DIHYDROXYACETONE METABOLIZING MICROORGANISMS

AND METHODS OF USING THEM

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application entitled “DIHYDROXYACETONE METABOLIZING MICROORGANISMS AND METHODS OF USING THEM,” having serial number 62/588,589, filed on November 20, 2017, which is entirely incorporated herein by reference.

STATEMENT REGARDING FEDERALY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant DE-PI0000031 awarded by The United States Department of Energy. The government has certain rights in the invention.

SEQUENCE LISTING

The Sequence Listing for this application is labeled“Seq-List.txt” which was created on November 6, 2017 and is 1 14 KB. The entire content of the sequence listing is incorporated herein by reference in its entirety.

BACKGROUND

Due to modern technology of extraction, the amount of natural gas produced in the USA during 2016 was 26.5 trillion cubic feet. Natural gas supplied about 27% of total energy used by the country (US-EIA). Due to the high rate of production, the cost of natural gas has fallen to $3.96 per thousand cubic feet (July 2017 industrial price) from a high value of $13.06 in July 2008. This incentivizes upgrading the inexpensive CH ₄ to value-added chemicals and liquid fuels. A first step in this process is generating fermentable carbon sources, such as dihydroxyacetone (DHA), from natural gas. Technology exists for such conversion (Fig. 1). DHA is a three carbon ketose and in its phosphorylated form (DHA-3-phosphate; DHA-P) is an intermediate of glycolysis. DHA can be catalytically produced from formaldehyde by the formose reaction (Deng et al., 2013; Gehrer et al., 1995; Matsumoto et al., 1984). DHA can be fermented to a number of chemicals and fuels, such as ethanol, butanol, lactate, and succinate, by appropriately engineered microbial biocatalysts (Fig. 1). Formaldehyde is currently industrially produced from methanol and methanol itself is produced from CH ₄ (see worldwide website: ihs.com/products/chemical-technology-pep-reviews-formaldehyd e-from- natural-gas-1974.html), leading to a chemical process from CH ₄ to fermentable sugar, DHA (Fig. 1).

Another attractive starting material for production of DHA is C0 ₂ and such a process is environmentally friendly. Formaldehyde can be produced chemically from C0 ₂ via methanol as an intermediate (Dong et al., 2017; Kothandaraman et al., 2016). In addition, formaldehyde can also be produced biologically from C0 ₂ with formate as an intermediate (Fig. 1) (Alissandratos & Easton, 2015). Rapid conversion of formaldehyde to DHA and DHA-P can serve as a new pathway for conversion of C0 ₂ to bio-products. Dickens and Williamson reported as early as 1958 that DHA can be produced biologically by transketolation of hydroxy pyruvate and formaldehyde (Dickens & Williamson, 1958). This transketolase is implicated in a unique pentose-phosphate dependent pathway (DHA cycle) in methanol utilizing yeast that fixes formaldehyde to xylulose-5-phosphate yielding DHA as an intermediate in the production of glyceraldehyde-3-phosphate in a cyclic mode (O'Connor & Quayle, 1979; Van Dijken et a!., 1978; Waites & Quayle, 1980). Dihydroxyacetone in the cytoplasm is phosphorylated by DHA kinase and/or glycerol kinase and the DHA-3-phosphate that enters glycolysis provides a route for utilization of CH ₄ and C0 ₂ by biological systems. Although formaldehyde can be produced by several microorganisms, the rate of production may not be high enough to support a biorefinery. Recently, Siegel, et al. described a computationally enhanced enzyme, formolase, that converts formaldehyde to DHA (Siegel et al., 2015). However, this pathway is yet to be demonstrated in a microorganism.

Although there are biological, chemical, and hybrid (chemical/biological) processes that can generate DHA at needed quantities from CH and C0 ₂ (Fig. 1), microbial biocatalysts that ferment DHA to bulk chemicals at high yield and productivity are lacking. A complicating factor is the inhibition of microbial growth by DHA due to its interaction with amino groups that induces DNA damage in cells that cannot rapidly metabolize DHA (Maillard reaction) (Petersen et al., 2004). Chemical conversion of DHA to compounds such as lactic acid, a starting material for PLA-based plastics, is known (Lux & Siebenhofer, 2013) but this process is expected to generate a mixture of D(-) and L(+)- isomers of lactic acid that requires expensive purification before use in the biodegradable plastics industry. Since fermentation of sugars by microorganisms is an efficient way of producing optically pure lactic acid, genetically modified microorganisms for production of D-lactic acid from DHA as the feedstock are desirable. In addition, once a fermentation process from DHA to lactate as a model system is developed, this process can be modified and applied to production of any one of several metabolic products of commercial interests that can serve as fuels and chemicals. SUMMARY

Genetic engineering to produce microorganisms that metabolize DHA provides a cost- effective approach for reducing production cost of biologically produced metabolites. Accordingly, genetically modified microorganisms that can increase the microorganisms’ ability to metabolize DHA as described herein.

The genes identified to be associated with the increased metabolism of DHA are ATP- dependent dihydroxyacetone kinase (dhaK), glycerol uptake facilitator ( glpF ), glycerol dehydrogenase ( gldA ), a-acetolactate decarboxylase ( budA ), acetolactate synthase (a/sS), pyruvate formate-lyase (pfIBA ), and methyl glyoxal synthase ( mgsA ). Accordingly, the present disclosure provides microorganisms that contain one or more genetic modifications to these genes. Such microorganisms metabolize DHA to metabolites of commercial interest, such as, D-lactate, butyrate, succinate, and ethanol.

In certain embodiments, the present disclosure provides microorganisms that metabolize DHA, wherein the microorganisms are genetically modified to increase the expression or activity of DhaK protein. The genetic modifications that increase the expression of DhaK include the expression via plasmids, mutations in the genomic DNA of microorganisms that result in the increased expression of DhaK, or mutations in the regulatory region of genes that cause overexpression of DhaK.

In further embodiments, in addition to the genetic modifications that increase expressions or activities of DhaK, the microorganisms are genetically modified to inactivate one or more genes selected from glpf, gldA, budA, alsS, pfIBA, and mgsA.

As such, the subject present disclosure provides microorganisms, for example, Escherichia coli, that are useful in the production of metabolites of interest by growing the microorganisms in media containing DHA. Accordingly, the materials and methods of the present disclosure can be used to produce metabolites used in a variety of applications.

Described herein are embodiments of one or more microorganisms comprising one or more genetic modifications resulting in an increased expression or activity of ATP-dependent dihydroxyacetone kinase (DhaK). In an embodiment, the microorganisms according to the present disclosure have one genetic modification. In an embodiment, the genetic modification resulting in the increased expression of DhaK comprises introducing into the microorganism a nucleotide sequence encoding a DhaK. In an embodiment, the DhaK is the DhaK from the bacterium Klebsiella oxytoca (SEQ ID NO: 50). In an embodiment, the DhaK is encoded by dhaK from the bacterium Klebsiella oxytoca comprises the native promoter and the protein encoding region. In an embodiment, the dhaK from the bacterium Klebsiella oxytoca comprising the native promoter and the protein encoding region comprises the sequence of (SEQ ID NO: 51). In an embodiment, the DhaK comprises a sequence selected from SEQ ID NOs: 28 to 49.

In an embodiment, described herein are microorganisms comprising one or more genetic modifications inactivating one or more genes encoding: glycerol uptake facilitator (GlpF), methylglyoxal synthase (MgsA), acetolactate decarboxylase (BudA), acetolactate synthase (AlsS), and pyruvate formate-lyase (PfIBA), and glycerol dehydrogenase (GldA).

In embodiments, described herein are microorganisms comprising one or more genetic modifications inactivating a combination comprising of genes selected from:

glpF and mgs A;

glpF and bud A;

glpF and alsS;

glpF and pfIBA;

glpf and gldA;

mgsA and budA]

mgs A and a/sS;

mgsA and pfIBA]

mgsA and gldA]

budA and alsS]

budA and pfIBA]

budA and gldA]

alsS and pfIBA]

alsS and gldA]

pfIBA and gldA]

glpF, mgsA and budA]

glpF, mgsA and alsS]

glpF, mgsA and pfIBA]

glpF, mgsA and gldA]

glpF, budA and alsS]

glpF, budA and pfIBA]

glpF, budA and gldA]

glpF, alsS and pfIBA]

glpF, alsS and gldA]

glpF, pfIBA and gldA]

alsS, pfIBA, and gldA]

budA, pfIBA, and gldA] budA, alsS, and gldA]

budA, alsS, and pfIBA]

mgs A, pfIBA, and gldA]

mgs A, alsS, and gldA]

mgs A, alsS, and pfIBA]

mgsA, budA, and gldA]

mgs A, budA, and pfIBA]

mgsA, budA, and a/sS;

glpF, alsS, pfIBA, and gldA;

glpF, budA, pfIBA, and gldA;

glpF, budA, alsS, and gldA;

glpF, budA, alsS, and pfIBA;

glpF, mgsA, pfIBA, and gldA;

glpF, mgsA, alsS, and gldA;

glpF, mgsA, alsS, and pfIBA;

glpF, mgsA, budA, and gldA;

glpF, mgsA, budA, and pfIBA;

glpF, mgsA, budA, and alsS;

mgsA, budA, alsS, and pfIBA;

mgsA, budA, alsS, and gldA;

mgsA, budA, pfIBA, and gldA;

mgsA, alsS, pfIBA, and gldA;

budA, alsS, pfIBA, and gldA;

mgsA, budA, alsS, pfIBA, and gldA]

glpF, budA, alsS, pfIBA, and gldA]

glpF, mgsA, alsS, pfIBA, and gldA]

glpF, mgsA, budA, pfIBA, and gldA]

glpF, mgsA, budA, alsS, and gldA]

glpF, mgsA, budA, alsS, and pfIBA] or

glpF, mgsA, budA, alsS, pfIBA, and gldA.

In embodiments, described herein are microorganisms comprising one or more genetic modifications inactivating a combination consisting of genes selected from:

glpF and mgsA;

glpF and budA;

glpF and alsS; glpF and pfIBA;

glpf and gldA;

mgsA and budA] mgs A and a/sS;

mgs A and pfIBA] mgs A and gldA] budA and a/sS;

budA and pfIBA ;

budA and gldA]

alsS and pfIBA]

alsS and gldA]

pfIBA and gldA]

glpF, mgsA and budA] glpF, mgsA and alsS] glpF, mgsA and pfIBA] glpF, mgsA and gldA] glpF, budA and alsS] glpF, budA and pfIBA] glpF, budA and gldA] glpF, alsS and pfIBA] glpF, alsS and gldA] glpF, pfIBA and gldA] alsS, pfIBA, and gldA] budA, pfIBA, and gldA] budA, alsS, and gldA] budA, alsS, and pfIBA] mgsA, pfIBA, and gldA] mgsA, alsS, and gldA] mgsA, alsS, and pfIBA] mgsA, budA, and gldA] mgsA, budA, and pfIBA] mgsA, budA, and alsS] glpF, alsS, pfIBA, and gldA; glpF, budA, pfIBA, and gldA; glpF, budA, alsS, and gldA; glpF, budA, alsS, and pfIBA; glpF, mgsA, pfIBA, and gldA;

glpF, mgsA, alsS, and gldA;

glpF, mgsA, alsS, and pfIBA;

glpF, mgsA, budA, and gldA;

glpF, mgsA, budA, and pfIBA;

glpF, mgsA, budA, and alsS;

mgsA, budA, alsS, and pfIBA;

mgsA, budA, alsS, and gldA;

mgsA, budA, pfIBA, and gldA;

mgsA, alsS, pfIBA, and gldA;

budA, alsS, pfIBA, and gldA;

mgsA, budA, alsS, pfIBA, and gldA]

glpF, budA, alsS, pfIBA, and gldA]

glpF, mgsA, alsS, pfIBA, and gldA]

glpF, mgsA, budA, pfIBA, and gldA]

glpF, mgsA, budA, alsS, and gldA]

glpF, mgsA, budA, alsS, and pfIBA] or

glpF, mgsA, budA, alsS, pfIBA, and gldA.

