forming). (Figure 3, steps A, O, N, E, F, G, H, I). In one aspect, the non-naturally occurring microbial organism includes a microbial organism having a butadiene pathway having at least one exogenous nucleic acid encoding butadiene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a malonyl-CoA:acetyl-CoA acyltransferase, an 3-oxoglutaryl-CoA reductase (ketone-reducing), a 3,5-dihydroxypentanoate kinase, a 3-hydroxy-5-phosphonatooxypentanoate kinase, a 3- hydroxy-5-[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate decarboxylase, a butenyl 4- diphosphate isomerase, a butadiene synthase and a 3-hydroxyglutaryl-CoA reductase (alcohol forming). (Figure 3, steps A, B, J, E, F, G, H, I).

In an additional embodiment, the invention provides a non-naturally occurring microbial organism having a butadiene pathway, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from the group consisting of acetyl-CoA to acetoacetyl-CoA, acetoacetyl-CoA to 3-hydroxybutyryl-CoA, 3-hydroxybutyryl-CoA to crotonyl-CoA, crotonyl- CoA to crotonaldehyde, crotonaldehyde to crotyl alcohol, crotyl alcohol to 2-betenyl-phosphate, 2-betenyl-phosphate to 2-butenyl-4-diphosphate, 2-butenyl-4-diphosphate to butadiene, erythrose-4-phosphate to erythritol-4-phosphate, erythritol-4-phosphate to 4-(cytidine 5'- diphospho)-erythritol, 4-(cytidine 5'-diphospho)-erythritol to 2-phospho-4-(cytidine 5'- diphospho)-erythritol, 2-phospho-4-(cytidine 5'-diphospho)-erythritol to erythritol-2,4- cyclodiphosphate, erythritol-2,4-cyclodiphosphate to l-hydroxy-2-butenyl 4-diphosphate, 1- hydroxy-2 -butenyl 4-diphosphate to butenyl 4-diphosphate, butenyl 4-diphosphate to 2-butenyl 4-diphosphate, l-hydroxy-2 -butenyl 4-diphosphate to 2-butenyl 4-diphosphate, 2-butenyl 4- diphosphate to butadiene, malonyl-CoA and acetyl-CoA to 3-oxoglutaryl-CoA, 3-oxoglutaryl- CoA to 3-hydroxyglutaryl-CoA to 3-hydroxy-5-oxopentanoate, 3-hydroxy-5-oxopentanoate to 3,5-dihydroxy pentanoate, 3,5-dihydroxy pentanoate to 3-hydroxy-5-phosphonatooxypentanoate, 3-hydroxy-5-phosphonatooxypentanoate to 3-hydroxy-5- [hydroxy(phosphonooxy)phosphoryl]oxy pentanoate, 3-hydroxy-5-

[hydroxy(phosphonooxy)phosphoryl]oxy pentanoate to butenyl 4-biphosphate, glutaconyl-CoA to crotonyl-CoA, glutaryl-CoA to crotonyl-CoA, 3-aminobutyryl-CoA to crotonyl-CoA, 4- hydroxybutyryl-CoA to crotonyl-CoA, crotonyl-CoA to crotonate, crotonate to crotonaldehyde, crotonyl-CoA to crotyl alcohol, crotyl alcohol to 2-butenyl-4-diphosphate, erythrose-4-phosphate to erythrose, erythrose to erythritol, erythritol to erythritol-4-phosphate, 3-oxoglutaryl-CoA to 3,5-dioxopentanoate, 3,5-dioxopentanoate to 5-hydroxy-3-oxopentanoate, 5-hydroxy-3- oxopentanoate to 3,5-dihydroxypentanoate, 3-oxoglutaryl-CoA to 5-hydroxy-3-oxopentanoate, 3,5-dioxopentanoate to 3-hydroxy-5-oxopentanoate and 3-hydroxyglutaryl-CoA to 3,5- dihydroxypentanoate. One skilled in the art will understand that these are merely exemplary and that any of the substrate-product pairs disclosed herein suitable to produce a desired product and for which an appropriate activity is available for the conversion of the substrate to the product can be readily determined by one skilled in the art based on the teachings herein. Thus, the invention provides a non-naturally occurring microbial organism containing at least one exogenous nucleic acid encoding an enzyme or protein, where the enzyme or protein converts the substrates and products of a butadiene pathway, such as that shown in Figures 1-3.

In alternative embodiments, this invention provides or uses non-naturally occurring microorganisms that express genes encoding enzymes that catalyze 1,3 -butadiene production, as shown in Figure 4. In some embodiments, pathways for the production of muconate are derived from central metabolic precursors. Muconate is a common degradation product of diverse aromatic compounds in microbes. Several biocatalytic strategies for making cz ^' s, czs-muconate have been developed. Engineered E. coli strains producing muconate from glucose via shikimate pathway enzymes have been developed in the Frost lab (U.S. Patent 5,487,987 (1996); Niu et al, Biotechnol Prog., 18:201-211 (2002)). These strains are able to produce 36.8 g/L of cis,cis- muconate after 48 hours of culturing under fed-batch fermenter conditions (22% of the maximum theoretical yield from glucose). Muconate has also been produced biocatalytically from aromatic starting materials such as toluene, benzoic acid and catechol. Strains producing muconate from benzoate achieved titers of 13.5 g/L and productivity of 5.5 g/L/hr (Choi et al., L Ferment. Bioeng. 84:70-76 (1997)). Muconate has also been generated from the effluents of a styrene monomer production plant (Wu et al., Enzyme and Microbiology Technology 35:598- 604 (2004)).

In some embodiments, the present invention provides a non-naturally occurring microbial organism that includes a microbial organism having a 1,3 -butadiene pathway which includes at least one exogenous nucleic acid encoding a 1,3-butadiene pathway enzyme expressed in a sufficient amount to produce 1,3-butadiene. The 1,3-butadiene pathway can be selected from (A) 1) trans, trans -muconate decarboxylase and 2) zra/?s-2,4-pentadienoate decarboxylase; (B) 1) cis, trans -muconate cz ^' s-decarboxylase and 2) tra/?s-2,4-pentadienoate decarboxylase; (C) 1) cis, trans -muconate zrans-decarboxylase 2) cz ^' s-2,4-pentadienoate decarboxylase; and (D) 1) cis, cis- muconate decarboxylase and 2) cz ^' s-2,4-pentadienoate decarboxylase, as indicated in the alternate pathways in Figure 4.

In some embodiments, the non-naturally occurring microbial organism having a 1,3- butadiene pathway can include two exogenous nucleic acids each encoding a 1,3-butadiene pathway enzyme. Thus, the two exogenous nucleic acids can encode a set selected from (A) 1) trans, trans -muconate decarboxylase and 2) zra/?s-2,4-pentadienoate decarboxylase; (B) 1) cis, trans -muconate cz ^' s-decarboxylase and 2) trans -2,4-pentadienoate decarboxylase; (C) 1) cis, trans -muconate zrans-decarboxylase 2) cz ^' s-2,4-pentadienoate decarboxylase; and (D) 1) cis, cis- muconate decarboxylase and 2) cz ^' s-2,4-pentadienoate decarboxylase, corresponding to the complete pathways shown in Figure 4. In some embodiments, the non-naturally occurring microbial organism having a 1,3-butadiene pathway has at least one exogenous nucleic acid that is a heterologous nucleic acid. In some embodiments, the non-naturally occurring microbial organism having a 1,3-butadiene pathway is in a substantially anaerobic culture medium.

