Unconventional Sources of Phosphorus or Sulfur

RELATED APPLICATIONS

This application claims the benefit of priority to United States Provisional Patent

Application serial number 61 870,469, filed August 27, 2013, which is hereby incorporated by reference.

BACKGROUND

In the fermentation industry, cell culture media is typically formulated to provide all nutrients necessary for the growth of a host cell line, with particular emphasis on meeting the cell line's requirements for carbon, nitrogen, phosphorus, sulfur, and other major nutrients. Some cell lines require additional components, including amino acids, trace minerals and metals, and complex growth factors. The presence of these nutrients provides a suitable growth environment for the organism of choice and, unfortunately, for any potential contaminating organisms. In this environment the production organism is required to compete directly with any contaminant organism in the cell culture.

Even in robust hosts, the combination of opportunistic infections of the culture and the metabolic burden resulting from the demands of product manufacture is a major concern in monoculture operations. Industrial robustness is typically considered a multigenic trait specific to the host strain and thus difficult to engineer predictably into organisms late in the development process. Addition of selective growth inhibitors, such as bacterial antibiotics, is one method used to create a more robust fermentation environment for host organisms that arc resistant to the growth inhibitor. However, antibiotic addition is often undesirable or unfeasible, and spontaneously resistant contaminations frequently result.

Accordingly, there exists a need for rationally engineered traits that, when engineered into a host organism, create a robust monoculture fermentation environment.

SUMMARY OF THE INVENTION

In certain embodiments, the invention relates to a genetically engineered organism, wherein the genetically engineered organism has been transformed by a nucleic acid molecule comprising any one of the sequences disclosed herein. In certain embodiments, the invention relates to a genetically engineered organism, wherein the genetically engineered organism has been transformed by a nucleic acid molecule; the nucleic acid molecule comprises a non-native gene; and the non-native gene encodes for a non-native enzyme selected from the group consisting of NAD:phosphite oxidoreductase (phosphite dehydrogenase), glycerol-3-phosphate dehydrogenase (sn- glycerol 3-phosphate: NAD(+) oxidoreductase, EC 1.1.1.8), glyceraldehyde-3-phosphate dehydrogenase, an organophosphatc degradation enzyme, a phosphodiesterase, a phospholipase, desulfurization enzyme, a dibcnzothiophene-5,5-dioxidc monooxygenase, a 2-hydroxybiphcnyl-2-sulfinatc sulfinolyase, a dibenzothiophene monooxygenase, and a NADH-FMN oxidoreductase.

In certain embodiments, the invention relates to a method, comprising the step of contacting any one of the aforementioned genetically engineered organisms with a substrate.

wherein

the substrate comprises a phosphorus-containing fraction and a non-phosphorus- containing fraction;

the phosphorus-containing fraction comprises, in an amount from about 10% by weight to about 100% by weight, a phosphorus-containing compound of any one of Formulas I-III;

the compound of formula I is

wherein, independently for each occurrence,

-H, alkyl, -OH, -OR ² , -SH, or -SR ²

R is -H, or alkyl;

Y is O or S;

Y is O or S; and

R ² is alkyl; the compound of formula II is

wherein, independently for each occurrence,

R ¹ is -H, oralkyl; and

Y ¹ is O or S;

the compound of formula III is

wherein, independently for each occurrence,

R ³ is -H, -OH, -OR ⁴ , -SH, -SR ⁴ , halo, alkyl, aryl, hetcroaryl, aralkyl, or hctcroaralkyl; and

R ⁴ is alkyl or aryl;

a native organism of the same species as the genetically engineered organism could not metabolize (i.e., use as a source of phosphorus) the phosphorus-containing compound; and

the genetically engineered organism converts the substrate to a product.

In certain embodiments, the invention relates to a method, comprising the step of contacting any one of the aforementioned genetically engineered organisms with a substrate,

wherein

the substrate comprises a sulfur-containing fraction and a non-sulfur-containing fraction;

the sulfur-containing fraction comprises, in an amount from about 10% by weight to about 100% by weight, a sulfur-containing compound of any one of Formulas IV-XI; the compound of formula IV is

wherein, independently for each occurrence,

R ⁵ is -H, -OH, -OR ⁷ , -SH, -SR ⁷ , R ⁷ , halo, alkyl, aryl, hctcroar l, aralkyl, heteroaralkyl, -SO ₂ H, -NHR ⁷ , or -NH-C(=0)-R ⁷ ;

R ⁶ is -H, -OH, -OR ⁷ , -SH, -SR ⁷ , R ⁷ , halo, alkyl, aryl, hctcroaryl, aralkyl, heteroaralkyl, -SO ₂ H, -NHR ⁷ , or -NH-C(=0)-R ⁷ ; and

R ⁷ is cycloalkyl, alkyl, or aryl, or any two R ⁷ , taken together, form a 5- or 6- niembcrcd ring;

the compound of formula V, formula VI, or formula VII, is

wherein, independently for each occurrence,

R ⁸ is -H, -OH, -OR ⁷ , -SH, -SR ⁷ , R ⁷ , halo, alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -SO ₂ H, -NHR ⁷ , or -NH-C(=0)-R ⁷ ;

R ⁷ is cycloalkyl, alkyl, or aryl, or any two R ⁷ , taken together, form a 5- or 6- membered ring; the compound of formula VIII, formula IX, or formula X is

wherein, independently for each occurrence,

R ⁹ is -H, -OH, -OR ⁷ , -SH, -SR ⁷ , R ⁷ , halo, alkyl, atyl, heteroaryl, aralkyl, heteroaralkyl, -SO ₂ H, -NH ₂ , -NHR ⁷ , or -NH-C(=0)-R ⁷ ;

R ⁷ is cycloalkyl, alkyl, or aryl, or any two R ⁷ , taken together, form a 5- or 6- membered ring;

R ¹⁰ is hydroxyalkyl, R ⁹ , or -<CH ₂ ) _x R ^s ; and

x is 1 , 2, 3, or 4;

the compound of formula XI is

wherein, independently for each occurrence,

R ⁹ is -H, -OH, -OR ⁷ , -SH, -SR ⁷ , R ⁷ , halo, alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -SO ₂ H, -NH ₂ , -NHR ⁷ , or -NH-C(=0)-R ⁷ ; and

R ⁷ is cycloalkyl, alkyl, or aryl, or any two R ⁷ , taken together, form a 5- or 6- membered ring;

a native organism of the same species as the genetically engineered organism could not metabolize (i.e., use as a source of sulfur) the sulfur-containing compound; and the genetically engineered organism converts the substrate to a product.

In certain embodiments, the invention relates to a product made by any one of the aforementioned methods.

In certain embodiments, the invention relates to a recombinant vector comprising a gene opcrably linked to a promoter, wherien the gene encodes an enzyme; and the enzyme is NAD:phosphite oxidoreductase (phosphite dehydrogenase), glyccrol-3-phosphatc dehydrogenase (sn-glyccrol 3-phosphatc: NAD(+) oxidoreductase, EC 1.1.1.8), glyccraldchydc-3-phosphatc dehydrogenase, an organophosphatc degradation enzyme, a phosphodiesterase, a phospholipase, desulfurization enzyme, a dibenzothiophene-5,5- dioxide monooxygenase, a 2-hydroxybiphcnyl-2-sulfinatc sulfinolyase, a dibenzothiophene monooxygenase, or a NADH-FMN oxidoreductase.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 depicts various DNA sequences of the invention.

Figure 2 tabulates various organisms of the invention and exemplary potential applications.

Figure 3 tabulates various organisms of the invention and exemplary potential applications.

Figure 4 tabulates various organophosphorus compounds useful as feedstocks in the invention, and the chemical formula of each compound.

Figure 5 tabulates various organosulfur compounds useful as feedstocks in the invention, and the chemical formula of each compound.

Figure 6 depicts the names and structures of various organophosphorus compounds useful as feedstocks in the invention.

Figure 7 depicts the names and structures of various organosulfur compounds useful as feedstocks in the invention.

