Engineered clostridium cells and methods for use thereof

Abstract

Disclosed herein is an engineered Clostridium tyrobutyricum cell, wherein the overexpression of a heterologous aldehyde / alcohol dehydrogenase (adhE2) and a heterologous carboxylic acid reductase increases production of biobutanol, and method of use thereof. Further disclosed herein is a method of biosynthesizing butanol, comprising fermenting the engineered C. tyrobutyricum cell with growth media and a carbohydrate-containing source.

Description

[0001] ENGINEERED CLOSTRIDIUM CELLS AND METHODS

[0002] FOR USE THEREOF

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to, and the benefit of, U. S. Provisional Application No.

[0005] 63 / 711,891, filed October 25, 2024, which is incorporated herein by reference in its entirety.

[0006] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0007] This invention was made with government support under Grant / Contract No. DE-AR0001512 awarded by the Department of Energy, Advanced Research Projects Agency -Energy (ARPA-E). The government has certain rights in the invention.

[0008] REFERENCE TO SEQUENCE LISTING

[0009] The sequence listing submitted on October 24, 2025, as an. XML file entitled “103362-058WOl_ST26” created on October 23, 2025, and having a file size of 35,024 bytes is hereby incorporated by reference pursuant to 37 C. F. R. § 1.52(e)(5).

[0010] BACKGROUND

[0011] Renewable, sustainable biofuel production is necessary to combat the growing crisis caused by greenhouse gas emissions contributing to climate change. Biobutanol is a 4-carbon alcohol with potential as a drop-in replacement to gasoline; biobutanol is less hydroscopic and has a lower vapor pressure than ethanol and it has a similar energy content to gasoline, 27 MJ / L versus 32 MJ / L, respectively. Biobutanol is traditionally produced via the acetone-butanol-ethanol (ABE) fermentation, typically producing products in a 3:6:1 ABE weight ratio when produced using the microbe Clostridium acetobutylicum. This fermentation is characterized by two distinct phases: the acetogenesis phase first produces acids acetate and butyrate, followed by the solventogenesis phase, producing products acetone, butanol, and ethanol. This complicated shift in metabolic pathways, along with the challenges presented by the spore-forming life cycles of traditional ABE producers, hinders the adoption of industrial biobutanol production. Finally, as already mentioned, the co-production of acetone, ethanol, acetate and butyrate as well as Clostridium acetobutylicum’’ s inherent butanol toxicity typically results in fermentations with low butanol yield, titer, and productivity. More efficient methods of producing biobutanol and necessary. The present application addresses this need. SUMMARY

[0012] In accordance with the purpose of the disclosed materials, methods, and kits, as embodied and described herein, the disclosed subject matter, in one aspect, relates to compositions and methods for biosynthesizing butanol. In some aspects, the disclosed subject matter relates to a recombinant microorganism (e.g. Clostridium tyrobutyricum) for the biosynthesis of butanol.

[0013] In one aspect, disclosed herein is a recombinant microorganism, wherein the recombinant microorganism is engineered cell to express or overexpress a heterologous carboxylic acid reductase (CAR) gene and a heterologous aldehyde / alcohol dehydrogenase E2 (adhE2) gene. Also disclosed herein is a method of biosynthesizing butanol, comprising fermenting the recombinant microorganism with a growth medium and a carbohydrate-containing source under conditions effective to obtain a butanol containing product. Further disclosed herein is a system useful for the biosynthesis of butanol, comprising a container suitable for fermentation, the disclosed recombinant microorganism, growth medium, and a carbohydrate-containing source.

[0014] In one embodiment, as disclosed herein is a recombinant microorganism, wherein the heterologous adhE2 comprises mutations that increase cofactor selectivity, butyraldehyde selectivity, or a combination thereof. In some embodiments, the adhE2 comprises an amino acid substitution selected from D485G, L488A, S601 A, V608S, or P619G in SEQ ID NO: 1.

[0015] In one embodiment, the heterologous CAR is from Mycobacterium marinum. In a further embodiment, the CAR comprises SEQ ID NO: 3 or a sequence at least 90% identical to SEQ ID NO: 3. In some embodiments, the CAR comprises a mutation that increases cofactor selectivity. In an additional embodiment, the CAR comprises an amino acid substitution selected from R507F and / or N508G in SEQ ID NO: 3.

[0016] In one embodiment, a heterologous sfp gene is overexpressed in the recombinant microorganism. In some embodiments, the sfp gene is derived from Bacillus subtilis. In a further embodiment, the sfp gene comprises SEQ ID NO: 8 or a sequence at least 90% identical to SEQ ID NO: 8.

[0017] In one embodiment, is the recombinant microorganism, wherein an additional tRNA construct is expressed. In a further embodiment, the additional tRNA construct expressed has at least 90% identity to SEQ ID NO: 10.

[0018] In one embodiment, as disclosed herein is the recombinant microorganism, wherein phosphite dehydrogenase (PTDH) is overexpressed. In a further embodiment, the PTDH comprises SEQ ID NO: 5 or a sequence at least 90% identical to SEQ ID NO: 5. In a further embodiment, the PTDH comprises a mutation that increases NADPH regeneration. In an additional embodiment, the PTDH comprises an amino acid substitution selected from G175A, A176R, and combinations thereof in SEQ ID NO: 5.

[0019] In some embodiments, the overexpression of the adhE2 gene, CAR gene, and / or PTDH gene is driven by a promoter endogenous to C. tyrobutyricum or C. acetobutylicum. In a further embodiment, the promoter is a THL, CAT1, or FLA promoter.

[0020] In one embodiment, as disclosed herein, the method of butanol biosynthesis comprising fermenting the recombinant microorganism with a growth medium and a carbohydrate-containing source is supplemented with at least one additional organic compound. In a further embodiment, the at least one additional organic compound is methyl viologen (MV). In an additional embodiment, the MV is supplemented after 12 hours. In another embodiment, the at least one additional organic compound is butyrate. In a further embodiment, the butyrate is supplemented after 24 hours. In an additional embodiment, the growth medium and carbohydrate-containing source are supplemented with both MV and butyrate. In one embodiment, the carbohydrate-containing source comprises a sugar selected from glucose, xylose, mannitol, and combinations thereof. In some embodiments, the growth medium is clostridial growth medium.

[0021] In one embodiment, the butanol yield is equal to or greater than 0.23 g / g sugar.

[0022] In one embodiment, as disclosed herein, the system comprising a container suitable for fermentation, the recombinant microorganism, growth medium, and a carbohydrate-containing source, further comprises methyl viologen and butyrate.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods.

[0025] FIGS. 1A-1C show sequence similarity logo of sequences generated from the Rosetta enzyme design. Fig. 1 A shows sequence logo for docking butyraldehyde and NADP+with ADD. Fig. IB shows sequence logo for docking butyraldehyde and NAD+with AAD. Fig. 1C shows sequence logo for docking acetaldehyde and NAD+. The size of the letter for each amino acid residue indicates its occurrence frequency observed in the simulation.

[0026] FIG. 2 shows a comparison of fermentation performance for various AAD mutant strains. FIGS. 3A-3F show fermentation kinetics for various AAD mutant strains. Fig. 3A shows fermentation kinetics for Ct_pC_WT. Fig. 3B shows fermentation kinetics for Ct_pC_WT with 50 pM MV added at 12 h. Fig. 3C shows fermentation kinetics of Ct_pC_P619G. Fig. 3D shows fermentation kinetics of Ct_pC_P619G with 50 pM MV added at 12 h. Fig. 3E shows fermentation kinetics for Ct_pC_ASG. Fig. 3F shows fermentation kinetics for Ct_pC_ASG with 50 pM MV added at 12 h.

[0027] FIGS. 4A-4B show fermentation kinetics for various AAD mutant strains. Fig. 4A shows C. tyrobutyricum overexpressing AAD D485G. Fig. 4B shows C. tyrobutyricum overexpressing AAD L488A.

[0028] FIGS. 5A-5D show active site models for the AAD wild-type and mutant. Fig. 5A shows the NAD(P)H-binding cavity for the wild-type AAD. Fig. 5B shows the active site close-up for the wild-type AAD. Fig. 5C shows the NAD(P)H-binding cavity for the AAD mutant S601A_V608S_P619G. Fig. 5D shows the active site close-up for the wild-type AAD S601A mutation.

[0029] FIGS. 6A-6D show active site models for the NAD(P)H-binding cavity. Fig. 6A shows the wild-type active site, Asp485. Fig. 6B shows the ADD mutant, D485G active site. Fig. 6C shows the wild-type Leu488 active site. Fig. 6D shows the ADD mutant, L488A, active site.

[0030] FIGS. 7A-7D show fermentation kinetics for varying promoter-gene combinations. Fig.

[0031] 7A shows fermentation kinetics for Ct_pC_WT. Fig. 7B shows fermentation kinetics for Ct_pT_WT. Fig. 7C shows fermentation kinetics for Ct_pC_P619G. Fig. 7D shows fermentation kinetics for Ct_pT_P619G.

[0032] FIG. 8 shows SDS-PAGE of cell protein extracts of various C. tyrobutyricum strains confirming the presence of ADD (94.4 kDa) except for the wild-type strain without expressing adhEZ.

[0033] FIG. 9 shows fermentation kinetics for Ct-amts. Fig. 9 (top) shows fermentation results for the control strain of C. tyrobutyricum containing the PMTL82151 adhE2 expression plasmid driven by the FLA promoter. Fig. 9 (middle) shows the test strain of C. tyrobutyricum containing two plasmids, the PMTL82151 adhE2 expression plasmid driven by the FLA promoter and a second PMTL82151 plasmid expressing a tRNA construct, mmCAR, and sfp driven by the CAT1 promoter. Fig. 9 (bottom) shows the test strain containing the two plasmids expressing adhE2 and tRNA, mmCAR, and sfp with 6 g / L butyrate supplemented at 24 hours.

[0034] DETAILED DESCRIPTION

[0035] Before the present compositions and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Any patents, applications, and publications as listed throughout this document are hereby incorporated by reference in their entirety herein.

[0036] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. 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. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.

[0037] Definitions

[0038] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

[0039] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of’ and “consisting of.” Similarly, the term “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.

[0040] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound”, “a composition”, or “a cancer”, includes, but is not limited to, two or more such compounds, compositions, or cancers, and the like.

[0041] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0042] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, 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, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of Tess than x’, less than y’, and Tess than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0043] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or subranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0044] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0045] The term “alcohol” includes for example 1 -propanol, isobutanol, 1 -butanol, 2-methyl 1-butanol, 3-methyl 1-butanol or 2-phenylethanol. The term “1-butanol” or “n-butanol” generally refers to a straight chain isomer with the alcohol functional group at the terminal carbon. The term “butyrate” or “butanoate” refers to a four-carbon short-chain fatty acid. Butyrate is the conjugate base of butyric acid. In some embodiments, the fermentation of an engineered C. tyrobutyricum cell is supplemented with butyrate. In a further embodiment, the butyrate is supplemented after 24 hours.

[0046] An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% or more increase so long as the increase is statistically significant.

[0047] As used herein, the term “conditions effective” as it relates to the fermentation of a fermentation mixture refers to a set of adjustable process parameters, such as pH, temperature, metabolite environment, and time, which can produce a desired product. For example, the incubation of a fermentation mixture (e.g., the recombinant microorganism and growth media) under conditions effective can convert a compound or compounds, such as single carbon compounds, to a target product, such as butanol, at an efficiency.

[0048] As used herein, the term “alcohol dehydrogenase” is intended to include the enzymes that catalyze the conversion of ethanol or butanol into acetaldehyde or butyraldehyde respectively. Commonly, the same enzyme catalyzes the reverse reaction from acetaldehyde to ethanol.

[0049] As used herein, the term “aldehyde dehydrogenase” is intended to include the enzymes that catalyze the oxidation of aldehydes. Aldehyde dehydrogenases convert aldehydes into carboxylic acids. Commonly, the same enzymes catalyze the reverse reaction.

[0050] As used herein, the term “aldehyde / alcohol dehydrogenase” or “AAD” refers to the enzymes that catalyze the conversion of butyryl-CoA, butyraldehyde, acetaldehyde, or acetyl-CoA to butanol or ethanol. Aldehyde / alcohol dehydrogenases play crucial roles for the production of ethanol and butanol. These enzymes can consist of an aldehyde dehydrogenase domain and an alcohol dehydrogenase domain. In some embodiments, an aldehyde / alcohol dehydrogenase adhE2 enzyme is overexpressed with a carboxylic acid reductase (CAR) enzyme. In some embodiments, the adhE2 is mutated to increase cofactor and / or butyraldehyde selectivity. In another embodiment, the adhE2 comprises an amino acid substitution selected from D485G, L488A, S601A, V608S, P619G, and combinations thereof, in SEQ ID NO: 1.

[0051] As used herein, the term “bifunctional” is intended to include enzymes that catalyze more than one biochemical reaction step. A specific example of a bifunctional enzyme is adhE2 which catalyzes both the alcohol dehydrogenase and aldehyde dehydrogenase reactions.

[0052] As used herein, the term “carboxylic acid reductase” or “CAR” refers to enzymes that catalyze the ATP and NADPH-dependent reduction of carboxylic acids to their corresponding aldehyde. In some embodiments, the carboxylic acid reductase (SEQ ID NO: 3) enzyme is derived from Mycobacterium marinum. In some embodiments, the CAR comprises SEQ ID NO: 3, or a sequence at least 90% identical to SEQ ID NO: 3. In additional embodiments, the CAR comprises a mutation that increases cofactor selectivity. In some embodiments, the CAR comprises an amino acid substitution selected from R507F, N508G, and combinations thereof in SEQ ID NO: 3.

[0053] As used herein, “phosphite dehydrogenase” or “PTDH” refers to an oxidoreductase enzyme that catalyzes the conversion of phosphite to phosphate using NAD+or NADP+as an electron acceptor. The reaction produces phosphate and reduced NADH or NADPH. PTDH exhibits high specificity for phosphite over other phosphorous-containing compounds. In some embodiments, PTDH is overexpressed to efficiently regenerate NADH and NADPH. In some embodiments, PTDH comprises SEQ ID NO: 5, or a sequence at least 90% identical to SEQ ID NO: 5. In some embodiments, the PTDH enzyme comprises a mutation that increases NADPH regeneration. In some embodiments, PTDH comprises an amino acid substitution selected from G175A and / or A176Rin SEQ ID NO: 5.