Figure 4 shows the conversion of muconate isomers to 1,3-butadiene by decarboxylase enzymes. Cis,cis-muconate; cis,trans-muconate; or trans,trans-muconate, is first decarboxylated to either cis-2,4-pentadienoate or trans-2,4-pentadienoate (Steps A, B, C and D of Figure 4). 2,4- Pentadienoate is subsequently decarboxylated to form 1,3-butadiene (Steps E, F of Figure 4).

In some embodiments the decarboxylation of any muconate isomer can serve as part of a pathway to 1,3-butadiene. The biological production of cis,cis-muconate is well-known in the art (Draths and Frost. J Am Chem Soc. 116:399-400 (1994); Niu et al. Biotechnol Prog. 18:201-211 (2002); Bang and Choi. J Ferm Bioeng. 79(4):381-383 (1995)). Isomers of muconate can be interconverted by muconate isomerase enzymes in the EC class 5.2.1. In some embodiments decarboxylation of either isomer of 2,4-pentadienoate will form 1,3 -butadiene. Isomers of 2,4-pentadienoate can alternatively be formed from starting materials other than muconate (e.g., introduction of second double bond via dehydrogenation of pent-2- enoate or pent-4-enoate; removal of CoA from 2,4-pentadienoyl-CoA via a hydrolase, synthetase, or transferase, dehydrogenation of 2,4-pentadienal or 2,4-pentadienol via an aldehyde or aldehyde/alcohol dehydrogenase, respectively). Isomers of 2,4-pentadienoate can be interconverted by isomerase enzymes in the EC class: 5.2.1.

Numerous decarboxylase enzymes have been characterized and shown to decarboxylate structurally similar substrates to muconate or 2,4-pentadienoate isomers. Exemplary enzymes include sorbic acid decarboxylase, aconitate decarboxylase (EC 4.1.1.16), 4-oxalocrotonate decarboxylase (EC 4.1.1.77), cinnamate decarboxylase and ferulic acid decarboxylase. These enzymes are applicable for use in the present invention to decarboxylate muconate and/or 2,4- pentadienoate as shown in Figure 4.

One decarboxylase enzyme with closely related function is sorbic acid decarboxylase which converts sorbic acid to 1,3-pentadiene. Sorbic acid decarboxylation by Aspergillus niger requires three genes: padAl, ohbAl, and sdrA (Plumridge et al. Fung. Genet. Bio, 47:683-692 (2010). PadAl is annotated as a phenylacrylic acid decarboxylase, ohbAl is a putative 4- hydroxybenzoic acid decarboxylase, and sdrA is a sorbic acid decarboxylase regulator.

Additional species have also been shown to decarboxylate sorbic acid including several fungal and yeast species (Kinderlerler and Hatton, Food Addit Contam., 7(5):657-69 (1990); Casas et al, Int J Food Micro., 94(l):93-96 (2004); Pinches and Apps, Int. J. Food Microbiol. 116: 182- 185 (2007)). For example, Aspergillus oryzae and Neosartorya fischeri have been shown to decarboxylate sorbic acid and have close homo logs to padAl, ohbAl, and sdrA.

sdrA XP_001818649.1 169768358 Aspergillus oryzae

padAl XP_001261423.1 119482790 Neosartorya fischeri ohbAl XP_001261424.1 119482792 Neosartorya fischeri sdrA XP_001261422.1 119482788 Neosartorya fischeri

Aconitate decarboxylase is another useful enzyme for this invention. This enzyme catalyzes the final step in itaconate biosynthesis in a strain of Candida and also in the filamentous fungus Aspergillus terreus. (Bonnarme et al. J Bacteriol. 177:3573-3578 (1995); Willke and Vorlop, Appl Microbiol. Biotechnol 56:289-295 (2001)) Aconitate decarboxylase has been purified and characterized from Aspergillus terreus. (Dwiarti et al, J. Biosci. Bioeng. 94(1): 29-33 (2002)) The gene and protein sequence for the cis-aconitic acid decarboxylase (CAD) enzyme were reported previously (EP 2017344 Al; WO 2009/014437 Al), along with several close homologs listed in the table below.

In some embodiments 4-Oxalocronate decarboxylase catalyzes the decarboxylation of 4- oxalocrotonate to 2-oxopentanoate. This enzyme has been isolated from numerous organisms and characterized. Genes encoding this enzyme include dmpH and dmpE in Pseudomonas sp. (strain 600) (Shingler et al, 174:711-724 (1992)), xylll and xyllll from Pseudomonas putida (Kato et al, Arch.Microbiol 168:457-463 (1997); Stanley et al, Biochemistry 39:3514 (2000); Lian et al, J.Am.Chem.Soc. 116:10403-10411 (1994)) and Reut_B5691 and Reut_B5692 from Ralstonia eutropha JMP134 (Hughes et al, 158:79-83 (1984)). The genes encoding the enzyme from Pseudomonas sp. (strain 600) have been cloned and expressed in E. coli (Shingler et al, 174:711-724 (1992)).

In some embodiments, a class of decarboxylases can be used to catalyze the conversion of cinnamate (phenylacrylate) and substituted cinnamate derivatives to their corresponding styrene derivatives. These enzymes are common in a variety of organisms and specific genes encoding these enzymes that have been cloned and expressed in E. coli include: pad 1 from Saccharomyces cerevisae (Clausen et al., Gene 142: 107-112 (1994)), pdc from Lactobacillus plantarum (Barthelmebs et al, 67: 1063-1069 (2001); Qi et al, Metab Eng 9:268-276 (2007); Rodriguez et al, J.Agric.Food Chem. 56:3068-3072 (2008)), pofK (pad) from Klebsiella oxytoca (Uchiyama et al., Biosci.Biotechnol.Biochem. 72: 116-123 (2008); Hashidoko et al.,

Biosci.Biotech.Biochem. 58:217-218 (1994)) , Pedicoccus pentosaceus (Barthelmebs et al., 67: 1063-1069 (2001)), and padC from Bacillus subtilis and Bacillus pumilus (Shingler et al, 174:711-724 (1992)). A ferulic acid decarboxylase from Pseudomonas fluorescens also has been purified and characterized (Huang et al, J.Bacteriol. 176:5912-5918 (1994)). Enzymes in this class are stable and do not require either exogenous or internally bound co-factors, thus making these enzymes ideally suitable for biotransformations (Sariaslani, Annu.Rev.Microbiol. 61 :51-69 (2007)).

Each of the decarboxylases listed above represents a possible suitable enzyme for the desired transformations shown in Figure 4. If the desired activity or productivity of the enzyme is not observed in the desired conversions (e.g., muconate to 2,4-pentadienoate, 2,4- pentadienoate to butadiene), the decarboxylase enzymes can be evolved using known protein engineering methods to achieve the required performance. Importantly, it was shown through the use of chimeric enzymes that the C-terminal region of decarboxylases appears to be responsible for substrate specificity (Barthelmebs, L.; Divies, C; Cavin, J.-F. 2001. Expression in Escherichia coli of Native and Chimeric Phenolic Acid Decarboxylases with Modified Enzymatic Activities and Method for Screening Recombinant E. coli Strains Expressing These Enzymes, Appl. Environ. Microbiol. 67, 1063-1069.). Accordingly, directed evolution experiments to broaden the specificity of decarboxylases in order to gain activity with muconate or 2,4-pentadienoate can be focused on the C-terminal region of these enzymes.