Figure 8 depicts a plasmid map of vector pNC273, which was used to construct strain NS392.

Figure 9 tabulates the sequence of pNC273.

Figure 10 depicts the growth of two organisms (modified Y. lipolytica NS392 (solid circles) and wild-type S. cerevisiae NS22 (open circles)) on three different growth media: potassium phosphite as the sole phosphorus source (left), potassium phosphate as the sole phosphorus source (middle), and potassium phosphate plus hygromyic as a control condition.

Figure 11 depicts the growth of NS22, wildrype S. cerevisiae, with phosphate or phosphite as phosphorus source in defined medium. Different 10-fold serial dilutions of the inocula were made to observe the possible occurence of a lag phase.

Figure 12 depicts the growth of NS435 with (a) phosphate (Pi), and (b) phosphite

(Pt).

Figure 13 depicts the growth of isolates after serial transfer of NS435 in phosphite media.

Figure 14 depicts a plasmid map of pNC360.

Figure 15 tabulates the sequence of pNC360.

Figure 16 depicts growth of NS435 with different concentrations of hypophosphite.

Figure 17 depicts NS252 + ptxD transformants patched on defined medium agar plates with phosphate (Pi, left) or phosphite (Pt, right) as phosphorus source. Untransforrned NS252 was also patched.

Figure 18 depicts a plasmid map of pNC351.

Figure 19 tabulates the sequence of pNC351.

DETAILED DESCRIPTION OF THE INVENTION

Overview

In certain embodiments, the invention relates to a genetically engineered host organism, wherein the genetically engineered host organism has a non-native ability to obtain a growth-limiting nutrient from a complex substrate; and the complex substrate could not have been metabolized or used as a nutrient by the native host organism. In certain embodiments, the non-native ability will provide the organism with a significant competitive advantage, and provide a major barrier to the success of contaminants in a fermentation.

In certain embodiments, organisms generally contain only a small amount of phosphorus and sulfur (e.g., about 3% and about 1% by mass of the cell, respectively). So, in order to grow, organisms need less of these growth-limiting nutrients as compared to, for example, nitrogen.

In certain embodiments, the genetically engineered host organism is a bacterium, a yeast, a fungus, an algae, a mammalian cell, or an insect cell. In certain embodiments, the genetically engeineered host organism is a bacterium or a yeast.

In certain embodiments, the invention relates to a method of using the above- mentioned genetically engineered host organism, comprising contacting the genetically engineered host organism with a modified cell culture medium. In certain embodiments, the invention relates to a method of using the above-mentioned genetically engineered host organism, comprising contacting the genetically engineered host organism with a modified cell culture medium, wherein the genetically engineered host organism converts the cell culture medium to a product. In certain embodiments, using this approach provides a unique and targeted manner to promote the growth of the desired genetically engineered host organism. In certain embodiments, the above-mentioned methods minimize the growth of contaminant organisms, provide a valuable competitive advantage, and allow management of production of a range of valuable products.

In certain embodiments, the inventive methods decrease or eliminate the need for use of prophylactic antibiotics in large scale yeast cultures. Avoiding unnecessary antibiotics is an important benefit due to emerging environmental considerations and societal pressures. Additionally, in certain embodiments, the technique can be applied to bacterial systems in which antibiotics may not be added. In certain embodiments, the technique can be applied to minimize the growth of wild yeast contaminants that arc natively resistant to many commonly used antibiotics.

In certain embodiments, the genetically engineered host organism is a yeast; and the product is cthanol, isobutanol, lactic acid, succinic acid, crythritol, an isoprcnoid, a lipid, and enzyme product, a bulk commodity chemical, or a high value specialty chemical.

In certain embodiments, the genetically engineered host organism is a bacterium; and the product is butanol, ethanol, isopropanol, 1 ,3-propanediol (PDO), 1 ,4-butanediol (BDO), succinic acid, itaconic acid, an enzyme product, a polyol, a protein product, a bulk commodity chemical, or a high value specialty chemical.

In certain embodiments, the inventive technology is applicable in the production of one or more commodities, fine chemicals, or pharmaceuticals. Definitions

As used herein, the term "biomass" refers to a primarily carbohydrate-containing material. Biomass can also refer to a polysaccharidc-containing material. It can also refer to a cellulose-, hcmicellulose-, or lignoccllulose-containing material. Biomass is commonly obtained from, for example, wood, plants, residue from agriculture or forestry, organic component of municipal and industrial wastes, primary sludges from paper manufacture, waste paper, waste wood (e.g., sawdust), agricultural residues such as corn husks, corn cobs, rice hulls, straw, bagasse, starch from corn, wheat oats, and barley, waste plant material from hard wood or beech bark, fibcrboard industry waste water, bagasse pity, bagasse, molasses, post-fermentation liquor, furfural still residues, aqueous oak wood extracts, rice hull, oats residues, wood sugar slops, fir sawdust, naphtha, corncob furfural residue, cotton balls, rice, straw, soybean skin, soybean oil residue, corn husks, cotton stems, cottonseed hulls, starch, potatoes, sweet potatoes, lactose, waste wood pulping residues, sunflower seed husks, hexose sugars, pentose sugars, sucrose from sugar cane and sugar beets, corn syrup, hemp, and combinations of the above.

"Dry weight" and "dry cell weight" mean weight determined in the relative absence of water. For example, reference to oleaginous cells as comprising a specified percentage of a particular component by dry weight means that the percentage is calculated based on the weight of the cell after substantially all water has been removed.

"Exogenous gene" is a nucleic acid that codes for the expression of an R A and/or protein that has been introduced into a cell (e.g., by transformation/transfection), and is also referred to as a "transgenc." A cell comprising an exogenous gene may be referred to as a recombinant cell, into which additional exogenous genc(s) may be introduced. The exogenous gene may be from a different species (and so heterologous), or from the same species (and so homologous), relative to the cell being transformed. Thus, an exogenous gene can include a homologous gene that occupies a different location in the genome of the cell or is under different control, relative to the endogenous copy of the gene. An exogenous gene may be present in more than one copy in the cell. An exogenous gene may be maintained in a cell as an insertion into the genome (nuclear or plastid) or as an episomal molecule.

"Expression vector" or "expression construct" or "plasmid" or "recombinant DNA construct" is a vehicle for introducing a nucleic acid into a host cell. The nucleic acid can be one that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a scries of specified nucleic acid elements that permit transcription and/or translation of a particular nucleic acid. The expression vector can be part of a plasmid, virus, or nucleic acid fragment, or other suitable vehicle. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.

"Inducible promoter" is a promoter that mediates transcription of an operably linked gene in response to a particular stimulus.

"In operable linkage" is a functional linkage between two nucleic acid sequences, such a control sequence (typically a promoter) and the linked sequence (typically a sequence that encodes a protein, also called a coding sequence). A promoter is in operable linkage with an exogenous gene if it can mediate transcription of the gene.

"L satc" is a solution containing the contents of lysed cells.

"Lysis" is the breakage of the plasma membrane and optionally the cell wall of a biological organism sufficient to release at least some intracellular content, often by mechanical, viral or osmotic mechanisms that compromise its integrity.

"Lysing" is disrupting the cellular membrane and optionally the cell wall of a biological organism or cell sufficient to release at least some intracellular content.

"Osmotic shock" is the rupture of cells in a solution following a sudden reduction in osmotic pressure. Osmotic shock is sometimes induced to release cellular components of such cells into a solution.

The terms "plasmid", "vector" and "cassette" refer to an extra chromosomal element often carrying genes that are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or rccombincd into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell. "Transformation cassette" refers to a specific vector containing a foreign gene and having elements, in addition to the foreign gene, that facilitate transformation of a particular host cell. "Expression cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.