[0054] As used herein, “methyl viologen”, “MV”, or “l,l imethyl-4, 4’ -bipyridinium dichloride” refers to a redox-active compound used as an electron carrier. MV may be used as an electron acceptor or mediator to facilitate or enhance redox reactions. In fermentation systems, MV, plays a specialized role as an artificial electron carrier and shuttles electrons between metabolic enzymes and external electron acceptors. MV also plays a role in the flux of NAD+ / NADP+and NADH / NADPH, particularly in combination with PTDH. In some embodiments, methyl viologen is an additional component in the fermentation reaction. In some embodiments, MV is supplemented after 12 hours. In some embodiments, MV and butyrate are additional components in the fermentation reaction.

[0055] Engineered Clostridium tyrobutyricum cells for biosynthesizing butanol

[0056] The present disclosure provides variant carboxylic acid reductase (CAR) enzymes and variant aldehyde / alcohol dehydrogenase enzymes, as well as their nucleic acid and protein sequences. Further encompassed by the disclosure are recombinant host cells and cell cultures that include the variant CAR, PTDH, and adhE2 enzymes, as well as sfp genes and heterologous tRNAs, for the production of alcohols, specifically butanol. In order for the production of alcohols from fermentable sugars or biomass to be commercially viable, the process must be optimized for efficient conversion and recovery of product. The present disclosure addresses this need by providing compositions and methods for improved production of butanol using engineered variant enzymes and engineered recombinant host cells. The host cells serve as biocatalysts resulting in high-titer production and yields of butanol. In the present application, ideally, the butanol yield is equal to or greater than 0.23 g / g sugar.

[0057] Carboxylic acid reductase (CAR) enzymes play a key role in biobutanol production. However, CARs require activation by phosphopantetheinyl transferases (PPTase). PPTases are responsible for post-translational modifications of acyl carrier proteins. CARs contain an acyl carrier domain that requires post-translational modification to be active. In some embodiments, the sfp gene derived from B. subtilis is co-expressed with a CAR to post-translationally modify and activate said CAR. In some embodiments, the sfp gene comprises SEQ ID NO: 8 or a sequence at least 90% identical to SEQ ID NO: 8. Additionally, the mmCAR is not codon optimized for expression in C. tyrobutyricum. In some embodiments, an additional tRNA construct is expressed. In some embodiments, the additional tRNA construct comprises a sequence at least 90% identical to SEQ ID NO: 10.

[0058] Expression control sequences are known in the art and include, for example, promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), and the like, that provide for the expression of the polynucleotide sequence in a host cell. Expression control sequences interact specifically with cellular proteins involved in transcription (Maniatis et al., Science, 236: 1237-1245 (1987)). Exemplary expression control sequences are described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology, Vol.

[0059] 185, Academic Press, San Diego, Calif. (1990). In some embodiments, overexpression of adhE2, CAR, and / or PTDH is driven by a promoter endogenous to C. tyrobutyricum or C. acetobutylicum. In some embodiments, the promoter driving expression of adhE2, CAR, and / or PTDH is a THL, CAT1, or FLA promoter.

[0060] The terms “altered level of expression” and “modified level of expression” are used interchangeably and mean that a polynucleotide, polypeptide, or hydrocarbon is present in a different concentration in an engineered host cell as compared to its concentration in a corresponding wild-type cell under the same conditions. In one aspect, the level of expression of the C. acetobutylicum adhE2 gene in C. tyrobutyricum is altered. In additional embodiments the level of expression of CAR and / or PTDH is also altered.

[0061] Butanol is hydrophobic and less volatile than ethanol. 1-Butanol has an energy density closer to gasoline. Butanol at 85 percent strength can be used in cars without any change to the engine (unlike ethanol) and it produces more power than ethanol and almost as much power as gasoline. Butanol is also used as a solvent in chemical and textile processes, organic synthesis and as a chemical intermediate. Furthermore, butanol also is used as a component of hydraulic and brake fluids and as a base for perfumes. In one aspect, recombinant microorganism is engineered to produce more butanol than a control. In another aspect, a method of butanol biosynthesis is disclosed, the method comprising fermenting recombinant microorganism with a growth medium and a carbohydrate-containing source under conditions effective to obtain a butanol containing product.

[0062] The native producers of 1 -butanol, such as Clostridium acetobutylicum, also produce byproducts such as acetone, ethanol, and butyrate as fermentation products. However, these microorganisms are relatively difficult to manipulate. Genetic manipulation tools for these organisms are not as efficient as those for user-friendly hosts such as E. coli and physiology and their metabolic regulation are much less understood, prohibiting rapid progress towards high-efficiency production. Furthermore, no native microorganisms have been identified to produce from glucose other higher alcohols such as isobutanol, 2-methyl 1- butanol, 3-methyl 1-butanol, and 2-phenylethanol to industrially relevant quantities, despite the small amounts that have been identified.

[0063] Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular adhE2 sequence is disclosed and discussed and a number of modifications that can be made to a particular sequence. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A- E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

[0064] It is understood that as discussed herein the use of the terms homology and identity mean the same thing as similarity. Thus, for example, if the use of the word homology is used between two non-natural sequences it is understood that this is not necessarily indicating an evolutionary relationship between these two sequences, but rather is looking at the similarity or relatedness between their nucleic acid sequences. Many of the methods for determining homology between two evolutionarily related molecules are routinely applied to any two or more nucleic acids or proteins for the purpose of measuring sequence similarity regardless of whether they are evolutionarily related or not.

[0065] In general, it is understood that one way to define any known variants and derivatives or those that might arise, of the disclosed genes and proteins herein, is through defining the variants and derivatives in terms of homology to specific known sequences. This identity of particular sequences disclosed herein is also discussed elsewhere herein. In general, variants of genes and proteins herein disclosed typically have at least, about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent homology to the stated sequence or the native sequence. Those of skill in the art readily understand how to determine the homology of two proteins or nucleic acids, such as genes. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.

[0066] Another way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U. S. A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

[0067] It is understood that any of the methods typically can be used and that in certain instances the results of these various methods may differ, but the skilled artisan understands if identity is found with at least one of these methods, the sequences would be said to have the stated identity, and be disclosed herein. For example, as used herein, a sequence recited as having a particular percent homology to another sequence refers to sequences that have the recited homology as calculated by any one or more of the calculation methods described above. For example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using the Zuker calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by any of the other calculation methods. As another example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using both the Zuker calculation method and the Pearson and Lipman calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by the Smith and Waterman calculation method, the Needleman and Wunsch calculation method, the Jaeger calculation methods, or any of the other calculation methods. As yet another example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using each of calculation methods (although, in practice, the different calculation methods will often result in different calculated homology percentages).

[0068] As discussed herein there are numerous variants of the adhE2 (SEQ ID NO: 1) protein and CAR (SEQ ID NO: 3) protein that are known and herein contemplated. In addition, to the known functional adhE2 strain variants there are derivatives of the adhE2 protein which also function in the disclosed methods and compositions. Protein variants and derivatives are well understood to those of skill in the art and can involve amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional variants. Insertions include amino and / or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 2 to 6 residues are deleted at any one site within the protein molecule. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example M13 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically of single residues but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. The mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place.

[0069] As this specification discusses various proteins and protein sequences it is understood that the nucleic acids that can encode those protein sequences are also disclosed. This would include all degenerate sequences related to a specific protein sequence, i.e. all nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences. Thus, while each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed protein sequence.

[0070] Also disclosed herein is a method of biosynthesizing butanol, the method comprising fermenting the disclosed recombinant microorganism with growth medium and carbohydrate-containing source. In some embodiments the growth medium is clostridial growth medium. In another embodiment, the carbohydrate-containing source is a sugar selected from glucose, xylose, mannitol, and combinations thereof. In some embodiments, the growth medium and carbohydrate-containing source are supplemented with at least one additional organic compound. In some embodiments, the at least one additional organic compound methyl viologen and / or butyrate. In a further embodiment, the MV is supplemented after 12 hours. In another embodiment, the butyrate is supplemented after 24 hours.

[0071] Also disclosed herein is a system useful for the biosynthesis of butanol, comprising a container suitable for fermentation, the disclosed recombinant microorganism, growth medium, and a carbohydrate containing source. In some embodiments, the system further comprises methyl viologen and butyrate.

[0072] The details of one or more embodiments of the invention are set forth in the accompanying drawings and description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0073] EXAMPLES

[0074] 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 the compounds, compositions, articles, devices, and / or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. 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 otherwise indicated, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

[0075] Example 1

[0076] Bioinformatic protein sequence database search methods and ligand / enzyme complex modeling via substrate docking studies were used to identify new aldehyde-alcohol dehydrogenase (AAD) and aldehyde ferredoxin-oxidoreductase (AOR) enzymes. Clostridium acetobutylicum adhEZ AAD enzyme (SEQ ID NO: 1) was mutated to increase butanol selectivity.

[0077] An in-silico mutation study was performed on the adhEZ enzyme to identify point mutations to the alcohol dehydrogenase domain that result in increased butanol selectivity. Ideal mutations allow for increased butyraldehyde affinity, decrease acetaldehyde affinity, while maintaining affinity to NADH cofactor.

[0078] Following the in-silico mutation study, in-vivo studies were used to determine the efficacy of various point mutations in adhEZ by transforming Clostridium tyrobutyricum with the p82151 plasmid containing the CAT promoter (SEQ ID NO: 9) for overexpressing various adhEZ mutants. The mutations were tested in vivo in serum bottle fermentations. Fermentations were done in Clostridial Growth Media (CGM) supplemented with 50 g / L glucose, for a duration of 144 hours. The candidates identified from the genetic enzyme ortholog (GEO) search were also evaluated in vivo with serum bottle fermentation. Initial and final fermentation samples were taken and analyzed.

[0079] An in-silico mutation study on the Clostridium acetobutylicum adhEZ enzyme was performed to determine which point mutations to the alcohol dehydrogenase would increase selectivity of cofactor NADPH. Again, the best mutations from were evaluated in-vivo in serum bottle fermentations.

[0080] All but one of the adhEZ mutants, P619I, resulted in increased butanol yield. 2,3 -butanediol (2,3-BDO) was also produced in the fermentation assays. 2,3-BDO production was confirmed by HPLC.

[0081] Example 2

[0082] To confirm the results of the small scale serum bottle fermentations, fermentation was performed in a 1 liter bioreactor using strain adhEZ S601A / V608S / P619G. The large scale fermentation resulted in very high acetate titers. However, these high acetate titers are likely due to the high control pH.

[0083] Additionally, Clostridium tyrobutyricum was transformed with the p82151 plasmid which contains the CAT promoter to overexpress adhEl and different carboxylic acid reductase (CAR) genes. A plasmid co-expressing adhEl and a CAR gene from N iowensis (known as niCAR) was designed. Fermentation assays with this plasmid reduced acetate production. Additional CAR genes of interest include mmCAR (SEQ ID NO: 3) and miCAR from M. marinum and M. immunogenum, respectively.

[0084] Example 3

[0085] Background:

[0086] C. tyrobutyricum was engineered to produce n-butanol by introducing a heterologous aldehyde-alcohol dehydrogenase (AAD) encoded by the adhEl gene from C. acetobutylicum. However, butanol biosynthesis in the engineered C. tyrobutyricum strain was limited by the available NADH cofactor pool and the co-production of butyrate. Additionally, the adhEl enzyme is non-selective in that it produces both ethanol and butanol from acetaldehyde and butyraldehyde substrates, respectively. C. tyrobutyricum wa further engineered to express phosphite dehydrogenase (PTDH) (SEQ ID NO: 5) for NADH cofactor regeneration and carboxylic acid reductase (CAR) (SEQ ID NO: 3) for butyrate reduction to butyraldehyde, which can be further reduced to butanol by AAD.

[0087] Fermentation assays showed improvements in butanol production by the single point mutant adhE'l P619G and triple-point mutant adhEl S601A / V608S / P619G compared to the wild type control. F ermentation with the mutant plasmid pMTL82151 -pC l adhEl P619G resulted in a butanol titer of 8.68 ± 0.13 g / liter, an increase in butanol titer of 197% when compared to the control pMTL82151-pCAT_adhE2.

[0088] Finally, the combined effects of the mutant AAD enzyme and the THL promoter. C. tyrobutyricum strains containing adhEl driven by the THL promoter have shown higher butanol titers compared to adhEl strains driven by the CAT1 promoter. Combining mutant adhEl with a stronger promoter resulted in higher butanol titer and yield. Fermentation with the mutant plasmid pMTL82151 -pTHL_at / AE2 P619G resulted in a butanol titer of ll.57± 0.15 g / liter, an increase in butanol titer of 170% when compared to the control pMTL82151-pTHL_adhE2.

[0089] Carboxylic Acid Reduction:

[0090] Additionally, Clostridium tyrobutyricum was transformed with the pMTL82151 plasmid containing the CAT promoter for co-overexpressing adhEl and various carboxylic acid reductase (CAR) genes. The most successful CAR enzyme for reassimilating butyrate was the CAR gene from M. marinum (known as mmCAR) (SEQ ID NO: 3). Due to the size of the mmCAR gene, this gene was expressed on the pMTL82151 plasmid along with the sfp gene (SEQ ID NO: 8) from B. subtilis, which is required for post-translational modification of the CAR gene and chloramphenicol resistance marker. adhEZ (SEQ ID NO: 2) was co-expressed from another plasmid containing the FLA promoter, and an erythromycin resistance marker. Both plasmids were conjugated into the C. tyrobutyricum wild-type strain. Additionally, the mmCAR gene was originally codon-optimized for E. coli, so 5 additional tRNAS (SEQ ID NO: 10), 3 of which are native to C. cellulolyticum, were expressed to avoid codon-bias in protein expression.

[0091] The mutant strain expressing both mmCAR and adhEZ was compared to the control which contained only the PMTL82151-pFLA_at / AE2 plasmid. Fermentation with the mutant strain containing the plasmid pMTL82151-pCAT_tRNA_mmCAR_sfp resulted in a butanol titer of 10.74 ± 1.45 g / liter and a yield of 0.20 ± 0.02 g / g glucose, an increase in butanol titer of 35% and an increase in butanol yield of 43% when compared to the control pMTL82151-pFLA_at / AE2. Finally, the ability of the mmCAR mutant to reassimilate butyrate was evaluated by adding approximately 6 g / liter butyrate to the serum bottle at the 24-hour time mark. Fermentation resulted in a butanol titer of 11.36 ± 0.33 g / liter and a yield of 0.24 ± 0.01 g / g glucose. Additionally, the final butyrate concentration of this fermentation was 6.62 ± 0.06 g / liter, indicating that little butyrate was produced.

