RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patent application serial number 61/905,454, filed on November 18, 2013, the contents of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Isoprene (CAS Number 78-79-5) is a 5-carbon hydrocarbon useful as a starting material for synthesizing a variety of chemicals. Isoprene may be used as a monomer or co- monomer for the production of higher value polymers. Examples of chemicals that can be produced using isoprene include polyisoprene, polybutylene, styrene-isoprene-styrene block co-polymers, and others. An example of an industry that uses isoprene is the synthetic rubber industry. The majority of isoprene is produced as a "by-product" during the processing of crude oil into usable fractions. For example, isoprene has typically been produced during the catalytic cracking of crude oil fractions. However, recently the use of catalytic crackers has diminished due to the availability of inexpensive natural gas, resulting in a reduced supply of the four and five carbon chain molecules that are found in crude oil, but not natural gas.

Although the direct conversion of dimethylallyl diphosphate to isoprene by isoprene synthase enzymes is known in the art, the present inventors have shown that isoprene can also be produced from two different alcohols, 3-methyl-2-buten-l-ol and 2-methyl-3-buten-2-ol, using a 2-methyl-3-buten-2-ol dehydratase such as, for example, linalool dehydratase- isomerase an enzyme isolated from Castellaniella defragrans strain 65Phen. Thus, improved simple, low-cost methods for converting 3-methyl-2-buten-l-ol and / or 2-methyl-3-buten-2- ol to isoprene leveraging the catalytic ability of linalool dehydratase-isomerase in the process are desirable.

SUMMARY OF THE INVENTION

The invention provides an immobilized enzyme system comprising a 2-methyl-3- buten-2-ol dehydratase enzyme immobilized on a support or a microorganism expressing a 2- methyl-3-buten-2-ol dehydratase enzyme immobilized on a support. The invention also provides methods of producing isoprene comprising the step of contacting 3-methyl-2-buten-

1- ol or 2-methyl-3-buten-2-ol with an immobilized enzyme system of the invention to effect the conversion of 3-methyl-2-buten-l-ol or 2-methyl-3-buten-2-ol to isoprene. In one embodiment, the invention provides a method of producing isoprene by contacting 3-methyl-

2- buten-l-ol or 2-methyl-3-buten-2-ol with an optionally immobilized, 2-methyl-3-buten-2-ol dehydratase, to effect the conversion of 3-methyl-2-buten-l-ol or 2-methyl-3-buten-2-ol to isoprene and optionally collecting the isoprene.

Embodiments of the present invention provide non-naturally occurring microbial organisms, i.e., microorganisms that include a heterologous 2-methyl-3-buten-2-ol dehydratase enzyme. The microorganisms include an exogenous nucleic acid encoding a 2- methyl-3-buten-2-ol dehydratase expressed at a sufficient level to catalyze the conversion of 2-methyl-3-buten-2-ol to isoprene. A preferred example of a 2-methyl-3-buten-2-ol dehydratase is linalool dehydratase-isomerase derived from Castellaniella defragrans strain 65Phen.

In one embodiment, the non-naturally occurring microorganisms are immobilized on a support. The cells may be immobilized through an adhesion process to supports such as wood chips, activated charcoal, plastic particles, etc. Alternatively, the cells may be immobilized through entrapment in a support, for example entrapment or encapsulation in alginate or polyvinyl alcohol matrices, or through covalent crosslinking to the support, for example crosslinking to chitosan or chitin using glutaraldehyde or another suitable crosslinking reagent.

In one embodiment, a support can be porous or nonporous, but is preferably porous. It can be continuous or non-continuous, flexible or nonflexible. A support can be made of a variety of materials including supports made of ceramic, glassy, metallic, organic polymeric materials, or combinations thereof. Examples of supports include macroporous and microporous supports including but not limited to: organic polymeric supports, such as particulate or beaded supports, woven and nonwoven webs (such as fibrous webs), microporous fibers, microporous membranes, hollow fibers or tubes. Porous materials are particularly desirable because they provide large surface areas. The porous support can be synthetic or natural, organic or inorganic. In one embodiment of the present invention, the 2-methyl-3-buten-2-ol dehydratase is recovered or purified from the non-naturally occurring microorganism. The enzyme may exist in a crude enzyme extract, e.g. , a whole-cell lysate or a lysate cleared of insoluble materials, or as a partially or substantially purified enzyme preparation. In one embodiment, the 2-methyl-3-buten-2-ol dehydratase enzyme is immobilized on a support. The enzyme may be a crude enzyme extract, partially purified, or substantially purified. The enzyme may be immobilized through crosslinking to the support, for example, crosslinking to chitosan or chitin using glutaraldehyde or another suitable crosslinking reagent. In another embodiment, the 2-methyl-3-buten-2-ol dehydratase is modified to contain an epitope to facilitate either purification or immobilization, or both.