Some of the decarboxylases required to catalyze the transformations in Figure 4 may exhibit higher activity on specific isomers of muconate or 2,4-pentadienoate. Isomerase enzymes can be applied to convert less desirable isomers of muconate and 2,4-pentadienoate into more desirable isomers for decarboxylation. Exemplary isomerases that catalyze similar

transformations and thus represent suitable enzymes for this invention include maleate cis-trans isomerase (EC 5.2.1.1), maleylacetone cis-trans isomerase (EC 5.2.1.2) and fatty acid cis-trans isomerase. Maleate cis-trans isomerase converts fumarate to maleate. This enzyme is encoded by the maiA gene from A lcaligenes faecalis (Hatakeyama, et al, 1997, Gene Cloning and

Characterization of Maleate cis-trans Isomerase from Alcaligenes faecalis, Biochem. Biophys. Research Comm. 239, 74-79) or Serratia marcescens (Hatakeyama et al., Biosci. Biotechnol. Biochem. 64: 1477-1485 (2000)). Similar genes that can be identified by sequence homology include those from Geobacillus stearothermophilus, Ralstonia pickettii 12D, and Ralstonia eutropha HI 6. Additional maleate cis-trans isomerase enzymes are encoded by the enzymes whose amino acid sequences are provided as sequence ID's 1 through 4 in ref (Mukouyama et al., US Patent 6,133,014). Maleylacetone cis, trans -isomerase catalyzes the conversion of 4- maleyl-acetoacetate to 4-fumaryl-acetyacetate, a cis to trans conversion. This enzyme is encoded by maiA in Pseudomonas aeruginosa (Fernandez-Canon et al., J Biol.Chem. 273:329-337 (1998))) and Vibrio cholera (Seltzer, J Biol.Chem. 248:215-222 (1973)). A similar enzyme was identified by sequence homology in E. coli 0157. The cti gene product catalyzes the conversion of cis- unsaturated fatty acids (UFA) to trans- UFA. The enzyme has been characterized in P. putida (Junker et al, J Bacteriol. 181 :5693-5700 (1999)). Similar enzymes are found in

Shewanella sp. MR-4 and Vibrio cholerae.

3-Hydroxyacid decarboxylase enzymes for formation of 1,3 -butadiene

Figure 5 shows the decarboxylative dehydration of 3-hydroxypent-4-enoate (3HP4) to 1,3 -butadiene, 3-Hydroxyacid decarboxylase enzymes catalyze the ATP-driven decarboxylation of 3 -hydroxy acids to alkene derivatives. 3-Hyroxyacid decarboxylase enzymes have recently been described that catalyze the formation of isobutylene, propylene and ethylene (WO

2010/001078 and Gogerty and Bobik, Appl. Environ. Microbiol, p. 8004-8010, Vol. 76, No. 24 (2010)). In some embodiments the invention uses similar enzymes to catalyze the conversion of 3-hydroxypent-4-enoate (3HP4) to 1,3 -butadiene, shown in Figure 5 and 3,5- dihydroxypentanoate to 3-butene-l-ol , shown in Figure 6. The 3-butene-l-ol product can then be converted to butadiene via chemical dehydration or biological dehydration via a 3-butene-l-ol dehydratase enzyme.

Conversion of 3-hydroxypent-4-enoate to butadiene is carried out by a 3-hydroxypent-4- enoate decarboxylase. Similarly, conversion of 3,5-dihydroxypentanoate to 3-butene-l-ol is carried out by a 3,5-dihydroxypentanoate decarboxylase. Such enzymes may share similarity to mevalonate pyrophosphate decarboxylase or diphosphomevalonate decarboxylase enzymes. One potential 3-hydroxypent-4-enoate decarboxylase is the Saccharomyces cerevisiae mevalonate diphosphate decarboxylase (ScMDD or ERG19) which was shown to convert 3-hydroxy-3- methylbutyrate (3-HMB) to isobutene (Gogerty and Bobik, 2010, Appl. Environ. Microbiol, p. 8004-8010, Vol. 76, No. 24). Two improved variants of the enzyme, ScMDDl (I145F) and ScMDD2 (R74H), were demonstrated to achieve 19-fold and 38-fold increases compared to the wild-type His-tagged enzyme. ERG 19 and additional enzymes candidates are provided below.

imvaD 146329706 YP_001209416.1 Dichelobacter nodosus

VCS1703A

MPTP_0700 332686202 YP_004455976.1 Melissococcus plutonius

ATCC 35311

RKLH11_3963 254513287 ZP_05125352.1 Rhodobacteraceae

bacterium KLH11

Figure 6 shows pathways to butadiene, 3-hydroxypent-4-enoate (3HP4), 2,4- pentadienoate and 3-butene-l-ol from 3-HP-CoA and/or acrylyl-CoA. Enzymes are A. 3- hydroxypropanoyl-CoA acetyltransferase, B. 3 -oxo-5-hydroxypentanoyl-Co A reductase, C. 3,5- dihydroxypentanoyl-CoA dehydratase, D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4- dienoyl-CoA synthetase, transferase and/or hydrolase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, I. 3- oxo-5-hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate dehydratase, K. 3- hydroxypropanoyl-CoA dehydratase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, M.

acrylyl-CoA acetyltransferase, N. 3-oxopent-4-enoyl-CoA reductase, O. 3-oxopent-4-enoyl-CoA synthetase, transferase and/or hydrolase, P. 3-oxopent-4-enoate reductase, Q. 5-hydroxypent-2- enoate dehydratase, R. 3-hydroxypent-4-enoyl-CoA dehydratase, S. 3-hydroxypent-4-enoate dehydratase, T. 3-hydroxypent-4-enoyl-CoA transferase, synthetase or hydrolase, U. 3,5- dihydroxypentanoate decarboxylase, V. 5-hydroxypent-2-enoate decarboxylase, W. 3-butene-l- ol dehydratase (or chemical conversion), X. 2,4-pentadiene decarboxylase, Y. 3-hydroxypent-4- enoate decarboxylase. 3-HP-CoA is 3-hydroxypropanoyl-CoA.

Several reactions shown in Figure 6 are catalyzed by alcohol dehydrogenase enzymes. These reactions include Steps B, I, N and P of Figure 6.

Exemplary genes encoding enzymes that catalyze the reduction of an aldehyde to alcohol (i.e., alcohol dehydrogenase or equivalently aldehyde reductase) include alrA encoding a medium-chain alcohol dehydrogenase for C2-C14 (Tani et al., Appl.Environ.Microbiol. 66:5231- 5235 (2000)), yqhD and fucO from E. coli (Sulzenbacher et al, 342:489-502 (2004)), and bdh I and bdh II from C. acetobutylicum which converts butyryaldehyde into butanol (Walter et al, 174:7149-7158 (1992)). YqhD catalyzes the reduction of a wide range of aldehydes using NADPH as the cofactor, with a preference for chain lengths longer than C(3) (Sulzenbacher et al, 342:489-502 (2004); Perez et al, J Biol.Chem. 283:7346-7353 (2008)). The adhA gene product from Zymomonas mobilisE has been demonstrated to have activity on a number of aldehydes including formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, and acrolein (Kinoshita et al., Appl Microbiol Biotechnol 22:249-254 (1985)). Additional aldehyde reductase candidates are encoded by bdh in C. saccharoperbutylacetonicum and Cbei_1722, Cbei_2181 and Cbei_2421 in C. Beijerinckii. Additional aldehyde reductase gene candidates in

Saccharomyces cerevisiae include the aldehyde reductases GRE3, ALD2-6 and HFD1, glyoxylate reductases GOR1 and YPL113C and glycerol dehydrogenase GCY1 (WO

2011/022651A1; Atsumi et al, Nature 451 :86-89 (2008)). The enzyme candidates described previously for catalyzing the reduction of methylglyoxal to acetol or lactaldehyde are also suitable lactaldehyde reductase enzyme candidates.