[0092] Phosphite Dehydrogenase for Cofactor Regeneration

[0093] Another strategy for increasing butanol production in C. tyrobutyricum was increasing the cofactor pool. Production of butanol requires NADH, and it has been shown that NADH is a limiting factor in butanol production in C. acetobutylicum. Supplementation of NADH is impractical due to the cost prohibitive nature of NADH, and instead cofactor regeneration is the optimal route. Several routes of NAD(P)H regeneration have been investigated, but overexpression of phosphite dehydrogenase (PTDH) (SEQ ID NO: 5) in C. tyrobutyricum has never been attempted.

[0094] PTDH oxidizes inorganic phosphite (hydrogen phosphonate) to phosphate, transforming NAD+ to NADH in the process. The PTDH gene originating from Pseudomonas stutzeri WW88 was codon optimized for C. tyrobutyricum and co-overexpressed with adhEZ, driven by the CAT1 promoter. Additionally, PTDH was mutated at G175A and A176R, two mutations that have been shown to greatly increase activity for both NAD+ and NADP+ substrates.

[0095] Fermentation for the PTDH mutant with adhEZ was first performed in a non-buffered fermentation media, as the preferred buffer containing calcium carbonate may cause phosphite to precipitate. Fermentation of the PTDH mutant with adhEZ was compared to the fermentation of the control, WT pCAT adhEZ. For both the control and the mutant, 30 mM of sodium phosphite was added to the fermentation medium. The fermentation for the mutant PTDH with adhE2 (pCAT_at / AE2_PTDH) produced 5.48 ± 0.15 g / liter butanol. This titer of butanol is even higher than that of the WT_pCAT_at / AE2 buffered fermentation, which results in 2.92 ± 0.19 g / liter butanol. Fermentation for the mutant PTDH was performed in buffered fermentation media. Fermentation with the mutant strain containing the plasmid pMTL82151 pCAT_at / AE2_PTDH resulted in a butanol titer of 13.10 ± 0.09 g / liter and a yield of 0.26 ± 0.01 g / g glucose. Fermentation of the PTDH mutant with adhEZ resulted in no C2 products, no acetate, no ethanol. This mutant successfully produced butanol, additionally, this success was achieved with the wild-type adhEZ enzyme driven by a relatively weaker promoter, pCATl.

[0096] Summary

[0097] Engineering of the adhEZ enzyme resulted in increased butanol titer by 170% when compared to the control pMTL82151 pTHL adhEZ. By overexpressing the CAR enzyme, the butanol titer was increased by 35% and the butanol yield was increased by 43% when compared to the control pMTL82151 pFLA adhEZ. Furthermore, results indicated that butanol titers can be increased by butyrate supplementation. Finally, fermentation overexpressing the PTDH with sodium phosphite supplementation resulted in a butanol titer of 13.10 ± 0.09 g / liter and a yield of 0.26 ± 0.01 g / g glucose, with no C2 products.

[0098] Example 4: In-Silico Analysis and Engineering of an Aldehyde / Alcohol Dehydrogenase for Alternative Cofactor Utilization and Selective Butanol Production

[0099] Biobutanol production by solventogenic Clostridia is limited by a low butanol titer and yield. To overcome this limitation, Clostridium tyrobutyricum was engineered to overexpress the adhEZ (SEQ ID NO: 2) gene encoding a bifunctional aldehyde / alcohol dehydrogenase (AAD) for converting acetyl-CoA / butyryl-CoA to acetaldehyde / butyraldehyde and then to ethanol / butanol. The goal was to increase butanol biosynthesis in C. tyrobutyricum by engineering AAD targeting of amino acid residues in the enzyme catalytic center that could increase butanol: ethanol ratios and alter cofactor specificity. In silico mutagenesis and analysis via Rosetta analysis showed that several AAD point mutations increased butanol production and selectivity over ethanol. C. tyrobutyricum strains overexpressing various AAD mutants were then created. Two AAD mutants, D485G and L488A, engineered to utilize NADPH as the cofactor, increased butanol production by over 100% in batch fermentation, with yields of 0.10-0.13 g / g (vs 0.05 g / g glucose for the wildtype AAD). Two additional AAD mutants, P619G and S601A V608S P619G, engineered for increased butanol selectivity, also gave higher butanol yields of 0.13-0.15 g / g. Butanol production further increased to 0.23 g / g when methyl viologen was added to the fermentation. In silico analysis was leveraged to guide rational engineering of AAD with higher selectivity and activity for butanol production.

[0100] Introduction

[0101] Climate change, driven by the burning of fossil fuels, is the largest existential crisis facing humanity, and new sustainable solutions must be discovered and implemented to address this challenge. Butanol produced via fermentation, i.e., biobutanol, is not a new process, but it is seeing renewed interest. Biobutanol is a potential alternative to gasoline as a fuel for internal combustion engines, and it has many advantages over the established biofuel ethanol. Butanol is less hygroscopic than ethanol and has a higher energy density than ethanol, two factors among many that make it a superior biofuel. Butanol is currently produced from petroleum processes, where it serves as a platform chemical to produce butyl acrylate and methacrylate esters. Butanol is naturally produced in solventogenic Clostridia via the acetone-butanol-ethanol (ABE) fermentation. The strain most widely adopted and studied for butanol production is Clostridium acetobutylicum, which typically yields acetone, butanol, and ethanol with a weight concentration ratio of 3:6:1. The coproduction of butanol and acetone, combined with low overall titers, makes the recovery cost of traditional butanol production commercially more expensive. To improve fuel production, the metabolic network has been engineered to produce butanol through the hot channel, i.e., directly from butyryl-CoA; however, these modifications were unable to eliminate ethanol and acetate production.

[0102] One alternative to the ABE fermentation for biobutanol production is investigating the potential of engineering the hyper butyrate-producing Clostridium tyrobutyricum, which can reassimilate acetate for butyrate biosynthesis via a butyryl-CoA / acetate CoA transferase. C. tyrobutyricum is also capable of tolerating butanol at high concentrations, greater than 15 g / L. These two factors make C. tyrobutyricum a promising microbial cell factory for butanol production. C. tyrobutyricum can be made to produce n-butanol by overexpressing a heterologous alcohol / aldehyde dehydrogenase (AAD) such as the one encoded by the adh Z (SEQ ID NO: 2) gene from C. acetobutylicum. It was shown that the engineered C. tyrobutyricum with a high metabolic flux toward butyryl-CoA-produced n-butanol from glucose at a high titer (>20 g / L) and yield (>0.20 g / g) in batch fermentation. High alcohol titers and no acetone production simplify downstream butanol processing for this Clostridium species.

[0103] However, metabolic engineering alone has not been able to address all of the needs for economically viable biobutanol production. In the case of engineered C. tyrobutyricum, butanol production in C. tyrobutyricum is made possible by over-expressing adhE2. The AAD enzyme encoded by adhE2 (SEQ ID NO: 2) possesses two domains, an aldehyde dehydrogenase (ALDH) and an alcohol dehydrogenase (ADH), where the ALDH domain catalyzes the conversion of butyryl-coenzyme A (butyryl-CoA) to butyraldehyde and the ADH domain catalyzes the conversion of butyraldehyde to butanol. In its native C. acelobulyUcum. the adhE2 gene is expressed under alcohol ogenic conditions for butanol production. Although metabolic engineering strategies have been able to make enhancements to butanol, it has been difficult to eliminate ethanol production entirely since AAD also converts acetyl-CoA to ethanol. Researchers have shown that it is possible to increase butanol selectivity by introducing point mutations into an AAD enzyme encoded by adhE \, which is highly similar to at / AE2-AAD. Additionally, introducing three mutations into the substrate binding site of the Zymomonas mobdiz ADH2 enzyme also significantly increased the enzyme activity for butanol biosynthesis.

[0104] Another potential route for increasing butanol biosynthesis is by engineering cofactor promiscuity into the AAD enzyme. Research has shown that a potential limiting factor to butanol production is the limited pool of an NADH cofactor. It was discovered that two separate AADs had separate cofactor activities, and when a point mutation was made to mimic the AAD specific to NADPH, the butanol titer from fermentation increased by over 50%. Therefore, engineering the AAD enzyme to be able to use both cofactors NADH and NADPH may potentially allow the enzyme to turnover more butyraldehyde to produce more butanol.

[0105] Finally, in silico screening strategies have been employed to aid in the design of more selective or active enzymes. One application that has found much success in enzyme design is the design protocol implemented in the Rosetta macromolecular modeling suite. Enzymes engage in interactions that stabilize the transient state, which in turn accelerate reactions. This principle was used as the basis for design in a particular Rosetta enzyme design protocol, where enzyme residues were optimized for transient or transition-state binding affinity and catalysis. The Rosetta protocol has been successful in de novo enzyme design, designing enzymes for use on alternative substrates and redesigning enzymes for alternative applications. For example, guided by the Rosetta macromolecular modeling suite, the specificity of ketoisovalerate decarboxylase enzyme was able to be reprogrammed for long-chain fatty acids, resulting in an over 100-fold increase in specificity for the targeted reaction. In another example, an NMN+-recycling phosphite dehydrogenase (PTDH) was engineered with a two-stage approach: first, Rosetta was used to rationally design amino acid residues directly contacting NMN+, and second, site-saturation mutagenesis was applied to target sites distant from NMN+within the cofactor-binding pocket. Using this approach paired with high-throughput screening with the Gor Ortho growth selection platform, allowed for the development of a PTDH to cycle NMN+with ~ 147-fold improved catalytic efficiency, thus demonstrating the capability of utilizing Rosetta to design enzymes for the use of an alternative cofactor.

[0106] Inspired by these successful attempts at enzyme engineering, it was investigated whether it is possible to engineer the / rE -encoded AAD to achieve higher butanol selectivity and increased activity on the alternative cofactor NADPH. First potential AAD mutants were screened in silico using RosettaDesign. Then the fermentation performance of C. tyrobutyricum transformed with promising adh Z mutants was evaluated via serum bottle fermentation and an enzymatic assay.

[0107] Results

[0108] In Silico Screening for Beneficial Mutations to AAD.

[0109] The Rosetta macromolecular modeling suite was used to aid in determining which mutations may be beneficial to increasing the enzyme selectivity. Alphafold2 was used to generate a protein model for AAD, and the enzymatic constraints were lifted from a crystallized Bacillus stearothermophilus glycerol dehydrogenase, where the coordinates of the bound substrates were derived from the glycerol position in the glycerol dehydrogenase. However, for the sake of simplicity, the substrates, either acetaldehyde or butyraldehyde, and not the reaction intermediates, were placed in the active site.

[0110] Designing for selectivity was complicated due to the multistate design nature of the problem. It is optimal to design the enzyme to enhance the binding of the substrate butyraldehyde and reduce the binding of acetaldehyde, all the while maintaining or enhancing the binding of the cofactor, whether that be NADH or NADPH. The discovery of mutations that can enhance the binding of butyraldehyde may also enhance the binding of acetaldehyde, which is undesired. Likewise, a mutation that may reduce the level of binding of acetaldehyde may reduce butyraldehyde binding as well. Typically, these situations are managed using a multistate design approach, where design is performed with all substrates considered. Unfortunately, there is currently no open-source, multistate enzyme design application within Rosetta. Therefore, the strategy employed involved single-state design for both substrates, butyraldehyde and acetaldehyde, and then identifying the top mutations for butyraldehyde binding that were not shared with enhanced acetaldehyde binding. These results were visualized by generating sequence similarity logos from the sequences of the designed AAD mutants (Fig. 1A). A similar approach was used to find the altered sequences for AAD using an alternative cofactor NADPH (Fig. IB). Fig. 1C shows sequence logo for docking acetaldehyde and NAD+. From these sequence similarity logos, mutated residues that were present in the AAD mutants designed for butyraldehyde binding that were not present for acetaldehyde binding were easily detected. From performing these design simulations, various mutations were identified that were then evaluated in the wet lab.

[0111] Increasing Butanol Selectivity of Mutant AAD in C. tyrobutyricum.

[0112] Various point mutations were made to the adh Z gene in the pCAT- / rE plasmid. These plasmids were transformed to the C. tyrobutyricum wild-type strain. The various plasmids and strains constructed and used are given in Table 1. The mutants were then evaluated in batch fermentations with CGM containing ~60 g / L glucose.

[0113] Fig. 2 shows the serum-bottle-batch fermentation results for the tested mutants. Except for Ct_pC_P619I, all strains overexpressing engineered AAD produced more butanol when compared to the control, Ct_pC_WT. A typical batch fermentation of strain Ct_pC_WT resulted in the production of 2.92 ± 0.19 g / L of butanol and 0.62 ± 0.01 g / L of ethanol, in addition to 1.75 ± 0.18 g / L and 16.57 ± 0.07 g / L of acetic and butyric acids, respectively. Ct_pC_P619G and Ct_pC_ASG produced much more butanol at 7.79 ± 0.67 g / L and 8.68 ± 0.13 g / L, increases of 167% and 197%, respectively, compared to Ct_pC_WT. With strain Ct_pC_P619G, the butanol yield (butanol produced / glucose consumed) and selectivity (butanol / all products) increased~2-fold to 0.15 ± 0.01 g / g and 0.38 ± 0.01 g / g, respectively (vs 0.05 ± 0.01 and 0.13 ± 0.01 for the Ct_pC_WT). Meanwhile, the butanol / ethanol ratio also increased to 9.08 ± 0.14 (vs 4.68 ± 0.31 for Ct_pC_WT) and alcohol / acid ratio increased to 0.75 ± 0.37 (vs 0.19 ± 0.04 for Ct_pC_WT). Clearly, the P619G mutation on the AAD increased its alcohol dehydrogenase activity as well as selectivity over ethanol and acids. AAD with S601A and V608S point mutations also showed significant effects on increasing butanol yield and selectivity; however, AAD with combined P619G, S601A, and V608S point mutations did not show any further impacts on increasing butanol production, which was likely due to limitation in available NADH required for butanol biosynthesis, as evidenced by the much lowered C4 / C2 ratios for Ct_pC_P619G and Ct_pC_ASG compared to Ct_pC_WT. Engineering Alternative Cofactor Utilization of AAD

[0114] Since butanol biosynthesis in Ct_pC_WT is limited by the NADH cofactor available for AAD-catalyzed reactions, changing AAD’s cofactor to use NADPH, which is more abundant than NADH in Clostridia, can increase butanol production by C. tyrobutyricum expressing an AAD capable of using both NADH and NADPH as cofactors. Additional mutant strains were thus created with the intention of engineering increased utilization of the alternative NADPH cofactor. Strains Ct_pC_D485G and Ct_pC_L488A were created; fermentations for the Ct_pC_D485G strain resulted in the production of 4.10 ± 0.64 g / L butanol and 0.43 ± 0.03 g / L ethanol, giving a butanol yield of 0.10 g / g and butanol: ethanol ratio of 9.7 ± 3.1. Even further improvements were achieved by the Ct_pC_L488A strain, resulting in a titer of 6.56 ± 0.03 g / L, a butanol yield of 0.125 ± 0.002 g / g glucose, and a butanol: ethanol ratio of 6.0 ± 0.7. The selectivity for these two mutants also increased to 0.28-0.31 ± 0.03 (vs 0.13 ± 0.01 for Ct_pC_WT). These results showed D485G and L488A point mutations increased butanol production by over 100% and confirmed that allowing AAD to use NADPH as a cofactor increases butanol biosynthesis in the engineered C. tyrobutyricum.