In one embodiment, the invention provides a method of producing isoprene by contacting 3-methyl-2-buten-l-ol or 2-methyl-3-buten-2-ol from any source including non- naturally occurring microorganisms expressing the alcohols as described previously, synthetically prepared alcohols, or alcohols obtained from the byproduct of other reactions, with an optionally immobilized, 2-methyl-3-buten-2-ol dehydratase to effect the conversion of 3-methyl-2-buten-l-ol or 2-methyl-3-buten-2-ol to isoprene and thereafter collecting the isoprene. In this embodiment the dehydratase may be in the form of a crude enzyme extract, partially purified, or substantially purified and may optionally further be immobilized on a support.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the manner in which the above recited features of the present disclosure can be understood in detail, a more particular description of the disclosure, briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.

Figure 1 shows the chemical structures of (a) 3-methyl-2-buten-l-ol, (b) 2-methyl-3- buten-2-ol and (c) isoprene. Figure 2 shows an E. co/z-codon-optimized nucleic acid sequence (SEQ ID NO: 1), including an artificial ribosome binding site and an amino-terminal 6-histidine epitope tag, for the linalool dehydratase-isomerase of Castellaniella defragrans strain 65Phen.

Figure 3 shows an E. co/z ^' -codon-optimized nucleic acid sequence (SEQ ID NO: 2), including an artificial ribosome binding site and an amino-terminal 6-histitidine epitope tag, for strawberry alcohol acyltransferase.

Figure 4 shows a gas chromatogram of a 1 ml sample of the headspace of a 20- milliliter vial containing 1 mM 3-methyl-2-buten-l-ol dissolved in Luria Bertani broth. Peak 1 is 3-methyl-2-buten-l-ol, with a retention time of 3.96 minutes.

Figure 5 shows a gas chromatogram of a 1 ml sample of the headspace of a 20- milliliter vial containing 1 mM 2-methyl-3-buten-2-ol dissolved in Luria Bertani broth. Peak 1 is 2-methyl-3-buten-2-ol with a retention time of 2.96 minutes.

Figure 6 shows a gas chromatogram of a 1 ml sample of the headspace of a 20- milliliter vial containing E. coli strain BL21 harboring plasmid pJ404-LDI cultured overnight on 1 mM 3-methyl-2-buten-l-ol in Luria Bertani broth supplemented with 100 μg/ml ampicillin. Peak 1 is 3-methyl-2-buten-l-ol, with a retention time of 3.96 minutes. Peak 2 is 2-methyl-3-buten-2-ol with a retention time of 2.96 minutes. Peak 3 is isoprene, with a retention time of 2.49 minutes.

Figure 7 shows a gas chromatogram of a 1ml sample of the headspace of a 20- milliliter vial containing E. coli strain BL21 harboring plasmid pJ404-LDI cultured overnight on 1 mM 2-methyl-3-buten-2-ol in Luria Bertani broth supplemented with 100 μg/ml ampicillin. Peak 1 is 2-methyl-3-buten-2-ol with a retention time of 2.96 minutes. Peak 2 is isoprene with a retention time of 2.49 minutes.

Figure 8 shows the results of GC/MS analysis of authentic isoprene.

Figure 9 shows the results of GC/MS analysis of the peak at 2.49 minutes from Example 1, verifying the identity of the peak as isoprene. DETAILED DESCRIPTION

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only, and are not restrictive of the invention, as claimed. In this application, the use of the singular includes the plural, the word "a" or "an" means "at least one", and the use of "or" means "and/or", unless specifically stated otherwise. Furthermore, the use of the term "including", as well as other forms, such as "includes" and "included", is not limiting. Also, terms such as "element" or "component" encompass both elements or components comprising one unit and elements or components that comprise more than one unit unless specifically stated otherwise. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.