"Promoter" is a nucleic acid control sequence that directs transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

"Recombinant" is a cell, nucleic acid, protein, or vector, which has been modified due to the introduction of an exogenous nucleic acid or the alteration of a native nucleic acid. Thus, e.g., recombinant cells can express genes that arc not found within the native (non-rccombinant) form of the cell or express native genes differently than those genes arc expressed by a non-rccombinant cell. Recombinant cells can, without limitation, include recombinant nucleic acids that encode for a gene product or for suppression elements such as mutations, knockouts, antisense, interfering RNA (RNAi) or dsRNA that reduce the levels of active gene product in a cell. A "recombinant nucleic acid" is a nucleic acid originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases, ligases, exonucleases, and endonucleases, or otherwise is in a form not normally found in nature. Recombinant nucleic acids may be produced, for example, to place two or more nucleic acids in operable linkage. Thus, an isolated nucleic acid or an expression vector formed in vitro by ligating DNA molecules that arc not normally joined in nature, are both considered recombinant for the purposes of this invention. Once a recombinant nucleic acid is made and introduced into a host cell or organism, it may replicate using the in vivo cellular machinery of the host cell; however, such nucleic acids, once produced rccombinantly, although subsequently replicated intraccllularly, arc still considered recombinant for purposes of this invention. Similarly, a "recombinant protein" is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid.

"Sonication" is a process of disrupting biological materials, such as a cell, by use of sound wave energy.

"Transformation" refers to the transfer of a nucleic acid fragment into a host organism or the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "recombinant", "transgenic" or "transformed" organisms. Thus, isolated polynucleotides of the present invention can be incorporated into recombinant constructs, typically DNA constructs, capable of introduction into and replication in a host cell. Such a construct can be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. Typically, expression vectors include, for example, one or more cloned genes under the transcriptional control of 5' and 3' regulatory sequences and a selectable marker. Such vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or dcvclopmcntally-rcgulatcd, or location-specific expression), a transcription initiation start site, a ribosomc binding site, a transcription termination site, and/or a polyadenylation signal.

Microbe Ensineerine

A. Overview

In certain embodiments of the invention, a microorganism is genetically modified to improve or provide de novo growth characteristics on a variety of feedstock materials.

Genes and gene products may be introduced into microbial host cells. Suitable host cells for expression of the genes and nucleic acid molecules arc microbial hosts that can be found broadly within the fungal or bacterial families and which grow over a wide range of temperature, pH values, and solvent tolerances.

Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct chimeric genes to produce the any one of the gene products of the instant sequences. These chimeric genes could then be introduced into appropriate microorganisms via transformation techniques to provide high- level expression of the enzymes.

For example, a gene encoding an enzyme can be cloned in a suitable plasmid, and the aforementioned starting parent strain as a host can be transformed with the resulting plasmid. This approach can increase the copy number of each of the genes encoding the enzymes and, as a result, the activities of these enzymes can be increased. The plasmid is not particularly limited so long as it can autonomously replicate in the microorganism. Vectors or cassettes useful for the transformation of suitable host cells arc well known in the art. Typically the vector or cassette contains sequences directing transcription and translation of the relevant gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5' of the gene harboring transcriptional initiation controls and a region 3' of the DNA fragment which controls transcriptional termination. It is most preferred when both control regions are derived from genes homologous to the transformed host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a production host.

Promoters, cDNAs, and 3'UTRs, as well as other elements of the vectors, can be generated through cloning techniques using fragments isolated from native sources (see for example Molecular Cloning: A Laboratory Manual, Sambrook ct al. (3d edition, 2001 , Cold Spring Harbor Press; and U.S. Pat. No. 4,683,202 (incorporated by reference)). Alternatively, elements can be generated synthetically using known methods (sec for example Gene. 1 95 Oct. 16; 164(1 ):49-53).

B. Homologous Recombination

Homologous recombination is the ability of complementary DNA sequences to align and exchange regions of homology. Transgenic DNA ("donor") containing sequences homologous to the genomic sequences being targeted ("template") is introduced into the organism and then undergoes recombination into the genome at the site of the corresponding genomic homologous sequences.

The ability to carry out homologous recombination in a host organism has many practical implications for what can be carried out at the molecular genetic level and is useful in the generation of an oleaginous microbe that can produced tailored oils. By its very nature homologous recombination is a precise gene targeting event, hence, most transgenic lines generated with the same targeting sequence will be essentially identical in terms of phenotype, necessitating the screening of far fewer transformation events. Homologous recombination also targets gene insertion events into the host chromosome, potentially resulting in excellent genetic stability, even in the absence of genetic selection. Because different chromosomal loci will likely impact gene expression, even from heterologous promotcrs/UTRs, homologous recombination can be a method of querying loci in an unfamiliar genome environment and to assess the impact of these environments on gene expression.

A particularly useful genetic engineering approach using homologous recombination is to co-opt specific host regulatory elements such as promotcrs UTRs to drive heterologous gene expression in a highly specific fashion.

Because homologous recombination is a precise gene targeting event, it can be used to precisely modify any nuclcotidc(s) within a gene or region of interest, so long as sufficient flanking regions have been identified. Therefore, homologous recombination can be used as a means to modify regulatory sequences impacting gene expression of RNA and/or proteins. It can also be used to modify protein coding regions in an effort to modify enzyme activities such as substrate specificity, affinities and Km, and thus affecting the desired change in metabolism of the host cell. Homologous recombination provides a powerful means to manipulate the host genome resulting in gene targeting, gene conversion, gene deletion, gene duplication, gene inversion and exchanging gene expression regulatory elements, such as promoters, enhancers and 3'UTRs.

Homologous recombination can be achieved by using targeting constructs containing pieces of endogenous sequences to "target" the gene or region of interest within the endogenous host cell genome. Such targeting sequences can cither be located 5' of the gene or region of interest, 3' of the genc/rcgion of interest or even flank the genc/rcgion of interest. Such targeting constructs can be transformed into the host cell either as a supercoiled plasmid DNA with additional vector backbone, a PCR product with no vector backbone, or as a linearized molecule. In some cases, it may be advantageous to first expose the homologous sequences within the transgenic DNA (donor DNA) with a restriction enzyme. This step can increase the recombination efficiency and decrease the occurrence of undesired events. Other methods of increasing recombination efficiency include using PCR to generate transforming transgenic DNA containing linear ends homologous to the genomic sequences being targeted.

C. Vectors and Vector Components

Vectors for transformation of microorganisms in accordance with the present invention can be prepared by known techniques familiar to those skilled in the art in view of the disclosure herein. A vector typically contains one or more genes, in which each gene codes for the expression of a desired product (the gene product) and is operably linked to one or more control sequences that regulate gene expression or target the gene product to a particular location in the recombinant cell.

This subsection is divided into subsections. Subsection I describes control sequences typically contained on vectors as well as novel control sequences provided by the present invention. Subsection 2 describes genes typically contained in vectors as well as novel codon optimization methods and genes prepared using them provided by the invention.

J . C ^" onirof Sequences

Control sequences are nucleic acids that regulate the expression of a coding sequence or direct a gene product to a particular location in or outside a cell. Control sequences that regulate expression include, for example, promoters that regulate transcription of a coding sequence and terminators that terminate transcription of a coding sequence. Another control sequence is a 3' untranslated sequence located at the end of a coding sequence that encodes a polyadenylation signal. Control sequences that direct gene products to particular locations include those that encode signal peptides, which direct the protein to which they are attached to a particular location in or outside the cell.

Thus, an exemplary vector design for expression of an exogenous gene in a microbe contains a coding sequence for a desired gene product (for example, a selectable marker, or an enzyme) in operable linkage with a promoter active in microalgac. Alternatively, if the vector docs not contain a promoter in operable linkage with the coding sequence of interest, the coding sequence can be transformed into the cells such that it becomes opcrably linked to an endogenous promoter at the point of vector integration.

The promoter used to express an exogenous gene can be the promoter naturally linked to that gene or can be a heterologous promoter.

A promoter can generally be characterized as cither constitutive or inducible.