[0115] Effect of Methyl Viologen on Fermentation Kinetics

[0116] Methyl viologen (MV) at 50 pM was added to serum bottle fermentations at 12 h to determine the effect of additional reducing potential on the fermentation kinetics of the AAD mutant strains. Supplementing the fermentation with MV can divert the electrons from hydrogen generation to NADH regeneration. The effects of the MV addition can be seen in Figs. 3 A-3F. The greatest effect was seen in the Ct_pC_WT, where the butyrate concentration reduced by approximately a third and the butanol production more than doubled. Beneficial effects of MV addition were also seen in the Ct_pC_P619G fermentation, resulting in a significant reduction in acetic and butyric acids, and a major increase in the butanol titer, from 8.68 ± 0.13 g / L to 12.74 ± 0.73 g / L. Additionally, beneficial effects were seen in the Ct_pC_ASG fermentation, where the butanol titer increased from 7.79 to 11.01 g / L. However, high butyrate production in this strain limited the butanol yield, where Ct_pC_ASG with MV and Ct_pC_P619G with MV yields were similar at 0.21 and 0.23 g / g glucose, respectively. These results confirmed that AAD with P619G, S601 A, and V608S point mutations was also beneficial to butanol biosynthesis when more NADH was available with MV addition.

[0117] Enzyme Activity Assays

[0118] Enzyme activity assays were performed to confirm that the increase in butanol production (titer, yield, and selectivity) from the mutants was in fact due to increased enzyme activity. These enzyme activity assay results are given in Table 2. Ct_pC_ASG had the highest activities for both butyraldehyde and acetaldehyde, with an average specific activity of 2605.7 ± 54.1 and 4286.8 ± 579.8 mU / mg, respectively, which were over 60-fold higher than those found in Ct_pC_WT (37.95 ± 7.25 mU / mg for butyraldehyde and 34.91 ± 6.25 mU / mg for acetaldehyde). Although Ct_pC_ASG’s activities were also much higher than those for Ct_pC_P619G, the butanol production performance in batch fermentation was comparable to that of Ct_pC_P619G. The lack of further improvement in butanol production with much increased alcohol dehydrogenase activities in Ct_pC_ASG suggested that butanol biosynthesis may be limited by the flux from acetyl-CoA to butyryl-CoA. Interestingly, the AAD mutant P619I was routinely predicted to be a high butanol selective enzyme from the in silico experiments, but both serum bottle fermentations and enzyme activity assays showed this to not be the case. The strain Ct_pC_P619I gave less butanol production in serum bottle fermentations, and in the enzyme assays, the mutant showed higher activity for acetaldehyde than it did for butyraldehyde, which might be the reason for lower butanol production.

[0119] The AAD mutants designed for NADPH utilization, mutants Ct_pC_D485G and Ct_pC_L488A, were also evaluated. For both mutants, an increased activity for the combination of butyraldehyde and the natural cofactor, NADH, was observed when compared to the wild-type enzyme, with Ct_pC_D485G and Ct_pC_L488A having activities of 63.2 ± 1.5 and 70.2 ± 0.6 mU / mg, respectively, whereas the Ct_pC_WT control strain had an enzyme activity of 37.9 ± 7.2 mU / mg. The AAD is a strictly NADH-dependent enzyme, but because the enzyme assays were performed using a whole cell lysate, there was a high amount of background activity consuming NADPH even in the control. Therefore, when these mutants are evaluated, it is sensible to compare the mutants to the control. Increased activity for combination of butyraldehyde and the alternative cofactor, NADPH, when compared to the control, was observed, with Ct_pC_D485G and Ct_pC_L488A having activities of 113.5 ± 1.8 and 222.0 ± 1.7 mU / mg, respectively, whereas the control had an activity of 81.9 ± 2.1 mU / mg. The increased enzyme activity with the NADPH cofactor contributed to the increased butanol production, especially for Ct_pC_L488A (Fig. 4B) with a much higher NADPH enzyme activity compared to the wild-type (Fig. 3A) and Ct_pC-D485G (Fig. 4A) because more reducing equivalents were available, resulting in decreased butyric and acetic acid production and increased butanol production.

[0120] Model Investigation for the Determination of Increased Butanol Selectivity

[0121] Visual inspection of the models generated from Rosetta facilitated the inference of the mutational effects on butanol selectivity. Models of the enzyme active site are given in Figs. 5A-5D. These images allow for many deductions. First, an increase in the size of the cofactor-binding chamber is clearly obvious when comparing Figs. 5A and 5C. This is due to the proline to glycine mutation at the residue position 619. Additionally, Fig. 5C clearly shows a hydrogen bond between the oxygen on the NADH cofactor and the hydrogen of the serine molecule at the amino acid position 608, and this hydrogen bond is absent in the wild-type. This hydrogen bond helps stabilize the cofactor in the binding chamber. Finally, size differences of the active site are clearly visible when comparing Figs. 5B and 5D. The mutation of the amino acid at position 601 from a serine residue to an alanine residue increased the size of the binding chamber (9.8 A vs 9.5 A) and allowed the substrate butyraldehyde to occupy beneficial conformations that were not possible otherwise. Further evaluation of the in silico mutation studies provides additional insights as to why specific mutations are beneficial. A helpful analysis tool in Rosetta is the residue energy breakdown function, which can decompose per residue score into inherent residue energies (one-body scores) and residue interaction scores. This breakdown demonstrates how mutating one residue may affect another residue and how the affected residue may in turn affect other residues. Using this function, the residue scores for two different models, mutant Ct_pC_ASG complexed with butyraldehyde and NAD+, and the wild-type AAD complexed with butyraldehyde and NAD+were decomposed (Table 3). Table 3 indicates that for both S601 A and P619G, there wasw a decrease in fa sol for the residue-NAD+interaction, meaning that the mutation resulted in a reduction in the desolvation energy between the residue and the NAD+cofactor. A reduction in the desolvation energy between the 601 residue and the butyraldehyde substrate was also observed due to the S601 A mutation. These insights indicate that a major factor in the increase in butyraldehyde activity from these mutations was due to the reduction in the desolvation energy in the active site. This agrees with past research that found butanol selectivity for an AAD was increased by increasing hydrophobicity of the binding site. However, a significant increase in the desolvation energy between the wild-type AAD complexed with NAD+and the V608S mutant complex with NAD+was observed. Although there was a significant increase in the desolvation energy, the mutation from valine to serine for the 608 residue results in a reduction in energy for the hbond sc and fa rep between the 601 residue and the NAD+cofactor, indicating a reduction in the repulsive van der Waals force and favorable hydrogen bonding between the two components, resulting in a net decrease in the energy of 1.53.

[0122] Model Investigation for the Determination of Increased NADPH Usage

[0123] Again, insights into the benefits of the D485G and L488A mutations as they pertain to NADPH cofactor utilization were gained by investigating the models generated by Rosetta. Images of these models are shown in Figs. 6A-6D. For the mutant D485G, there is no longer an interaction between the carboxylic end of the aspartate and the carboxylic end of the NADPH cofactor (Figs.

[0124] 6A-6B). However, the reasoning for the increased enzyme activity for the L488A mutants is not readily apparent from the models generated by Rosetta (Figs. 6C-6D). Again, information provided from Rosetta’s residue energy breakdown provides key insights (Table 3). By mutating lysine to alanine at the 488 position, the repulsive energy between the amino acid at this position and the tyrosine at the position at 493 was greatly reduced. Therefore, the mutation at the 488 position does not directly affect interaction with the NADPH cofactor; instead, it greatly reduces steric hindrance with a neighboring residue. Combined Effect of the Stronger Promoter and Mutant AAD.

[0125] The adhEl (SEQ ID NO: 2) gene was driven by the call (SEQ ID NO: 9) promoter, so that any effect of the mutations could be easily observed. The mutations resulted in significant increases in the butanol titer and yield, but increases were still comparable to only that of the wild-type adhEl driven by the thiolase promoter, pTHL- / vE. The single point mutant AAD-P619G was then over-expressed, driven by the thiolase promoter to determine if even further increases in thiolase butanol titer and yield are achievable by incorporating the stronger promoter. This was in fact the case; strain Ct_pT_P619G achieved a butanol titer of 11.57 ± 0.15 g / L and a yield of 0.20 ± 0.002 g / g glucose, compared to a butanol titer of 6.78 ± 0.06 g / L and a yield of 0.13 ± 0.001 g / g glucose achieved by the control Ct_pT_WT (Figs. 7A-7D, Table 1). A key observation when comparing Ct_pT_P619G to strain Ct_pT_WT was the acetate to butyrate ratios. For the Ct_pT_WT strain, the acetate: butyrate ratio was 0.45, whereas for the Ct_pT_P619G strain, the acetate: butyrate ratio was 1.36. Increasing the acetate: butyrate ratio for increasing AAD alcohol dehydrogenase activity is also observable for mutants driven by the catl promoter. It is possible that the exhaustion of the C4 flux being converted to butanol leads to accumulation of CoA-SH, which in turn inhibits the thiolase enzyme and prevents further flux toward C4 products. This is consistent with the finding that butanol biosynthesis might become limited by the flux from acetyl-CoA to butyryl-CoA when alcohol dehydrogenase activities were increased tremendously.

[0126] Discussion

[0127] C. tyrobutyricum butanol production is due to the overexpression of the heterologous adhE encoding a bidomain aldehyde / alcohol dehydrogenase. Overexpression of adhEl in C. tyrobutyricum, resulted in 20.5 g / L of n-butanol with a high yield of 0.33 g / g using mannitol as the substrate. However, this strain suffered from excessive amounts of acetate and butyrate production. A metabolic engineering approach was then applied to try to address this problem. C. tyrobutyricum Acall::adhEl was created by knocking out call -encoding butyryl -Co A / acetate CoA transferase with adhE'l insertion on the genome, and the strain was able to produce 26.2 g / L n-butanol with a yield of 0.23 g / g. Although butyrate production was nearly eliminated in this strain, considerable amounts of acetate and ethanol persisted. The present application did not knockout the cal but the observed acetate production was also high. Therefore, the high amount of acetate production might be attributable to limited NADH availability.

[0128] The present application addresses these two issues, ethanol coproduction and limited cofactor availability, by engineering AAD to selectively produce butanol and / or to have high activity for the NADPH cofactor. These mutants were discovered by performing a docking study in the Rosetta macromolecular modeling suite, where amino acids near the active site were allowed to mutate to reduce the energy of the enzyme-substrate complex. This docking study proved valuable, discovering two point-mutations to the adhEZ gene, both of which significantly improved the titer and yield when compared to the strain overexpressing the wild-type. Additionally, the in silico studies discovered two AAD point mutations that enabled NADPH utilization. In the present application, the substrate (either acetaldehyde for the reduction reaction resulting in the production of ethanol, or butyraldehyde for the reduction reaction resulting in the production of butanol) and the cofactor (either NADH or NADPH) were docked. But by docking a reaction intermediate that more closely resemble the transient state, mutations that would lower the energy of the transientstate complex even further could be predicted, resulting in even higher butanol-producing mutants.

[0129] To better understand the underlying principles behind the increased butanol production for the AAD mutants, the Rosetta docking data using the residue energy breakdown function was utilized. This showed that beneficial mutations would result in changing an associated amino acid that would reduce the desolvation energy between the amino acid and the substrate, supporting the assessment that beneficial mutations are those that increase hydrophobicity in the active site. Further improvements to the butanol titer and yield produced by the mutant strains could potentially be made by investigating the effect of combining the mutant adhEZ genes with different promoters. Other promoters such as the fl a and thl promoters outperform the cat\ promoter, resulting in increased butanol yields. It has also been demonstrated that adhEZ is driven by the inducible lac promoter.

[0130] Additionally, the ability to engineer the ADH domain of the AAD enzyme to utilize the alternative cofactor NADPH was demonstrated. The AAD is a strictly NADH-dependent enzyme, and research has shown that in similar Clostridia, the NADPH pool is 70-fold that of the NADH pool. Being able to utilize this great driving force to produce more butanol can be a great asset. For this reason, simulations were performed and resulted in the generation of AAD point mutants D485G and L488A. By inspecting the models generated by Rosetta and analyzing the breakdown in residue energies, the likely causes for the increase in enzyme activity of these two NADPH-utilizing AAD mutants was determined. It should be noted that for the enzyme assays performed, there still appeared to be a high amount of activity for the NADPH cofactor even for the wild-type enzyme. This was likely due to background effects, because of the use of the whole cell lysate. In future work, it would be prudent to rerun these assays using purified enzymes to verify that the mutations D485G and L488A have indeed changed the AAD cofactor dependency from NADH for the wild-type to NADPH. It is important to note how the results of the present application compare to those of similar studies. There were two particular AAD mutation studies of interest. Cho et al. (2019) engineered the AAD encoded by the / iEI to increase selectivity for butanol production in C. acetobutylicum M5. Site-saturation mutagenesis was performed on several sites in the / vEI gene, where it was discovered that the mutation to the 619-residue position from methionine to glycine increased the butanol yield by 20%. In this case, the at / Z / E I genes were driven by the native C. acetobutylicum thiolase promoter. The in silico design study of the present application showed that mutating the 619 position to glycine resulted in some of the lowest substrate-binding scores. However, the mutation was infrequently sampled and, therefore, cannot be visualized in the sequence logo. So, based on the in silico design experiments and inspired by the work performed by Cho et al., a mutant containing the 619-glycine mutation was evaluated. Changing the 619-residue from proline to glycine in AAD encoded by adhE2 driven by the native thiolase promoter gave a 54% increase in butanol production. As for the mutations intended to utilize an alternative cofactor NADPH, the results of the present application are compared to the results from experiments performed by Jang et al. (2012), who studied butanol production by C. acetobutylicum and achieved a 4% increase in the butanol yield by utilizing a mutated / rE I gene possessing a D485G mutation. The same mutation, but to the adhE2 gene resulted in a 100% increase in the butanol yield, and a 160% increase in the butanol yield was realized with the L488A mutation. However, comparing the results from Jang et al. (2012) to the results of the present study is not an equivalent comparison because the control strain in Jang et al. was already a highly proficient butanol producer. This comparison though does show the promise of utilizing in silico experiments to predict beneficial mutations. Table 6 summarizes the comparison of the present application with that of Jang et al.