In embodiments of the present disclosure, the microorganism is Escherichia coli, Gluconobacter oxydans, Gluconobacter asaii, Achromobacter delmarvae, Achromobacter viscosus, Achromobacter lacticum, Agrobacterium tumefaciens, Agrobacterium radiobacter, Alcaligenes faecalis, Arthrobacter citreus, Arthrobacter tumescens, Arthrobacter paraffineus, Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans, Aureobacterium saperdae, Azotobacter indicus, Brevibacterium ammoniagenes, divaricatum, Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacterium globosum, Brevibacterium fuscum, Brevibacterium etoglutamicum, Brevibacterium helcolum, Brevibacterium pusillum, Brevibacterium testaceum, Brevibacterium roseum, Brevibacterium immariophilium, Brevibacterium linens, Brevibacterium protopharmiae, Corynebacterium acetophilum, Corynebacterium glutamicum, Corynebacterium callunae, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Enterobacter aerogenes, Erwinia amylovora, Erwinia carotovora, Erwinia herbicola, Erwinia chrysanthemi, Flavobacterium peregrinum, Flavobacterium fucatum, Flavobacterium aurantinum, Flavobacterium rhenanum, Flavobacterium sewanense, Flavobacterium breve, Flavobacterium meningosepticum, Micrococcus sp. CCM825, Morganella morganii, Nocardia opaca, Nocardia rugosa, Planococcus eucinatus, Proteus rettgeri, Propionibacterium shermanii, Pseudomonas synxantha, Pseudomonas azotoformans, Pseudomonas fluorescens, Pseudomonas ovalis, Pseudomonas stutzeri, Pseudomonas acidovolans, Pseudomonas mucidolens, Pseudomonas testosteroni, Pseudomonas aeruginosa, Rhodococcus erythropolis, Rhodococcus rhodochrous, Rhodococcus sp. ATCC 15592, Rhodococcus sp. ATCC 19070, Sporosarcina ureae, Staphylococcus aureus, Vibrio metschni ovii, Vibrio tyrogenes, Actinomadura madurae, Actinomyces violaceochromogenes, Kitasatosporia parulosa, Streptomyces coelicolor, Streptomyces flavelus, Streptomyces griseolus, Streptomyces lividans, Streptomyces olivaceus, Streptomyces tanashiensis, Streptomyces virginiae, Streptomyces antibioticus, Streptomyces cacaoi, Streptomyces lavendulae, Streptomyces viridochromogenes, Aeromonas salmonicida, Bacillus pumilus, Bacillus circulans, Bacillus thiaminolyticus, Bacillus licheniformis, Bacillus subtilis, Bacillus amyloliquifaciens, Bacillus coagullans, Escherichia freundii, Microbacterium ammoniaphilum, Serratia marcescens, Salmonella typhimurium, Salmonella schottmulleri, or Xanthomonas citri. In embodiments, the parent or wild-type microorganism is a microorganisms as listed above.

In embodiments, microorganisms as described herein, when cultured in a medium containing DHA, can produce an increased amount of a metabolite compared to the amount of the metabolite produced by the parental microorganism cultured in the medium containing DHA.

Described herein are methods of culturing or growing a microorganism in a medium comprising DHA.

Culturing or growing as described herein can be under conditions that allow for the production of a metabolite of interest. The culturing or growing can be a batch process, fed- batch process or a continuous process. In embodiments, the culturing or growing can be a fed-batch process and DHA is introduced into the culture medium in a fed-batch manner at a concentration of: 250 mM to 500 mM, 275 mM to 350 mM, about 325 mM, or about 333 mM.

Methods as described herein can further comprise recovering the metabolite of interest.

Described herein are embodiments of compositions comprising a microorganism of as described herein and a medium as described herein. In an embodiment, the medium can comprise DHA.

Described herein are engineered cells. Engineered cells as described herein can comprise a microorganism containing therein one or more genetic modifications resulting in an increased expression of ATP-dependent dihydroxyacetone kinase (DhaK), increased activity of DhaK, or both, thereby providing the engineered cell increased DHA metabolism compared to the wild-type non-modified organism.

The one or more genetic modifications contained therein resulting in increased expression can comprise one or more expression vectors for exogenous expression of one or more recombinant enzymes relating to DHA metabolism, the one or more expression vectors comprising a nucleotide sequence encoding a recombinant DhaK protein.

In an embodiment, the nucleotide sequence encoding a recombinant DhaK comprises SEQ ID NO: 50. In an emdodiment, the nucleotide sequence encoding a recombinant DhaK is derived from the bacterium Klebsiella oxytoca and comprises the native promoter and the native protein encoding region. In an embodiment, the nucleotide sequence encoding a recombinant DhaK is derived from the bacterium Klebsiella oxytoca and comprises the native promoter and the protein encoding region comprises the sequence of SEQ ID NO: 51. In certain embodiments, the nucleotide sequence encoding a recombinant DhaK comprises a sequence selected from SEQ ID NOs: 28 to 49.

In emdodiments, the one or more genetic modifications resulting in increased activity of DhaK comprises inactivating one or more genes encoding: glycerol uptake facilitator (GlpF), methylglyoxal synthase (MgsA), acetolactate decarboxylase (BudA), acetolactate synthase (AlsS), and pyruvate formate-lyase (PfIBA), and glycerol dehydrogenase (GldA).

In embodiments, the one or more genetic modifications resulting in increased activity of DhaK can comprise inactivating:

glpF and mgs A;

glpF and bud A;

glpF and alsS;

glpF and pfIBA;

glpf and gldA;

mgsA and budA]

mgs A and a/sS;

mgsA and pfIBA]

mgsA and gldA]

budA and alsS]

budA and pfIBA]

budA and gldA]

alsS and pfIBA]

alsS and gldA]

pfIBA and gldA]

glpF, mgsA and budA]

glpF, mgsA and alsS]

glpF, mgsA and pfIBA]

glpF, mgsA and gldA]

glpF, budA and alsS] glpF, bud A and pflBA]

glpF, bud A and gldA]

glpF, alsS and pflBA]

glpF, alsS and gldA]

glpF, pflBA and gldA]

alsS, pflBA, and gldA ;

budA, pflBA, and gldA ;

budA, alsS, and gldA]

budA, alsS, and pflBA ;

mgs A, pflBA, and gldA]

mgs A, alsS, and gldA]

mgs A, alsS, and pflBA]

mgs A, budA, and gldA]

mgs A, budA, and pflBA]

mgsA, budA, and alsS]

glpF, alsS, pflBA, and gldA;

glpF, budA, pflBA, and gldA;

glpF, budA, alsS, and gldA;

glpF, budA, alsS, and pflBA;

glpF, mgsA, pflBA, and gldA;

glpF, mgsA, alsS, and gldA;

glpF, mgsA, alsS, and pflBA;

glpF, mgsA, budA, and gldA;

glpF, mgsA, budA, and pflBA;

glpF, mgsA, budA, and alsS;

mgsA, budA, alsS, and pflBA;

mgsA, budA, alsS, and gldA;

mgsA, budA, pflBA, and gldA;

mgsA, alsS, pflBA, and gldA;

budA, alsS, pflBA, and gldA;

mgsA, budA, alsS, pflBA, and gldA] glpF, budA, alsS, pflBA, and gldA] glpF, mgsA, alsS, pflBA, and gldA] glpF, mgsA, budA, pflBA, and gldA] glpF, mgsA, budA, alsS, and gldA] glpF, mgsA, budA, alsS, and pflBA] or glpF, mgs A, bud A, alsS, pfIBA, and gldA.

In embodiments, the microorganism of engineered cells as described herein is Escherichia coli, Gluconobacter oxydans, Gluconobacter asaii, Achromobacter delmarvae, Achromobacter viscosus, Achromobacter lacticum, Agrobacterium tumefaciens, Agrobacterium radiobacter, Alcaligenes faecalis, Arthrobacter citreus, Arthrobacter tumescens, Arthrobacter paraffineus, Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans, Aureobacterium saperdae, Azotobacter indicus, Brevibacterium ammoniagenes, divaricatum, Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacterium globosum, Brevibacterium fuscum, Brevibacterium ketoglutamicum, Brevibacterium helcolum, Brevibacterium pusillum, Brevibacterium testaceum, Brevibacterium roseum, Brevibacterium immariophilium, Brevibacterium linens, Brevibacterium protopharmiae, Corynebacterium acetophilum, Corynebacterium glutamicum, Corynebacterium callunae, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Enterobacter aerogenes, Erwinia amylovora, Erwinia carotovora, Erwinia herbicola, Erwinia chrysanthemi, Flavobacterium peregrinum, Flavobacterium fucatum, Flavobacterium aurantinum, Flavobacterium rhenanum, Flavobacterium sewanense, Flavobacterium breve, Flavobacterium meningosepticum, Micrococcus sp. CCM825, Morganella morganii, Nocardia opaca, Nocardia rugosa, Planococcus eucinatus, Proteus rettgeri, Propionibacterium shermanii, Pseudomonas synxantha, Pseudomonas azotoformans, Pseudomonas fluorescens, Pseudomonas ovalis, Pseudomonas stutzeri, Pseudomonas acidovolans, Pseudomonas mucidolens, Pseudomonas testosteroni, Pseudomonas aeruginosa, Rhodococcus erythropolis, Rhodococcus rhodochrous, Rhodococcus sp. ATCC 15592, Rhodococcus sp. ATCC 19070, Sporosarcina ureae, Staphylococcus aureus, Vibrio metschnikovii, Vibrio tyrogenes, Actinomadura madurae, Actinomyces violaceochromogenes, Kitasatosporia parulosa, Streptomyces coelicolor, Streptomyces flavelus, Streptomyces griseolus, Streptomyces lividans, Streptomyces olivaceus, Streptomyces tanashiensis, Streptomyces virginiae, Streptomyces antibioticus, Streptomyces cacaoi, Streptomyces lavendulae, Streptomyces viridochromogenes, Aeromonas salmonicida, Bacillus pumilus, Bacillus circulans, Bacillus thiaminolyticus, Bacillus licheniformis, Bacillus subtilis, Bacillus amyloliquifaciens, Bacillus coagullans, Escherichia freundii, Microbacterium ammoniaphilum, Serratia marcescens, Salmonella typhimurium, Salmonella schottmulleri, or Xanthomonas citri.

Described herein are methods of metabolizing dihydroxyacetone (DHA), comprising: providing a plurality of engineered cells, each of the plurality being an engineered cell as described herein; providing a medium comprising DHA (or medium and DHA separately then combining the two); and culturing the plurality of engineered cells in the medium comprising DHA to metabolize DHA and produce a DHA metabolite of interest. The culturing is a batch process, fed-batch process, or a continuous process. The culturing can be a fed-batch process and DHA is introduced into the culture medium in a fed-batch manner at a concentration of: 250 mM to 500 mM, 275 mM to 350 mM, about 325 mM, or about 333 mM. Methods as described herein can further comprise isolating the metabolite of interest.

Described herein are kits. In embodiments, a kit can comprise one or more microorganisms; and one or more expression vectors comprising one or more recombinant DhaK coding sequences, one or more expression vectors inactivating one or more genes encoding: glycerol uptake facilitator (GlpF), methylglyoxal synthase (MgsA), acetolactate decarboxylase (BudA), acetolactate synthase (AlsS), and pyruvate formate-lyase (PfIBA), and glycerol dehydrogenase (GldA), one or more expression vectors decreasing activity of proteins: glycerol uptake facilitator (GlpF), methylglyoxal synthase (MgsA), acetolactate decarboxylase (BudA), acetolactate synthase (AlsS), and pyruvate formate-lyase (PfIBA), and glycerol dehydrogenase (GldA), or a combination thereof.

In embodiments, the one or more one or more recombinant DhaK coding sequences have about 90% to about 100% sequence identify with SEQ ID NOs 28-51. In embodiments, the one or more expression vectors inactivating one or more genes encoding: glycerol uptake facilitator (GlpF), methylglyoxal synthase (MgsA), acetolactate decarboxylase (BudA), acetolactate synthase (AlsS), and pyruvate formate-lyase (PfIBA), and glycerol dehydrogenase (GldA) inactivate

glpF and mgs A;

glpF and bud A;

glpF and alsS;

glpF and pfIBA;

glpf and gldA;

mgsA and budA]

mgs A and a/sS;

mgsA and pfIBA]

mgsA and gldA]

budA and alsS]

budA and pfIBA]

budA and gldA]

alsS and pfIBA]

alsS and gldA]

pfIBA and gldA]

glpF, mgsA and budA]

glpF, mgsA and alsS] glpF, mgs A and pflBA]

glpF, mgs A and gldA]

glpF, budA and a/sS;

glpF, budA and pflBA]

glpF, budA and gldA]

glpF, alsS and pflBA]

glpF, alsS and gldA]

glpF, pflBA and gldA]

alsS, pflBA, and gldA ;

budA, pflBA, and gldA ;

budA, alsS, and gldA]

budA, alsS, and pflBA]

mgs A, pflBA, and gldA] mgs A, alsS, and gldA]

mgs A, alsS, and pflBA] mgsA, budA, and gldA] mgs A, budA, and pflBA] mgsA, budA, and alsS] glpF, alsS, pflBA, and gldA;

glpF, budA, pflBA, and gldA;

glpF, budA, alsS, and gldA;

glpF, budA, alsS, and pflBA;

glpF, mgsA, pflBA, and gldA;

glpF, mgsA, alsS, and gldA;

glpF, mgsA, alsS, and pflBA;

glpF, mgsA, budA, and gldA;

glpF, mgsA, budA, and pflBA; glpF, mgsA, budA, and alsS;

mgsA, budA, alsS, and pflBA; mgsA, budA, alsS, and gldA;

mgsA, budA, pflBA, and gldA; mgsA, alsS, pflBA, and gldA;

budA, alsS, pflBA, and gldA;

mgsA, budA, alsS, pflBA, and gldA] glpF, budA, alsS, pflBA, and gldA] glpF, mgsA, alsS, pflBA, and gldA] glpF, mgs A, bud A, pfIBA, and gldA]

glpF, mgs A, bud A, alsS, and gldA]

glpF, mgsA, budA, alsS, and pfIBA] or

glpF, mgs A, budA, alsS, pfIBA, and gldA.