Constitutive promoters are generally active or function to drive expression at all times (or at certain times in the cell life cycle) at the same level. Inducible promoters, conversely, are active (or rendered inactive) or are significantly up- or down-regulated only in response to a stimulus. Both types of promoters find application in the methods of the invention. Inducible promoters useful in the invention include those that mediate transcription of an opcrably linked gene in response to a stimulus, such as an exogenously provided small molecule, temperature (heat or cold), lack of phosphorus or sulfur in culture media, etc. Suitable promoters can activate transcription of an essentially silent gene or upregulatc, preferably substantially, transcription of an opcrably linked gene that is transcribed at a low level.

Inclusion of termination region control sequence is optional, and if employed, then the choice is be primarily one of convenience, as the termination region is relatively interchangeable. The termination region may be native to the transcriptional initiation region (the promoter), may be native to the D A sequence of interest, or may be obtainable from another source. Sec, for example, Chen and Orozco, Nucleic Acids Res. ( 1988) 16:841 1.

2. Genes and Codon Optimization

Typically, a gene includes a promoter, coding sequence, and termination control sequences. When assembled by recombinant DNA technology, a gene may be termed an expression cassette and may be flanked by restriction sites for convenient insertion into a vector that is used to introduce the recombinant gene into a host cell. The expression cassette can be flanked by DNA sequences from the genome or other nucleic acid target to facilitate stable integration of the expression cassette into the genome by homologous recombination. Alternatively, the vector and its expression cassette may remain unintcgratcd (e.g., an cpisomc), in which case, the vector typically includes an origin of replication, which is capable of providing for replication of the heterologous vector DNA.

A common gene present on a vector is a gene that codes for a protein, the expression of which allows the recombinant cell containing the protein to be differentiated from cells that do not express the protein. Such a gene, and its corresponding gene product, is called a selectable marker or selection marker. Any of a wide variety of selectable markers can be employed in a transgene construct useful for transforming the organisms of the invention.

For optimal expression of a recombinant protein, it is beneficial to employ coding sequences that produce mRNA with codons optimally used by the host cell to be transformed. Thus, proper expression of transgenes can require that the codon usage of the transgene matches the specific codon bias of the organism in which the transgene is being expressed. The precise mechanisms underlying this effect are many, but include the proper balancing of available aminoacylated tRNA pools with proteins being synthesized in the cell, coupled with more efficient translation of the transgenic messenger RNA (mRNA) when this need is met. When codon usage in the transgene is not optimized, available tRNA pools arc not sufficient to allow for efficient translation of the heterologous mRNA resulting in ribosomal stalling and termination and possible instability of the transgenic mRNA.

D. Expression of Two or More Exogenous Genes

Further, a genetically engineered microorganism may comprise and express more than one exogenous gene. One or more genes can be expressed using an inducible promoter, which allows the relative timing of expression of these genes to be controlled. Expression of the two or more exogenous genes may be under control of the same inducible promoter or under control of different inducible promoters. In the latter situation, expression of a first exogenous gene can be induced for a first period of time (during which expression of a second exogenous gene may or may not be induced) and expression of a second or further exogenous gene can be induced for a second period of time (during which expression of a first exogenous gene may or may not be induced). Provided herein arc vectors and methods for engineering microbes to grow on non-traditional growth media.

E. Transformation

Cells can be transformed by any suitable technique including, e.g., biolistics, clcctroporation, glass bead transformation and silicon carbide whisker transformation. Any convenient technique for introducing a transgene into a microorganism can be employed in the present invention. Transformation can be achieved by, for example, the method of D. M. Morrison (Methods in Enzymology 68, 326 (1979)), the method by increasing permeability of recipient cells for DNA with calcium chloride (Mandcl, M. and Higa, A., J. Mol. Biol., 53, 159 ( 1970)), or the like.

Examples of expression of transgenes in oleaginous yeast (e.g., Yarrowia lipolytica) can be found in the literature (see, for example, Bordes ct al., J Microbiol Methods, Jun. 27 (2007)). Examples of expression of exogenous genes in bacteria such as E. coli are well known; sec for example Molecular Cloning: A Laboratory Manual, Sambrook ct al. (3d edition, 2001, Cold Spring Harbor Press).

Vectors for transformation of microorganisms in accordance with the present invention can be prepared by known techniques familiar to those skilled in the art. In one embodiment, an exemplary vector design for expression of a gene in a microorganism contains a gene encoding an enzyme in operable linkage with a promoter active in the microorganism. Alternatively, if the vector does not contain a promoter in operable linkage with the gene of interest, the gene can be transformed into the cells such that it becomes operably linked to an endogenous promoter at the point of vector integration. The vector can also contain a second gene that encodes a protein. Optionally, one or both gene(s) is arc followed by a 3' untranslated sequence containing a polyadenylation signal. Expression cassettes encoding the two genes can be physically linked in the vector or on separate vectors. Co-transformation of microbes can also be used, in which distinct vector molecules are simultaneously used to transform cells (see for example Protist 2004 December; 1 5{4):381-93). The transformed cells can be optionally selected based upon the ability to grow in the presence of the antibiotic or other selectable marker under conditions in which cells lacking the resistance cassette would not grow.

Phosphortis-Containine Compounds in Feedstocks

In certain embodiments, the invention relates to use of an atypical phosphorus- containing feedstock comprising, consisting essentially of, or consisting of a phosphorus- containing compound of any one of Formulas I-III. In certain embodiments, a non- genetically engineered organism, i.e., a native organism, could not metabolize (i.e., use as a source of phosphorus) the phosphorus-containing compounds in the feedstock.

In certain embodiments, the invention relates to any one of the aforementioned phosphorus-containing feedstocks, wherein the phosphorus-containing compound is a compound of formula I or a salt thereof:

wherein, independently for each occurrence,

R is -H, alkyl, -OH, -OR ² , -SH, or -SR ² ;

R ¹ is -H, or alkyl;

Y is O or S;

Y ¹ is O or S; and

R ² is alkyl. In certain embodiments, the invention relates to any one of the aforementioned phosphorus-containing feedstocks, wherein the phosphorus-containing compound is a compound of formula II or a salt thereof:

wherein, independently for each occurrence,

R ¹ is -H, or alkyl; and

Y' is O or S.

In certain embodiments, the invention relates to any one of the aforementioned phosphorus-containing feedstocks, wherein the phosphorus-containing compound is a compound of formula III or a salt thereof:

wherein, independently for each occurrence,

R ³ is -H, -OH, -OR ⁴ , -SH, -SR ⁴ , halo, alkyl, aryl, heteroaryl, aralkyl, or hctcroaralkyl; and

R ⁴ is alkyl or aryl.

In certain embodiments, the invention relates to any one of the aforementioned phosphorus-containing feedstocks, wherein the phosphorus-containing compound is selected from the group consisting of:

Sulfur-Containinp Compounds in Feedstocks

In certain embodiments, the invention relates to use of an atypical sulfur-containing feedstock comprising, consisting essentially of, or consisting of a sulfur-containing compound of_any one of Formulas IV-XI. In certain embodiments, a non-genetically engineered organism, i.e., a native organism, could not metabolize (i.e., use as a source of sulfur) the sulfur-containing compounds in the feedstock.

In certain embodiments, the invention relates to any one of the aforementioned sulfur-containing feedstocks, wherein the sulfur-containing compound is a compound of formula IV or a salt thereof:

wherein, independently for each occurrence,

R ⁵ is -H, -OH, -OR ⁷ , -SH, -SR ⁷ , R ⁷ , halo, alkyl, aryl, hcteroaryl, aralkyl, hetcroaralkyl,

-SO ₂ H, -NHR ⁷ , or -NH-C(=0)-R ⁷ ;

R ⁶ is -H, -OH, -OR ⁷ , -SH, -SR ⁷ , R ⁷ , halo, alkyl, aryl, heteroaryl, aralkyl, hetcroaralkyl,

-SO ₂ H, -NHR ⁷ , or -NH-C(=0)-R ⁷ ; and

R ⁷ is cycloalkyl, alkyl, or aryl, or any two R ⁷ , taken together, form a 5- or 6- mcmbcrcd ring. In certain embodiments, the invention relates to any one of the aforementioned sulfur-containing feedstocks, wherein the sulfur-containing compound is a compound of formula V, formula VI, or formula VII, or a salt thereof:

wherein, independently for each occurrence,

R ⁸ is -H, -OH, -OR ⁷ , -SH, -SR ⁷ , R ⁷ , halo, alkyl, aryl, hctcroaryl, aralkyl, hctcroaralkyl,

-SO ₂ H, -NHR ⁷ , or -NH-C(=0)-R ⁷ ;

R ⁷ is cycloalkyl, alkyl, or aryl, or any two R ⁷ , taken together, form a 5- or 6- membered ring.