Enzymes exhibiting 4-hydroxybutyrate dehydrogenase activity (EC 1.1.1.61) also fall into this category. Such enzymes have been characterized in Ralstonia eutropha (Bravo et al, J Forens Sci, 49:379-387 (2004)), Clostridium kluyveri (Wolff et al, Protein Expr.Purif. 6:206-212 (1995)) and Arabidopsis thaliana (Breitkreuz et al, J Biol Chem, 278:41552-41556 (2003)). The A. thaliana enzyme was cloned and characterized in yeast (Breitkreuz et al., J.Biol. Chem. 278:41552-41556 (2003)). Yet another gene is the alcohol dehydrogenase adhi from Geobacillus thermoglucosidasius (Jeon et al, J Biotechnol 135:127-133 (2008)).

Another exemplary aldehyde reductase is methylmalonate semialdehyde reductase, also known as 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31). This enzyme participates in valine, leucine and isoleucine degradation and has been identified in bacteria, eukaryotes, and mammals. The enzyme encoded by P84067 from Thermus thermophilus HB8 has been structurally characterized (Lokanath et al., J Mol Biol, 352:905-17 (2005)). The reversibility of the human 3-hydroxyisobutyrate dehydrogenase was demonstrated using isotopically-labeled substrate (Manning et al, Biochem J, 231 :481-4 (1985)). Additional genes encoding this enzyme include 3hidh in Homo sapiens (Hawes et al., Methods Enzymol, 324:218-228 (2000)) and Oryctolagus cuniculus (Hawes et al, supra; Chowdhury et al, Biosci.Biotechnol Biochem. 60:2043-2047 (1996)), mmsB in Pseudomonas aeruginosa and Pseudomonas putida, and dhat in Pseudomonas putida (Aberhart et al, J Chem.Soc.[Perkin 1] 6: 1404-1406 (1979); Chowdhury et al., Biosci.Biotechnol Biochem. 60:2043-2047 (1996); Chowdhury et al., Biosci.Biotechnol Biochem. 67:438-441 (2003)). Several 3-hydroxyisobutyrate dehydrogenase enzymes have been characterized in the reductive direction, including mmsB from Pseudomonas aeruginosa (Gokarn et al, US Patent 739676, (2008)) and mmsB from Pseudomonas putida.

There exist several exemplary alcohol dehydrogenases that reduce a ketone to a hydroxyl functional group. Two such enzymes from E. coli are encoded by malate dehydrogenase (mdh) and lactate dehydrogenase (ldhA). In addition, lactate dehydrogenase from Ralstonia eutropha has been shown to demonstrate high activities on 2-ketoacids of various chain lengths including: lactate, 2-oxobutyrate, 2-oxopentanoate and 2-oxoglutarate (Steinbuchel et al., Eur.J.Biochem. 130:329-334 (1983)). Conversion of alpha-ketoadipate into alpha-hydroxyadipate can be catalyzed by 2-ketoadipate reductase, an enzyme reported to be found in rat and in human placenta (Suda et al, Arch.Biochem.Biophys. 176:610-620 (1976); Suda et al,

Biochem.Biophys.Res.Commun. 77:586-591 (1977)). An additional oxidoreductase is the mitochondrial 3-hydroxybutyrate dehydrogenase (bdh) from the human heart which has been cloned and characterized (Marks et al, J.Biol.Chem. 267:15459-15463 (1992)). Alcohol dehydrogenase enzymes of C. beijerinckii (Ismaiel et al, J.Bacteriol. 175:5097-5105 (1993)) and T. brockii (Lamed et al, Biochem.J. 195: 183-190 (1981); Peretz et al, Biochemistry. 28:6549- 6555 (1989)) convert acetone to isopropanol. Methyl ethyl ketone reductase catalyzes the reduction of MEK to 2-butanol. Exemplary MEK reductase enzymes can be found in

Rhodococcus ruber (Kosjek et al, Biotechnol Bioeng. 86:55-62 (2004)) and Pyrococcus furiosus (van der et al, Eur.J.Biochem. 268:3062-3068 (2001)).

A number of organisms can catalyze the reduction of 4-hydroxy-2-butanone to 1,3- butanediol, including those belonging to the genus Bacillus, Brevibacterium, Candida, and Klebsiella among others, as described by Matsuyama et al. ( (1995)). A mutated Rhodococcus phenylacetaldehyde reductase (Sar268) and a Leifonia alcohol dehydrogenase have also been shown to catalyze this transformation at high yields (Itoh et al., Appl. Microbiol Biotechnol. 75: 1249-1256 (2007)).

Alcohol dehydrogenase enzymes that reduce 3-oxoacyl-CoA substrates to their corresponding 3-hyroxyacyl-CoA product are also relevant to the pathways depicted in Figure 6. 3-Oxoacyl-CoA dehydrogenase enzymes (EC 1.1.1.35) convert 3 -oxoacyl-Co A molecules into 3-hydroxyacyl-CoA molecules and are often involved in fatty acid beta-oxidation or

phenylacetate catabolism. For example, subunits of two fatty acid oxidation complexes in E. coli, encoded by fadB and fadJ, function as 3-hydroxyacyl-CoA dehydrogenases (Binstock et al, Methods Enzymol. 71 Pt C:403-411 (1981)). Given the proximity in E. coli of paaH to other genes in the phenylacetate degradation operon (Nogales et al, 153:357-365 (2007)) and the fact that paaH mutants cannot grow on phenylacetate (Ismail et al., Eur.J Biochem. 270:3047-3054 (2003)), it is expected that the E. coli paaH gene also encodes a 3-hydroxyacyl-CoA

dehydrogenase. Acetoacetyl-CoA reductase participates in the acetyl-CoA fermentation pathway to butyrate in several species of Clostridia and has been studied in detail (Jones et al, Microbiol Rev. 50:484-524 (1986)). The enzyme from Clostridium acetobutylicum, encoded by hbd, has been cloned and functionally expressed in E. coli (Youngleson et al, J Bacteriol. 171 :6800-6807 (1989)). Yet other genes demonstrated to reduce acetoacetyl-CoA to 3-hydroxybutyryl-CoA are phbB from Zoogloea ramigera (Ploux et al., Eur.J Biochem. 174: 177-182 (1988)) and phaB from Rhodobacter sphaeroides (Alber et al., Mol.Microbiol 61 :297-309 (2006)). The former gene is NADPH-dependent, its nucleotide sequence has been determined (Peoples et al, Mol.Microbiol 3:349-357 (1989)) and the gene has been expressed in E. coli. Substrate specificity studies on the gene led to the conclusion that it could accept 3-oxopropionyl-CoA as a substrate besides acetoacetyl-CoA (Ploux et al, Eur.J Biochem. 174: 177-182 (1988)). Additional genes include phaB in Paracoccus denitrificans, Hbdl (C -terminal domain) and Hbd2 (N-terminal domain) in Clostridium kluyveri (Hillmer and Gottschalk, Biochim. Biophys. Acta 3334: 12-23 (1974)) and HSD17B10 in Bos taurus (Wakil et al, J Biol.Chem. 207:631-638 (1954)). The enzyme from Paracoccus denitrificans has been functionally expressed and characterized in E. coli (Yabutani et al., FEMS Microbiol Lett. 133:85-90 (1995)). A number of similar enzymes have been found in other species of Clostridia and in Metallosphaera sedula (Berg et al, Science. 318: 1782-1786 (2007)). The enzyme from Candida tropicalis is a component of the peroxisomal fatty acid beta- oxidation multifunctional enzyme type 2 (MFE-2). The dehydrogenase B domain of this protein is catalytically active on acetoacetyl-CoA. The domain has been functionally expressed in E. coli, a crystal structure is available, and the catalytic mechanism is well-understood (Ylianttila et al, Biochem Biophys Res Commun 324:25-30 (2004); Ylianttila et al, J Mol Biol 358: 1286- 1295 (2006)).