In embodiments, the one or more expression vectors decreasing activity of proteins: glycerol uptake facilitator (GlpF), methylglyoxal synthase (MgsA), acetolactate decarboxylase (BudA), acetolactate synthase (AlsS), and pyruvate formate-lyase (PfIBA), and glycerol dehydrogenase (GldA) decrease activity of enyzyme products of:

glpF and mgsA;

glpF and budA;

glpF and alsS;

glpF and pfIBA;

glpf and gldA;

mgsA and budA]

mgsA and a/sS;

mgsA and pfIBA]

mgsA and gldA]

budA and alsS]

budA and pfIBA]

budA and gldA]

alsS and pfIBA]

alsS and gldA]

pfIBA and gldA]

glpF, mgsA and budA]

glpF, mgsA and alsS]

glpF, mgsA and pfIBA]

glpF, mgsA and gldA]

glpF, budA and alsS]

glpF, budA and pfIBA]

glpF, budA and gldA]

glpF, alsS and pfIBA]

glpF, alsS and gldA]

glpF, pfIBA and gldA]

alsS, pfIBA, and gldA]

budA, pfIBA, and gldA]

budA, alsS, and gldA] budA, alsS, and pflBA]

mgs A, pflBA, and gldA]

mgs A, alsS, and gldA]

mgs A, alsS, and pflBA]

mgs A, budA, and gldA]

mgsA, budA, and pflBA ;

mgsA, budA, and a/sS;

glpF, alsS, pflBA, and gldA;

glpF, budA, pflBA, and gldA;

glpF, budA, alsS, and gldA;

glpF, budA, alsS, and pflBA;

glpF, mgsA, pflBA, and gldA;

glpF, mgsA, alsS, and gldA;

glpF, mgsA, alsS, and pflBA;

glpF, mgsA, budA, and gldA;

glpF, mgsA, budA, and pflBA;

glpF, mgsA, budA, and alsS;

mgsA, budA, alsS, and pflBA;

mgsA, budA, alsS, and gldA;

mgsA, budA, pflBA, and gldA;

mgsA, alsS, pflBA, and gldA;

budA, alsS, pflBA, and gldA;

mgsA, budA, alsS, pflBA, and gldA]

glpF, budA, alsS, pflBA, and gldA]

glpF, mgsA, alsS, pflBA, and gldA]

glpF, mgsA, budA, pflBA, and gldA]

glpF, mgsA, budA, alsS, and gldA]

glpF, mgsA, budA, alsS, and pflBA] or

glpF, mgsA, budA, alsS, pflBA, and gldA.

Other embodiments of kits are described herein, and can comprise one or more engineered cells as described herein; and a culture medium suitable for growing one or more engineered cells. Kits can further comprise dihydroxyacetone (DHA).

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1. A chemical or biological process for production of DHA from C0 ₂ or CH ₄ and further fermentation of DHA to products of commercial interest. Figs. 2A-2B. Fermentative growth of E. coli strain TG1 13 with or without DHA kinase encoding plasmids. Plasmid pDC4, E. coli dhaKLM ; plasmid pDC1 17d, K. oxytoca dhaK. Cultures were grown in LB medium supplemented with DHA (10 or 30 g.L ^-1 ) at 37°C with pH control at 7.0. Figure 2A, 1 1 1 mM DHA; Figure 2B, 333 mM DHA.

Fig. 3. Fed-batch fermentation of DHA by E. coli strain TG1 13 (pDC1 17d). Fermentation was started with 333 mM DHA in LB medium at 37°C. At 29 hrs, an additional 333 mM DHA was added to the cultures. pH of the cultures was controlled at 7.0 with 2N KOH.

Figs. 4A-4B. Fermentation of glucose or DHA in LB medium by K. variicola at 37°C and pH 7.0. Figure 4A, glucose (275 mM); Figure 4B, DHA (333 mM).

Fig. 5. Fed-batch fermentation of DHA in LB by K. variicola strain LW225. Fermentation was started with 333 mM DHA in LB at 37°C and pH 7.0. At 23, 34 and 46 h, additional 333 mM DHA was added to a total of 1.33 M (120 g.L ¹ ). All of the added DHA was fermented at 60 h.

Figs. 6A-6B. Fermentation of DHA to succinate or ethanol by engineered E. coli derivatives. Figure 6A - Fermentation of DHA in LB by E. coli strain KJ122 (pDC1 17d) to succinate was started with 87 mM DHA at 37°C and pH 7.0. At various times (24, 42.5, 71.5, 93.5 and 145 h), 100 mM DHA was added to the fermentation to a total of 590 mM. Figure 6B - Fermentation of DHA in LB by an ethanologenic E. coli strain SE2378 (pDC1 17d) was started with 99 mM DHA at 37°C and pH 7.0. At 21.5 h, an additional 181 mM DHA was added to the fermentation for a total of 280 mM.

Fig. 7. Fermentation pathway for Dihydroxyacetone of E. coli. The“X” over the arrows leading to glycerol and acetyl-coA represent deletion of the gldA and pfIB, respectively in the mutant strains. DHA, dihydroxyacetone; DHA-P, dihydroxyacetone-3-phosphate; GlpF, glycerol uptake facilitator; GldA, glycerol dehydrogenase; DhaD, dihydroxyacetone oxidoreductase; TPI, triose-phosphate isomerase; FRD, fumarate reductase; D-LDH, D- lactate dehydrogenase; PFI, pyruvate formate-lyase; ADH, alcohol dehydrogenase; PTA, phosphotransacetylase; ACK, acetate kinase.

Fig. 8. Sensitivity of K. variicola to DHA. Strain LW200 was inoculated into pH- controlled (7.0) fermentations (37°C) with the indicated concentration of DHA in LB. Cell density and lactate concentration were determined during a 48 h period and the highest values are presented.

Fig. 9. Metabolic and inhibitory pathways of DHA in E. coli. DHA added to culture medium is transported by glycerol facilitator (GlpF) and by other unidentified transporters. If the concentration of DHA in the medium is higher than the rate of transport, DHA that accumulates at the cell surface interacts with externally exposed components of the cell leading to growth inhibition. Once DHA enters the cell, it is phosphorylated by DHA kinase to non-toxic DHA-phosphate. If the DHA kinase activity is lower than the rate of DHA transport, excess DHA in the cytoplasm interacts with cellular components such as DNA and protein leading to growth inhibition. For rapid growth and fermentation of DHA to a product of choice, a balance among various reactions, transport, kinase level and activity, and ATP availability, needs to exist to overcome the toxicity of DHA.

Fig. 10 Inhibition of the growth of E. coli by DHA. Strain TG1 13(pDC1 17d) or LW416 (TG1 13, AglpF, pDC1 17d) was inoculated into pH-controlled (7.0) fermentations (37 °C) with the indicated concentration of DHA in LB. Cell density and lactate concentration were determined during a 48-h period, and the highest values are presented. Reported values for LW416 with 456 mM DHA are from 96-h incubation. Dashed lines indicate TG1 13 (pDC1 17d); solid lines indicate LW416; circles indicate cell density; squares indicate D-lactate titer.

BRIEF DESCRIPTION OF THE SEQUENCES

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit (unless the context clearly dictates otherwise), between the upper and lower limits of that range, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, inorganic chemistry, material science, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the compositions and compounds disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is in atmosphere. Standard temperature and pressure are defined as 25 °C and 1 atmosphere.

Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.

As used herein, the singular forms“a”,“an” and“the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms“including”,“includes”,“having”,“has”,� ��with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term“comprising”. Thus, for example, reference to“a support” includes a plurality of supports. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

The phrases“consisting essentially of or“consists essentially of indicate that the claim encompasses embodiments containing the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claim.

The term“about” or“approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system and on the parameter being measured. Where particular values are described in the application and claims, unless otherwise stated the term“about” meaning within an acceptable error range for the particular value should be assumed. In the context of compositions containing amounts of ingredients where the term“about” is used, these compositions contain the stated amount of the ingredient with a variation (error range) of 0-10% around the value (X±10%).

in the present disclosure, ranges are stated in shorthand, so as to avoid having to set out at length and describe each and every value within the range. Any appropriate value within the range can be selected, where appropriate, as the upper value, lower value, or the terminus of the range. For example, a range of 0.1 -1.0 represents the terminal values of 0.1 and 1.0, as well as the intermediate values of 0.2, Q.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges encompassed within 0.1-10, such as 0.2-0.5, 0.2-0.8, 0.7-1.0, etc. Values having at least two significant digits within a range are envisioned, for example, a range of 5-10 indicates all the values between 5.0 and 10.0 as well as between 5.00 and 10.00 including the terminal values. Also, when ranges are used herein, combinations and subcombinations of ranges (e.g., subranges within the disclosed range), and the specific embodiments therein, are included.

Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of molecular biology, medicinal chemistry, and/or organic chemistry. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described herein.

As used in the specification and the appended claims, the singular forms“a,”“an,” and “the” may include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to“a support” includes a plurality of supports. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

As used in the specification and the appended claims, the singular forms“a,”“an,” and “the” may include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to“a support” includes a plurality of supports. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

As used herein,“control” is an alternative subject or sample used in an experiment for comparison purposes and included to minimize or distinguish the effect of variables other than an independent variable.

As used herein,“overexpressed” or“overexpression” refers to an increased expression level of an RNA or protein product encoded by a gene as compared to the level of expression of the RNA or protein product in a normal or control cell.

As used herein, “expression” refers to the process by which polynucleotides are transcribed into RNA transcripts. In the context of mRNA and other translated RNA species, “expression” also refers to the process or processes by which the transcribed RNA is subsequently translated into peptides, polypeptides, or proteins.

As used herein,“nucleic acid” and“polynucleotide” generally refer to a string of at least two base-sugar-phosphate combinations and refers to, among others, single-and double- stranded DNA, DNA that is a mixture of single-and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double- stranded or a mixture of single- and double-stranded regions. In addition, polynucleotide as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. “Polynucleotide” and “nucleic acids” also encompasses such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia. For instance, the term polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein.“Polynucleotide” and“nucleic acids” also includes PNAs (peptide nucleic acids), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids. Natural nucleic acids have a phosphate backbone, artificial nucleic acids may contain other types of backbones, but contain the same bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are“nucleic acids” or "polynucleotide" as that term is intended herein.

As used herein,“deoxyribonucleic acid (DNA)” and“ribonucleic acid (RNA)” generally refer to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. RNA may be in the form of a tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, RNAi (RNA interference construct), siRNA (short interfering RNA), or ribozymes.

As used herein,“nucleic acid sequence” and“oligonucleotide” also encompasses a nucleic acid and polynucleotide as defined above.

As used herein,“DNA molecule” includes nucleic acids/polynucleotides that are made of DNA.

As used herein,“wild-type” is the typical form of an organism, variety, strain, gene, protein, or characteristic as it occurs in nature, as distinguished from mutant forms that may result from selective breeding or transformation with a transgene.

As used herein, “identity,” is a relationship between two or more polypeptide or polynucleotide sequences, as determined by comparing the sequences. In the art,“identity” also refers to the degree of sequence relatedness between polypeptide as determined by the match between strings of such sequences. “Identity” can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. I/I/., Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., Eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991 ; and Carillo, H., and Lipman, D., SIAM J. Applied Math. 1988, 48: 1073. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are codified in publicly available computer programs. The percent identity between two sequences can be determined by using analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, Madison Wis.) that incorporates the Needelman and Wunsch (J. Mol. Biol., 1970, 48: 443-453) algorithm (e.g., NBLAST, and XBLAST). The default parameters are used to determine the identity for the polypeptides or polynucleotides of the present disclosure.

As used herein,“heterologous” refers to compounds, molecules, nucleotide sequences (including genes), and polypeptide sequences (including peptides and proteins) that are different in both activity (function) and sequence or chemical structure. As used herein, “heterologous” can also refer to a gene or gene product that is from a different organism. For example, a human GTP cyclohydrolase or a synthase can be said to be heterologous when expressed in yeast.

As used herein,“homologue” refers to a polypeptide sequence that shares a threshold level of similarity and/or identity as determined by alignment of matching amino acids. Two or more polypeptides determined to be homologues are said to be homologues. Homology is a qualitative term that describes the relationship between polypeptide sequences that is based upon the quantitative similarity.

As used herein,“paralog” refers to a homologue produced via gene duplication of a gene. In other words, paralogs are homologues that result from divergent evolution from a common ancestral gene.

As used herein,“orthologues” refers to homologues produced by speciation followed by divergence of sequence but not activity in separate species. When speciation follows duplication and one homologue sorts with one species and the other copy sorts with the other species, subsequent divergence of the duplicated sequence is associated with one or the other species. Such species specific homologues are referred to herein as orthologues.

As used herein,“xenologs” are homologues resulting from horizontal gene transfer.

As used herein,“similarity” is a quantitative term that defines the degree of sequence match between two compared polypeptide sequences.

As used herein, “cell,” "cell line," and "cell culture" include progeny. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological property, as screened for in the originally transformed cell, are included.

As used herein,“culturing” refers to maintaining cells under conditions in which they can proliferate and avoid senescence as a group of cells. “Culturing” can also include conditions in which the cells also or alternatively differentiate.

As used herein, "organism", "host", and "subject" refers to any living entity comprised of at least one cell. A living organism can be as simple as, for example, a single isolated eukaryotic cell or cultured cell or cell line, or as complex as a mammal, including a human being, and animals (e.g., vertebrates, amphibians, fish, mammals, e.g., cats, dogs, horses, pigs, cows, sheep, rodents, rabbits, squirrels, bears, primates (e.g., chimpanzees, gorillas, and humans). "Subject" may also be a cell, a population of cells, a tissue, an organ, or an organism, preferably to human and constituents thereof.

As used herein,“gene” refers to a hereditary unit corresponding to a sequence of DNA that occupies a specific location on a chromosome and that contains the genetic instruction for a characteristic(s) or trait(s) in an organism. As used herein,“synthetic gene” can refer to a recombinant gene comprising one or more coding sequences for a protein of interest, or a synthetically purified protein that is not naturally occurring in its purified state.