[0131] The approach of the present application engineered for increased butyraldehyde selectivity and NADPH utilization. For increasing selectivity, the present application had many differences to Cho et al. (2019). Predominantly, the approach of the present application is more high-throughput than that of Cho et al. (2019). Instead of performing site randomized mutagenesis and screening over 60 mutants, the present approach utilized Rosetta design to selectively guide mutations. The present application used biochemical data to evaluate the mutations. Although sufficient for a first look, structural studies, such as cocrystallization with substrates, allow further mechanistic understanding. As previously mentioned, the design protocol was performed by modeling the binding of the substrate while applying enzymatic constraints. Substrates were chosen as the docked moiety, because there was limited information available for the reaction intermediate. In summary, in silico mutation studies using the Rosetta design protocol were performed to predict beneficial mutations to the / rE -encoding AAD enzyme which would increase selectivity for butanol production in C. tyrobutyricum fermentations. These results predicted several AAD point mutants, which showed an increase in the butanol titer and selectivity and the ability to utilize the alternative NAPDH cofactor when compared to the wild-type. Fermentations using the AAD mutant L488A resulted in an over 100% increase in butanol production (6.6 g / L vs 2.9 g / L) and 160% increase in the butanol yield (0.13 g / g vs 0.05 g / g). Fermentations using the AAD mutant P619G resulted in a nearly 200% increase in butanol production (8.7 g / L vs 2.9 g / L) and yield (0.15 g / g vs 0.05 g / g). Furthermore, the butanol / ethanol ratio from P619G also increased 93% (9.1 vs 4.7). It was also shown that the addition of 50 pM MV in the fermentation further increased the butanol yield to 0.23 g / g. It was then shown that driving the expression of the single point mutant adh Z by the stronger thl promoter results in an even higher butanol titer and yield. The fermentation results of the Ct_pT_P619G and the Ct_pT_WT control were compared; fermentations for the Ct_pT_P619G strain resulted in a 70% increase in butanol production (11.57 g / L vs 6.78 g / L) and a 51% increase in yield (0.20 g / g vs 0.13 g / g).

[0132] For all the fermentations using beneficial AAD mutants, increased acetate: butyrate ratios were observed. This increase in acetate production may be due to stifled C4 flux, resulting from thiolase inhibition due to CoA accumulation. This bottleneck may be addressed by incorporating a mutated thiolase enzyme that is less inhibited by CoA-SH32,33 or by overexpressing a strong 3-hydroxybutyryl-CoA dehydrogenase enzyme that can pull flux to C4 products. Studies have shown that coexpressing the NADPH-dependent 3 -hydroxybutyryl- CoA dehydrogenase encoded by hbdl from Clostridium kluyveri with adhE2 in C. tyrobutyricum increased the flux from acetyl-CoA to butyryl-CoA and butanol production by rebalancing the redox cofactors through increasing the NADP+ / NADPH turnover. Also, butanol production via mutated AAD may be further increased by using noncanonical cofactors to increase the reducing equivalents required for butanol biosynthesis.

[0133] Methods

[0134] Bacterial Strains and Cultivation.

[0135] Escherichia coli DH5a was used for plasmid propagation and E. coli CA434 served as the donor strain for the conjugation process. Luria-Bertani (LB) broth and solid LB agar plates were supplemented with 30 pg / mL chloramphenicol (Cm) and used for routine E. coli cultivation. C. tyrobutyricum ATCC 25755 was cultivated under anaerobic conditions at 37 °C using either a liquid Reinforced Clostridial Medium (RCM; Difco, Detroit, MI) or solid RCM (with 15 g / L agar). 30 pg / mL of thiamphenicol (Tm) and 250 pg / mL of D-cycloserine were supplemented when required.

[0136] Construction of Recombinant Plasmids.

[0137] The plasmids used and constructed are given in Table 1. The primers used are listed in Table 4. KOD One PCR Master Mix (Code No. KMM-101, Toyobo Research Reagents, Osaka, Japan) was used for PCR gene amplification. The adhE2 gene (GenBank AF321779.1) was amplified from genomic DNA from C. acetobutylicum and was cloned into the pMTL82151 plasmid under the control of the various promoters with a chloramphenicol (Cm) resistance marker. Point mutations were performed using their respective primers in PCR amplification, and then the linearized plasmids were closed via Gibson Assembly using the Gibson Assembly Master Mix (no. 5510A, New England Biolabs, Ipswich, MA).

[0138] Transformation and Confirmation of Recombinant Strains.

[0139] The Clostridium modular plasmid pMTL82151 and its derivatives were used for gene expression. The recombinant plasmids for overexpressing the adhE2 gene were transformed into C. tyrobutyricum via conjugation. Briefly, the recombinant plasmids were first transformed into E. coli DH5a for amplification and then extracted and transformed into E. coli CA434, which was then cultivated in LB medium with 30 pg / mL Cm until the OD600 reached 1.5-2.0. About 3 mL of E. coli CA434 cells were centrifuged and washed twice (with 1 mL PBS lx) for antibiotic removal. Following a cell wash, the E. coli donor cells were mixed with 0.4 mL of the C. tyrobutyricum ATCC 25755 recipient cells, which were grown on RCM overnight to an OD600 of 2.0-3.0. The cell mixture was spread onto a well-dried RCM agar plate and incubated under anaerobic conditions at 37 °C. 1 mL of RCM was used to wash the agar plate to collect the transconjugants after 24 h of cell growth, then the cells were spread onto two RCM plates containing 25 pg / mL Tm and 200 pg / mL D-cycloserine. This is to eliminate the residual donor E. coli. Transformant colonies were generally observed after 48-96 h of incubation. Positive transformants selected from the agar plates were confirmed by colony PCR using the appropriate primers and gel electrophoresis to verify the presence of targeted DNA fragments. SDS-PAGE was performed with cell lysates to confirm the expressions of AAD (MW 94.4 kDa) in the mutant strains transformed with plasmids containing adhE2 (Fig. 8). The confirmed mutant strains were maintained in CGM containing 15% glycerol and 30 pg / mL thiamphenicol or 40 pg / mL erythromycin and stored at -80°C.

[0140] Fermentation Kinetics Studies.

[0141] Batch fermentation kinetics of C. tyrobutyricum mutants were studied in serum bottles with clostridial growth medium (CGM) containing (per liter): 4 g tryptone, 2 g yeast extract, 2 g (NHi^SCh, 0.5 g KH2PO4, 1 g K2HPO43H2O, 40 g CaCCh (pH buffer), and trace elements. 5 ml of 660 g / L glucose-H2O was added to each bottle containing 43 mL of the medium to bring the glucose concentration to 60 g / L, which was then inoculated with 2 mL of cells from an overnight culture in RCM, to bring the total volume to 50 mL. The fermentation was conducted at 37 °C and sampled regularly to monitor cell growth, substrate (glucose) consumption, and production of acetic acid, butyric acid, ethanol, and butanol during the fermentation. The fermentation kinetics data are summarized in Table 5.

[0142] Analytical Methods.

[0143] Cell density as the optical density at 600 nm (OD600) was monitored with a UV-vis spectrophotometer (UV-1601, Shimadzu, Kyoto, Japan). Glucose, acetate, ethanol, butyrate, and butanol present in the fermentation broth were analyzed by high-performance liquid chromatography (HPLC) with a reflective index detector (Shimadzu RID- 10 A) and an organic acid analysis column (Bio-Rad HPX-87H) at 65 °C with 0.01 M H2SO4 at 0.6 mL / min. Butanol and ethanol concentrations were also determined by gas chromatography (GC, Shimadzu GC-2014) with a Phenomenex ZB-FFAP capillary column.

[0144] Assay of Alcohol Dehydrogenase Activity.

[0145] Ethanol dehydrogenase and butanol dehydrogenase activities were measured from a purified cell lysate, following the procedures described below. Cells cultured in serum bottles containing CGM were harvested at an OD600 of 6.0 and washed with 10 mL of 0.1 M Tris-HCl, pH 7.6. Pellets were resuspended with lysis buffer to bring the cell concentration to 1 g / mL, then anaerobically lysed using a Model 100 sonic dismembrator (Fisher Scientific, PA, USA), and centrifuged at 14,000g for 25 min at 4 °C. The Bradford method was used to measure total protein concentration, and then an assay was performed with a Spectramax 250 spectrophotometer (GE Healthcare, IL, USA), monitoring NAD(P)H absorbance at 340 nm. Reactions were performed in 96-well plates containing 0.1 M Tris-HCl (pH 7.6), 300 pM NADH, and 50 mM butyraldehyde or 150 mM acetaldehyde. 25 pL of the protein extract was added to 975 pL of the reaction mixture to initiate the reaction. The definition of one unit of alcohol dehydrogenase activity is defined as the amount of enzyme necessary to consume 1 pmol of NADH per min. All experiments were performed in triplicate unless otherwise stated.

[0146] Molecular Modeling.

[0147] The homology model for the mutant at / AE2-AAD was generated by using Alphafold2. Visualization of protein structures was performed using the Schrodinger PyMOL software. Coordinates for NAD+and the substrate used in docking were extracted from the cocrystallized nicotinamide cofactor and glycerol from PDBs 1JQA and 1JQ5, respectively. Ligands were prepared using the Biochemical Library following the protocol described elsewhere. The Rosetta docking protocol involved mutation from the AAD structure, repeated rounds of stochastic rigidbody perturbation by translation and rotation for NALL, acetaldehyde or butyraldehyde, and side-chain repacking and minimization to optimize active-site rotamers. Simulations followed the protocol as described by Black et al. (2020) with minor deviations. Briefly, the Monte Carlo (MC) method was used to sample all moves, coordinate restraints were employed to maintain the NAD+and acetaldehyde or the NAD+and butyraldehyde in a catalytically competent pose, and total flexibility for substrate and protein backbone torsions was permitted to elucidate the prime binding conformation. One thousand enzyme design trials were run for each combination of substrates. Then, models were sorted based on specific criteria. Of these top 100 models, sequence logos were generated to visualize the most conserved amino acids for each amino acid residue, and consistent mutations for butyraldehyde binding but not acetaldehyde binding were created in vivo.

[0148] Example 5: Overexpression Of Carboxylic Acid Reductase In C. Tyrobutyricum And Its Effect On Butyrate Assimilation

[0149] Butanol production in C. tyrobutyricum via overexpression of the aldehyde / alcohol dehydrogenase (AAD) gene adhE2 native to C. acetobutylicum is accompanied by a significant production of butyric acid. The present application discloses the assimilation of this butyrate for butanol production via a carboxylic acid reductase (CAR) enzyme. Several CAR enzymes were investigated for their ability to reduce butyrate to butyraldehyde, then the mmCAR enzyme was co-overexpressed with AAD in C. tyrobutyricum. The resulting fermentation achieved a butanol yield of 0.20 g / g glucose, an over forty percent increase when compared to the control. Yields were even further increased in this strain with butyrate supplementation.

[0150] Introduction

[0151] Renewable, sustainable biofuel production is necessary to combat the growing crisis caused by greenhouse gas emissions contributing to climate change. Biobutanol is a 4-carbon alcohol with potential as a drop-in replacement to gasoline; biobutanol is less hydroscopic and has a lower vapor pressure than ethanol and it has a similar energy content to gasoline, 27 MJ / L versus 32 MJ / L, respectively. Biobutanol is traditionally produced via the acetone-butanol -ethanol (ABE) fermentation, typically producing products in a 3:6:1 ABE weight ratio when produced using the microbe Clostridium acetobutylicum. This fermentation is characterized by two distinct phases: the acetogenesis phase first produces acids acetate and butyrate, followed by the solventogenesis phase, producing products acetone, butanol, and ethanol. This complicated shift in metabolic pathways, along with the challenges presented by the spore-forming life cycles of traditional ABE producers, hinders the adoption of industrial biobutanol production. Finally, as already mentioned, the co-production of acetone, ethanol, acetate and butyrate as well as Clostridium acetobutylicum’ s inherent butanol toxicity typically results in fermentations with low butanol yield, titer, and productivity. Researchers have aimed to address these production issues by investigating biobutanol production in other microbes, one such microbe being Clostridium tyrobutyricum.

[0152] Clostridium tyrobutyricum is a Gram-positive, strictly anaerobic bacterium known for its acidogenic properties, predominantly producing acetic and butyric acids from glucose. It is considered a hyper butyrate-producing microorganism, with a unique metabolic capability to reassimilate acetate for butyrate biosynthesis via the butyryl -Co A / acetate CoA transferase (CoAT) pathway. This mechanism contrasts with the more commonly observed phosphotransbutyrylase-butyrate kinase (PTB-BK) pathway, which is prevalent among other clostridial species and microbes.

[0153] C. tyrobutyricum has been engineered to overexpress a heterologous alcohol / aldehyde dehydrogenase encoded by the adhE2 gene native to C. acetobutylicum. This modification enables the conversion of butyryl-CoA into n-butanol, enhancing its potential for biofuel production. The engineered C. tyrobutyricum demonstrates a high metabolic flux directed toward butyryl-CoA, leading to n-butanol production at a yield greater than 0.30 g / g and a titer exceeding 20 g / L in glucose fermentation.

[0154] A notable advantage of this recombinant strain is its exclusive production of butanol without the co-formation of acetone, resulting in a significantly higher C4 / C2 ratio. This streamlined metabolic process simplifies downstream processing and purification, as butanol becomes the sole major fermentation product, making the strain an attractive candidate for industrial applications in biofuel production.