Any of the natural or non-naturally occurring microbial organisms described herein can be cultured to produce and/or secrete the biosynthetic butadiene. A library containing butadiene producers can be cultured for the biosynthetic production of butadiene as is known in the art and described herein, in order to produce butadiene for detection with the apparatuses and methods described herein.

For the production of butadiene, the microbes are cultured in a medium with carbon source and other essential nutrients. It is sometimes desirable and can be highly desirable to maintain anaerobic conditions in the fermenter to reduce the cost of the overall process. Such conditions can be obtained, for example, by first sparging the medium with nitrogen and then sealing the wells or fermenters. For strains where growth is not observed anaerobically, then microaerobic or substantially anaerobic conditions can be applied. Exemplary anaerobic conditions have been described previously and are well-known in the art. Exemplary aerobic and anaerobic conditions are described, for example, in United State publication 2009/0047719, filed August 10, 2007. For industrial BD production using the strains identified by the methods and apparatuses described herein, fermentations can be performed in a batch, fed-batch or continuous manner.

If desired, the pH of the medium can be maintained at a desired pH, in particular neutral pH, such as a pH of around 7 by addition of a base, such as NaOH or other bases, or acid, as needed to maintain the culture medium at a desirable pH. The growth rate can be determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time.

The growth medium can include, for example, any carbohydrate source which can supply a source of carbon to the non-naturally occurring microorganism. Such sources include, for example, sugars such as glucose, xylose, arabinose, galactose, mannose, fructose, sucrose and starch. Other sources of carbohydrate include, for example, renewable feedstocks and biomass. Exemplary types of biomasses that can be used as feedstocks in the screening methods include cellulosic biomass, hemicellulosic biomass and lignin feedstocks or portions of feedstocks. Such biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch. Given the teachings and guidance provided herein, those skilled in the art will understand that renewable feedstocks and biomass other than those exemplified above also can be used for culturing the microbial organisms for the production of butadiene.

In addition to renewable feedstocks such as those exemplified above, the butadiene microbial organisms also can be modified for growth on syngas as its source of carbon. In this specific embodiment, one or more proteins or enzymes are expressed in the butadiene producing organisms to provide a metabolic pathway for utilization of syngas or other gaseous carbon source.

Synthesis gas, also known as syngas or producer gas, is the major product of gasification of coal and of carbonaceous materials such as biomass materials, including agricultural crops and residues. Syngas is a mixture primarily of H2 and CO and can be obtained from the gasification of any organic feedstock, including but not limited to coal, coal oil, natural gas, biomass, and waste organic matter. Gasification is generally carried out under a high fuel to oxygen ratio. Although largely H2 and CO, syngas can also include C0 ₂ and other gases in smaller quantities. Thus, synthesis gas provides a cost effective source of gaseous carbon such as CO and, additionally, C0 ₂ .

The Wood-Ljungdahl pathway catalyzes the conversion of CO and H2 to acetyl-CoA and other products such as acetate. Organisms capable of utilizing CO and syngas also generally have the capability of utilizing C0 ₂ and C0 ₂ /H ₂ mixtures through the same basic set of enzymes and transformations encompassed by the Wood-Ljungdahl pathway. H2-dependent conversion of C0 ₂ to acetate by microorganisms was recognized long before it was revealed that CO also could be used by the same organisms and that the same pathways were involved. Many acetogens have been shown to grow in the presence of C0 ₂ and produce compounds such as acetate as long as hydrogen is present to supply the necessary reducing equivalents (see for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, New York, (1994)). This can be summarized by the following equation: 2 C0 ₂ + 4 H ₂ + n ADP + n Pi→ CH ₃ COOH + 2 H ₂ 0 + n ATP.

Hence, non-naturally occurring microorganisms possessing the Wood-Ljungdahl pathway can utilize C0 ₂ and H ₂ mixtures as well for the production of acetyl-CoA and other desired products. The Wood-Ljungdahl pathway is well known in the art and consists of 12 reactions which can be separated into two branches: (1) methyl branch and (2) carbonyl branch. The methyl branch converts syngas to methyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branch converts methyl-THF to acetyl-CoA. The reactions in the methyl branch are catalyzed in order by the following enzymes or proteins: ferredoxin oxidoreductase, formate dehydrogenase, formyltetrahydrofolate synthetase, methenyltetrahydrofolate cyclodehydratase,

methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolate reductase. The reactions in the carbonyl branch are catalyzed in order by the following enzymes or proteins: methyltetrahydrofolatexorrinoid protein methyltransferase (for example, AcsE), corrinoid iron- sulfur protein, nickel-protein assembly protein (for example, AcsF), ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase and nickel-protein assembly protein (for example, CooC). Following the teachings and guidance provided herein for introducing a sufficient number of encoding nucleic acids to generate a butadiene pathway, those skilled in the art will understand that the same engineering design also can be performed with respect to introducing at least the nucleic acids encoding the Wood-Ljungdahl enzymes or proteins absent in the host organism. Therefore, introduction of one or more encoding nucleic acids into the microbial organisms such that the modified organism contains the complete Wood- Ljungdahl pathway will confer syngas utilization ability, and libraries of such modified microbes can be screened using the apparatuses and methods described herein.

Additionally, the reductive (reverse) tricarboxylic acid cycle coupled with carbon monoxide dehydrogenase and/or hydrogenase activities can also be used for the conversion of CO, C02 and/or H2 to acetyl-CoA and other products such as acetate. Organisms capable of fixing carbon via the reductive TCA pathway can utilize one or more of the following enzymes: ATP citrate - lyase, citrate lyase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate:

ferredoxin oxidoreductase, succinyl-CoA synthetase, succinyl-CoA transferase, fumarate reductase, fumarase, malate dehydrogenase, NAD(P)H: ferredoxin oxidoreductase, carbon monoxide dehydrogenase, and hydrogenase. Specifically, the reducing equivalents extracted from CO and/or H2 by carbon monoxide dehydrogenase and hydrogenase are utilized to fix C02 via the reductive TCA cycle into acetyl-CoA or acetate. Acetate can be converted to acetyl-CoA by enzymes such as acetyl-CoA transferase, acetate kinase/phosphotransacetylase, and acetyl- CoA synthetase. Acetyl-CoA can be converted to the butadiene, glyceraldehyde-3 -phosphate, phosphoenolpyruvate, and pyruvate, by pyruvate: ferredoxin oxidoreductase and the enzymes of gluconeo genesis. Following the teachings and guidance provided herein for introducing a sufficient number of encoding nucleic acids to generate a butadiene pathway, those skilled in the art will understand that the same engineering design also can be performed with respect to introducing at least the nucleic acids encoding the reductive TCA pathway enzymes or proteins absent in the host organism. Therefore, introduction of one or more encoding nucleic acids into the microbial organisms such that the modified organism contains the complete reductive TCA pathway will confer syngas utilization ability, and libraries of such modified microbes can be screened using the apparatuses and methods described herein.