As used herein, the term“recombinant” generally refers to a non-naturally occurring nucleic acid, nucleic acid construct, or polypeptide. Such non-naturally occurring nucleic acids may include natural nucleic acids that have been modified, for example that have deletions, substitutions, inversions, insertions, etc., and/or combinations of nucleic acid sequences of different origin that are joined using molecular biology technologies (e.g., a nucleic acid sequences encoding a fusion protein (e.g., a protein or polypeptide formed from the combination of two different proteins or protein fragments), the combination of a nucleic acid encoding a polypeptide to a promoter sequence, where the coding sequence and promoter sequence are from different sources or otherwise do not typically occur together naturally (e.g. , a nucleic acid and a constitutive promoter), etc.). Recombinant also refers to the polypeptide encoded by the recombinant nucleic acid. Non-naturally occurring nucleic acids or polypeptides include nucleic acids and polypeptides modified by man.

As used herein, “plasmid” as used herein refers to a non-chromosomal double- stranded DNA sequence including an intact“replicon” such that the plasmid is replicated in a host cell.

As used herein, the term“vector” is used in reference to a vehicle used to introduce an exogenous nucleic acid sequence into a cell. A vector may include a DNA molecule, linear or circular (e.g. plasmids), which includes a segment encoding a polypeptide of interest operatively linked to additional segments that provide for its transcription and translation upon introduction into a host cell or host cell organelles. Such additional segments may include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from yeast or bacterial genomic or plasmid DNA, or viral DNA, or may contain elements of both.

As used herein, "operatively linked" or “operatively coupled” indicates that the regulatory sequences useful for expression of the coding sequences of a nucleic acid are placed in the nucleic acid molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition can also be applied to the arrangement of coding sequences and transcription control elements (e.g. promoters, enhancers, and termination elements), and/or selectable markers in an expression vector.

As used herein,“cDNA” refers to a DNA sequence that is complementary to a RNA transcript in a cell. It is a man-made molecule. Typically, cDNA is made in vitro by an enzyme called reverse-transcriptase using RNA transcripts as templates.

As used herein, the term“transfection” refers to the introduction of an exogenous and/or recombinant nucleic acid sequence into the interior of a membrane enclosed space of a living cell, including introduction of the nucleic acid sequence into the cytosol of a cell as well as the interior space of a mitochondria, nucleus, or chloroplast. The nucleic acid may be in the form of naked DNA or RNA, it may be associated with various proteins or regulatory elements (e.g., a promoter and/or signal element), or the nucleic acid may be incorporated into a vector or a chromosome.

As used herein,“transformation” or“transformed” refers to the introduction of a nucleic acid (e.g., DNA or RNA) into cells in such a way as to allow expression of the coding portions of the introduced nucleic acid.

As used herein,“stable expression,”“stable incorporation,”“stable transfection” and the like refer to the integration of an exogenous gene into the genome of a host cell, which can allow for long term expression of the exogenous gene.

As used herein,“transient expression,”“transient transfection,” and the like refer to the introduction of an exogenous gene into a host cell that does not result in stable incorporation of the gene into the host cell.

As used herein “chemical” refers to any molecule, compound, particle, or other substance that can be a substrate for an enzyme in the enzymatic pathway described herein and/or a carboxylesterase enzyme or biochemical pathway. A“chemical” can also be used to refer to a metabolite of a carboxylic ester. As such,“chemical” can refer to nucleic acids, proteins, organic compounds, inorganic compounds, metabolites etc.“Chemical” can also refer to the product produced by the carboxylesterase action.

As used herein“biologically coupled” refers to the association of or interaction between two or more physically distinct molecules, groups of molecules compounds, organisms, or particles where the association is directly or indirectly mediated between the two or more physically distinct molecules, groups of molecules compounds, organisms or particles via a biologic molecule or compound. This can include direct binding between two biologic molecules and signal transduction pathways.

As used herein,“biological communication” refers to the communication between two or more molecules, compounds, or objects that is mediated by a biologic molecule or biologic interaction. As used herein,“biologic molecule,”“biomolecule,” and the like refer to any molecule that is present in a living organism and includes without limitation, macromolecules (e.g. proteins, polysaccharides, lipids, and nucleic acids) as well as small molecules (e.g. metabolites and other products produced by a living organism).

As used herein, “regulation” refers to the control of gene or protein expression or function.

As used herein,“promoter” refers to the DNA sequence(s) that control or otherwise modify transcription of a gene and can include binding sites for transcription factors, RNA polymerases, and other biomolecules and substances (e.g. inorganic compounds) that can influence transcription of a gene by interaction with the promoter. Typically these sequences are located at the 5’ end of the sense strand of the gene, but can be located anywhere in the genome.

As used herein,“native” refers to the endogenous version of a molecule or compound relative to the host cell or population being described.

As used herein,“non-naturally occurring” refers to a non-native version of a molecule or compound or non-native expression or presence of a molecule or compound within a host cell or other composition. This can include where a native molecule or compound is influenced to be expressed or present at a different location within a host, at a non-native period of time within a host, or is otherwise in an altered environment, even when considered within the host. Non-limiting examples include where a protein that is expressed only in the nucleus of a cell is expressed in the cytoplasm of the cell or when a protein that is only normally expressed during the embryonic stage of development is expressed during the adult stage.

As used herein,“encode” refers to the biologic phenomena of transcribing DNA into an RNA that, in some cases, can be translated into a protein product. As such, when a protein is said herein to be encoded by a particular nucleotide sequence, it is to be understood that this refers to this biologic relationship between DNA and protein. It is well established that RNA can be translated into protein based on the triplet code where 3 nucleotides represent an amino acid. This term also includes the idea that DNA can be transcribed into RNA molecules with biologic functions, such as ribozymes and interfering RNA species. As such, when a RNA molecule is said to be encoded by a particular nucleotide sequence it is to be understood that this is referring to the transcriptional relationship between the DNA and RNA species in question. As such“encoding nucleotide” refers to herein as the nucleotide which can give rise through transcription, and in the case of proteins, translation a functional RNA or protein.

As used herein “codon optimized” or “codon optimization” refers to a codon modification or making modifications to the codons for amino acids in a polypeptide such that they reflect the codon usage bias of the cell type that the polypeptide is expressed in. Modifications to the codons can be made using techniques generally known in the art.

As used herein, the term “metabolite produced by a microorganism” refers to a metabolite of commercial interest. Metabolites that can be produced from the microorganisms of the present disclosure and commercially used depend on the parent microorganism genetically modified to produce the microorganism of the present disclosure. For example, a microorganism producing succinate can be genetically modified according to the present disclosure to produce a microorganism capable producing succinate by metabolizing DHA as a carbon source. The metabolites envisioned to be produced by the microorganism of interest include ethanol, succinate, malate, lactate, acetate, and formate. Additional examples of metabolites that can be produced by a microorganism are well known to a person of ordinary skill in the art and such embodiments are within the purview of the present disclosure.

As described herein, the phrase“parental microorganism” refers to the microorganism to which the genetic modifications according to the present disclosure are performed to produce the DHA metabolizing microorganism of the present disclosure. Accordingly, the characteristics of DHA metabolizing microorganisms of the present disclosure are represented in relation to the parental microorganisms. For example, a parental microorganism may metabolize DHA to a product of interest to a certain level; however, when genetically modified according to the present disclosure, the resultant microorganism metabolizes DHA to the product of interest at a higher level compared to that of the parental microorganism.

Accordingly, for example, if the wild-type E. coli is genetically modified according to the present disclosure to produce an E. coli that metabolizes DHA to a product of interest, the wild-type E. coli is the parental microorganism of the resultant DHA metabolizing E. coli. Similarly, if an E. coli containing an initial genetic modification is further genetically modified according to the present disclosure to produce a DHA metabolizing E. coli, the E. coli strain containing the initial genetic modification is the parental strain of the resultant DHA metabolizing E. coli.

The term“microorganism” used herein refers to organisms recognized in the art as “microorganisms”. Microorganisms contemplated in the present disclosure include bacteria, filamentous fungi, and yeast. Additional examples of microorganism that can be used according to the present disclosure are well known to a person of ordinary skill in the art and such embodiments are within the purview of the present disclosure.

A“native gene” or“an endogenous gene” is a gene that is naturally found in a host microorganism; whereas, an “exogenous gene” is a gene introduced into a host microorganism and which was obtained from a microorganism other the host microorganism. Likewise, a“native promoter” or“endogenous promoter” is a promoter that is naturally found in a host microorganism. In contrast,“exogenous promoter” or“heterologous promoter” is a promoter introduced into a host microorganism via a genetic construct and which was obtained from a microorganism different from host microorganism.

The non-italicized abbreviations as used herein refer to the corresponding protein; whereas italicized abbreviations used herein refer to the corresponding gene. For example, the term“BudA” refers to a-acetolactate decarboxylase protein and the term“budA" refers to the gene encoding the a-acetolactate decarboxylase protein.

As used herein, “coding sequence” or“coding region” refers to the portions] of a gene’s DNA or RNA that codes for protein. For example, a DhaK coding sequence can be the portion of a dhaK gene that codes for the DhaK protein. In certain aspects, a DhaK coding sequence can comprise the exons of a dhaK gene spliced together in a recombinant DNA sequence or vector.

Discussion

DHA can be produced from an abundant source of natural gas by chemical processes with formaldehyde as an intermediate. Carbon dioxide, a by-product of various industries including ethanol/butanol biorefineries, can also be converted to formaldehyde and then to DHA. DHA, upon entry into a cell and phosphorylation to DHA-3-phosphate enters the glycolytic pathway, and can be fermented to several products. However, DHA is inhibitory to microbes due to its chemical interaction with cellular components.

World-wide natural gas production in 2016 was 3.55 trillion cubic meters, and the natural gas flared is estimated to contribute about 350 million tons of C0 ₂ . The global warming potential of CH ₄ is several orders of magnitude higher than that of C0 ₂ . Upgrading CH ₄ to chemicals and liquid fuels converts low-cost natural gas to high-value products and traps it from release into atmosphere. Current chemical technology can produce dihydroxyacetone (DHA) from CH provided a microorganism can ferment this growth-inhibitory sugar. Here we report metabolically engineered microorganisms that ferment DHA to products. Combining the existing technology of chemical conversion of CH to DHA and the fermentation of this sugar is a strategy to transform inexpensive CH4 to chemicals and liquid fuels.

Accordingly, the present disclosure provides microorganisms comprising one or more genetic modifications resulting in an increased expression or catalytic activity of DhaK. The one or more genetic modifications according to the present disclosure produce microorganisms that exhibit, compared to the parental microorganisms, increased metabolism of DHA. Non-limiting examples of DhaK that can be used in the present disclosure are provided by proteins identified by the UniProt entries: A0A1 S7RJ83 (SEQ ID NO: 29), M1 PGD2 (SEQ ID NO: 30), A0A0P0ADK4 (SEQ ID NO: 31), N1 LHI3 (SEQ ID NO: 32), A0A0D6SX59 (SEQ ID NO: 33), A0A0M2NGK1 (SEQ ID NO: 34), A0A0G8CNG7 (SEQ ID NO: 35), A0A0G8F707 (SEQ ID NO: 36), A0A1X6QHI3 (SEQ ID NO: 37), A0A1X6PRC2 (SEQ ID NO: 38), A0A1 S8G149 (SEQ ID NO: 39), A0A1 R4IR08 (SEQ ID NO: 40), L0DGU5 (SEQ ID NO: 41), A0A1 W7M7M6 (SEQ ID NO: 42), W8H5T6 (SEQ ID NO: 43), A0A0K2RG10 (SEQ ID NO: 44), A0A0D5AH36 (SEQ ID NO: 45), A0A164XR98 (SEQ ID NO: 46), A0A164KEB5 (SEQ ID NO: 47), A0A0P9Z768 (SEQ ID NO: 48) and A0A1V0WK58 (SEQ ID NO: 49). These UniProt protein entries identify homologs of DhaK. Additional homologs of DhaK are well known to a person of ordinary skill in the art and use of such homologs is within the purview of the present disclosure. Also, a person of ordinary skill in the art can identify homologs of DhaK in additional organisms and use of such homologs is also within the purview of the present disclosure.

In certain embodiments, dhakor DhaK coding sequences can be derived from a parent sequence of organisms and/or sequences as described herein. Sequences derived from a parent sequence can maintain at least the sequences necessary to express a functional protein (for example the sequence for the active site of the enzyme), and a sequence derived from a parent sequence can maintain about 50% to about 100% sequence identity with the parental gene or parental coding sequence, about 60% to about 90% sequence identity with the parental gene or parental coding sequence, or about 70% to about 80% sequence identity with the parental gene or parental coding sequence. It would be within the skill of the art for a skilled artisan to derive and express a sequence of interest for microorganisms, methods, and kits as described herein for the same purpose of microorganisms, methods, and kits as described herein.