In certain embodiments, the invention relates to any one of the aforementioned sulfur-containing feedstocks, wherein the sulfur-containing compound is a compound of formula VIII, formula IX, or formula X or a salt thereof:

wherein, independently for each occurrence,

R ⁹ is -H, -OH, -OR ⁷ , -SH, -SR ⁷ , R ⁷ , halo, alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl,

-SO2H, -NH ₂ , -NHR ⁷ , or -NH-C(=0)-R ⁷ ;

R ⁷ is cycloalkyl, alkyl, or aryl, or any two R ⁷ , taken together, form a 5- or 6- mcmbcrcd ring;

R ¹⁰ is hydroxyalkyl, R ⁹ , or -(CH ₂ ) _X R ⁹ ; and

x is 1 , 2, 3, or 4.

In certain embodiments, the invention relates to any one of the aforementioned sulfur-containing feedstocks, wherein the sulfur-containing compound is a compound of formula XI or a salt thereof:

wherein, independently for each occurrence,

R ⁹ is -H, -OH, -OR ⁷ , -SH, -SR ⁷ , R ⁷ , halo, alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl,

-SO ₂ H, -NH ₂ , -NHR ⁷ , or -NH-C(=0)-R ⁷ ; and

R ⁷ is cycloalkyl, alkyl, or aryl, or any two R ⁷ , taken together, form a 5- or 6- mcmbcrcd ring.

In certain embodiments, the invention relates to any one of the aforementioned sulfur-containing feedstocks, wherein the sulfur-containing compound is selected from the group consisting of:

Exemplary Isolated Nucleic Acid Molecules and Vectors

In certain embodiments, the invention relates to an isolated nucleic acid molecule, wherein the nucleic acid molecule encodes an enzyme that provides the organism with the ability to assimilate a phosphorus source or a sulfur source that otherwise would not have been accessible to the native organism; and the enzyme is NAD:phosphite oxidorcductase (phosphite dehydrogenase), glycerol-3 -phosphate dehydrogenase (sn-glyccrol 3-phosphate: NAD(+) oxidoreductase, EC 1.1.1.8), glyceraldehyde-3-phosphate dehydrogenase, an organophosphate degradation enzyme, a phosphodiesterase, a phospholipase, dcsulfurization enzyme, a dibcnzothiophcnc-5,5-dioxidc monooxygenase, a 2- hydroxybiphenyl-2-sulfinatc sulfinolyase, a dibenzothiophene monooxygenase, or a NADH-FMN oxidorcductase.

In certain embodiments, the invention relates to an isolated nucleic acid molecule, wherein the nucleic acid molecule is selected from the group consisting of Del/tia acidoorans phosphodiesterase pdeA, Ertterobacter aerogenes ltpdABDE gpdQ, Flavobacterium opdA without periplasmic leader sequence, Psendomonas aeruginosa PAOl phoA, Pseudomonas onteilii Cl l hoc A, Pseudomonas stutzeri WM88 hlxABCDEFHGIJKLMN, Pseudomonas stutzeri WM88 plxABCDE, Rhodococcus dszD, and Rhodococcus dszABC.

In certain embodiments, the invention relates to an isolated nucleic acid molecule comprising any one of the sequences disclosed herein. In certain embodiments, the invention relates to an isolated nucleic acid molecule having at least 85% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the invention relates to an isolated nucleic acid molecule having at least 90% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the invention relates to an isolated nucleic acid molecule having at least 95% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the invention relates to an isolated nucleic acid molecule having at least 99% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the invention relates to an isolated nucleic acid molecule having any one of the sequences disclosed herein.

A recombinant vector comprising any one of the aformcntioncd nucleic acid molecules opcrably linked to a promoter.

In certain embodiments, the invention relates to a recombinant vector comprising any one of the sequences disclosed herein. In certain embodiments, the invention relates to a recombinant vector having at least 85% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the invention relates to a recombinant vector having at least 90% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the invention relates to a recombinant vector having at least 95% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the invention relates to a recombinant vector having at least 99% sequence homology with any one of the sequences disclosed herein.

Exemplary Genetically Engineered Oreanisms of the Invention

In certain embodiments, the invention relates to a genetically engineered organism, wherein the genetically engineered organism has been transformed by a nucleic acid molecule or a recombinant vector comprising any one of the sequences disclosed herein. In certain embodiments, the nucleic acid molecule or recombinant vector has at least 85% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the nucleic acid molecule or recombinant vector has at least 90% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the nucleic acid molecule or recombinant vector has at least 95% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the nucleic acid molecule or recombinant vector has at least 99% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the invention relates to a genetically engineered organism, wherein the genetically engineered organism has been transformed by a nucleic acid molecule or a recombinant vector having any one of the sequences disclosed herein.

In certain embodiments, the invention relates to a genetically engineered organism, wherein the genetically engineered organism has been transformed by a nucleic acid molecule; the nucleic acid molecule comprises a non-native gene; and the non-native gene encodes for a non-native enzyme selected from the group consisting of NAD:phosphite oxidorcductase (phosphite dehydrogenase), glyccrol-3-phosphatc dehydrogenase (sn- glyccrol 3-phosphate: NAD(+) oxidorcductase, EC 1.1.1.8), glyceraldehyde-3-phosphate dehydrogenase, an organophosphatc degradation enzyme, a phosphodiesterase, a phospho lipase, desulfurization enzyme, a dibenzothiophene-5,5-dioxide monooxygenase, a 2-hydroxybiphcnyl-2-sulfinatc sulfinolyase, a dibenzothiophene monooxygenase, and a NADH-FM oxidorcductase.

In certain embodiments, the invention relates to any one of the aforementioned genetically engineered organisms, wherein the non-native gene is selected from the group consisting of dszABC, dszA, dszABCD, ds∑B, dszC, dszD, gpdQ, hocA, lxA, txABCDEEHGUKLMN, hlxB, hlxC, hlxD, htxE, txF, htxG, htxH, htxl, htxJ, htxK, htxL, htxM, htxN, opdA, ophA, pde, pdeA, phoA, plxABCDE, ptxD, gpA, ugpAECB, ugpB, ugpC, ugpE, tipdA, updABDE, updB, updD, and pdE.

Any organism may be used as a source of the non-native gene, as long as the organisms has the desired enzymatic activity The non-native gene can each be obtained from chromosomal DNA of any one of the aforementioned microorganisms by isolating a DNA fragment complementing auxotrophy of a variant strain lacking the enzymatic activity. Alternatively, if the nucleotide sequence of these gene of the organism has already been elucidated (Biochemistry, Vol. 22, pp.5243-5249, 1983; J. Biochem. Vol. 95, pp.909- 916, 1984; Gene, Vol. 27, pp.193-199, 1984; Microbiology, Vol. 140, pp.1817- 1828, 1994; Mol. Gene Genet. Vol. 218, pp.330-339, 1989; and Molecular Microbiology, Vol. 6, pp317-326, 1992), the genes can be obtained by PCR using primers synthesized based on each of the elucidated nucleotide sequences, and the chromosome DNA as a template.

In certain embodiments, the invention relates to any one of the aforementioned genetically engineered organisms, wherein the non-native gene is selected from the group consisting of De!fiia acidoorans phosphodiesterase pdeA, E ierobacter aerogenes updABDE gpdQ, Flavobacterium opdA without periplasmic leader sequence, Pseudomonas aeruginosa PAO l phoA, Pseudomonas monieUii C l l hocA, Pseudomonas stutzeri WM88 htxABCDEFHGUKLMN, Pseudomonas stutzeri W 88 ptxABCDE, Rhodococcus dszD, and Rhodococcus dszABC.