As used herein, the term "non-naturally occurring" when used in reference to a microbial organism or microorganism of the invention is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary metabolic polypeptides include enzymes or proteins that convert 2-methyl-3-buten-2-ol to isoprene.

A metabolic modification refers to a biochemical reaction that is altered from its naturally occurring state. Therefore, non-naturally occurring microorganisms can have genetic modifications to nucleic acids encoding metabolic polypeptides or, functional fragments thereof. Exemplary metabolic modifications are disclosed herein.

As used herein, the term "isolated" when used in reference to a microbial organism is intended to mean an organism that is substantially free of at least one component as the referenced microbial organism is found in nature. The term includes a microbial organism that is removed from some or all components as it is found in its natural environment. The term also includes a microbial organism that is removed from some or all components as the microbial organism is found in non-naturally occurring environments. Therefore, an isolated microbial organism is partly or completely separated from other substances as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments. Specific examples of isolated microbial organisms include partially pure microbes, substantially pure microbes and microbes cultured in a medium that is non-naturally occurring.

As used herein, the terms "microbe," "microbial," "microbial organism" or

"microorganism" are intended to mean any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The term also includes cell cultures of any species that can be cultured for the production of a biochemical. "Exogenous" as it is used herein is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the microbial organism. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism. The source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity following introduction into the host microbial organism. Therefore, the term "endogenous" refers to a referenced molecule or activity that is present in the host.

Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the microbial organism. The term "heterologous" refers to a molecule or activity derived from a source other than the referenced species whereas "homologous" refers to a molecule or activity derived from the host microbial organism. Accordingly, exogenous expression of an encoding nucleic acid of the invention can utilize either or both a heterologous or homologous encoding nucleic acid.

The non-naturally occurring microbial organisms of the invention can contain stable genetic alterations, which refer to microorganisms that can be cultured for greater than five generations without loss of the alteration. Generally, stable genetic alterations include modifications that persist greater than 10 generations, particularly stable modifications will persist more than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely.

Those skilled in the art will understand that the genetic alterations, including metabolic modifications exemplified herein, are described with reference to a suitable host organism such as Escherichia coli and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway. However, given the complete genome sequencing of a wide variety of organisms and the high level of skill in the area of genomics, those skilled in the art will readily be able to apply the teachings and guidance provided herein to essentially all other organisms. For example, the E. coli metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species. Such genetic alterations include, for example, genetic alterations of species homologs, in general, and in particular, orthologs, paralogs or non-orthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent and are responsible for substantially the same or identical functions in different organisms. For example, mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologs for the biological function of hydrolysis of epoxides. Genes are related by vertical descent when, for example, they share sequence similarity of sufficient amount to indicate they are homologous, or related by evolution from a common ancestor. Genes can also be considered orthologs if the proteins that they code for share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable. Genes that are orthologous can encode proteins with sequence similarity of about 25% to 100% amino acid sequence identity. Genes encoding proteins sharing an amino acid similarity less that 25% can also be considered to have arisen by vertical descent if their three-dimensional structure also shows similarities. Members of the serine protease family of enzymes, including tissue plasminogen activator and elastase, are considered to have arisen by vertical descent from a common ancestor.

Orthologs include genes or their encoded gene products that through, for example, evolution, have diverged in structure or overall activity. For example, where one species encodes a gene product exhibiting two functions and where such functions have been separated into distinct genes in a second species, the three genes and their corresponding products are considered to be orthologs. For the production of a biochemical product, those skilled in the art will understand that the orthologous gene harboring the metabolic activity to be introduced or disrupted is to be chosen for construction of the non-naturally occurring microorganism. An example of orthologs exhibiting separable activities is where distinct activities have been separated into distinct gene products between two or more species or within a single species. A specific example is the separation of elastase proteolysis and plasminogen proteolysis, two types of serine protease activity, into distinct molecules as plasminogen activator and elastase. A second example is the separation of mycoplasma 5'-3' exonuclease and Drosophila DNA polymerase III activity. The DNA polymerase from the first species can be considered an ortholog to either or both of the exonuclease or the polymerase from the second species and vice versa.