In one embodiment of the present disclosure, the genetic modification resulting in the increased expression of DhaK comprises introducing into the microorganism a nucleotide sequence, for example, a DNA or RNA sequence, comprising a dhaK, synthetic dhaK, coding sequence for a DhaK, or synthetic coding sequence for a DhaK. In certain embodiments, a dhaK, synthetic dhaK, or DhaK coding sequence present in a DNA vector introduced into a microorganism is identical to the dhaK or DhaK coding sequence present in the genome of the microorganism, i.e., the DNA vector provides extra copies of the endogenous dhaK or DhaK coding sequence. In certain other embodiments, a dhaK, synthetic dhaK, , synthetic dhaK, coding sequence for a DhaK, or DhaK coding sequence present in a DNA vector is different from the dhaK or DhaK coding sequence present in the genome of the microorganism, i.e., the DNA vector provides an exogenous homolog of the dhaK or DhaK coding sequence present in the genome of the microorganism. In further embodiments, a dhaK or DhaK coding sequence is not present in the genome of the microorganism into which a dhaK or DhaK coding sequence in a DNA vector is introduced, i.e., the DNA sequence is the only source of a dhaK or DhaK coding sequence in the microorganism. In specific embodiments, a dhaK gene or DhaK coding sequence in a DNA vector introduced into a microorganism is the dhaK gene or DhaK coding sequence from the bacterium Klebsiella oxytoca (SEQ ID NO: 50). In particular embodiments, a dhaK or DhaK coding sequence in a DNA vector introduced into a microorganism (which is synthetic or non-naturally occurring) is the dhaK comprising the native promoter as well as the protein encoding region (i.e. coding sequence) from the bacterium Klebsiella oxytoca. An example of such DNA vector comprising the native promoter as well as the protein encoding region from the bacterium Klebsiella oxytoca is given by (SEQ ID NO: 51). In specific embodiments, a dhaK gene or DhaK coding sequence in a DNA vector introduced into a microorganism is a synthetic recombinant dhaK gene or DhaK coding sequence from the bacterium Klebsiella oxytoca (SEQ ID NO: 50).

Any of the genes encoding DhaK proteins identified above with the Uniprot entries can also be used without or with the corresponding native promoters. For a particular Uniprot entry or another DhaK of interest, a skilled artisan can identify the corresponding gene or protein coding sequence, including the corresponding promoter sequence (or otherwise suitable promoter sequence), and prepare and use the appropriate DNA vector.

Examples of DNA vector or synthetic/artificial (i.e. not-naturally occurring, or not- naturally occurring in the microorganism in which the plasmid is to be expressed) DNA vectors comprising the dhaK or DhaK coding sequence includes a plasmid, cosmid, yeast artificial chromosome (YAC), 2-micron DNA. Additional examples of DNA vectors suitable for the expression of a gene of interest in a microorganism are well known to a person of ordinary skill in the art and such embodiments are within the purview of the present disclosure.

An example of a typical DNA vector or artificial/synthetic DNA vector suitable for the expression of a gene of interest into a microorganism can comprise an origin of replication, a promoter which can drive the expression of the gene, synthetic gene, or coding sequence, one or more selectable markers, and one or more restriction enzyme cleavage sites for cloning the gene of interest into the DNA vector. The promoter can be an inducible promoter or a constitutive promoter, and can be a promotor that is specific for the microorganisms into which the DNA vector is introduced into. The selectable markers can be an antibiotic resistance gene or a gene providing for a missing biochemical function in the microorganism. Additional examples of promoters as well as selectable markers are well known to a person of ordinary skill in the art and such embodiments are within the purview of the present disclosure. In one embodiment, the DNA vector comprising the dhaK, synthetic dhaK, synthetic dhaK, coding sequence for a DhaK, or DhaK coding sequence is incorporated into the genome of the microorganism. In another embodiment, the DNA vector comprising the dhaK, synthtitc dhaK, synthetic dhaK, coding sequence for a DhaK, or DhaK coding sequence is present as an extra-genomic genetic material.

In a particular embodiment, the microorganism is a bacterium and the DNA vector is a plasmid carrying the dhaK, synthetic dhaK, DhaK coding sequence, or synthetic DhaK coding sequence. In a particular embodiment, the microorganism is a bacterium and the DNA vector is an artificial/synthetic plasmid carrying the dhaK, synthetic dhaK, DhaK coding sequence, or synthetic DhaK coding sequence

In further embodiments, the present disclosure provides microorganisms comprising one or more genetic modifications resulting in the increased expression or activity of DhaK and further comprising one or more genetic modifications inactivating one or more genes encoding: glycerol uptake facilitator (GlpF), methyl glyoxal synthase (MgsA), acetolactate decarboxylase (BudA), acetolactate synthase (AlsS), and pyruvate formate-lyase (PfIBA), and glycerol dehydrogenase (GldA).

Homologs of glpF, mgsA, budA, alsS, pfIBA, and gldA in various bacteria are well known and a person of ordinary skill in the art can determine appropriate homologs of these genes or proteins to be used in particular embodiments. Such embodiments are within the purview of this present disclosure.

In certain embodiments, just one of genes glpF, mgsA, budA, alsS, pfIBA, and gldA is inactivated. In other embodiments, any combination of two, three, four, or five genes from glpF, mgsA, budA, alsS, pfIBA, and gldA can be inactivated in the microorganisms of the present disclosure. Such combinations include:

glpF and mgsA;

glpF and budA;

glpF and alsS;

glpF and pfIBA;

glpf and gldA;

mgsA and budA]

mgsA and a/sS;

mgsA and pfIBA

mgsA and gldA]

budA and alsS]

budA and pfIBA]

budA and gldA] alsS and pflBA]

alsS and gldA]

pflBA and gldA]

glpF, mgsA and budA] glpF, mgs A and a/sS;

glpF, mgs A and pflBA] glpF, mgs A and gldA] glpF, budA and alsS] glpF, budA and pflBA] glpF, budA and gldA] glpF, alsS and pflBA] glpF, alsS and gldA] glpF, pflBA and gldA] alsS, pflBA, and gldA] budA, pflBA, and gldA] budA, alsS, and gldA] budA, alsS, and pflBA] mgs A, pflBA, and gldA] mgs A, alsS, and gldA] mgs A, alsS, and pflBA] mgsA, budA, and gldA] mgs A, budA, and pflBA] mgsA, budA, and alsS] glpF, alsS, pflBA, and gldA] glpF, budA, pflBA, and gldA] glpF, budA, alsS, and gldA] glpF, budA, alsS, and pflBA] glpF, mgsA, pflBA, and gldA] glpF, mgsA, alsS, and gldA ] glpF, mgsA, alsS, and pflBA] glpF, mgsA, budA, and gldA] glpF, mgsA, budA, and pflBA] glpF, mgsA, budA, and alsS] mgsA, budA, alsS, and pflBA] mgsA, budA, alsS, and gldA] mgsA, budA, pflBA, and gldA] mgsA, alsS, pfIBA, and gldA]

budA, alsS, pfIBA, and gldA ;

mgsA, budA, alsS, pfIBA, and gldA]

glpF, budA, alsS, pfIBA, and gldA]

glpF, mgsA, alsS, pfIBA, and gldA]

glpF, mgsA, budA, pfIBA, and gldA]

glpF, mgsA, budA, alsS, and gldA]

glpF, mgsA, budA, alsS, and pfIBA] or

glpF, mgsA, budA, alsS, pfIBA, and gldA as well as any combination thereof.

Microorganisms produced according to the instant disclosure can have one or more genes inactivated by various methods known in the art. Deletion/inactivation of a gene indicates that the genetic modification of the gene results in inactivation of the enzymatic activity of the polypeptide produced by the gene. For example, a gene can be inactivated by the introduction into the gene of insertions, deletions, random mutations, frameshift mutations, point mutations, insertion of one or more stop codons or a combination thereof. Thus, certain aspects of the present disclosure provide insertion of at least one stop codon (e.g., one to ten or more stop codons) into the gene. Some aspects of the present disclosure provide for the insertion or deletion of 1 , 2, 4, 5, 7, 8, 10, 1 1 , 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29 or more bases to introduce a frameshift mutation into a gene. Yet other embodiments of the subject application provide for the introduction of one or more point mutations (e.g., 1 to 30 or more) within a gene while other aspects of the present disclosure provide for the total or complete deletion of a gene. Mutations and/or deletions in the promoter region of a gene resulting in the inactivation of the gene can also be performed. In each of these aspects of the present disclosure, metabolic pathways are inactivated by the inactivation of the enzymatic activity of the polypeptide(s) encoded by the inactivated gene(s).

As a skilled artisan would recognize, inactivation of genes as described herein can also be accomplished by other means, such as siRNA/shRNA knockdown (which can be accomplished using expression vectors encoding siRNA, for example, tailored to a gene of interest), increased degradation of RNA transcripts, blocking of RNA transcription, or increased degradation of protein.

In further embodiments, the present disclosure provides microorganisms, wherein the parental microorganisms produce metabolites of interest using a carbon source different from DHA. Such metabolites of interest include ethanol, butyric acid, lactic acid, and succinic acid. Additional embodiments of metabolites of interest that can be produced by microorganisms are known to a skilled artisan and such embodiments are within the purview of the present disclosure. When such parental microorganisms that produce metabolites of interest are genetically modified according to the present disclosure, particularly, by overexpressing or increasing catalytic activity of DhaK, and optionally, further inactivating one or more genes from glpF, mgsA, budA, alsS, pfIBA, and gldA, the resultant microorganisms produce the metabolite of interest using DHA as a carbon source.

Non-limiting examples of the genetically modified bacterial microorganisms according to the subject present disclosure include Escherichia coli, Klebsiella spp., K. oxytoca, K. variicola, Gluconobacter oxydans, Gluconobacter asaii, Achromobacter delmarvae, Achromobacter viscosus, Achromobacter lacticum, Agrobacterium tumefaciens, Agrobacterium radiobacter, Alcaligenes faecalis, Arthrobacter citreus, Arthrobacter tumescens, Arthrobacter paraffineus, Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans, Aureobacterium saperdae, Azotobacter indicus, Brevibacterium ammoniagenes, Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacterium globosum, Brevibacterium fuscum, Brevibacterium ketoglutamicum, Brevibacterium helcolum, Brevibacterium pusillum, Brevibacterium testaceum, Brevibacterium roseum, Brevibacterium immariophilium, Brevibacterium linens, Brevibacterium protopharmiae, Corynebacterium acetophilum, Corynebacterium glutamicum, Corynebacterium callunae, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Enterobacter aerogenes, Erwinia amylovora, Erwinia carotovora, Erwinia herbicola, Erwinia chrysanthemi, Flavobacterium peregrinum, Flavobacterium fucatum, Flavobacterium aurantinum, Flavobacterium rhenanum, Flavobacterium sewanense, Flavobacterium breve, Flavobacterium meningosepticum, Micrococcus sp. CCM825, Morganella morganii, Nocardia opaca, Nocardia rugosa, Planococcus eucinatus, Proteus rettgeri, Propionibacterium shermanii, Pseudomonas synxantha, Pseudomonas azotoformans, Pseudomonas fluorescens, Pseudomonas ovalis, Pseudomonas stutzeri, Pseudomonas acidovolans, Pseudomonas mucidolens, Pseudomonas testosteroni, Pseudomonas aeruginosa, Rhodococcus erythropolis, Rhodococcus rhodochrous, Rhodococcus sp. ATCC 15592, Rhodococcus sp. ATCC 19070, Sporosarcina ureae, Staphylococcus aureus, Vibrio metschnikovii, Vibrio tyrogenes, Actinomadura madurae, Actinomyces violaceochromogenes, Kitasatosporia parulosa, Streptomyces coelicolor, Streptomyces flavelus, Streptomyces griseolus, Streptomyces lividans, Streptomyces olivaceus, Streptomyces tanashiensis, Streptomyces virginiae, Streptomyces antibioticus, Streptomyces cacaoi, Streptomyces lavendulae, Streptomyces viridochromogenes, Aeromonas salmonicida, Bacillus pumilus, Bacillus circulans, Bacillus subtilis, Bacillus thiaminolyticus, Escherichia freundii, Microbacterium ammoniaphilum, Serratia marcescens, Salmonella typhimurium, Salmonella schottmulleri, or Xanthomonas citri. In preferred embodiments, the microorganism of the present disclosure is E. coli or Klebsiella spp. , particularly, K. oxytoca or K. variicola. Unlike other microbial systems, the microorganisms of the subject present disclosure can be employed in production of metabolites of interest using growth media containing DHA. In certain embodiments, the DHA metabolizing microorganisms of the present disclosure are metabolically evolved for desirable characteristics, for example, synthesis of metabolites of interest.

The microorganism of the present disclosure, when cultured in a medium containing DHA produces an increased amount of a metabolite of interest compared to the amount of the metabolite produced by the parental microorganism cultured in the medium containing DHA. Accordingly, a further embodiment of the present disclosure provides a method of culturing or growing a microorganism of the present disclosure in a medium containing DHA under conditions that allow the production of a metabolite of interest. In certain embodiments, the methods further comprise recovering, purifying, or otherwise isolating the metabolite[s] of interest.

The culturing or growing can be performed in a batch process, a fed batch process, or a continuous process. In preferred embodiments, DHA can be introduced into the culture medium in a fed-batch manner at a DHA concentration at the feeding points from 250 mM to 500 mM, preferably, between 275 mM to 350 mM, and more preferably about 325 mM, and even more preferably, about 333 mM.

Methods of producing different culture/growth media and conditions that allow culturing/growing of the microorganisms of interest are well known to a person of ordinary skill in the art and such embodiments are within the purview of the present disclosure. Methods of recovering products of interest from culture/growth media are also well known in the art and such embodiments are within the purview of the present disclosure.