In certain embodiments, the invention relates to any one of the aforementioned genetically engineered organisms, wherein the genetically engineered organism is a species of the genus Acetobacter, Acinetobacter, Afcaligenes, Arxula, Aspergillus, Auranliochytrium, Bacillus, Candida, Chlamydomonas, Clostridium, Corynebaclerium, Escherichia, Hansenula, Isochrysis, Kluyveromyces, Lactococcus, Micrococcus, Nannochloropsis, Ogataea, Paracocctis, Pavlova, Penicilfiiim, Pichia, Pseudomonas, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharo yces, Streptococcus, Streptomyces, Synechococcus, Tetraselmis, Thermoanaerobacter, Thermoanaerobacteriu , Trichoderma, Xanthaomonas, or Yarrowia.

In certain embodiments, the invention relates to any one of the aforementioned genetically engineered organisms, wherein the genetically engineered organism is a species of the genus Aspergillus, Bacillus, Chlamydomonas, Corynebaclerium, Escherichia, Hansenula, Kluyveromyces, Saccharomyces, Synechococcus, or Yarrowia.

In certain embodiments, the invention relates to any one of the aforementioned genetically engineered organisms, wherein the genetically engineered organism is selected from the group consisting of Acetobacter, Acinetobacter calcoaceticus, Alcaligenes eutropha, Arxula adeninivorans, Aspergillus nidulans, Aspergillus niger, Aspergillus orzyae, Aspergillus terreus, Auranliochytrium spp., Bacillus licheniforms, Bacillus methanolicus. Bacillus stearothermophilus, Bacillus subtilis, Candida utilis, Chlamydomonas reinhardtii, Clostridium acetobutylicum, Clostridium thermocellum, Corynebaclerium gltilamicum, Escherichia colt, Hansenula polymorpha, Isochrysis spp., Kluyveromyces laclis, Kluyveromyces marxianus, Lactococcus lactis, Micrococcus lysodeiklicus, Nannochloropsis spp. , Ogataea, Paracoccus denitriflcans, Pavlova spp., Penicillium chrysoge um, Pichia gttilliermondii, Pichia pastoris, Pichia stipitis, Pseudomonas ptilida, Rhizop s spp., Rhodosporidium spp., Rhodolortila spp., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Streptococcus lactis, Streplomyces, Synechococcus elongatus, Tetrasel is spp., Thermoanaerobacter spp., Thermoanaerobacter him spp., Trichoderma reesei, Xanthaomonas campeslris, or Yarrowia lipofytica.

In certain embodiments, the invention relates to any one of the aforementioned genetically engineered organisms, wherein the genetically engineered organism is selected from the group consisting of Aspergillus niger, Bacillus subtilis, Chlamydomonas reinhardtii, Corynebacteriitm glutamicum, Escherichia coli, Hansemila po!ymorpha, Kluyveromyces marxianus, Saccharomyces cerevisiae, Synechococcus elongatus, or Yarrowia lipofytica. Exemplary Methods of the Invention

In certain embodiments, the invention relates to a method, comprising the step of contacting any one of the aforementioned genetically engineered organisms with a substrate,

wherein

the substrate comprises a phosphorus-containing fraction and a non-phosphorus- containing fraction;

the phosphorus-containing fraction comprises, in an amount from about 10% by weight to about 100% by weight, a phosphorus-containing compound of any one of Formulas I-III;

a native organism of the same species as the genetically engineered organism could not metabolize (i.e., use as a source of phosphorus) the phosphorus-containing compound; and

the genetically engineered organism converts the substrate to a product.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the phosphorus-containing compounds have a low molecular weight. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the phosphorus-containing compounds have a molecular weight between about 30 Da and about 800 Da. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the phosphorus-containing compounds have a molecular weight between about 40 Da and about 600 Da. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the phosphorus-containing compounds have a molecular weight of about 40 Da, about 50 Da, about 60 Da, about 70 Da, about 80 Da, about 90 Da, about 100 Da, about 1 10 Da, about 120 Da, about 130 Da, about 140 Da, about 150 Da, about 160 Da, about 170 Da, about 180 Da, about 190 Da, about 200 Da, about 220 Da, about 240 Da, about 260 Da, about 280 Da, about 300 Da, about 320 Da, about 340 Da, about 360 Da, about 380 Da, about 400 Da, about 420 Da, about 440 Da, about 460 Da, about 480 Da, about 500 Da, about 520 Da, about 540 Da, bout 560 Da, about 580 Da, or about 600 Da.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the phosphorus-containing compounds have less than 12 carbon atoms. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the phosphorus-containing compounds have less than 8 carbon atoms. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the phosphorus-containing compounds have 1 , 2, 3, 4, 5, 6, or 7 carbon atoms.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the phosphorus-containing compounds are between about 8% and about 75% phosphorus by weight. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the phosphorus-containing compounds are between about 15% and about 47% phosphorus by weight. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the phosphorus-containing compounds arc about 8%, about 10%, about 12%, about 14%, about 16%, about 18%, about 20%, about 22%, about 24%, about 26%, about 28%, about 30%, about 32%, about 34%, about 36%, about 38%, about 40%, about 42%, about 44%, about 46%, about 48%, about 50%, about 52%, about 54%, about 56%, about 58%, about 60%, about 62%, about 64%, about 66%, about 68%, about 70%, about 72%, or about 74% phosphorus by weight.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the phosphorus-containing compounds have an octanol-water partition coefficient (log P) less than about 5. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the phosphorus-containing compounds have an octanol-watcr partition coefficient (log P) from about -0.5 to about 5. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the phosphorus-containing compounds have an octanol-water partition coefficient (log P) of about -0.5, about 0, about 0.5, about 1 , about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, or about 4.5.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the phosphorus-containing compounds are soluble in water at about 20 °C at a concentration of between about 0.01 g/L to about 1000 g L. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the phosphorus- containing compounds arc soluble in water at about 20 °C at a concentration of about 0.01 g L, about 0.05 g L, about 0.1 g/L, about 0.5 g/L, about 1 g/L, about 5 g/L, about 10 g/L, about 15 g/L, about 20 g/L, about 25 g/L, about 30 g/L, about 35 g L, about 40 g/L, about 45 g/L, about 50 g/L, about 55 g/L, about 60 g/L, about 65 g/L, about 70 g L, about 75 g L, about 80 g/L, about 85 g/L, about 90 g L, about 95 g/L, or about 100 g/L.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the phosphorus-containing compounds move through the cell membrane by passive transport. Passive transport includes diffusion, facilitated diffusion, and filtration.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the phosphorus-containing compounds move through the cell membrane by active transport, such as, for example, via an ATP-Binding Cassette (ABC) transporter or other known transmembrane transporter.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the phosphorus-containing compounds arc transported through the cell membrane.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the phosphorus-containing compounds are substantially non-biocidal.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the phosphorus-containing compounds are substantially biodegradable.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the phosphorus-containing fraction comprises the phosphorus-containing compound in about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% by weight.

In certain embodiments, the invention relates to a method, comprising the step of contacting any one of the aforementioned genetically engineered organisms with a substrate,

wherein

the substrate comprises a phosphorus-containing fraction and a non-phosphorus- containing fraction ;

the phosphorus-containing fraction comprises, in an amount from about 10% by weight to about 100% by weight, a phosphorus-containing compound selected from the group consisting of:

the genetically engineered organism converts the substrate to a product.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the phosphorus-containing fraction comprises the phosphorus-containing compound in about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% by weight. In certain embodiments, the invention relates to a method, comprising the step of contacting any one of the aforementioned genetically engineered organisms with a substrate,

wherein

the substrate comprises a phosphorus-containing fraction and a non-phosphorus- containing fraction;

the phosphorus-containing fraction consists essentially of a phosphorus-containing compound selected from the group consisting of

the genetically engineered organism converts the substrate to a product.