Accordingly, given the teachings and guidance provided herein, those skilled in the art will understand that a non-naturally occurring microbial organism can be produced that secretes the biosynthesized BD when grown on a carbon source such as a carbohydrate. All that is required is to engineer in one or more of the required enzyme or protein activities to achieve biosynthesis of the desired compound or intermediate including, for example, inclusion of some or all of the butadiene biosynthetic pathways. Accordingly, a non-naturally occurring microbial organism that produces and/or secretes butadiene when grown on a carbohydrate or other carbon source and produces and/or secretes any of the intermediate metabolites shown in the butadiene pathway when grown on a carbohydrate or other carbon source can be provided and libraries of such modified microbes can be screened using the apparatuses and methods described herein.

The butadiene producing microbial organisms can initiate synthesis from an intermediate, for example, acetoacetyl-CoA, 3-hydroxybutyryl-CoA, crotonyl-CoA, crotonaldehyde, crotyl alcohol, 2- betenyl-phosphate, 2-butenyl-4-diphosphate, erythritol-4-phosphate, 4-(cytidine 5'- diphospho)- erythritol, 2-phospho-4-(cytidine 5'-diphospho)-erythritol, erythritol-2,4- cyclodiphosphate, 1- hydroxy-2-butenyl 4-diphosphate, butenyl 4-diphosphate, 2-butenyl 4- diphosphate, 3- oxoglutaryl-CoA, 3-hydroxyglutaryl-CoA, 3-hydroxy-5-oxopentanoate, 3,5- dihydroxy pentanoate, 3-hydroxy-5-phosphonatooxypentanoate, 3-hydroxy-5- [hydroxy(phosphonooxy)phosphoryl]oxy pentanoate, crotonate, erythrose, erythritol, 3,5- dioxopentanoate or 5-hydroxy-3-oxopentanoate.

The non-naturally occurring microbial organisms can be constructed using methods well known in the art as exemplified herein to exogenously express at least one nucleic acid encoding a butadiene pathway enzyme or protein in sufficient amounts to produce butadiene. It is understood that the microbial organisms are cultured under conditions sufficient to produce butadiene. Following the teachings and guidance provided herein, the non-naturally occurring microbial organisms can achieve biosynthesis of butadiene resulting in intracellular

concentrations between about 0.001-2000 mM or more. Generally, the intracellular concentration of butadiene is between about 3-1500 mM, particularly between about 5-1250 mM and more particularly between about 8-1000 mM, including about 10 mM, 100 mM, 200 mM, 500 mM, 800 mM, or more. Intracellular concentrations between and above each of these exemplary ranges also can be achieved from the non-naturally occurring microbial organisms, and libraries of such modified microbes can be screened using the apparatuses and methods described herein.

In some embodiments, culture conditions include anaerobic or substantially anaerobic growth or maintenance conditions. Exemplary anaerobic conditions have been described previously and are well known in the art. Exemplary anaerobic conditions for fermentation processes are described herein and are described, for example, in U.S. publication

2009/0047719, filed August 10, 2007. Any of these conditions can be employed with the non- naturally occurring microbial organisms as well as other anaerobic conditions well known in the art. Under such anaerobic or substantially anaerobic conditions, the butadiene producers can synthesize butadiene at intracellular concentrations of 5-10 mM or more as well as all other concentrations exemplified herein. It is understood that, even though the above description refers to intracellular concentrations, butadiene producing microbial organisms can produce butadiene intracellularly and/or secrete the product into the culture medium, and further the butadiene will partition to the headspace as butadiene gas. Either the soluble or gas form of BD can be detected and/or measured by the apparatuses and methods described herein.

The culture conditions can include, for example, liquid culture procedures as well as fermentation and other large scale culture procedures. As described herein, particularly useful yields of the biosynthetic BD can be obtained under anaerobic or substantially anaerobic culture conditions. And libraries of such modified microbes or of such varied fermentation conditions can be screened using the apparatuses and methods described herein.

As described herein, one exemplary growth condition for achieving biosynthesis of butadiene includes anaerobic culture or fermentation conditions. In certain embodiments, the non-naturally occurring microbial organisms can be sustained, cultured or fermented under anaerobic or substantially anaerobic conditions. Briefly, anaerobic conditions refer to an environment devoid of oxygen. Substantially anaerobic conditions include, for example, a culture, batch fermentation or continuous fermentation such that the dissolved oxygen concentration in the medium remains between 0 and 10% of saturation. Substantially anaerobic conditions also includes growing or resting cells in liquid medium or on solid agar inside a sealed chamber maintained with an atmosphere of less than 1% oxygen. The percent of oxygen can be maintained by, for example, sparging the culture with an N2/C02 mixture or other suitable non-oxygen gas or gases.

The culture conditions described herein can be scaled up and grown continuously for manufacturing of butadiene. Exemplary growth procedures include, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. All of these processes are well known in the art. Fermentation procedures are particularly useful for the biosynthetic production of commercial quantities of butadiene of the microbes identified or developed using the methods and apparatuses described herein. Generally, and as with non-continuous culture procedures, the continuous and/or near-continuous production of butadiene will include culturing a non- naturally occurring butadiene producing organism in sufficient nutrients and medium to sustain and/or nearly sustain growth in an exponential phase. Continuous culture under such conditions can be include, for example, growth for 1 day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous culture can include longer time periods of 1 week, 2, 3, 4 or 5 or more weeks and up to several months. Alternatively, the microbial organisms can be cultured for hours, if suitable for a particular application. It is to be understood that the continuous and/or near-continuous culture conditions also can include all time intervals in between these exemplary periods. It is further understood that the time of culturing the microbial organism is for a sufficient period of time to produce a sufficient amount of product for a desired purpose.

In alternative embodiments, any fermentation procedure can be used to practice the invention, and fermentation procedures are well known in the art. Briefly, fermentation for the biosynthetic production of butadiene can be utilized in, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. Examples of batch and continuous fermentation procedures are well known in the art. And using the methods and apparatuses described herein one can rapidly screen a library of microbes grown under a variety of fermentation conditions to identify those conditions that enhance BD production.

After identification of the microbes producing butadiene or the enhanced methods of fermentation by screening libraries according to the methods and apparatuses described herein, further detailed characterizations can be performed at non-HTS conditions. Suitable purification and/or assays to quantify the production of butadiene can be performed using well known methods. Suitable replicates such as triplicate cultures can be grown for each engineered strain to be tested. For example, product and byproduct formation in the engineered production host can be monitored. The final product and intermediates, and other organic compounds, can be analyzed by methods such as HPLC (High Performance Liquid Chromatography), GC-MS (Gas Chromatography-Mass Spectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) or other suitable analytical methods using routine procedures well known in the art. The release of product in the fermentation broth can also be tested with the culture supernatant. Byproducts and residual glucose can be quantified by HPLC using, for example, a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al, Biotechnol. Bioeng. 90:775-779 (2005)), or other suitable assay and detection methods well known in the art. The individual enzyme or protein activities from the exogenous DNA sequences used to create the screened library can also be assayed using methods well known in the art. For typical Assay Methods, see Manual on Hydrocarbon Analysis (ASTM Manula Series, A.W. Drews, ed., 6th edition, 1998, American Society for Testing and Materials, Baltimore, Maryland.

The butadiene can be separated from other components in the culture using a variety of methods well known in the art. Such separation methods include, for example, extraction procedures as well as methods that include continuous liquid-liquid extraction, pervaporation (a membrane-based method for the separation of mixtures of liquids by partial vaporization through a non-porous or porous membrane), membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, and ultrafiltration. All of the above methods are well known in the art.

Creating Libraries

Described herein and below in more detail are exemplary methods that have been developed for the mutagenesis and diversification of genes to target desired properties of specific enzymes. Such methods are well known to those skilled in the art. Any of these can be used to alter and/or optimize the activity of a butadiene pathway enzyme or protein to create libraries suitable for the high-throughputs screening methods and apparatuses described herein of the present invention.