[0155] The present application discloses enhanced butanol production in C. tyrobutyricum fermentation through acetate assimilation following a multi-step, enzymatic cascade. First, acetate is converted to butyrate via the CoAT pathway, then butyrate is converted to butyraldehyde, where butyraldehyde is converted to butanol via the alcohol dehydrogenase domain of the aldehydealcohol dehydrogenase (AAD) enzyme encoded by the adhE2 (SEQ ID NO: 2) gene. The butyrate to butyraldehyde conversion step is unique, as it is catalyzed by the Mycobacterium marinum carboxylic acid reductase (CAR) (SEQ ID NO: 3). CARs catalyze the reduction of a carboxylic acid substrate to its corresponding aldehyde by first activating the carboxylate using ATP and then facilitating the reduction step with NADPH as the hydride donor. CAR genes are typically co- expressed with an sfp gene, which encodes an enzyme which activates the CAR enzyme by transferring a phosphopantetheinyl group to a conserved serine residue within the CAR protein. This modification converts CAR from its inactive apo-form to the active holo-form, enabling it to catalyze the reduction of carboxylic acids to aldehydes.

[0156] The present application discloses an engineered strain of C. tyrobutyricum to overexpress the heterogenous adhEZ (SEQ ID NO: 2), mmCAR (SEQ ID NO: 4), and sfp (SEQ ID NO: 8) genes to enhance the n-butanol production. The adhEZ and the mmCAR / .s i? synthetic operons were expressed on two separate plasmids, identifying the effect of different promoters on the plasmids. Additionally, the effect of overexpressing a construct encoding tRNAs (SEQ ID NO: 10) corresponding to codons rare to C. tyrobutyricum was investigated to assist in the translation of the heterogenous mmCAR and sfp genes. In another embodiment, the mmCAR enzyme is mutated to increase CAR activity and the effect this mutant had on butanol production was identified. Finally, CAR activity was confirmed through the in-vitro biotransformation of butyrate to butanol using cell lysate of C. tyrobutyricum expressing the adhEZ and mmCAR and sfp genes.

[0157] Materials & Methods

[0158] Bacterial strains and cultivation

[0159] All strains used are listed in Table 7. For general plasmid propagation the E. coli strain DH5a (New England BioLabs Inc., Ipswich, MA) was used. E. coli CA434 served as the donor strain for the conjugation process. Luria-Bertani (LB) broth, or solid LB agar plate, supplemented with 30 pg / mL chloramphenicol (Cm), was used for routine E. coli cultivation. C. tyrobutyricum ATCC 25755 was cultivated under anaerobic conditions at 37 °C using either liquid Reinforced Clostridial Medium (RCM; Difco, Detroit, MI) or solid RCM (with 15 g / L agar). 30 pg / mL thiamphenicol (Tm) and 250 pg / mL D-cycloserine were supplemented when required.

[0160] Plasmid construction and transformation

[0161] The plasmids used are listed in Table 7. The adhEZ gene (Genebank AF321779.1) and the promoter pFLA (GenBank AE001437) were amplified from genomic DNA from C. acetobutylicum. Promoters pCATl (GenBank CP137758, Region 2340603-2340928) and pTHL(GenBank CP137758, Region 2908102-2908282) were amplified from genomic DNA from C. tyrobutyricum. pMTL82151 plasmids were assembled via Gibson Assembly using the Gibson Assembly Master Mix (#5510A, New England Biolabs, Ipswich, MA). The pZE expression plasmids containing the Mycobacterium marinum CAR (mmCAR, NCBI RefSeq WP_019305838.1) gene, Nocardia iowensis CAR (niCAR, NCBI RefSeq WP_218468927.1) gene, and Bacillus subtilis 4'-phosphopantetheinyl transferase (sfp, NCBI RefSeq WP 015715234.1) gene, codon-optimized for E. coli expression, were kindly provided from Dr. Kunjapur, University of Delaware. The Mycobacterium immunogenum CAR (miCAR, NCBI RefSeq WP 019305838.1) gene was cloned from genomic DNA purchased from DSMZ. The pZE plasmids were directly used for gene expression in Rosetta-gami™ 2, the pZE miCAR ^? plasmid was created by cloning the miCAR gene in place of the mmCAR gene via Gibson Assembly.

[0162] The CAR and sfp genes were PCR-amplified then cloned into the pMTL82151 plasmid with the CAT1 promoter and thiamphenicol resistance marker, the tRNA construct was synthesized using TWIST Bioscience (Twist Bioscience, San Fransisco, CA), codon optimized for C. tyrobutyricum, and plasmids were assembled via Gibson Assembly. Plasmids were transformed into NEB DH5 alpha (New England BioLabs Inc., Ipswich, MA) for plasmid propagation and colony screening.

[0163] Expression in E. coli

[0164] The pZE expression plasmids were transformed into E. coli Rosetta-gami™ 2, Expression was performed in TB medium, where kanamycin for pZE plasmid selection and chloramphenicol for pRARE2 plasmid selection were supplemented. Aerobic growth at 37 °C with shaking at 200 rpm brought the cell to an approximate 0.6 OD600, then tetracycline was injected to a final concentration of 1 pg / ml and the temperature was reduced to 20 °C for overnight protein expression. Cells harvest by centrifuging and resuspension in 25 mm Tris-HCl pH 8.0 was then performed. Finally, cell lysate was prepared by sonication on ice proceeded by centrifugation. C. tyrobutyricum conjugation

[0165] C. tyrobutyricum plasmid transformation was performed via conjugation following established protocols with modifications. E. coli CA434 containing shuttle plasmid was cultivated in LB medium with 30 pg / mL Cm supplement. After OD600 reached 1.5-2.0, 3 mL E. coli CA434 cells were centrifuged and washed twice (with 1 mL PBS IX) for antibiotic removal. Following cell wash, the E. coli donor cells and 0.4 mL of the C. tyrobutyricum ATCC 25755 recipient cells were mixed. The C. tyrobutyricum ATCC 25755 required reaching an OD600 of 2.0-3.0 after overnight growth on RCM. The cell mixture was spread onto a well-dried RCM agar plate and incubated under anaerobic conditions at 37°C. One mL of RCM was used to wash the agar plate to collect the transconjugants after 24 h of cell growth, then the cells were spread onto two RCM plates containing 25 pg / mL Tm and 200 pg / mL D-cycloserine. This is to eliminate residual donor E. coli. Transformant colonies were generally observed after 48-96 h of incubation. Fermentation kinetics

[0166] Unless otherwise noted, batch fermentations with C. tyrobutyricum mutants were carried out in serum bottles containing clostridial growth medium (CGM) which contains the following (per liter): 4 g tryptone, 2 g yeast extract, 2 g (NH^SCh, 0.5 g KH2PO4, 1 g K2HPO4 GH2O, 40 g CaCCh (pH buffer), and trace elements. 5 ml of 660g / L glucose H2O was added to each bottle containing 43 mL of the medium to bring the glucose concentration to 60 g / L, which was then inoculated with 2 mL of cells from an overnight culture in RCM, to bring the total volume to 50 mL. The fermentation was carried out at 37 °C, and samples were taken at regular intervals to monitor cell growth, substrate (glucose) consumption and production of butanol, ethanol, acetic acid and butyric acid during the fermentation. All fermentations were performed in duplicate. Analytical techniques

[0167] Cell growth was determined by measuring the optical density at 600 nm (OD600) using a UV-vis spectrophotometer (UV-1601, Shimadzu, Kyoto, Japan). Glucose, acetate, ethanol, butyrate, and butanol concentrations in the fermentation broth were analyzed using an HPLC. The HPLC system consisted of an automatic injector (Shimadzu SIL-lOAi), a pump (Shimadzu LC-lOAi), an organic acid analysis column (Bio-Rad HPX-87H), a column oven at 65 °C (Shimadzu CTO-10A), and a reflective index detector (Shimadzu RID-10A). The eluent was 0.01 M H2SO4 with a flow rate of 0.6 mL / min.

[0168] CAR enzyme assay

[0169] CAR enzyme assays were performed according to the procedure described with modifications. Unless otherwise specified, assays were performed in 100 mM Tris- HC1 pH 7.5 prepared at 30 °C, ImM ATP, 0.25 mM NADPH, 10 mM MgCh,10 pL of clarified cell lysate with a concentration of approximately 20 mg / mL, and 5 mM carboxylic acid substrate in a total volume of 200 mL. After measurement of total protein concentrations by the Bradford method. Reactions were performed in triplicate in a 96-well microtiter plate by using a Spectromax 250 spectrophotometer (GE Healthcare, IL, USA), monitoring decreases in absorbance at 340 nm. at 30°C, over 5 or 10 min after a 5 min preincubation at 30C.

[0170] CAR butyrate conversion reaction

[0171] Biotransformations with the addition of the cofactor regenerating system for NAD(P)H were performed in the presence of 100 mM Tris-HCl buffer (pH 7.0), 10 mM or 100 mM butyric acid, 2 mM NADH, 2 mM NADPH, 2 mM ATP, 20 mM MgCh, and 20 mM sodium phosphite. The reaction was initiated by adding 100 pL of cell lysate at 20 mg / ml concentration to the reaction medium. After approximately 20 h incubation, the reaction mixtures were centrifuged, and the reaction products were analyzed using GC. Results

[0172] Demonstration of CAR activity in enzyme assay E. coli

[0173] Various CAR genes were expressed in E. coli and the cell lysates were assayed to evaluate the genes’ ability to specifically reduce butyrate. Genes evaluated were: Nocardia iowensis CAR (NCBI RefSeq WP 218468927.1), Mycobacterium marinum CAR (NCBI RefSeq WP 019305838.1), and Mycobacterium marinum CAR (NCBI RefSeq WP 043078362.1). The results of the enzyme assays are shown in Table 8. The three enzymes were assayed along with a control of the E. coli cell lysate without the pZE plasmid present. Two substrates were evaluated, benzoic acid and butyric acid. Benzoic acid was assayed to ensure the enzymes expressed properly and had catalytic activity; all three enzymes are active on benzoic acid. As can be seen in Table 8, all three enzymes were active on benzoic acid, and were over 10 times more active compared to the control. M. immunogenum CAR displayed the highest activity for both substrates.

[0174] Demonstration of mmCAR activity in enzyme assay C. tyrobutyricum

[0175] Enzyme activity assays were performed to demonstrate the activity of the mmCAR expressed in C. tyrobutyricum. Three samples were evaluated for these assays. One sample was clarified cell lysate from C. tyrobutyricum containing an adhEl expression plasmid and a CAR expression plasmid with the tRNA construct. The second sample was clarified cell lysate from C. tyrobutyricum containing an adhEl expression plasmid and a CAR expression plasmid without the tRNA construct, and the final sample was a control containing only the adhEl expression plasmid. The results of these enzyme assays are shown in Table 9. Evident from the table both species containing CAR displayed significant activity when compared against the control. However, normalizing both species containing CAR by the control results, makes apparent that the species containing the tRNA construct has approximately twice the activity than the species without the tRNA construct. This is another example of the efficacy of the tRNA construct in heterologous gene expression in C. tyrobutyricum.

[0176] mmCAR expression in C. tyrobutyricum

[0177] After confirming enzyme activity towards butyric acid, these CAR enzymes were overexpressed along with adhEl' in C. tyrobutyricum fermentations. Serum Bottle fermentation results are shown in Fig. 9. Fig. 9 shows the fermentation results for the following strains: the control strain - C. tyrobutyricum containing the PMTL82151 adhEl expression plasmid driven by the FLA promoter with an erythromycin resistance marker; the test strain - C. tyrobutyricum containing two plasmids, the PMTL82151 adhEl expression plasmid driven by the FLA promoter with an erythromycin resistance marker and a second PMTL82151 plasmid expressing a tRNA construct and mmCAR and sfp driven by the CAT1 promoter with a chloramphenicol resistance marker; and the test strain with 6 g / L butyrate supplementation at 24 hours. The test strain shows significant increases in butanol titer and yield when compared against the control strain; the test strain achieved a titer of 10.74 ± 1.45 g / L and yield of 0.20 ± 0.028 g / g glucose whereas the control strain resulted in a titer of 6.67 ± 0.04 and yield of 0.14 ± 0.001 g / g glucose. A decrease in butyrate titer, 10.67 ± 0.06 g / L and 5.14 ± 1.68 g / L was observed for the control and test strain, respectively.

[0178] A follow-up fermentation was then performed with the test strain, where butyrate was supplemented to bring the butyrate concentration to 6 g / L at 24 hours. A decrease in butyrate concentration from 24 to 48 hours was observed for this fermentation, with gradual increase in butyrate concentration following (Fig. 9). This fermentation resulted in the highest titer and yield, 11.36 ± 0.33 g / L and 0.24 ± 0.008 g / g glucose. One major point to note from this fermentation is the final acetate concentration, 4.48 ± 0.09 g / L, compared to the final acetate concentration for the test strain fermentation without butyrate addition, 2.80 ± 0.32 g / L. The increase in acetate concentration for the fermentation with butyrate supplementation is likely due to an inhibition of the CAT 1 -catalyzed reaction. The acetate increase may also be a consequence of the increased need for ATP.

[0179] In both test strain fermentations, with and without the supplementation of butyrate, there was still butyrate production. Although there were significant improvements in the yield and titer for the test strain, this was not conclusive in determining CAR activity. An in-vitro assay was performed to investigate the CAR activity.

[0180] mmCAR fermentations with PTDH

[0181] Phosphite dehydrogenase containing two, point-mutations, G175A / A176R, (referred to as PTDH-AR) was co-overexpressed with adhEZ, mmCAR, and sfp to try to address the potential bottleneck of the limited NADPH cofactor pool. First, the optimum amount of phosphite loading for butanol production was determined by running fermentations with 7.5-, 15-, 30-, 45-, and 60-mM concentrations of sodium phosphite dibasic pentahydrate. The results of these experiments are given in Table 10. Apparent from the table, the optimum phosphite concentration is around 30 mM, resulting in butanol yields of 0.20 ± 0.00. For fermentations performed with 30 mM phosphite there were also no appreciable concentrations of ethanol nor acetate. However, there seemed to be no benefit to mmCAR expression when comparing co-overexpression of adhEZ, PTDH, mmCAR, and sfp to co-overexpression of adhEZ and PTDH alone; the Ct-pC-PA fermentation with 30mM phosphite supplementation resulted in a butanol yield of 0.25 ± 0.04 versus a yield of 0.20 ± 0.00 for Ct-atms with 30mM phosphite supplementation. mmCAR fermentations with PTDH and acetate or butyrate supplementation

[0182] Next, the impact of PTDH expression on the ability of CAR enzymes to reassimilate acids for butanol production was investigated. Two fermentations were performed, one with acetate and one with butyrate supplementation, where acid was added at the start of the fermentation to bring the initial concentration of the respective acid to 6g / L. Results for these fermentations are shown in Table 10. There is no apparent increase in butanol titer for either fermentation with acid supplementation, but it is important to note that there was no acetate remaining in the fermentation with initial acetate supplementation.