In certain aspects of the present disclosure, fermentation of DHA to D-lactate by E. coli strain TG1 13 was inefficient and growth was inhibited by 30 g. L ¹ DHA. An ATP-dependent DHA kinase from Klebsiella oxytoca (pDC1 17d) permitted growth of strain TG1 13 in a medium with 30 g.L ¹ DHA and in a fed-batch fermentation, the D-lactate titer of TG1 13 (pDC1 17d) was 580 ± 21 mM at a yield of 0.92 g.g ^-1 DHA fermented. Klebsiella variicola strain LW225 with a higher glucose flux, compared to E. coli, produced 81 1 ± 26 mM D-lactic acid at an average volumetric productivity of 2.0 g.L ¹ h ¹ . This may have required a balance between transport of the triose and utilization by the microorganism. Using other engineered E. coli strains, DHA can also be fermented to succinic acid and ethanol, demonstrating the potential of converting CH ₄ and C0 ₂ to value-added chemicals and fuels by a combination of chemical/biological processes.

Described herein are methods of metabolizing dihydroxyacetone (DHA). Methods as described herein can comprise providing a plurality of microorganisms or engineered cells as described herein (a homologous plurality or heterologous plurality); providing a medium comprising DHA; and culturing the plurality of engineered cells in the medium comprising DHA to metabolize DHA and produce a DHA metabolite of interest. The culturing can be a batch process, fed-batch process, or a continuous process. The culturing can be a fed-batch process and DHA is introduced into the culture medium in a fed-batch manner at a concentration of: 250 mM to 500 mM, 275 mM to 350 mM, about 325 mM, or about 333 mM. Methods as described herein can comprise isolating the metabolite of interest by a method as known in the art (for example filtering, HPLC, evaporation, distillation, and the like). In certain embodiments, metabolites of interest are lactate, L-lactate, D-lactate, succinate, ethanol, and butanol. In certain embodiments, metabolites of interest are lactate, L-lactate, and D-lactate. In an embodiment, a metabolite of interest is succinate. In an embodiment, metabolites of interest are ethanol and butanol.

Also described herein are kits. In an embodiment, kits as described herein can comprise one or more microorganism and one or more expression vectors, the one or more expression vectors encoding a DhaK protein, inactivating one or more genes encoding: glycerol uptake facilitator (GlpF), methylglyoxal synthase (MgsA), acetolactate decarboxylase (BudA), acetolactate synthase (AlsS), and pyruvate formate-lyase (PfIBA), and glycerol dehydrogenase (GldA), one or more expression vectors decreasing activity of proteins: glycerol uptake facilitator (GlpF), methylglyoxal synthase (MgsA), acetolactate decarboxylase (BudA), acetolactate synthase (AlsS), and pyruvate formate-lyase (PfIBA), and glycerol dehydrogenase (GldA), or a combination thereof. Engineered cells can be created using such kits according to methods as known in the art, such as electroporation and transfection with cationic lipids.

In certain embodiments, also described herein are kits comprising one or more microorganisms or engineered cells as described herein and culture medium. The kit can further comprise DHA, either as a component of the medium or separate component to be mixed with the medium by the user.

While embodiments of the present disclosure are described in connection with the Examples and the corresponding text and figures, there is no intent to limit the disclosure to the embodiments in these descriptions. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure.

EXAMPLES Now having described the embodiments of the disclosure, in general, the examples describe some additional embodiments. While embodiments of the present disclosure are described in connection with the example and the corresponding text and figures, there is no intent to limit embodiments of the disclosure to these descriptions. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure.

EXAMPLE 1 - MATERIALS AND METHODS

Embodiments of materials and methods according to the present disclosure as described herein the present example:

Materials

Analytical grade chemicals were used. Organic and inorganic chemicals including DHA dimer were from Fisher Scientific (Pittsburgh, PA). Biochemicals were from Sigma- Aldrich Co. (St. Louis, MO). Molecular biology reagents and supplies were from New England Biolabs (Ipswich, MA), Invitrogen (Carlsbad, CA) or Clontech (Mountain view, CA). Plasmid DNA extraction kits were from Zymo Research (Irvine, CA). DNA and RNA clean up kits were from QIAGEN (Valencia, CA). DNA oligonucleotides for PCR were from Life Technologies (Carlsbad, CA).

Methods

Strains, media and growth conditions

Bacterial strains, plasmids, and primers are listed in Tables 1 , 2 and 3, respectively. Bacterial cultures were grown in LB medium described previously (Patel et al., 2006). Mineral salts medium was AM1 medium (Martinez et al., 2007). Low-phosphate medium was a modification of a medium described by Jin and Lin (Jin & Lin, 1984) and contained, MOPS (50 mM), KH2PO4 (1 mM), KCI (40 mM), NaCI (34 mM), (NH ₄ ) ₂ S0 ₄ (15 mM), MgS0 ₄ .7H ₂ 0 (0.8 mM), casamino acids (1 g.L ¹ ), trace mineral solution from AM 1 medium (1 ml.L ¹ ) and required amount of DHA. Ampicillin (100 mg.L ¹ ), Kanamycin (50 mg.L ¹ ), X-Gal (25 mg.L ¹ ) and IPTG (5 mM) were added to the media as needed for selection. Ampicillin concentration was increased to 1.5 g.L ¹ for Klebsiella spp. Anaerobic cultures were grown in 13 x 100 mm screw cap tubes filled to the top. Fermentations were in 500 ml vessels with 250 ml of medium as described previously with pH control at 37°C (Beall et al., 1991). Fermentations started aerobically due to the air in the gas phase and the cultures were mixed by a magnetic stirrer (200 RPM) for base addition. As the cell density increased to an OD420 nm of about 0.5, limitation of 0 ₂ resulted in anaerobic condition (severe 0 ₂ -Iimitation) and initiation of fermentative growth.

Table 1. Embodiments of bacterial strains and plasmids according to the present disclosure

Table 2. Embodiments of plasmids according to the present disclosure

Table 3. Embodiments of primers according to the present disclosure

Strain constructions

E. coli mutants

E. coli strains TG1 13, KJ122, and SE2378 were described previously (Grabar et al., 2006; Jantama et al., 2008; and Kim et al., 2007). Strain LW290 is a derivative of strain TG1 13 with a deletion o gldA, which eliminates glycerol dehydrogenase. This strain was constructed using the method described by Datsenko and Wanner (Datsenko & Wanner, 2000). The primers used to amplify the kanamycin resistance gene flanked by FRT sequences and the 5’- and 3’-ends of gldA were 353 and 354. The PCR amplified product was electroporated into strain TG113 and kanamycin-resistant transformants were selected (strain LW290) and this strain carries a deletion of 1 ,012 bp DNA within the gldA. Strain LW410 was derived from strain TG1 13 and carries a 572 bp internal deletion of glpF and was constructed using the method of Datsenko and Wanner (Datsenko & Wanner, 2000). Details of construction of strain LW410 are presented below.

Construction of E. coli strains LW290 and LW410.

For construction of E.coli strain LW290, a AgldA derivative of strain TG1 13 primers 353 and 354 were used to amplify the kanamycin-resistance gene flanked by FRT sequences and the 5' and 3' ends of gldA. The PCR-amplified product was electroporated into strain TG1 13, and kanamycin-resistant transformants were selected (strain LW290). This strain carries a deletion of 1 ,012 bp of DNA within the gldA.

Construction of E. coli strain LW410 Strain LW410, a derivative of strain TG1 13, carries a deletion of glpF encoding glycerol facilitator. PCR-primers 451 and 452 were used to amplify the entire coding region of glpF from the genome of E. coli strain MG 1655 and the amplified fragment was cloned into plasmid vector pCR2.1-TOPO (plasmid pLW79). Using plasmid pLW79 as template and primers 453 and 454, the glpF flanking DNA and the plasmid backbone were PCR amplified. This linear DNA lacking a 572 bp internal fragment of glpF was ligated with a PCR product that carries a tetracycline resistance gene cassette (tef) flanked by FRT-sequences from plasmid pLOI2065 (primers 455 and 456) (plasmid pLW80). A linear fragment generated from plasmid pLW80 using primers 451 and 452 that contains the tet gene flanked by FRT and glpF was electroporated into strain TG1 13 and the tetracycline resistant transformants were selected. One of the FRT sequences and tet gene were removed using FLP-recombinase (Datsenko & Wanner, 2000) resulting in strain LW410. Deletion of glpF in strain LW410 was verified by DNA sequence and by phenotype using the absorbance based cell shrink and re-swelling assay of Heller et al. for GlpF activity (Heller et al., 1980).

K. variicola strain LW225

K. variicola strain LW225, a mutant of wild type strain AC1 lacking butanediol synthesis pathway enzymes, a-acetolactate decarboxylase ( budA ) and acetolactate synthase (a/sS), and pyruvate formate-lyase ( pfIBA ), was constructed to eliminate side reactions at the pyruvate node for production of D-lactate. Details of strain construction are presented below.

Construction of K. variicola strain LW225

Strain LW225 carries a deletion of the genes budA, alsS, and pfIBA encoding acetolactate decarboxylase, acetolactate synthase, and pyruvate formate-lyase, respectively, to eliminate side reactions at the pyruvate node. As a first step, a 1 ,500 bp budA-alsS gene fragment was amplified from K. variicola wild type strain AC1 genomic DNA with primers bupro070910 F and bud070910 R2. The amplified product, after confirmation by sequencing the DNA, was ligated into plasmid vector pUC19 that was hydrolyzed with HinCII (pMSR-141). The kanamycin resistance gene cassette with FRT was obtained from plasmid pLOI251 1 after Smal digestion (1 ,228 bp). A 758 bp internal fragment of the budA-alsS gene fragment was removed from plasmid pMSR-141 after Blpl and Bglll hydrolysis and the ends are filled in using the Klenow fragment of DNA polymerase (Klenow fill-in). The kanamycin resistance cassette with FRT fragment was inserted at this site (pMSR-141-Km). Using this plasmid as template, a PCR product containing buc/A-kan-FRT (1 ,953bp) was generated with the primer pair bupro070910 F, bud070910 R2. This buc/A-kan-FRT linear DNA fragment (1 ,953 bp) was introduced into strain AC1 by electroporation (18KV/cm, 25 pF, 200W using Bio-Rad Gene Pulser XCell; Hercules, CA) and transformants were selected on LB-agar with kanamycin (50 mg.L ¹ ). Competent cells of AC1 were prepared by washing a mid-exponential phase culture grown in LB 3 times with 10 % glycerol. AC1- buc/A-FRT-kan-FRT (strain MR900) was confirmed by its fermentation profile.

To remove the kanamycin resistance cassette, plasmid pCP20 was introduced into strain MR900 and transformants were selected on LB-agar with ampicillin (500 mg.L ¹ ) and chloramphenicol (40 mg.L ¹ ) at 30°C. After incubation of transformants at 42°C, kanamycin sensitive strain MR901 was obtained. To generate a ApfIBA derivative of strain MR901 , a 3, 127 bp pfIBA gene fragment was amplified using strain AC1 genomic DNA as the template and primers newpfIBA F and newpfIBA R. The amplified product was ligated into plasmid vector pUC19 after hydrolysis by EcoRI and Hindlll (pMSR-144). Kanamycin resistance gene cassette with FRT was ligated into plasmid pMSR-144 after hydrolysis with Nrul that removed 2,356 bp pfIBA DNA (pMSR-144-Km). A 3,635 bp fragment from pKD46 after hydrolysis with EcoRV and Stul was ligated with a 1 ,998 bp pfIBA- km fragment obtained after amplifying the DNA from plasmid pMSR-144-Km with primers newpfIBA F and newpfIBA R (pMSR-145). This temperature sensitive plasmid, pMSR-145 was introduced into strain MR901 and selected on LB-agar with kanamycin (50 mg.L ¹ ) at 30°C. After incubation of transformants at 42°C to eliminate the plasmid, mutant strains that lacked PFL activity were identified by their fermentation profile (strain MR902). Based on PCR amplification of remaining pfl DNA in these PFL strains, a pfIBA deletion mutant was identified and the kanamycin-resistance cassette was removed using FLP-recombinase. The (budA-alsS)- FRT, ApfIBA- FRT derivative of strain AC1 was maintained as strain LW225.

Construction of plasmids pDC4, pDC1 17d and pLW63

TA-cloning of DNA inserts was performed according to Manufacturer’s instructions with 4 pi of gel-purified PCR-product, 1 pi salt solution and 1 mI of commercial pCR2.1-TOPO vector. The cloning reaction was allowed to proceed for 30 min at room temperature before transforming 2 pL of cloning reaction into CaCI ₂ competent E. coli Top10 (Invitrogen) or Stellar™ cells (Clontech). Transformants were selected on LB-agar supplemented with kanamycin, X-Gal and IPTG. White colonies were screened by colony PCR and the DNA insert was confirmed by DNA Sequencing.

Plasmid pDC4 carries the dhaKLM operon (PEP-dependent DHA kinase) from E. coli with the native promoter in plasmid vector pTOPO. The dhaKLM DNA (4,283 bp) was amplified from E. coli MG1655 genomic DNA using primers 15 and 16 and cloned into plasmid vector pCR2.1-TOPO (TA-cloning). It includes a 553 bp DNA upstream of the dhaK start codon (ATG) and a 557 bp DNA sequence downstream of the dhaM stop codon of the dhaKLM operon.