In contrast, paralogs are homologs related by structure or ancestry but with different functions. These might arise by, for example, duplication of a gene followed by

evolutionary divergence to produce proteins with similar or common, but not identical functions. Paralogs can originate or derive from the same species or from a different species. For example, microsomal epoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase (epoxide hydrolase II) can be considered paralogs because they represent two distinct enzymes, co-evolved from a common ancestor, that catalyze distinct reactions and have distinct functions in the same species. Paralogs are proteins from the same species with significant sequence similarity to each other suggesting that they are homologous, or related through co-evolution from a common ancestor. Groups of paralogous protein families include HipA homologs, luciferase genes, peptidases, and others.

A non-orthologous gene displacement is a non-orthologous gene from one species that can substitute for a referenced gene function in a different species. Substitution includes, for example, being able to perform substantially the same or a similar function in the species of origin compared to the referenced function in the different species. Although generally, a non-orthologous gene displacement will be identifiable as structurally related to a known gene encoding the referenced function, less structurally related but functionally similar genes and their corresponding gene products nevertheless will still fall within the meaning of the term as it is used herein. Functional similarity requires at least some structural similarity in the active site or binding region of a non-orthologous gene product compared to a gene encoding the function sought to be substituted. Therefore, examples of non-orthologous genes include paralogs or unrelated genes.

Therefore, in identifying and constructing the non-naturally occurring microbial organisms of the invention having a 2-methyl-3-buten-2-ol dehydratase activity, those skilled in the art will understand with applying the teaching and guidance provided herein to a particular species that the identification of metabolic modifications can include identification and inclusion or inactivation of orthologs. To the extent that paralogs and/or non-orthologous gene displacements are present in the referenced microorganism that encode an enzyme catalyzing a similar or substantially similar metabolic reaction, those skilled in the art also can utilize these evolutionally related genes. Orthologs, paralogs and non-orthologous gene displacements can be determined by methods well known to those skilled in the art. As defined herein, enzymes or genes that are described or claimed as being "derived from" an organism include any homologs, paralogs, non-orthologous gene displacements that have substantially similar activity.

The methods and techniques utilized for culturing or generating the microorganisms disclosed herein are known to the skilled worker trained in microbiological and recombinant DNA techniques. Methods and techniques for growing microorganisms (e.g., bacterial cells), transporting isolated DNA molecules into the host cell and isolating, cloning and sequencing isolated nucleic acid molecules, knocking out expression of specific genes, etc., are examples of such techniques and methods. These methods are described in many items of the standard literature, which are incorporated herein in their entirety: "Basic Methods In Molecular Biology" (Davis, et al, eds. McGraw-Hill Professional, Columbus, OH, 1986); Miller, "Experiments in Molecular Genetics" (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1972); Miller, "A Short Course in Bacterial Genetics" (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1992); Singer and Berg, "Genes and Genomes" (University Science Books, Mill Valley, CA, 1991); "Molecular Cloning: A Laboratory Manual," 2nd Ed. (Sambrook, et al, eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989); "Handbook of Molecular and Cellular Methods in Biology and

Medicine" (Kaufman, et al., eds., CRC Press, Boca Raton, FL, 1995); "Methods in Plant Molecular Biology and Biotechnology" (Glick and Thompson, eds., CRC Press, Boca Raton, FL, 1993); and Smith-Keary, "Molecular Genetics of Escherichia coi (The Guilford Press, New York, NY, 1989).

In the present disclosure, we have developed a system for the conversion of two different alcohols, 3-methyl-2-buten-l-ol and 2-methyl-3-buten-2-ol, to isoprene using linalool dehydratase-isomerase (see Example 1 and Figures 6 and 7), an enzyme first identified in Castellaniella defragrans strain 65Phen. 3-methyl-2-buten-l-ol, 2-methyl-3- buten-2-ol and isoprene are hemiterpenoids (5-carbon molecules, Figure 1).

The natural substrates of linalool dehydratase-isomerase are acyclic monoterpenes geraniol, linalool and myrcene. Monoterpenes are 10-carbon molecules that are derived from a repeating 5-carbon subunit. The bi-functional linalool dehydratase-isomerase catalyzes the isomerization of geraniol and linalool in a reversible reaction as well as the interconversion of linalool and myrcene through a reversible hydration / dehydration reaction. The enzyme is believed to help Castellaniella defragrans strain 65Phen to use myrcene as a sole source of carbon by first hydrating the myrcene to linalool, and then isomerizing linalool to geraniol. Geraniol is then further metabolized to acetyl-CoA and other metabolites.