[0183] Effect of mmCAR mutant on butanol fermentation

[0184] Testing was performed to determine the effect of an mmCAR mutant on butanol fermentation performance. The R507F / N508G mutant was created to mutate the amino acid residues analogous to the one mutated in a different species of bacteria. Researchers found that mutating residues at positions 505 and 506 to amino acids phenylalanine and glycine in a different bacterial species increased the enzyme activity by 8.26-fold. Mutant mmCAR was cooverexpressed with mutant at / AE2_P619G and PTDH driven by the thiolase promoter. Results of the test fermentation and the control fermentation are shown in Table 11. All chemical species show a minor increase in titer for the test strain.

[0185] CAR butyrate conversion reaction

[0186] To confirm the activity of the CAR enzyme, biotransformation reactions were performed to convert butyrate to butanol in-vitro. Results of these experiments are shown in Table 12. Two experiments were performed at different butyrate concentrations. One experiment was performed with an initial butyrate concentration of 1 mM, another experiment was performed with an initial butyrate concentration of 10 mM. The test samples contained the C. tyrobutyricum clarified cell lysate possessing the PMTL82151 pCAT_at / AE2_PTDH and PMTL82152 pCAT mmCAR ^? plasmids. The control sample contained the C. tyrobutyricum clarified cell lysate possessing the PMTL82151 pC AT_at / AE2_PTDH plasmid. The presence of the PTDH enzyme allows for the in-vitro regeneration of NAD(P)H cofactors, so the only limiting reagent in the reaction is ATP. Therefore, the ATP concentration was used to calculate the maximum theoretical yield. Samples with an initial excess of butyrate showed a significant increase in butanol concentration in the final measurement, showing the production of butanol. Additionally, there was a significant production of ethanol. Based on the initial limiting concentration of ATP, the production of alcohols reached a yield of 0.98 ± 0.018 and 0.131 ± 0.007 mole / mole ATP for ethanol and butanol, respectively. Acetate, butyrate, ethanol, and for some samples, butanol were initially measured, stemming from their presence in the cell lysate. The ethanol yield is calculated based on the initial acetate concentration, however, because the whole cell lysate also contains the enzyme for CoAT, it is possible that the excess butyrate present drives acetate production via CoAT, resulting in high final ethanol titers.

[0187] At low butyrate concentrations no butanol production was observed, but ethanol production was still present. Since all enzymes in the metabolic pathway are present in the cell lysate, its theoretically possible alcohol can be produced via CoAT then via at / AE2, however, this has not been witnessed in fermentations with acid supplementation without CAR. Also, there is virtually no ethanol production in the test sample. These results indicate that mmCAR is present and active, and capable of reassimilating butyrate for butanol production in-vitro.

[0188] Discussion

[0189] There is incredible value in reducing organic acids into their respective aldehydes and alcohols. Carboxylic acids have been reduced via chemical means, but the addition of problematic chemicals including sodium borohydride and lithium aluminum hydride typically make such means impractical.

[0190] Both enzyme classes, aldehyde oxidoreductases (AORs) and carboxylic acid reductases (CARs), catalyze the reduction of carboxylic acids to the respective aldehydes. AORs reversibly oxidize organic aldehydes to their respective acids, with the reaction typically favoring the production of the oxidized product due to thermodynamic considerations. Although AORs have been shown to reduce acids to aldehydes, their primary utility lies in the oxidation of aldehydes. In contrast, carboxylic acid reductases (CARs) reduce carboxylic acids to aldehydes by utilizing adenosine triphosphate (ATP) and NADPH, producing adenosine monophosphate (AMP), pyrophosphate (PPi), and NADP+ as byproducts. Unlike AORs, the hydrolysis of ATP in CAR-catalyzed reactions is highly thermodynamically favorable, effectively driving the reduction of acids to aldehydes.

[0191] By coupling the CAR reaction with an alcohol dehydrogenase, a complete route from the carboxylic acids to the respective alcohol can be formed. Previous research identified multiple CARs that reduced a wide selection of bifunctional carboxylic acids. These CARs showed the ability to produce the corresponding alcohol by reducing the respective acid both in-vitro and in-vivo. The CAR reaction was coupled with an aldo-keto reductase and cofactor regenerating systems for ATP and NADPH to reduce 4-HB to 1,4-butanediol (1,4-BDO) and adipic acid to 1,6-hexanediol (1,6-HDO), achieving conversions ranging from 50-76% in-vitro and 50-95% conversion in-vivo. Researchers previously demonstrated in E. coli the ability to convert endogenously produced butyrate to butanol by expressing mmCAR, sfp, and adh2 encoding alcohol dehydrogenase from S. cerevisiae. Overexpression of these genes in pseudo-operon configuration resulted in 100% bioconversion efficiency converting butyrate to butanol when supplemented with 1 g / L of butyrate. Research then demonstrated ~2 g / L butanol production under fed-batch fermentation by co-cultivating an upstream strain (butyrate-producing) with a downstream strain (butyrate to butanol converting).

[0192] The present application utilizes mmCAR and sfp, but differs from prior work with the utilization of the adhEZ AAD gene from C. acetobutylicum for reducing butyraldehyde to butanol. Additionally, in the present application the mmCAR and sfp genes were not codon optimized, instead additional co-overexpression of a tRNA construct was relied on to mitigate codon usage bias.

[0193] The tRNA operon from nucleotide bases 2576947 to 2578424 in the Clostridium tyrobutyricum strain KCTC 5387 genome was selected to roughly model the tRNA construct after. The direct upstream region was included to include any regulatory elements it may contain. Whereas the genomic tRNA element encodes for 15 tRNAs, the plasmid element only encodes for 5. Two tRNAs native to C. tyrobutyricum, Arg-anticodon- ACG and Arg-anticodon-TCG were left. However, 3 of the 5 codons in the construct were native to C. cellulolyticum, Ala-anticodon-GGC and Val-anticodon-GAC, and Arg-anticodon-CCG. All three of these tRNAs are not present in the native C. tyrobutyricum genome. Foreign tRNAs were overexpressed as the similarity of the two organisms would make for more harmonious expression. A quick check of the codon usage for the various CAR genes elucidates the usefulness of the tRNA construct. A codon usage table for the various CAR genes is found in Table 14.

[0194] Table 14 gives the codons translated by the tRNAs expressed in the tRNA construct, and it includes the usage of these codons in 4 genes, each CAR gene and the BS2 optimized for M. immunogenum. The BS2 protein has been shown to be an effective biosensor in C. tyrobutyricum. First note the difference between the CAR enzymes that were codon optimized for E. coli and the one which was not. Both mmCAR and niCAR were provided pre-optimized for E. coli expression, whereas miCAR was extracted from M. immunogenum genomic DNA. Therefore, the tRNA that translates codon ARG-CGG has limited utility in the mmCAR and niCAR enzyme translation, the same can be said for the tRNA that translates ARG-CGA. Important to note is the high utility of the tRNAs that translate codons Ala-GCC and Val-GTC, where these codons are highly repeated in all three genes. A final note, C. tyrobutyricum is unable to natively translate the three codons Ala-GCC, Val-GTC, and Arg-CGG because it does not have the tRNAs that contains the anticodons.

[0195] In primary metabolism fermentations such as the butanol fermentation in C. tyrobutyricum, the amount of ATP produced is strongly linked to product distribution. Besides the glycolytic pathway, the only other source of ATP is through acetate and production, the production of solvents, ethanol and butanol, results in significantly less ATP formation.

[0196] Utilization of ATP for butyrate reduction can be limited due to high butanol production, or likewise high amounts of acetate may be generated to supply necessary ATP. A slight increase in acetate production was observed in the serum bottle fermentation results where butyrate was supplemented. This can be due to butyryl-CoA / acetate CoA transferase inhibition, or possibly due to overproduction of acetate for necessary ATP generation. By incorporating a cofactor regenerating system this issue can be mitigated. Polyphosphate kinase is such an enzyme, where the Polyphosphate kinase 2 (PPK2) catalyzes the breakdown of inorganic polyphosphate (polyP) to synthesize nucleoside triphosphates such as ATP.

[0197] Although mmCAR has been shown to have limited activity on acetic acid, changing cellular concentrations of acetic acid may drive its reduction. The increase in ethanol concentration, 3.11 ± 0.65 g / L for the strain overexpressing mmCAR, versus 0.79 ± 0.79g / L for the control strain, may be due to CAR activity on the acetic acid, resulting in increased ethanol production.

[0198] Conclusion

[0199] The present application effectively established a butyrate assimilation pathway via the CAR enzyme, increasing butanol production in C. tyrobutyricum fermentations. Butanol yield was increased in fermentation overexpressing CAR, resulting in an over 40 percent increase in yield compared to the control. Yields were further increased by 20 percent in CAR strains with butyrate supplementation. This CAR activity was confirmed in vitro. Additionally, the effectiveness of a tRNA construct for heterogenous gene overexpression was evaluated. Tables

[0200] Table 1. Plasmids and strains constructed and used in overexpressing adhE2 and its variants plasmid / strain description

[0201] plasmids

[0202] pMTL82151 Clostridium-E.coli shuttle plasmid, containing: pBPl g-positive replicon, catP resistance marker, ColEl g-negative replicon, TraJ conjugal transfer function, and MCS

[0203] pCAT-at / AE2 pMTL82151 plasmid with the adhE2 gene driven by the C. tyrobutyricum catl promoter cloned into the MCS region via Gibson assembly

[0204] pTHL-at / AE2 pMTL82151 plasmid with the adhE2 gene driven by the C. tyrobutyricum thl promoter cloned into the MCS region via Gibson assembly strains

[0205]

[0206] u, proA2, with Inc. Pb conjugative plasmid

[0207]

[0208] C. tyrobutyricum ATCC 25755; wild-type stain; same as KCTC 5387

[0209] Ct_pC_WT C. tyrobutyricum transformed with pC

[0210]

[0211] Ct_pC_D485G C. tyrobutyricum transformed with pCAT-at / AE2 containing D485G point mutation

[0212] Ct_pC_L488A C. tyrobutyricum transformed with pCAT-at / AE2 containing L488A point mutation

[0213] Ct_pC_P619G C. tyrobutyricum transformed with pCAT-at / AE2 containing P619G point mutation

[0214] Ct_pC_P619I C. tyrobutyricum transformed with pCAT-at / AE2 containing P619I point mutation

[0215] Ct_pC_AS C. tyrobutyricum transformed with pCAT-at / AE2 containing S601A and V608S point mutations

[0216] Ct_pC_ASG C. tyrobutyricum transformed with pCAT-at / AE2 containing S601A, V608S, and P619G point mutations

[0217] Ct_pC_PSG C. tyrobutyricum transformed with pCAT-at / AE2 containing S601P, V608S, and P619G point mutations

[0218] Ct_pT_WT C. tyrobutyricum transformed with pT

[0219]

[0220] plasmid / strain description

[0221] Ct_pT_P619G C. tyrobutyricum transformed with pTHL- / rE containing P619G point mutation

[0222] Table 2. Enzyme Activities of whole cell lysate protein extracts of various strains

[0223] Strain Ethanol dehydrogenase Butanol dehydrogenase

[0224]

[0225] C. tyrobutyricum 14.4 ± 1.7 NA 14.4 ± 2.7 41.5 ± 0.6 Ct_pC_WT 34.9 ± 6.2 NA 37.9 ± 7.2 81.9 ± 2.1 Ct_pC AS 41.5 ± 21.7 NA 80.2 ± 18.6 NA

[0226] Ct_pC P619I 87.2 ± 11.4 NA- 66.1 ± 1.0 NA

[0227] Ct_pC P619G 93.6 ± 13.2 NA 242.8 ± 59.7 NA

[0228] Ct_pC ASG 4287 ± 580 NA 2606 ± 54 NA Ct_pC_D485G NA NA 63.2 ± 1.5 113.5 ± 1.8 Ct pC_L488A NA NA 70.2 ± 0.6 222.0 ± 1.7aU is given in pinole NADH consumed per minute.