For example, known mutations that increase the activity of a protein or enzyme can be introduced into an encoding nucleic acid molecule. Additionally, optimization methods can be applied to increase the activity of an enzyme or protein and/or decrease an inhibitory activity, for example, decrease the activity of a negative regulator.

One such optimization method is directed evolution. Directed evolution is a powerful approach that involves the introduction of mutations targeted to a specific gene in order to improve and/or alter the properties of an enzyme. Improved and/or altered enzymes can be identified through the development and implementation of sensitive high-throughput screening assays that allow the automated screening of many enzyme variants (for example, >104).

Iterative rounds of mutagenesis and screening typically are performed to afford an enzyme with optimized properties. Computational algorithms that can help to identify areas of the gene for mutagenesis also have been developed and can significantly reduce the number of enzyme variants that need to be generated and screened. Numerous directed evolution technologies have been developed (for reviews, see Hibbert et al, Biomol. Eng 22: 11-19 (2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical and biotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press; Otten and Quax. Biomol. Eng 22: 1-9 (2005).; and Sen et al, Appl Biochem.Biotechnol 143:212-223 (2007)) to be effective at creating diverse variant libraries, and these methods have been successfully applied to the improvement of a wide range of properties across many enzyme classes. Enzyme characteristics that have been improved and/or altered by directed evolution technologies include, for example: selectivity/specificity, for conversion of non-natural substrates; temperature stability, for robust high temperature processing; pH stability, for bioprocessing under lower or higher pH conditions; substrate or product tolerance, so that high product titers can be achieved; binding (Km), including broadening substrate binding to include non-natural substrates; inhibition (Ki), to remove inhibition by products, substrates, or key intermediates; activity (kcat), to increases enzymatic reaction rates to achieve desired flux; expression levels, to increase protein yields and overall pathway flux; oxygen stability, for operation of air sensitive enzymes under aerobic conditions; and anaerobic activity, for operation of an aerobic enzyme in the absence of oxygen.

A number of exemplary methods have been developed for the mutagenesis and diversification of genes to target desired properties of specific enzymes. Such methods are well known to those skilled in the art. Any of these can be used to alter and/or optimize the activity of a butadiene pathway enzyme or protein. Such methods include, but are not limited to EpPCR, which introduces random point mutations by reducing the fidelity of DNA polymerase in PCR reactions (Pritchard et al, J. Theor.Biol. 234:497-509 (2005)); Error-prone Rolling Circle Amplification (epRCA), which is similar to epPCR except a whole circular plasmid is used as the template and random 6-mers with exonuclease resistant thiophosphate linkages on the last 2 nucleotides are used to amplify the plasmid followed by transformation into cells in which the plasmid is re-circularized at tandem repeats (Fujii et al., Nucleic Acids Res. 32:el45 (2004); and Fujii et al, Nat. Protoc. 1 :2493-2497 (2006)); DNA or Family Shuffling, which typically involves digestion of two or more variant genes with nucleases such as DNase I or EndoV to generate a pool of random fragments that are reassembled by cycles of annealing and extension in the presence of DNA polymerase to create a library of chimeric genes (Stemmer, Proc. Natl. Acad. Sci. U.S.A. 91 : 10747-10751 (1994); and Stemmer, Nature 370:389-391 (1994)); Staggered Extension (StEP), which entails template priming followed by repeated cycles of 2 step PCR with denaturation and very short duration of annealing/extension (as short as 5 sec) (Zhao et al., Nat. Biotechnol. 16:258-261 (1998)); Random Priming Recombination (RPR), in which random sequence primers are used to generate many short DNA fragments complementary to different segments of the template (Shao et al., Nucleic Acids Res 26:681-683 (1998)).

Additional methods include Heteroduplex Recombination, in which linearized plasmid DNA is used to form heteroduplexes that are repaired by mismatch repair (Volkov et al, Nucleic Acids Res. 27:el8 (1999); and Volkov et al, Methods Enzymol. 328:456-463 (2000)); Random Chimeragenesis on Transient Templates (RACHITT), which employs DNase I fragmentation and size fractionation of single stranded DNA (ssDNA) (Coco et al., Nat. Biotechnol. 19:354- 359 (2001)); Recombined Extension on Truncated templates (RETT), which entails template switching of unidirectionally growing strands from primers in the presence of unidirectional ssDNA fragments used as a pool of templates (Lee et al, J. Molec. Catalysis 26: 119-129

(2003)); Degenerate Oligonucleotide Gene Shuffling (DOGS), in which degenerate primers are used to control recombination between molecules; (Bergquist and Gibbs, Methods Mol. Biol. 352: 191-204 (2007); Bergquist et al, Biomol. Eng. 22:63-72 (2005); Gibbs et al, Gene 271 :13- 20 (2001)); Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY), which creates a combinatorial library with 1 base pair deletions of a gene or gene fragment of interest

(Ostermeier et al, Proc. Natl. Acad. Sci. U.S.A. 96:3562-3567 (1999); and Ostermeier et al, Nat. Biotechnol. 17: 1205-1209 (1999)); Thio-Incremental Truncation for the Creation of Hybrid Enzymes (THIO-ITCHY), which is similar to ITCHY except that phosphothioate dNTPs are used to generate truncations (Lutz et al, Nucleic Acids Res. 29:E16 (2001)); SCRATCHY, which combines two methods for recombining genes, ITCHY and DNA shuffling (Lutz et al, Proc. Natl. Acad. Sci. U.S.A. 98: 11248-11253 (2001)); Random Drift Mutagenesis (RNDM), in which mutations made via epPCR are followed by screening/selection for those retaining usable activity (Bergquist et al, Biomol. Eng. 22:63-72 (2005)); Sequence Saturation Mutagenesis (SeSaM), a random mutagenesis method that generates a pool of random length fragments using random incorporation of a phosphothioate nucleotide and cleavage, which is used as a template to extend in the presence of "universal" bases such as inosine, and replication of an inosine- containing complement gives random base incorporation and, consequently, mutagenesis (Wong et al, Biotechnol. J. 3:74-82 (2008); Wong et al, Nucleic Acids Res. 32:e26 (2004); and Wong et al, Anal. Biochem. 341 : 187-189 (2005)); Synthetic Shuffling, which uses overlapping oligonucleotides designed to encode "all genetic diversity in targets" and allows a very high diversity for the shuffled progeny (Ness et al, Nat. Biotechnol. 20: 1251-1255 (2002));

Nucleotide Exchange and Excision Technology NexT, which exploits a combination of dUTP incorporation followed by treatment with uracil DNA glycosylase and then piperidine to perform endpoint DNA fragmentation (Muller et al, Nucleic Acids Res. 33:el 17 (2005)).