The 3-methyl-2-buten-l-ol or 2-methyl-3-buten-2-ol may be from any source. That is, the 3-methyl-2-buten-l-ol or 2-methyl-3-buten-2-ol may be derived from a

thermochemical process. The 3-methyl-2-buten-l-ol or 2-methyl-3-buten-2-ol may be derived from a bio-based process. The 3-methyl-2-buten-l-ol or 2-methyl-3-buten-2-ol may be produced on-purpose by a fermentation-based process. The 3-methyl-2-buten-l-ol or 2- methyl-3-buten-2-ol may be substantially pure, for example, greater than 90% 3-methyl-2- buten-l-ol or 2-methyl-3-buten-2-ol by weight. Or, the 3-methyl-2-buten-l-ol or 2-methyl- 3-buten-2-ol may be partially purified or unpurified and present at low concentrations, for example, 1% by weight or less.

The 2-methyl-3-buten-2-ol dehydratase enzyme activity may be supplied to the reaction as a non-naturally occurring microorganism or as an enzyme extract. The enzyme extract may be a crude extract, e.g., a whole-cell lysate, or it may be a partially or substantially purified enzyme. In one embodiment, the enzyme's purity is less than about 50%. In one embodiment, the enzyme's purity is greater than about 50%. In one embodiment the enzyme's purity is greater than about 75%, 85%, 95% or 99%. In a preferred embodiment, the 2-methyl-3-buten-2-ol dehydratase is supplied to the reaction as non-naturally occurring microorganism that has been immobilized on a substrate. In another preferred embodiment, the 2-methyl-3-buten-2-ol dehydratase is supplied to the reaction as an enzyme extract that has been immobilized on a substrate. Immobilization may permit the reuse or prolonged use of the linalool dehydratase-isomerase in the isoprene production process. In any of the embodiments, the 2-methyl-3-buten-2-ol dehydratase enzyme activity may be supplied by linalool dehydratase-isomerase of Castellaniella defragrans strain 65Phen.

EXAMPLE 1

MICROORGANISM FOR THE PRODUCTION OF ISOPRENE FROM 3-METHYL-2-BUTEN-1-OL

This working example shows the production of isoprene from 3 -methyl-2 -buten-l-ol and 2-methyl-3-buten-2-ol by a non-naturally occurring microorganism expressing one or more exogenous genes of an isoprene biosynthetic pathway.

The plasmid pJ404-LDI was constructed by DNA2.0 (Menlo Park, CA) using the E coli-codon-optimized sequence (SEQ ID NO: 1) of the linalool dehydratase-isomerase (LDI) of Castellaniella defragrans strain 65Phen. The LDI coding sequence was codon-optimized for expression in E. coli, synthesized and inserted into the plasmid expression vector pJexpress404. The resulting plasmid, pJ404-LDI, was electroporated into E. coli BL21 electrocompetent cells. Plasmid pJ404-SAAT was constructed by DNA2.0 (Menlo Park, CA) using the codon-optimized sequence (SEQ ID NO: 2) of the strawberry alcohol acyltransferase (SAAT). The SAAT coding sequence was codon-optimized for expression in E. coli, synthesized and inserted into the plasmid expression vector pJexpress404. The resulting plasmid, pJ404-SAAT, was electroporated into E. coli BL21 electrocompetent cells. pJ404- SAAT was used as a negative control.

Transformants of BL21 harboring either pJ404-LDI or pJ404-SAAT were selected on Luria-Bertani (LB)-agar plates (10 g/L yeast extract, 5 g/L Bacto Tryptone, 10 g/L sodium chloride, 15 g/L Bacto Agar) containing 100 μg/ml ampicillin. A single colony of BL21 harboring pJ404-LDI or pJ404-SAAT from the LB-agar plates was used to inoculate 10 ml of LB broth (10 g/L yeast extract, 5 g/L Bacto Tryptone, 10 g/L sodium chloride) containing 100 μg/ml ampicillin contained in 125-mL Erlenmeyer flasks. Flasks were incubated for 16 hours at 37 °C in a rotary shaking incubator. After 16 hours, the resulting cultures were diluted using fresh LB broth containing 100 μg/ml ampicillin to an optical density of 0.16 at 600 nm. 50 ml of the diluted cultures were placed in 300-ml Erlenmeyer flasks and incubated at 37 °C in a rotary shaking incubator until the optical density at 600 nm reached approximately 0.6, typically 90 minutes. 4 ml of the resulting cultures were then placed into 20 ml gas chromatography headspace vials. 3- methyl-2-buten-l-ol or 2-methyl-3-buten-2-ol was added to a final concentration of 1 mM, IPTG (Isopropyl β-D-l-thiogalactopyranoside) was added to 0.1 mM, and the cultures were grown for an additional 16 hours at 37 °C with shaking.