Plasmid pDC1 14cL that carries the dhaK from K. oxytoca ( dhaK _K o ) (ATP-dependent DHA kinase from Klebsiella oxytoca strain M5A1) was constructed using TA-cloning and blue- white selection. A 2354 bp DNA containing the dhaK _K o M5A1 was amplified by PCR with the genomic DNA as template and primers 783 and 784. Orientation of the inserted DNA and DNA sequence were verified by PCR and sequencing, respectively. Plasmid pDC1 17d carries the dhaK with its native promoter from K. oxytoca strain M5A1 in plasmid vector pBR322. Plasmid pDC1 17d was constructed using CPEC method (Quan & Tian, 201 1). Plasmid pBR322 was linearized by PCR using primers 803 and 418 without the tet coding region. The dhaK _K o Insert (2,589 bp) was amplified from plasmid pDC114cL using primers 804 and 805 and this includes a 235 bp native DNA sequence upstream of the start codon of the dhaK gene. This amplified fragment was inserted into linearized plasmid pBR322 DNA and transformed into E. coli strain Top10. Ampicillin-resistant and tetracycline sensitive transformants were screened by colony PCR and an appropriate plasmid was confirmed by DNA sequencing.

For construction of plasmid pLW63, dhaK _K o was amplified by PCR from plasmid pDC1 17d and inserted into pTrc99a for expression from its tac promoter. As a first step, a tetracycline resistance gene was inserted into pTrc99a (pLW60) for transformation into K. variicola due to its innate resistance for ampicillin. The tetracycline-resistance gene was amplified by PCR from pLOI2065 using primers, 377 and 378. The 1 ,875 bp fragment was inserted into a PCR amplified pTrc99a (primers 375 and 376; 3,121 bp backbone of the plasmid without the ampicillin resistance gene) by the CPEC method. The resulting plasmid pLW60 served as the vector for cloning a promoterless dhaK _K o from plasmid pDC1 17d. Plasmid pLW60 DNA was amplified by PCR using primers 175 and 176 to generate a 4,916 bp fragment with the ends at the MCS of the plasmid pLW60. A promoterless dhaK was amplified by PCR from plasmid pDC1 17d using primers 393 and 394. After digestion of the linearized plasmid vector fragment by Ncol and the dhaK fragment by Ncol and Eco53KI, the two fragments were ligated and transformed into E. coli Top10 (plasmid pLW63). Tetracycline- resistant transformants were selected and the plasmid was verified by PCR and DNA sequencing.

Enzyme assays

DHA kinase activity was determined in crude extracts of cultures grown in 250 ml of LB + DHA (30 g.L ¹ ) in fermenters with pH control (7.0) or under aerobic conditions (2.8 L Fernbach flask; 200 RPM) at 37°C to mid-exponential phase of growth. Cells harvested by centrifugation at 4,200 x g for 10 min at 4°C were washed once with 20 ml of HEPES buffer (50 mM; pH 7.5). Cells collected by centrifugation (5,900 x g; 5 min) were resuspended in 2 ml of HEPES buffer. Cells were passed through a French pressure cell operating at 20,000 PSI and the extract was centrifuged at 17,500 x g for 10 min to remove cell debris. The supernatant was centrifuged again at 39,000 x g for 20 min to remove large vesicles. This supernatant served as the extract for enzyme assay. Protein concentration was determined as per Bradford (Bradford, 1976).

DHA kinase was assayed using ATP or PEP as phosphate donor in a coupled assay as described previously (Gutknecht et al., 2001 ; Johnson et al., 1984). One ml assay mixture for ATP-dependent activity contained HEPES buffer (50 mM; pH 7,5), MgCI ₂ (2.55 mM), NADH (0.25 mM), ATP or PEP (1 mM), glycerol-3-phosphate dehydrogenase (rabbit muscle, 1.7 units; Sigma-Aldrich) and cell extract. Initial rate of oxidation of NADH after addition of DHA (1 mM) was determined in the coupled reaction at 340 nm. One unit of enzyme activity is one pmole. min ^-1 mg protein ^-1 .

Analytical methods

Organic acids, ethanol, and DHA were determined using an Agilent (1200) HPLC equipped with dual detectors (UV and refractive index, in series) and a BioRad Aminex HPX- 87H column (45°C; 4 mM H ₂ S0 ₄ as the mobile phase, 0.4 ml. min ^-1 flow rate) (Underwood et al., 2002). Optical purity of lactic acid was determined using an Agilent HPLC (1090) equipped with Chirex 3126(D)-penicillamine column (150 x 4.6 mm; Phenomenex, Torrance, CA) and variable wavelength detector. The eluent was 2 mM CuS0 ₄ at 0.6 ml. min ^-1 (Chauliac et al., 2015).

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

Following are examples which illustrate procedures for practicing the present disclosure. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 2 - GENETIC MODIFICATIONS THAT PRODUCE DHA METABOLIZING

MICROORGANISMS

During anaerobic growth in glucose-containing medium E. coli produces acetate, ethanol, lactate, formate, H ₂ , C0 ₂ , and small amount of succinate as fermentation products (Fig. 7). Although DHA is not in the glucose fermentation pathway, it is an intermediate of glycerol metabolism in E. coli especially during anaerobic condition (Gonzalez et al., 2008). DHA produced by glycerol dehydrogenase (gldA) is phosphorylated to DHA-P by a PEP- dependent kinase encoded by dhaKLM that is not associated with transport. Level of DHA kinase was reported to be very low during aerobic growth and increased during 0 ₂ -Iimitation condition in glycerol- grown cells (Durnin et al., 2009) and thus limiting the glycerol-DHA-DHA- P pathway to anaerobic growth condition. Dihydroxyacetone-3-phosphate, an intermediate of glycolysis, is expected to yield the same fermentation products as seen with glucose fermentation (Fig. 7). In this pathway, DHA added to the medium is transported by a facilitated diffusion channel (GlpF). In E. coli and other enteric bacteria, GlpF helps transport glycerol in an energy-independent manner. Since the GlpF channel can also transport glyceraldehyde and to a lesser extent erythritol and ribitol (Heller et al., 1980), it is likely DHA is also transported by this facilitator. Using cell shrinkage and reswelling assay for glycerol uptake demonstrated by Lin and co-workers (Heller et al., 1980), the rate of facilitated diffusion of DHA by a glpF mutant, strain LW410, was found to be about half (-0.04 AU.sec ¹ ) of the value of the parent, strain TG1 13 (-0.08 AU.sec ¹ ). In addition to GlpF, DHA transport systems also exist in E. coli based on the growth and fermentation of DHA by the glpF mutant (Fig. 10). The nature of these alternate transport systems is yet to be established and these could be the same non-GIpF transporters reported for glycerol in E. coli (Heller et al., 1980).

Upon phosphorylation, DHA-P enters the glycolysis pathway and is converted to pyruvate with associated ATP and NADH production. Thus, there are only two additional steps that are unique for DHA metabolism in E. coli, transport and phosphorylation. Fermentation of two DHA molecules to one each of acetate and ethanol would yield a net 3 ATPs while fermentation to two lactates results in a net yield of 2ATPs. These ATP yields are the same as that of glucose (2 DHA equivalents) fermentation by this bacterium. This shows that anaerobic growth of E. coli with DHA as a fermentable C-source is not constrained energetically or by redox balance.

EXAMPLE 3 - E. COLI DOES NOT GROW IN DHA-MINIMAL MEDIUM

Wild type E. coli (B, ATCC1 1303; C, ATCC8739; K-12, W31 10 and W, ATCC9637) did not grow with DHA as a carbon source in mineral salts medium under aerobic condition. This was expected apparently due to the very low level of DHA kinase in aerobically grown cells (Durnin et al., 2009). However, similar results were also obtained under anaerobic growth condition in DHA-mineral salts medium. DHA can interact with medium components such as phosphate and generate methylglyoxal, a highly reactive growth inhibitor (Riddle & Lorenz, 1968; Riddle & Lorenz, 1973). Lowering the phosphate concentration to 1 mM (Jin & Lin, 1984) did not overcome growth inhibition of E. coli by DHA (10 g. L ¹ ). Rapid conversion of DHA to non-toxic products, such as DHA-P, is expected to minimize the inhibitory effect of DHA on microbial biocatalysts. To understand the physiological constraints in the anaerobic metabolism of DHA in E. coli, a lactate-producing derivative of E. coli, strain TG1 13 (Grabar et al., 2006) was used in this study. The reported average specific and volumetric productivities of D-lactate for strain TG1 13 in mineral salts medium with glucose are 1.0 (g. r ¹ g cells ^-1 ) and 1.92 (g.L ¹ h ^_1 ), respectively, over a 24 h period. The high glucose flux to D- lactate in this strain is expected to minimize potential rate-limiting step(s) from the glycolytic pathway in DHA fermentation. In addition, strain TG1 13 carries a deletion of mgsA encoding methylglyoxal synthase that catalyzes DHA-P dependent methylglyoxal synthesis and thus eliminating production of this inhibitor by the bacterium from DHA-P.

EXAMPLE 4 - DHA FERMENTATION BY E. COLI STRAIN TG1 13

Due to the interaction of DHA with phosphate in mineral salts medium and generation of inhibitory compounds, E. coli strain TG1 13 was grown in rich medium with DHA (1 1 1 mM; 10 g.L ¹ ). Strain TG1 13 grew in this medium utilizing the nutrients in LB but fermented DHA at a very low level (Figs. 2A-2B, Table 4). About 40 mM DHA was consumed during the first 24 hours and the D-LA yield was 0.66 g.g ¹ of consumed DHA. Since there are only two steps between medium DHA and the glycolytic intermediate DHA-P, and the DHA transport is apparently facilitated diffusion, the rate-limiting step is apparently DHA kinase level in the cell. Strain TG1 13 grown in LB with DHA and harvested at late-exponential phase of fermentative growth did contain DHA kinase activity but at a very low level (about 0.1 unit; pmole.min Lmg protein ^-1 ) (Table 5). However, this DHA kinase activity is higher than previously reported values for glycerol-grown E. coli culture apparently due to higher level of DHA in the cytoplasm, an inducer of dhaKLM (Durnin et al., 2009; Sprenger et al., 1989). Due to the inability of strain TG1 13 to ferment DHA, the copy number of dhaKLM operon with its native promoter was increased by introducing plasmid pDC4. Under similar condition, strain TG1 13 (pDC4) grew to a higher cell density and fermented almost all of the 1 1 1 mM DHA added to the LB medium (Table 4; Figs. 2A-2B) although the DHA kinase activity of this culture at midexponential phase of growth was not different from the value of strain TG1 13 without the plasmid (Table 5). The reason for this difference in fermentation of DHA (1 1 1 mM) by the two cultures with comparable DHA kinase activity is not apparent.

Increasing the concentration of DHA to 333 mM (30 g.L ¹ ) completely abolished fermentative growth of strain TG1 13 with or without plasmid pDC4 (Fig. 2B). Culture density of strain TG1 13 or TG1 13 (pDC4) increased initially from the 0 ₂ in the gas phase of the fermenter utilizing the nutrients in LB to an OD of about 0.3 (420 nm) at about 5 hours. As the 0 ₂ became limiting due to increasing cell density, further growth stopped and DHA was not fermented. Only about 7 mM D-lactate was produced by these cultures (Table 4). At the 333 mM DHA concentration, the rate of transport of DHA may be higher than the rate of conversion to DHA-P by the low DHA kinase activity (about 0.1 unit). This imbalance in the transport and phosphorylation could lead to a higher DHA pool in the cytoplasm triggering production of inhibitory compounds, as seen by accumulation of brown colored compounds in the medium as well as potential direct interaction of DHA with cellular components (Maillard reaction) (Petersen et a!., 2004). An alternative possibility that DHA-P is produced at a higher rate than glycolytic flux and the accumulating DHA-P is converted to methylglyoxal by methylglyoxal synthase can be ruled out since strain TG1 13 is an mgsA deletion mutant.

Table 4. Growth and DHA fermentation characteristics of E. coli strain TG1 13 with DHA-kinase plasmids

Fermentations were in LB medium with the indicated concentration of DHA at pH 7.0 and 37°C. Culture pH was controlled by automatic addition of 2N KOH. The highest cell density in 24 hour fermentations is reported. The amount of DHA removed and D-lactate produced were determined after 24 h fermentations. Lactate yield is g.g ^-1 of DHA consumed.

*DHA at the concentration of 333mM (30 g.L ¹ ) led to production of brown colored compound(s) in the medium in the cultures of TG1 13 and TG1 13(pDC4).

Table 5. DHA kinase activities of E. coli and K. variicola

Plasmid pDC4 carries E. coli dhaKLM encoding PEP-dependent DHA-kinase. Plasmid pDd 17d carries K. oxytoca dhaK encoding an ATP-dependent DHA-kinase. Plasmid pLW63 carries the K. oxytoca dhaK with trc promoter.