In certain embodiments, the invention relates to a method comprising the step of contacting any one of the aforementioned genetically engineered organisms with a substrate,

wherein

the substrate consists of a phosphorus-containing fraction and a non-phosphorus- containing fraction; the phosphorus-containing fraction consists of a phosphorus-containing compound

selected from the group consisting of

the genetically engineered organism converts the substrate to a product.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the genetically engineered organism sequesters the product.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein a plurality of genetically engineered organisms is used.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the substrate does not comprise an antibiotic.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein a non-genetically engineered organism, i.e., a native organism, could not metabolize (i.e., use as a source of phosphorus) the phosphorus-containing compound.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the substrate comprises lignocellulosic material, glucose, xylose, sucrose, acetic acid, formic acid, lactic acid, butyric acid, a free fatty acid, dextrose, glycerol, fructose, lactose, galactose, mannose, rhamnose, or arabinose, or a combination thereof.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the pH of the substrate is from about 2.5 to about 10.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the genetically engineered organism is contacted with the substrate at a temperature of from about 15 °C to about 80 °C.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the genetically engineered organism is contacted with the substrate over a time period of from about 6 h to about 10 d.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the genetically engineered organism is contacted with the substrate in a fermentor.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the genetically engineered organism is contacted with the substrate in an industrial-size fermentor.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein a plurality of genetically engineered organisms is contacted with a plurality of substrates in a plurality of fermentors, wherein the plurality of fermentors are arranged in parallel.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the product is ethanol, isopropanol, lactic acid, an isoprenoid, a lipid, a high-value specialty chemical, butanol, 1 ,3-propanediol, 1,4-butanediol, succinic acid, an expressed protein product, an enzyme product, a polyol, a pharmaceutical product, itaconic acid, or a high value specialty chemical.

In certain embodiments, the invention relates to a method, comprising the step of contacting any one of the aforementioned genetically engineered organisms with a substrate,

wherein the substrate comprises a sulfur-containing fraction and a non-sulfur-containing fraction;

the sulfur-containing fraction comprises, in an amount from about 10% by weight to about 100% by weight, a sulfur-containing compound of any one of Formulas IV-XI;

a native organism of the same species as the genetically engineered organism could not metabolize (i.e., use as a source of sulfur) the sulfur-containing compound; and

the genetically engineered organism converts the substrate to a product.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the sulfur-containing compounds have a low molecular weight. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the sulfur-containing compounds have a molecular weight between about 30 Da and about 800 Da. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the sulfur-containing compounds have a molecular weight between about 40 Da and about 600 Da. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the sulfur-containing compounds have a molecular weight of about 40 Da, about 50 Da, about 60 Da, about 70 Da, about 80 Da, about 90 Da, about 100 Da, about 1 10 Da, about 120 Da, about 130 Da, about 140 Da, about 150 Da, about 160 Da, about 170 Da, about 180 Da, about 1 0 Da, about 200 Da, about 220 Da, about 240 Da, about 260 Da, about 280 Da, about 300 Da, about 320 Da, about 340 Da, about 360 Da, about 380 Da, about 400 Da, about 420 Da, about 440 Da, about 460 Da, about 480 Da, about 500 Da, about 520 Da, about 540 Da, bout 560 Da, about 580 Da, or about 600 Da.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the sulfur-containing compounds have less than 12 carbon atoms. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the sulfur-containing compounds have less than 8 carbon atoms. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the sulfur-containing compounds have 1, 2, 3, 4, 5, 6, or 7 carbon atoms.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the sulfur-containing compounds have an octanol-watcr partition coefficient (log P) less than about 5. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the sulfur-containing compounds have an octanol-watcr partition coefficient (log P) from about -0.5 to about 5. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the sulfur-containing compounds have an octanol-water partition coefficient (log P) of about - 0.5, about 0, about 0.5, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, or about 4.5.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the sulfur-containing compounds are soluble in water at about 20 °C at a concentration of between about 0.01 g/L to about 1000 g/L. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the sulfur-containing compounds are soluble in water at about 20 °C at a concentration of about 0.01 g/L, about 0.05 g L, about 0.1 g L, about 0.5 g L, about 1 g L, about 5 g/L, about 10 g L, about 15 g L, about 20 g L, about 25 g/L, about 30 g/L, about 35 g L, about 40 g/L, about 45 g/L, about 50 g/L, about 55 g/L, about 60 g L, about 65 g/L, about 70 g/L, about 75 g L, about 80 g L, about 85 g L, about 90 g/L, about 95 g L, or about 100 g/L.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the sulfur -containing compounds move through the cell membrane by passive transport. Passive transport includes diffusion, facilitated diffusion, and filtration.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the sulfur-containing compounds move through the cell membrane by active transport, such as, for example, via an ATP-Binding Cassette (ABC) transporter or other known transmembrane transporter.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the sulfur-containing compounds are transported through the cell membrane.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the sulfur-containing compounds arc substantially non-biocidal.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the sulfur-containing compounds arc substantially biodegradable.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the sulfur-containing fraction comprises the phosphorus-containing compound in about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% by weight.

In certain embodiments, the invention relates to a method, comprising the step of contacting any one of the aforementioned genetically engineered organisms with a substrate,

wherein

the substrate comprises a sulfur-containing fraction and a non-phosphorus- containing fraction ;

the sulfur-containing fraction comprises, in an amount from about 10% by weight to about 100% by weight, a sulfur-containing compound selected from the group consisting of:

the genetically engineered organism converts the substrate to a product.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the sulfur-containing fraction comprises the sulfur-containing compound in about 10%, about 1 %, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% by weight.

In certain embodiments, the invention relates to a method, comprising the step of contacting any one of the aforementioned genetically engineered organisms with a substrate,

wherein

the substrate comprises a sulfur-containing fraction and a non- sulfur-containing fraction;

the sulfur-containing fraction consists essentially of a sulfur-containing compound

the genetically engineered organism converts the substrate to a product.

In certain embodiments, the invention relates to a method comprising the step of contacting any one of the aforementioned genetically engineered organisms with a substrate,

wherein the substrate consists of a sulfur-containing fraction and a non-sulfur-containing fraction;

the sulfur-containing fraction consists of a sulfur-containing compound selected

the genetically engineered organism converts the substrate to a product. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the genetically engineered organism sequesters the product.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein a plurality of genetically engineered organisms is used.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the substrate does not comprise an antibiotic.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein a non-gcnctically engineered organism, i.e., a native organism, could not metabolize (i.e., use as a source of sulfur) the sulfur-containing compound.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the substrate comprises lignocellulosic material, glucose, xylose, sucrose, acetic acid, formic acid, lactic acid, butyric acid, a free fatty acid, dextrose, glycerol, fructose, lactose, galactose, mannose, rhamnose, or arabinosc, or a combination thereof.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the pH of the substrate is from about 2.5 to about 10.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the genetically engineered organism is contacted with the substrate at a temperature of from about 15 °C to about 80 °C.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the genetically engineered organism is contacted with the substrate over a time period of from about 6 h to about 10 d.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the genetically engineered organism is contacted with the substrate in a fermentor.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the genetically engineered organism is contacted with the substrate in an industrial-size fermentor.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein a plurality of genetically engineered organisms is contacted with a plurality of substrates in a plurality of fermentors, wherein the plurality of fermentors are arranged in parallel.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the product is ethanol, isopropanol, lactic acid, an isoprenoid, a lipid, a high-value specialty chemical, butanol, 1 ,3-propanediol, 1,4-butanediol, succinic acid, an expressed protein product, an enzyme product, a polyol, a pharmaceutical product, itaconic acid, or a high value specialty chemical.

Exemplary Products

In certain embodiments, the invention relates to a product made by any one of the aforementioned methods. EXEMPLIFICATION

The following examples are provided to illustrate the invention. It will be understood, however, that the specific details given in each example have been selected for purpose of illustration and arc not to be construed as limiting the scope of the invention. Generally, the experiments were conducted under similar conditions unless noted.