Isoprene was measured using headspace analysis on an Agilent 7890A GC equipped with a CTC-PAL autosampler and a FID. Headspace vials (20 ml) were incubated at 50 °C with agitation at 500 rpm for 2 minutes. Then 1 ml of the headspace was removed using a heated headspace syringe at 50 °C and injected into the GC inlet (250 °C, split of 20: 1).

Samples were analyzed using a FID detector set at 300 °C, with a helium carrier gas flow rate of 2 ml/min through a DB-624 30 m x 530 μιη x 3 μιη column (J&W Scientific), and an oven program of 85 °C for 5.25 minutes. The isoprene concentration in samples was calculated from calibration curves generated from isoprene calibration gas standards analyzed under the same GC/FID method. The isoprene product was also confirmed by headspace GC/MS using an Agilent 7890A GC equipped with a 5975C MSD and a CTC-PAL autosampler. Headspace vials were incubated at 85 °C with agitation at 600 rpm for 5 minutes. Then 1 ml of the headspace was removed using a heated headspace syringe at 85 °C and injected into the GC inlet (250 °C, split of 25: 1). The GC/MS method used helium as the carrier gas at 1 ml/min through a HP-5MS 30 m x 250 μιη x 0.25 μιη column (J&W Scientific), an oven program of 35 °C for 4 minutes, then ramped 25 °C/min to 150 °C, a MS source temperature of 230 °C, and a quadrupole temperature of 150 °C. The mass spectrometer was operated in scan mode from 25 to 160 mass units. The isoprene peak was identified by the NIST 1 1 MS Library, as well as comparison against an authentic sample (135 ppm isoprene, 135 ppm carbon dioxide in dry nitrogen gas, Matheson TRIGAS, Houston, TX). 3-methyl-2-buten-l-ol and 2-methyl-3-buten-2-ol were measured using headspace analysis on an Agilent 7890A GC equipped with a CTC-PAL autosampler and a FID.

Headspace vials (20 ml) were incubated at 85 °C with agitation at 600 rpm for 5 minutes. Then 1 ml of the headspace was removed using a heated headspace syringe at 85 °C and injected into the GC inlet (250 °C, split of 25: 1). Samples were analyzed using a FID detector set at 350 °C, with a helium carrier gas flow rate of 3 ml/min through at DB-624 30 m x 530 μιη x 3 μιη column (J&W Scientific), and an oven program of 90 °C, then ramping 20 °C/min to 230 °C for 3 minutes. The 3-methyl-2-buten-l-ol and 2-methyl-3-buten-2-ol concentrations in samples were calculated from calibration curves generated from diluted standards of each compound analyzed under the same GC/FID method. The results of this example are presented in Figures 4, 5, 6, 7, 8 and 9. LB broth containing 1 mM 3-methyl-2-buten-l-ol without E. coli cells showed a peak at 3.96 minutes corresponding to 3-methyl-2-buten-l-ol (Figure 4). LB broth containing 1 mM 2-methyl-3- buten-2-ol without E. coli cells showed a peak at 2.96 minutes (Figure 5). Similarly, cultures containing 1 mM 3-methyl-2-buten-l-ol with BL21 cells harboring pJ404-SAAT showed a peak at 3.96 minutes corresponding to 3-methyl-2-buten-l-ol, and an additional peak corresponding to the aldehyde 3-methyl-2-buten-l-al (prenal, data not shown). When 2- methyl-3-buten-2-ol was substituted for 3-methyl-2-buten-l-ol, BL21 cells harboring pJ404- SAAT showed only a single peak at 2.96 minutes corresponding to 2-methyl-3-buten-2-ol.

In contrast, cultures containing 1 mM 3-methyl-2-buten-l-ol with BL21 cells harboring pJ404-LDI converted 3-methyl-2-buten-l-ol to 2-methyl-3-buten-2-ol and isoprene, corresponding to peaks at 2.96 minutes and 2.49 minutes, respectively (Figure 6). Likewise, cultures containing 1 mM 2-methyl-3-buten-2-ol with BL21 cells harboring pJ404- LDI converted the 2-methyl-3-buten-2-ol to isoprene, corresponding to the peak at 2.49 minutes.