*UD, undetectable; activity was less than 0.01 pmole. min ^-1 mg protein ^-1

EXAMPLE 5 - FERMENTATION OF DHA BY E. COLI STRAIN TG1 13 WITH ATP-

DEPENDENT DHA KINASE

DHA kinase from K. pneumoniae supported fermentative growth of E. coli in glycerol medium (Sprenger ef a/., 1989). Since strain TG1 13 (pDC4) with PEP-dependent DHA kinase failed to grow at 30 g.L ^-1 DHA, a gene encoding ATP-dependent DHA kinase was cloned with its native promoter from K. oxutoca strain M5A1 ( dhaK plasmid pDC1 17d) and introduced into strain TG1 13. Strain TG1 13 (pDC1 17d) grew to an OD of 6.9 and fermented 333 mM DHA in 24 h with no detectable brown colored compound in the medium (Fig. 2B; Table 4). In this strain, the ATP-dependent DHA kinase activity was about 1 unit, about 10-times higher than the level of PEP-dependent activity of strain TG1 13 (Table 5). Apparently, this higher level of DHA kinase activity is needed to support growth of E. coli in a medium with 30 g.L ^-1 DHA. It should be noted that, in addition to the enzyme, a higher level of a phosphate donor, PEP or ATP, may also be required to rapidly remove DHA as DHA-P and to mitigate its inhibitory effect on cells. ATP-dependent DHA kinase is suitable here, since conversion of DHA to pyruvate generates two ATPs, while the same set of reactions may generate only one PEP. This two fold-higher level of phosphate donors (ATPs) in the cytoplasm can support higher DHA kinase activity and can offset the inhibitory effect of the higher DHA concentration in the medium. In a fed-batch fermentation, strain TG1 13 (pDC1 17d) produced 580 ± 21 mM D-lactate (52 ± 1.9 g.L ^-1 ) in 55 hours after an initial lag of about 10 h (Fig. 3). The average volumetric productivity of D-lactate for this culture over a 34 h period was 1.24 g.L ^-1 h ^-1 . This value is about 70% of the volumetric productivity reported for strain TG1 13 with glucose in mineral salts medium (Grabar et al., 2006). The lactate yield was 0.94 g.g ^-1 DHA fermented. These results show that the DHA-kinase and not the glycolytic flux is apparently the rate-limiting reaction in the conversion of DHA to fermentation products. Strain TG1 13 (pDC1 17d) also produced very low but detectable amounts of glycerol (27 ± 5 mM), especially during late stationary phase of growth, catalyzed by glycerol dehydrogenase operating in the reverse direction. Deletion of gldA (strain LW290) eliminated glycerol production during DHA fermentation.

Increasing the DHA concentration in the medium above 30 g.L ^-1 resulted in lack of growth of all E. coli cultures. This suggests that phosphorylation of DHA, either by DHA kinase or ATP availability is unable to keep up with the rate of entry of DHA resulting in accumulation of DHA in the cytoplasm that is growth inhibitory. The highest amount of DHA fermented by strain TG1 13 (pDC1 17d) in a fed-batch mode was about 0.62 ± 0.02 M in 55 h to produce 0.58 ± 0.02 M D-lactate (yield of 0.94 g. g ^-1 ), although strain TG1 13 is known to ferment 0.67 M glucose to higher than 1 M lactate in mineral salts medium in about 48 h (Grabar et al., 2006). Declining specific productivity of the aging culture may account for this low titer. Further increase in the DHA kinase level and/or glycolytic flux to raise the ATP level in the cell to support higher kinase activity may be needed to reach the D-lactate titer of strain TG1 13 on glucose.

EXAMPLE 6 - FERMENTATION OF DHA BY K. VARIICOLA The experiments with E. coli suggested that fermentation of DHA can be limited at two steps; DHA kinase or glycolytic flux. To distinguish between the two, K. varicola (strain AC1) that has a higher glucose flux compared to E. coli was selected for DHA fermentation. Specific rate of glucose consumption by strain AC1 (wild type) in rich medium was determined to be 6.2 ± 0.35 (g.ir ¹ . g dry weight of cells ^-1 ) and in glucose mineral salts medium this value only slightly declined (5.09 ± 1.22 g.h ^-1 g cells ^-1 ). Glucose flux of a homolactate producing derivative of strain AC1 , strain MR902, was calculated to be 5.9 ± 1.6 (g.h ^-1 g cells ^-1 ) when grown in LB medium with glucose and this value increased when strain MR902 was grown in mineral salts medium (7.2 ± 0.0.9 g.h ^-1 g cells ^-1 ). Average volumetric productivity of D-lactate for strain MR902 was 4.4 g.L ^-1 .h ^-1 in rich medium. These values are about twice the productivity for a lactate producing E. coli grown under similar conditions with glucose (Zhou et al., 2006a; Zhou et al., 2006b). Due to the higher glucose flux and lactate productivity, glycolytic flux is not expected to limit DHA fermentation in K. variicola strain LW225. In addition, Klebsiella spp. also produces an ATP-dependent DHA kinase from the chromosomal dhaK.

Although strain LW225 had a higher glucose flux, the growth rate of this strain in DHA containing medium was lower than that of an LB-glucose culture (Figs. 4A-4B). Fermentative growth rate of the culture with glucose was 0.84 ir ¹ compared to a m value of 0.25 ir ¹ for a DHA culture. Specific productivity of lactate with glucose was 5.4 g.ir ¹ .gcells ¹ while the specific productivity with DHA as C-source was about 35% of the glucose value (1.9 g.h ¹ .g cells ¹ ). These results suggest that strain LW225 also has a limiting step in DHA utilization, most probably at the DHA-kinase activity, as seen with E. coli strain TG1 13. Under fermentative condition, the ATP- and PEP-dependent DHA kinase activities of strain LW225 were 1.7 and 1.8 units, respectively (Table 5), suggesting that in K. variicola, DHA kinase activity is limited not be the enzyme level but by the availability of the co-substrate ATP and PEP.

In contrast to E. coli, K. varicola fermented 30 g. L ¹ DHA using the native DHA kinase(s) (Figs. 2A-2B and 4A-4B). Even with this higher level of DHA kinase activity K. variicola was unable to grow when the DHA concentration was increased to above 300 mM (Fig. 8) as seen with E. coli TG1 13(pDC1 17d). This inhibition is apparently due to an imbalance between the transport of DHA into the cytoplasm and the ability of the cell to provide ATP/PEP at a rate needed to detoxify DHA by conversion to DHA-P. Due to this growth inhibition by higher concentrations of DHA, strain LW225 fermentations were run in fed-batch mode (Fig. 5). Under this fermentation condition, a D-lactate titer of 81 1 ± 26 mM (72 g.L ¹ ) was reached in 60 h. Average volumetric productivity of lactate was 2.0 g.L ¹ h ¹ . This is about 35% of the D-lactate productivity (5.6 g.L ¹ h ¹ ) of K. variicola with glucose as the C-source. During late stationary phase, glycerol was also detected as a co-product, possibly a result of energy imbalance. Introducing plasmid pDC1 17d encoding ATP-dependent DHA kinase into strain LW225 did not significantly alter the fermentation profile of K. variicola suggesting that the native chromosomal copy of DHA kinase is sufficient to support growth and fermentation of DHA by strain LW225. Unexpectedly, presence of plasmid pDC1 17d in strain LW225 lowered the DHA kinase activity with either ATP or PEP as substrate (Table 5). Plasmid vector pBR322 without dhaK had no effect on the DHA-kinase activity of strain LW225. The decrease in DHA-kinase activity is related to the presence of plasmid-borne dhaK encoding the ATP- dependent DHA kinase from its native promoter and the physiological reason for this decrease in enzyme activity is not clear. Replacing the native promoter in plasmid pDC1 17d with an inducible trc promoter (LW225 with plasmid pLW63) lowered the DHA kinase activity also even in the absence of inducer. At an optimum level of IPTG (25 mM), the DHA kinase activity reached the level of the parent without the plasmid and this did not provide an additional advantage to the cell. Increasing IPTG concentration above 25 mM inhibited growth, probably due to ATP limitation caused by the DHA kinase activity.

These results show that DHA can be readily fermented to D-lactate either by E. coli strain TG1 13 (pDC1 17d) or by K. variicola strain LW225 under appropriate fermentation condition. A reduction in the rate of transport of DHA to match the flux rate of intermediary metabolism and supply of ATP/PEP could overcome the toxicity of DHA while also improving energy balance and D-lactate productivity (Fig. 9).

EXAMPLE 7 - FERMENTATION OF DHA TO SUCCINIC ACID BY STRAIN

KJ122(PDC1 17D)

Fermentation of DHA to D-lactic acid raised the possibility that DHA, produced from CH ₄ , can be fermented to any one of several products that are currently produced from hexoses and pentoses by various microbial biocatalysts. To demonstrate this potential, strain KJ122, an E. coli strain that produces succinic acid as the major fermentation product (Jantama et al., 2008) was transformed with plasmid pDC1 17d and the transformants fermented DHA to succinate (Fig. 6A). In this fed-batch fermentation, about 0.5 M DHA was converted to about 0.3 M succinate (38 g.L ^-1 ) and the succinate yield was 0.86 g.g ^-1 DHA consumed. The conversion efficiency was 66% of the theoretical yield. About 85 mM acetate was also produced by this culture, mostly during the growth phase. Further metabolic engineering to minimize acetate is expected to increase the succinate titer and productivity.

EXAMPLE 8 - FERMENTATION OF DHA TO ETHANOL BY E. COLI STRAIN

SE2378(PDC1 17D)

The abundance of natural gas and its current low price in USA raised the possibility of converting CH ₄ to liquid fuels (gas to liquids) that can be more readily used as a transportation fuel. As discussed earlier, technology exists for the conversion of natural gas to DHA provided an efficient microbial biocatalyst can be developed for fermentation of DHA to ethanol. Ethanologenic strains are known for E. coli and K. oxytoca (Jarboe et al., 2010). One of these ethanologens, E. coli strain SE2378 was transformed with plasmid pDC1 17d to ferment DHA to ethanol. The results presented in Fig. 6B show that DHA can be effectively fermented to ethanol at a yield of 0.39 g.g ^-1 (77% of the theoretical yield). This is comparable to the yield of 0.41 g.g ^-1 glucose fermented by strain SE2378 (Kim et al., 2007). Other engineered ethanologenic bacterial and yeast strains, appropriately engineered for DHA fermentation has the potential to increase the titer and yield of ethanol from DHA from natural gas. EXAMPLE 9 - DHA METABOLIZING MICROORGANISMS OF THE PRESENT

DISCLOSURE

Although DHA is inhibitory to growth and fermentation of E. coli, a rate-limiting step was identified as the activity of DHA kinase. By introducing an ATP-dependent DHA kinase, the inhibitory effect of DHA was mitigated in E. coli. A fed-batch fermentation process also overcame the toxicity of DHA. With these modifications, DHA was fermented by E. coli and K. variicola to D-lactic acid. Using appropriate engineered E. coli derivatives, DHA also was fermented to succinic acid or ethanol, as needed (Table 6). Further metabolic evolution of these microbial biocatalysts is anticipated to increase product titer, yield and productivity. These results show that DHA, produced from C0 ₂ , natural gas or biodiesel byproduct glycerol, is a valuable feedstock for fermentation to desired product(s).

Table 6. Fermentation of DHA by various E. coli derivatives to metabolites of interest.

EXAMPLE 10 - DELETING GLPF CAN INCREASE TOLERANCE OF E. COLI TO DHA.

The results presented suggest that a balance between the rate of transport and the conversion of DHA to DHA-P is needed for effective fermentation of DHA to products (Fig. 9). Any deviation from this leads to the accumulation of DHA in the cytoplasm and inhibition of growth. As discussed above, with a kinase that uses ATP as the phosphate donor, the optimum concentration of DHA for fermentation by E. coli strain TG1 13(pDC1 17d) was shifted to 333 mM from 1 1 1 mM DHA for a strain with PEP-dependent DHA kinase (Table 7 below and Figs. 2A-2B). An alternate way of shifting the balance toward DHA kinase and DHA-P is to lower DHA transport. Toward this objective, glpF was deleted from the chromosome of TG1 13(pDC1 17d). Deleting glpF (strain LW416), and thus eliminating one of the DHA transporters, increased DHA tolerance to about 450 mM, compared with a tolerance of 333 mM DHA for the glpF+ parent with the ATP-dependent DHA kinase (Fig. 10). Both the parent and glpF mutant, strain LW416, grew and fermented DHA to D-lactate at about the same rate up to about 350 mM DHA. At about 450 mM DHA, TG1 13 (pDC1 17d) did not grow, while the glpF mutant grew but at a rate that was about 30% of the value of the 333- mMDHA culture. The final cell density of the 450-mM DHA culture was about 60% of the 333-mM DHA culture. Due to the lower cell density, the average volumetric productivity of D-lactate of the 450-mM DHA culture was about 30% of the same culture with 350 mM DHA (1 .4 g-L-Th-1 ). These results show that by lowering DHA transport, the internal DHA concentration can be set in balance with the ATP- dependent DHA kinase activity at 450 mM DHA. The lower growth rate of this culture suggests that the rate of transport at DHA concentrations >450 mM is still higher than the in vivo kinase activity that detoxifies DHA. Additional mutations in the yet to be identified transporter(s) can help establish a DHA pool that is in balance with the kinase activity.

These results are in agreement with the working model that a balance between transport and conversion of DHA to DHA-P is critical to sustain growth and fermentation of DHA to products (Fig. 9). This can be achieved by manipulating the kinase and transport. An alternative process-based approach to fed-batch fermentation is a continuous feed of DHA at the optimum concentration, and this is expected to support the fermentation of this those that can be derived from CH4 to a higher concentration of the desired product by engineered microbial biocatalysts.

Table 7. Growth and DHA Fermentation of E.Coli

Cells, highest cell density (OD420) in 24-h fermentations in LB with the indicated amount of DHA; DHA, amount of DHA consumed; D-LA, highest amount of D-lactate produced; yield, g D-LA g of DHA consumed ¹ . It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated within the scope of the invention without limitation thereto.

Unless defined otherwise, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of separating, testing, and constructing materials, which are within the skill of the art. Such techniques are explained fully in the literature.

It should be emphasized that the above-described embodiments are merely examples of possible implementations. Many variations and modifications may be made to the above-described embodiments without departing from the principles of the present disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims. REFERENCES

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