Example 1 - Use of phosphite as phosphorus source for ptx -expressing Yarrowia tipol tica

A) Expression of bacterial ptxD in Yarrowia lipolytica

Vector pNC273 was used to construct strain NS392. See Figure 8.

Vector pNC273 was restriction digested with enzyme Pmel, and the linear fragment containing hygR and ptxD under control of the Y. lipolytica TEFI promoter and Y. lipolytica CYC1 terminator. Transformation was via standard protocols (Chen D.C. et al., Appl Microbiol Biotcchnol. 1997 Aug; 48(2):232-5). See Figure 9.

B) Competition experiment between /ptx -expressing Y. lipolytica and wildtype

S. cerevisiae

Yarrowia lipolytica NS392 (ptxD HYG ^R ) and Saccharomyces cerevisiae NS22 (wildtype, HYG ^S ) were pre-cultured overnight in defined medium with 10 mM potassium phosphate (NS22) or 10 mM potassium phosphite (NS392). After pre-culture growth, cells were washed twice in water and added to three experimental media conditions at an OD _{600 mm} of 0.06 for NS22 and 0.6 for NS392. The three media conditions were with potassium phosphite as sole phosphorus source, potassium phosphate as sole phosphorus source, and with potassium phosphate plus hygromycin as a control condition to select to HYG ^R Y. lipolytica and against HYG ^S S. cerevisiae. See Figure 10.

Defined Media composition

Macro nutrients

Glucose monohydrate 44

Urea 3

MgSO ₄ -7H ₂ 0 0.5

Potassium hydrogen phthalate 1

Disodium phthalate 4.25

Vitamins mg/L

Biotin 0.05

Thiamine 1.0 D-Pantothcnic acid 1.0

Nicotinic acid 1.0

myo-inositol 25

Pyridoxine 1.0

5 p-Aminobcnzoic acid 0.2

Micro elements mg/L

EDTA 15

CaCl ₂ 6H ₂ 0 7.5

(NH ₄ ) ₂ FeSO ₄ -6H ₂ 0 3.0

CuSO 5H ₂ 0 0.2

ZnSO ₄ -7H ₂ 0 1.0

MnSO ₄ H ₂ 0 0.5

Na ₂ Mo0 ₄ -2H ₂ 0 0.2

To this base medium, add either g/L

H ₂ P0 1.3

H ₂ P0 ₃ 1.2 Example 2 - Use of hypophosphite as phosphorus source for ptxD-expressing Yarrowia Upolytica

Y. Upolytica expressing ptxD also grows on hypophosphite. As shown in Table 1, NS 18, wildype Y. Upolytica, does not grow on phosphite or hypophosphite, and neither does NS 184, a Y. Upolytica strain engineered for increased lipid production. NS324 (created by transforming NS18 with pNC273) and NS392 (created by transforming NS I 84 with pNC273) arc able to grow on both phosphite and hypophosphite. However, E. coli W31 10, which has the native ability to convert phosphite to phosphate, is unable to grow on hypophosphite. Additionally, W31 10 was unable to grow on hypophosphite pre-incubated in defined yeast medium, suggesting that hypophosphite is not degraded to phosphite by incubation in medium alone. Additional measurements of growth with phosphate, phosphite, and hypophosphite are shown below in Table 2 for strain NS324.

Tabic 2

Example 3 - Use of phosphite as phosphorus source for ptxD-expressing Saccharomyces cerevisiae

Wildtype S. cerevisiae was shown to not grow with phosphite supplied as phosphorus source (Figure 1 1). Plasmid pNC273, containing ptxD under control of the Y. lipolytica TEF1 promoter, was transformed into S. cerevisiae NS22. Despite evidence of functional plxD expression in Y. lipolytica with the same vector, and prior evidence of Y. lipolytica TEFI promoter function in .V. cerevisiae expressing an antibiotic resistance marker, no growth was seen with this transformed construct with phosphite as phosphorus source. Subsequently, plxD was placed under control of the S. cerevisiae TEFI promoter in vector pNC360. With this transformed strain, NS435, growth was observed with phosphite as phosphorus source, although a lag phase was present. (Figure 12). To reduce the lag phase, NS435 was serial transferred 10 times in 5 mL defined medium with I mM potassium phosphite replacing potassium phosphate. Serial transfers were performed after cultures reached stationary phase, with approximately 70 generations occuring during the transfers. From the final serial passage whole culture was streaked to single colonics on phosphite containing solid agar medium. Scrveral of these isolates were grown in defined medium with 1 niM phosphate, washed, and cvalatcd in medium with 10 mM phosphite for growth rate and lag phase (Figure 13). Of these, a top performing isolate was retained and designated NS473.

Example 4 - Use of hypophosphite as phosphorus source for ptxD-expressing Saccharomyces cerevisiae

S. cerevisiae functionally expressing ptxD also surprisingly grows on hypophosphite. As shown in Table 3, NS22, wildype S. cerevisiae, docs not grow on phosphite or hypophosphite. NS435 (created by transforming NS22 with pNC360) is able to grow on both phosphite and hypophosphite. However, E. coli W31 10, which has the native ability to convert phosphite to phosphate, is unable to grow on hypophosphite. Additionally, W31 10 was unable to grow on hypophosphite prc-incubatcd in defined yeast medium, suggesting that hypophosphite is not degraded to phosphite by incubation in medium alone. Additional measurements of growth with hypophosphite arc shown below in Figure 16 for strain NS435.

Tabic 3

Example 5 - Use of phosphite as phosphorus source for ptxD-expressing Arxala adeninivorans

A. adeninivorans strain NS252, a wildtypc strain, was transformed with plasmid pNC351 , containing the ptxD gene under control of the A. adeninivorans PG 1 promoter. Transformation was performed with an clectrotransformation protocol with selection on defined medium plates with 1 mM potassium phosphite as phosphorus source (sec below). Colonics grew on plates spread with cells from the NS252 + pNC351 transformation, and 25 of these colonies were patched onto phosphate and phosphite defined media agar plates, and evaluated for the presence of ptxD via colony PCR. 24 of the 25 the putative transformants were positive for ptxD by colony PCR, and 25 of 25 displayed rapid growth on phosphite plates. Arxula adeninivoram transformation protocol

1. Inoculate 5 mL of YPD media in a 14-mL culture tube with A. adeninivorans strain NS252 from a YPD plate and put it in the 37 °C drum roller for overnight incubation.

2. Add about 2.5 mL of the overnight liquid culture into a 250-mL flask containing 22.5 mL of fresh YPD and incubate in the 37 °C shaker for 3.5-4 hrs.

3. Centrifuge the culture at 3000 rpm for 3 mins. Discard the supernatant, wash the cells with water followed by ccntrifugation and discard the supernatant again.

4. To the cell pellet, add 2 mL of 100 mM lithium acetate solution and 40 μL of 2 M dithiothrcitol. Transfer into an Eppcndorf tube.

5. Tape the tube on the 37 °C wheel and let it incubate for an hour.

6. Centrifuge at 10000 rpm for 10 seconds, discard the supernatant.

7. Wash cells with 1 mL water, and mix by gentle pipetting.

8. Centrifuge, discard supernatant, wash with cold 1 M sorbitol, mix by pipetting, centrifuge, discard supernatant.

9. Add 2 mL of cold 1 M sorbitol to cell pellet, place it on ice.

10. Into the prc-chillcd 0.2-cm clcctroporation cuvettes, add 40 μL of the cells and 5 μL. of DNA to be transformed, ideally at > 100 μg/ml DNA concentration.

1 1. Elcctroporatc at 25 μF, 200 ohms, 1.5 kV, ~4.9-5.0 ms time constant.

12. Recover transformed cells using 1 mL YPD at 37 °C overnight.

13. Plate 100 μL·5 0 μL. of the recovered culture onto appropriate selective plates, and incubate at 30 °C or 37 °C until colony formation.

INCORPORATION BY REFERENCE

All of the U.S. patents and U.S. published patent applications cited herein are hereby incorporated by reference. EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents arc intended to be encompassed by the following claims.