Further methods include Sequence Homology-Independent Protein Recombination (SHIPREC), in which a linker is used to facilitate fusion between two distantly related or unrelated genes, and a range of chimeras is generated between the two genes, resulting in libraries of single-crossover hybrids (Sieber et al, Nat. Biotechnol. 19:456-460 (2001)); Gene Site Saturation Mutagenesis™ (GSSM™), in which the starting materials include a supercoiled double stranded DNA (dsDNA) plasmid containing an insert and two primers which are degenerate at the desired site of mutations (Kretz et al., Methods Enzymol. 388:3-11 (2004)); Combinatorial Cassette Mutagenesis (CCM), which involves the use of short oligonucleotide cassettes to replace limited regions with a large number of possible amino acid sequence alterations (Reidhaar-Olson et al. Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al. Science 241 :53-57 (1988)); Combinatorial Multiple Cassette Mutagenesis (CMCM), which is essentially similar to CCM and uses epPCR at high mutation rate to identify hot spots and hot regions and then extension by CMCM to cover a defined region of protein sequence space (Reetz et al, Angew. Chem. Int. Ed Engl. 40:3589-3591 (2001)); the Mutator Strains technique, in which conditional ts mutator plasmids, utilizing the mutD5 gene, which encodes a mutant subunit of DNA polymerase III, to allow increases of 20 to 4000-X in random and natural mutation frequency during selection and block accumulation of deleterious mutations when selection is not required (Selifonova et al, Appl. Environ. Microbiol. 67:3645-3649 (2001)); Low et al, J. Mol. Biol. 260:359-3680 (1996)).

Additional exemplary methods include Look-Through Mutagenesis (LTM), which is a multidimensional mutagenesis method that assesses and optimizes combinatorial mutations of selected amino acids (Rajpal et al, Proc. Natl. Acad. Sci. U.S.A. 102:8466-8471 (2005)); Gene Reassembly, which is a DNA shuffling method that can be applied to multiple genes at one time or to create a large library of chimeras (multiple mutations) of a single gene (TUNABLE GENEREASSEMBLY™ (TGR™) technology supplied by Verenium Corporation, San Diego, CA), in silico PROTEIN DESIGN AUTOMATION™ (PDA™) (Xencor, Monrovia, California), which is an optimization algorithm that anchors the structurally defined protein backbone possessing a particular fold, and searches sequence space for amino acid substitutions that can stabilize the fold and overall protein energetics, and generally works most effectively on proteins with known three-dimensional structures (Hayes et al., Proc. Natl. Acad. Sci. U.S.A. 99: 15926- 15931 (2002)); and Iterative Saturation Mutagenesis (ISM), which involves using knowledge of structure/function to choose a likely site for enzyme improvement, performing saturation mutagenesis at chosen site using a mutagenesis method such as Stratagene QUIKCHANGE™ (Stratagene; San Diego CA), screening/selecting for desired properties, and, using improved clone(s), starting over at another site and continue repeating until a desired activity is achieved (Reetz et al, Nat. Protoc. 2:891-903 (2007); and Reetz et al, Angew. Chem. Int. Ed Engl.

45:7745-7751 (2006)).

In alternative embodiments, practicing the invention comprises use of any conventional technique commonly used in molecular biology, microbiology, and recombinant DNA, which are within the skill of the art. Such techniques are known to those of skill in the art and are described in numerous texts and reference works (See e.g., Sambrook et al, "Molecular Cloning: A

Laboratory Manual," Second Edition, Cold Spring Harbor, 1989; and Ausubel et al, "Current Protocols in Molecular Biology," 1987). [0119] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionaries of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole.

As used herein, the singular terms "a," "an," and "the" include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

It is intended that every maximum numerical limitation given throughout this

specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. [0124] All documents cited are, in relevant part, incorporated herein by reference. However, the citation of any document is not to be construed as an admission that it is prior art with respect to the present invention.

In alternative embodiments, the term "headspace" refers to the vapor/air mixture trapped above a solid or liquid sample in a sealed vessel.

In alternative embodiments, the terms "high throughput screening" and "HTS" refer to measuring BD in at least 96 samples in 1, 2, 3 or 4 hours or less. In alternative embodiments, the sample volume is less than 0.1, 0.2, 0.5, 1.0, or 2.0 mL or less.

In alternative embodiments, all component or composition levels are in reference to the active level of that component or composition, and are exclusive of impurities, for example, residual solvents or by-products, which may be present in commercially available sources.

Enzyme components weights are based on total active protein. All percentages and ratios are calculated by weight unless otherwise indicated. All percentages and ratios are calculated based on the total composition unless otherwise. The following examples, and the figures, are intended to clarify the invention, and to demonstrate and further illustrate certain preferred embodiments and aspects without restricting the subject of the invention to the examples and figures.

EXAMPLES

Example 1. An Exemplary Screening Plate Assay

A 96-well plate of cultures producing butadiene is prepared by growing cells expressing a butadiene biosynthetic pathway. A screening plate is prepared containing a solution of maleimide or maleimide and a Diels- Alder catalyst such as lanthanum chloride, LaC13 (Spino et al (1997) Can. J. Chem. 75: 1047-1054). See for example the apparatus of Figures Gl, G2 and H. The membrane placed at the bottom of the screening plate well or the integral membrane bottom of the screening plate allows gas to diffuse from the culture plate through the membrane into the screening plate wells (see Schrader et al (2008) 80(11):4014-9). The screening plate is attached above the culture plate and the two plates are sealed by a gasket, e.g. rubber, silicone, to isolate each well. A sterile plate seal or lid is attached to the top of the screening plate to maintain sterility of the culture. Volatile butadiene produced in a well of the cell culture plate diffuses into the corresponding well of the screening plate.

Maleimide reacts with butadiene to form a product which does not absorb at 270 nm, whereas the unreacted maleimide absorbs in the UV at 270 nm. After the screening plate is incubated with the culture plate, unreacted maleimide is measured. Preferably the unreacted maleimide is measured without operator intervention, as by a plate reader. In some cases it may be preferable to dissemble the apparatus, and isolate the upper plate before reading. In other cases it may be preferable to transfer the maleimide solution to a 96-well microtiter plate to measure absorption at 270 nm. Total butadiene formed can be determined by calculating the difference between a control well, which contained a non-butadiene producing cell, and the butadiene forming cell culture. Maleimide can be detected by other means, including those discussed herein.

To reduce evaporation of liquid in upper plate, the plates can be incubated in humidified enclosure, such a humidified shaker. Example 2. An Exemplary Filter Paper Assay

In one embodiment, a maleimide solution is applied to a filter paper. The filter paper is placed over a culture plate of butadiene -producing cells. See Figures E and F. Volatile butadiene reacts with maleimide on the maleimide-impregnated filter paper. After the filter paper is reacted with butadiene, any unreacted maleimide on the filter paper is reacted with a thiol or amine containing dye or fiuorescent dye by applying a solution containing the unreacted dye. The filter paper can be read directly using a plate reader or is imaged by a CCD camera to detect reacted dye. Butadiene producing cell cultures are identified by a decrease in dye or fluorescent dye on the filter paper.

To reduce evaporation of liquid in filter paper, the plates can be incubated in humidified enclosure, such a humified shaker.

Figure E presents one embodiment of a high-throughput assay using a filter paper over a microtiter plate. BD-sensitive film (e.g. filter paper as described herein) in which the BD reaction can be detected spectrophotometrically allows screening of large numbers of colonies on solid media for BD production. Individual colonies surviving mutagenesis and selection or screening, or engineered colonies, are transferred to colony growth dishes, e.g. Petri dish, that can easily accommodate a colony matrix array and a BD-reactant membrane (i.e. chemochromic sensor) of similar shape. If desired during or following an appropriate growth period, the agar plates can be made to go anaerobic to order to induce BD production by transfer to an anaerobic glove box. The sensor layer can be applied and the colonies produce BD. At the end of the desired production period, the sensor layers are analyzed for the location of "dots" where BD reacted with the chemical reagent on the sensor layer. If necessary, the sensors layers can be removed and the BD-sensing reaction developed prior to detection of, and quantitation of, the reactive dots. The dots correspond to the microbial colony that evolved or improved BD- production under the conditions tested. The identified clones can be transferred from the original plate to liquid media for further cultivation and additional characterization.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.