These results demonstrate that E. coli cells harboring pJ404-LDI isomerize 3-methyl- 2-buten-l-ol to 2-methyl-3-buten-2-ol and dehydrate 2-methyl-3-buten-2-ol to isoprene.

Figure 8 presents the GC/MS analysis of an authentic isoprene sample; Figure 9 presents the GC/MS analysis of the peak with a 2.49-minute retention time, with the same fragmentation pattern as authentic isoprene shown in Figure 8.

EXAMPLE 2 ENZYME PREPARATION FOR THE CONVERSION OF

3 -METHYL-2-BUTEN- 1 -OL OR 2-METHYL-3-BUTEN-2-OL TO ISOPRENE

This working example shows the production of isoprene from 2-methyl-3-buten-2-ol by an enzyme preparation of linalool dehydratase-isomerase.

A single colony of BL21 harboring pJ404-LDI or pJ404-SAAT from LB-agar plates was used to inoculate 50 ml of LB broth (10 g/L yeast extract, 5 g/L Bacto Tryptone, 10 g/L sodium chloride) containing 100 μg/ml ampicillin contained in 250-mL Erlenmeyer flasks. Flasks were incubated for 6 hours at 37 °C in a rotary shaking incubator. After 6 hours, the cultures were diluted five-fold to a final volume of 250 mL using fresh LB broth containing 100 μg/ml ampicillin and 0.1 mM IPTG. The initial optical density of the cultures was approximately 0.6 at 600 nm. The cultures were incubated with shaking at 37 °C for an additional two hours, at which time the cells were collected by centrifugation for 10 minutes at 5,000 x g and 4 °C. The cell pellet was resuspended in BugBuster Master Mix (Novagen) following the manufacturer's instructions. The cell suspension was incubated at room temperature on a rotating mixer for 20 minutes. Cellular debris was removed by

centrifugation for 10 minutes at 20,000 x g and 4 °C. The supernatant was transferred to a fresh tube and used to assay enzyme activity.

Enzyme assays were performed as follows. 0.15 mL of the cell lysates containing the linalool dehydratase-isomerase from pJ404-LDI was added to 0.35 mL of a reaction buffer containing 50 mM Tris buffer, pH 7.2, 2 mM dithiothreitol, and varying amounts of 2- methyl-3-buten-2-ol contained in a 20-milliliter headspace vial. The headspace vials were incubated at 37 °C for 1 hour, then analyzed for isoprene production using the GC/MS protocols described above. The results are provided in Table 1, below. Control reactions performed with the strawberry alcohol acyltransferase expressed from pJ404-SAAT showed no detectable isoprene (results not shown). Linalool dehydratase-isomerase catalyzes the conversion of 2-methyl-3-buten-2-ol to isoprene in a concentration-dependent manner. The low background levels of isoprene present in the "No Enzyme Extract" controls at 2.5 mM 2- methyl-3-buten-2-ol and above either reflect isoprene contamination present in the 2-methyl- 3-buten-2-ol or the thermal decomposition of 2-methyl-3-buten-2-ol to isoprene in the GC inlet only detectable at higher 2-methyl-3-buten-2-ol concentrations.

TABLE 1

Isoprene Produced (ppmv)

Initial 232-MB ^* , mM

No Enzyme Extract + LDI ^† Enzyme Extract

0.01 N.D. 0.3

0.25 N.D 9.0

1.0 N.D. 52.8

2.5 0.13 99.9

5.0 0.28 339.8

10.0 0.64 509.3

* 232-MB = 2-methyl-3-buten-2-ol

† LDI = linalool dehydratase-isomerase

EXAMPLE 3 IMMOBILIZATION OF WHOLE-CELL BIOCATALYST

EXPRESSING LINALOOL DEHYDRATASE-ISOMERASE

A single colony of BL21 harboring pJ404-LDI or pJ404-SAAT from LB-agar plates was used to inoculate 5 ml of LB broth (10 g/L yeast extract, 5 g/L Bacto Tryptone, 10 g/L sodium chloride) containing 100 μg/ml ampicillin contained in 14 mL polypropylene round- bottom tubes and incubated overnight at 30 °C in a rotary shaking incubator. The cultures were diluted 100-fold in 100 mL of LB broth containing 100 mg/ml ampicillin contained in 500-mL Erlenmeyer flasks and incubated for 16 hours at 30 °C in a rotary shaking incubator. Fifty milliliters of the cultures were used to inoculate 800 mL of MM29 medium

supplemented with 10 g/L Tryptone and 5 g/L yeast extract in a New Brunswick BioFlo 115 operated at 37 °C, pH 7.0 controlled with 9 % NH40H, and dissolved oxygen at 20% or greater. IPTG was added to 0.1 mM final concentration. The culture was allowed to grow an additional 18 hours (time or OD).

The medium MM29 was prepared by adding the following chemicals to

approximately 800 mL of deionized water: 0.66 g ( H ₄ ) ₂ S0 ₄ , 1.2 g Na ₂ HP0 ₄ , 3 g NH ₄ C1, 0.25 g K ₂ S0 ₄ , and 1 mL of a concentrated micronutrients solution. Sufficient water is added to bring the volume to 990 mL, and the medium is sterilized by autoclave. 10 mL of a divalent cation solution is added after the medium has cooled. The micronutrients solution consists of 1 liter of deionized water, 0.173 g sodium selenite, 0.004 g ( Η ) ₆ Μθ ₇ θ ₂₄ ·4Η ₂ 0, 0.025 g H ₃ BO ₃ , 0.007 g CoCl ₂ -6H ₂ 0, 0.003 g CuS0 ₄ -5H ₂ 0, 0.016 g MnCl ₂ -4H ₂ 0, and 0.003 g ZnS0 ₄ -7H ₂ 0. After mixing the chemicals in the water, the pH is adjusted to 3.0 with 3 M HC1 to fully dissolve the chemicals. The divalent cation solution contains, per L, 40 g MgCl ₂ -6H ₂ 0, 0.3 g FeS0 ₄ -7H ₂ 0, and 7 g CaCl ₂ -2H ₂ 0 dissolved in deionized water.

After 18 h, the cells were collected by centrifugation at 4 °C, 5,000 x g, for 20 minutes. The cells were resuspended in 300 mL of sterile 1 g/L sodium chloride in water followed by centrifugation at 4 °C, 5,000 x g, for 20 minutes. This washed cell pellet was resuspended in 350 mL of a sterile alginate solution (35 g/L sodium alginate, 1 g/L sodium chloride in water). The cells in the sodium alginate solution were loaded into 60-mL syringes and extruded through 20-guage needles as droplets into 250 mL of a sterile, 40 g/L calcium chloride solution in water at approximately 24 °C. The alginate beads were then held at 4 °C for 1 hour to ensure gelation, then washed with 1-L of sterile, ice-cold deionized water. The beads were then resuspended in 250 mL of sterile 150 mM sodium chloride and stored at 4 °C until use. Beads formed using this protocol were 2 to 4 millimeters in diameter.

The beads containing BL21 cells harboring pJ404-LDI were assayed for 2-methyl-3- buten-2-ol dehydratase activity in a manner similar to the enzyme assays. Either 5 or 15 beads were transferred to 20-milliliter headspace vials containing 0.5 mL of 50 mM Tris pH 7.8, 1 mM dithiothreitol and 2.5 mM 2-methyl-3-buten-2-ol. Alternatively, either 5 or 15 beads were transferred to 20-milliliter headspace vials containing 0.5 mL of Luria Bertani broth supplemented with 2.5 mM 2-methyl-3-buten-2-ol. Reactions performed in the absence of beads were used as a negative control. The headspace vials were incubated at 37 °C overnight. The results of this experiment are presented in Table 2, below. Immobilized whole cells of BL21 harboring plasmid pJ404-LDI express 2-methyl-3-buten-2-ol dehydratase activity. It is believed that the Luria Bertani broth allowed the cells to further grow during the course of the experiment, accounting for the higher isoprene concentrations.

TABLE 2

Sample Isoprene (ppmv)

Tris Buffer, No Beads 0.2

Tris Buffer, 5 Beads 62.2

Tris Buffer, 15 Beads (2 hour incubation) 34.4

Luria Bertani Broth, No Beads 0.1

Luria Bertani Broth, 5 Beads 206.7

Luria Bertani Broth, 15 Beads 427.2 While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.