[0229] Table 3. Residue Energy Breakdowns for Mutated Residues and Substrate Combinations. enzyme Resl Res2 fa atr fa re fa s Ik bal Ik ball fa ele hbond total p ol 1 iso c sc AAD__WT 601-SER butyraldeh -0.29 0.00 0.10 0.04 -0.07 0.00 0.0 -0.22 yde

[0230] AAD WT 601-SER NAD+-0.05 0.00 0.15 0.00 -0.05 0.08 0.0 0.13 AAD S601A 601-ALA butyraldeh -0.03 0.00 0.00 0.00 0.00 0.03 0.0 0.00 V608S P619G yde

[0231] AAD__S601A__ 601-ALA NAD+-0.01 0.00 0.00 0.00 0.00 0.05 0.0 0.04 V608S P619G

[0232] AAD WT 608-VAL NAD+-4.74 3.62 2.65 1.04 -1.16 -0.47 0.0 0.96 AAD S601A 608-SER NAD+-3.51 1.78 3.25 0.66 -1.07 -1.06 -0.6 -0.57 V608S P619G

[0233] AAD WT 619-PRO NAD+-0.16 0.00 0.00 0.00 0.00 0.05 0.0 -0.11 AAD_S601A_ 619-GLY NAD+-0.22 0.00 -0.1 0.00 -0.01 0.10 0.0 -0.20 V608S P619G

[0234] AAD WT 488-LYS 493-TYR -4.51 8.984 0.60 1.471 -0.83 -2.05 0 2.63 AAD__L488A 488-ALA 493-TYR -2.23 0.313 0.93 1.628 -0.74 -2.19 0 -3.23 fa atr— attractive energy between two atoms on different residues separated by a distance d; fa rep— repulsive energy between two atoms on different residues separated by a distance d; fa sol— Gaussian exclusion implicit solvation energy between protein atoms in different residues; Ik ball— anisotropic energy for desolvating a polar atom; Ik ball iso— isotropic energy for desolvating a polar atom; fa elec— energy of interaction between two nonbonded charged atoms separated by a distance d. Table 4. Primers used

[0235]

[0236] Table 5. Fermentation Results for Various C. tyrobutyricum Mutants overexpressing adhE2 and its variants

[0237]

[0238]

[0239] Table 6. Comparison of AAD Mutation Studies in Clostridia for Enhanced Butanol Production

[0240]

[0241]

[0242] Table 7. Plasmids and strains constructed and used

[0243]

[0244] pTHL- adhE2- pMTL82151 plasmid with the at / AE2-P619G mutant Present P619G PTDH-AR and PTDH-AR genes driven by the C. tyrobutyricum application THL promoter cloned into the MCS region via Gibson

[0245] assembly pZE CAR sfp E. coli car & sfp expression plasmid, kanR resistance Dr. Kunjapur marker and tetO inducible promoter

[0246] pZE miCAR sfp pZE CAR sfp plasmid containing Mycobacterium Present immunogenum car and sfp application pZE mmCAR sfp pZE CAR sfp plasmid containing Mycobacterium Dr. Kunjapur marinum car and sfp

[0247] pZE niCAR sfp pZE CAR sfp plasmid containing Nocardia iowensis Dr. Kunjapur car and sfp

[0248] E. coli DH5a gyrA96 deoR nupG purB20 (p80dlacZAM15 A(lacZYA- New England argF)U169, hsdR17(rK-mK+), - BioLabs

[0249] E. coli CA434 hsd20(rB~, m8-), recA13, rpsL20, leu,proA2, with IncPb (Williams et al., conjugative plasmid R702 1990) Rosetta-gami™ 2 A(ara-leu)7697 AlacX74 AphoA PvuII phoR araD139 Novagen ahpC galE galK rpsL (DE3) F'[lac+laclqpro] gor522:: Tn!0 trxB pRARE2 (Cam8, Str8, Tet8)

[0250] RG-miCAR_sfp Rosetta-gami™ 2 transformed with pZE miCAR sfp Present plasmid application RG-mmCAR_sfp Rosetta-gami™ 2 transformed with pZE mmCAR sfp Present plasmid application RG-niCAR_sfp Rosetta-gami™ 2 transformed with pZE niCAR sfp Present plasmid application C. tyrobutyricum ATCC 25755; Wild type stain; same as KCTC 5387 ATCC

[0251] Ct-pF-WT C. tyrobutyricum transformed with pFL

[0252]

[0253] Ct-pT-PA-P619G C. tyrobutyricum ATCC 25755 transformed with Present pTHL- adhE2-P619G_PTDH- AR plasmid application Ct-ams C. tyrobutyricum transformed with plasmids pFLA- Present adhE2 and pCAT-mmCAR sfp application Ct-atms C. tyrobutyricum transformed with plasmids pFLA- Present adhE2 and pCAT-tRNA_mmCAR_sfp application Ct-aptms C. tyrobutyricum transformed with plasmids pCAT- Present adhE2-PTDH-AR and pCAT-tRNA_mmCAR_sfp application Ct-a-Gptms-FG C. tyrobutyricum transformed with plasmids pTHL- Present adhE2-P619G_PTDH-AR and pCAT-tRNA mmCAR- application R507F / N508G_sfp Table 8. Enzyme specific activity of whole cell lysate protein extract (E. coll)

[0254] CAR Enzyme Benzoic Acid (mU / mg) Butyric Acid (mU / mg) M. marinum 37.57 ± 0.22 11.27 ± 0.25

[0255] M. immunogenum 38.09 ± 0.22 17.48 ± 0.15

[0256] N. iowensis 25.85 ± 1.37 9.19 ± 0.14

[0257] Control 2.85 ± 0.04 3.31 ± 0.01

[0258] U is given in pmole NADPH consumed per minute.

[0259] Table 9. Enzyme specific activity of whole cell lysate protein extract (C. tyrobutyricum)

[0260] C. tyrobutyricum mutant Butyric Acid (mU / mg) Activity Normalized

[0261] w / tRNA Construct 59.15 ± 1.17 36.71 ± 1.17

[0262] w / o tRNA Construct 41.28 ± 6.00 18.83 ± 6.00

[0263] Control 22.43 ± 3.17 0

[0264] U is given in pmole NADPH consumed per minute.

[0265] Table 10. Phosphite loading for Ct-aptms fermentations

[0266] Phosphite Glucose BuOH Butyrate EtOH Acetate BuOH C4 / C2 AL / BuOH / loading consumed (g / L) (g / L) (g / L) (g / L) Yield (g / g)

[0267] acid EtOH(mM> fe'D

[0268]

[0269] (g / g)(g / g)7.5 51.41 9.15 10.62 0.76 0.28 0.18 19.04 0.91 12.07 15 52.34 10.09 8.78 1.17 0.16 0.19 14.20 1.26 8.61 30 51.66 ± 10.41 6.29 ± 0.00* 0.00* 0.20 ± Inf. 1.70± Inf.

[0270] 0.97 ± 0.36 0.17 0.00 0.30 45 48.27 7.03 7.68 1.80 0.00* 0.15 8.17 1.15 3.90 60 47.11 5.99 7.53 1.97 0.00* 0.13 6.87 1.06 3.04 Table 11. Acid supplementation in Ct-aptms fermentations

[0271] Suppiemen Glucose BuO Butyrat EtO Acetat BuO C4 / C AL / BuOH t consume H e H e H 2 acid /

[0272] d (g / L) (g / L) Yield (g / g) (g / g

[0273] (g / L) (g / L) (g / L) EtOH

[0274]

[0275] No acid 51.66 10.09 8.78 0.00 0.00 0.20 Inf 1.70 Inf addition

[0276] 6 g / L 53.56 6.22 12.90 0.34 0.35 0.12 27.49 0.49 18.42 acetate

[0277] 6 g / L 50.38 9.70 10.16 0.00* 0.36 0.20 55.51 0.92 Inf. butyrate

[0278] Table 12. Effect of mutated mmCAR on fermentation

[0279] Strain Glucose BuOH Butyrate EtOH Acetate BuOH C4 / C2 AL / BuOH / consumed (g / L) (g / L) (g / L) (g / L) Yield (g / g)

[0280] <g / L) <g / g>

[0281]

[0282] Ct-pT-PA- 53.64 8.33 2.97 1.58 5.71 0.16 1.55 1.14 5.29 P619G

[0283] (OmM

[0284] phosphite)

[0285] Ct-a- 46.06 8.91 3.71 1.80 5.81 0.19 1.66 1.12 4.95 Gptms-FG

[0286] (OmM

[0287] phosphite)

[0288] Table 13. In-vitro biotransformation reactions performed with C. tyrobutyricum cell lysate overexpressing CAR, AAD, and PTDH

[0289]

[0290]

[0291] Table 14. Codon usage for CAR enzymes, relative frequency, (absolute count)

[0292] Gene Ala-GCC Val-GTC Arg-CGG Arg-CGT Arg-CGA M. marinum CAR 0.28 (34) 0.32 (28) (F(6) 0.54 (42)

[0293]

[0294] M. immunogenum CAR 0.39 (48) 0.33 (32) 0.31 (18) 0.21 (15) 0.04 (3) N. iowensis CAR 0.18 (27) 0.21 (18) (T(0) 0.62 (53) o" O) BS2(Mi) 0.33 (2) 0.50 (6) 6~(0) 0.33 (1) 6" 0) SEQUENCES

[0295] SEQ ID NO:1 (adhE2) MKVTNQKELKQKLNELREAQKKFATYTQEQVDKIFKQCAIAAAKERINLAKLAVEETGI GLVEDKIIKNHFAAEYIYNKYKNEKTCGIIDHDDSLGITKVAEPIGIVAAIVPTTNPTSTAI FKSLISLKTRNAIFFSPHPRAKKSTIAAAKLILDAAVKAGAPKNIIGWIDEPSIELSQDLMS EADIILATGGPSMVKAAYSSGKPAIGVGAGNTPAIIDESADIDMAVSSIILSKTYDNGVIC ASEQSILVMNSIYEKVKEEFVKRGSYILNQNEIAKIKETMFKNGAINADIVGKSAYIIAKM AGIEVPQTTKILIGEVQSVEKSELFSHEKLSPVLAMYKVKDFDEALKKAQRLIELGGSGH TSSLYIDSQNNKDKVKEFGLAMKTSRTFINMPSSQGASGDLYNFAIAPSFTLGCGTWGG NSVSQNVEPKHLLNIKSVAERRENMLWFKVPQKIYFKYGCLRFALKELKDMNKKRAFI VTDKDLFKLGYVNKITKVLDEIDIKYSIFTDIKSDPTIDSVKKGAKEMLNFEPDTIISIGGG SPMDAAKVMHLLYEYPEAEIENLAINFMDIRKRICNFPKLGTKAISVAIPTTAGTGSEATP FAVITNDETGMKYPLTSYELTPNMAIIDTELMLNMPRKLTAATGIDALVHAIEAYVSVM ATDYTDELALRAIKMIFKYLPRAYKNGTNDIEAREKMAHASNIAGMAFANAFLGVCHS MAHI< LGAMHHVPHGIACAVLIEEVII< YNATDCPTI< QTAFPQYI< SPNAI< RI< YAEIAEYL NLKGTSDTEKVTALIEAISKLKIDLSIPQNISAAGINKKDFYNTLDKMSELAFDDQCTTAN PRYPLISELKDIYIKSF SEQ ID NO: 2 (adhE2 nucleotide) ATGAAAGTTACAAATCAAAAAGAACTAAAACAAAAGCTAAATGAATTGAGAGAAG CGCAAAAGAAGTTTGCAACCTATACTCAAGAGCAAGTTGATAAAATTTTTAAACAAT GTGCCATAGCCGCAGCTAAAGAAAGAATAAACTTAGCTAAATTAGCAGTAGAAGAA ACAGGAATAGGTCTTGTAGAAGATAAAATTATAAAAAATCATTTTGCAGCAGAATA TATATACAATAAATATAAAAATGAAAAAACTTGTGGCATAATAGACCATGACGATT CTTTAGGCATAACAAAGGTTGCTGAACCAATTGGAATTGTTGCAGCCATAGTTCCTA CTACTAATCCAACTTCCACAGCAATTTTCAAATCATTAATTTCTTTAAAAACAAGAA ACGCAATATTCTTTTCACCACATCCACGTGCAAAAAAATCTACAATTGCTGCAGCAA AATTAATTTTAGATGCAGCTGTTAAAGCAGGAGCACCTAAAAATATAATAGGCTGG ATAGATGAGCCATCAATAGAACTTTCTCAAGATTTGATGAGTGAAGCTGATATAATA TTAGCAACAGGAGGTCCTTCAATGGTTAAAGCGGCCTATTCATCTGGAAAACCTGCA ATTGGTGTTGGAGCAGGAAATACACCAGCAATAATAGATGAGAGTGCAGATATAGA TATGGCAGTAAGCTCCATAATTTTATCAAAGACTTATGACAATGGAGTAATATGCGC TTCTGAACAATCAATATTAGTTATGAATTCAATATACGAAAAAGTTAAAGAGGAATT TGTAAAACGAGGATCATATATACTCAATCAAAATGAAATAGCTAAAATAAAAGAAA CTATGTTTAAAAATGGAGCTATTAATGCTGACATAGTTGGAAAATCTGCTTATATAA TTGCTAAAATGGCAGGAATTGAAGTTCCTCAAACTACAAAGATACTTATAGGCGAA GTACAATCTGTTGAAAAAAGCGAGCTGTTCTCACATGAAAAACTATCACCAGTACTT GCAATGTATAAAGTTAAGGATTTTGATGAAGCTCTAAAAAAGGCACAAAGGCTAAT AGAATTAGGTGGAAGTGGACACACGTCATCTTTATATATAGATTCACAAAACAATA AGGATAAAGTTAAAGAATTTGGATTAGCAATGAAAACTTCAAGGACATTTATTAAC ATGCCTTCTTCACAGGGAGCAAGCGGAGATTTATACAATTTTGCGATAGCACCATCA TTTACTCTTGGATGCGGCACTTGGGGAGGAAACTCTGTATCGCAAAATGTAGAGCCT AAACATTTATTAAATATTAAAAGTGTTGCTGAAAGAAGGGAAAATATGCTTTGGTTT AAAGTGCCACAAAAAATATATTTTAAATATGGATGTCTTAGATTTGCATTAAAAGAA TTAAAAGATATGAATAAGAAAAGAGCCTTTATAGTAACAGATAAAGATCTTTTTAA ACTTGGATATGTTAATAAAATAACAAAGGTACTAGATGAGATAGATATTAAATACA GTATATTTACAGATATTAAATCTGATCCAACTATTGATTCAGTAAAAAAAGGTGCTA AAGAAATGCTTAACTTTGAACCTGATACTATAATCTCTATTGGTGGTGGATCGCCAA TGGATGCAGCAAAGGTTATGCACTTGTTATATGAATATCCAGAAGCAGAAATTGAA AATCTAGCTATAAACTTTATGGATATAAGAAAGAGAATATGCAATTTCCCTAAATTA GGTACAAAGGCGATTTCAGTAGCTATTCCTACAACTGCTGGTACCGGTTCAGAGGCA ACACCTTTTGCAGTTATAACTAATGATGAAACAGGAATGAAATACCCTTTAACTTCT TATGAATTGACCCCAAACATGGCAATAATAGATACTGAATTAATGTTAAATATGCCT AGAAAATTAACAGCAGCAACTGGAATAGATGCATTAGTTCATGCTATAGAAGCATA TGTTTCGGTTATGGCTACGGATTATACTGATGAATTAGCCTTAAGAGCAATAAAAAT GATATTTAAATATTTGCCTAGAGCCTATAAAAATGGGACTAACGACATTGAAGCAA GAGAAAAAATGGCACATGCCTCTAATATTGCGGGGATGGCATTTGCAAATGCTTTCT TAGGTGTATGCCATTCAATGGCTCATAAACTTGGGGCAATGCATCACGTTCCACATG GAATTGCTTGTGCTGTATTAATAGAAGAAGTTATTAAATATAACGCTACAGACTGTC CAACAAAGCAAACAGCATTCCCTCAATATAAATCTCCTAATGCTAAGAGAAAATAT GCTGAAATTGCAGAGTATTTGAATTTAAAGGGTACTAGCGATACCGAAAAGGTAAC AGCCTTAATAGAAGCTATTTCAAAGTTAAAGATAGATTTGAGTATTCCACAAAATAT AAGTGCCGCTGGAATAAATAAAAAAGATTTTTATAATACGCTAGATAAAATGTCAG AGCTTGCTTTTGATGACCAATGTACAACAGCTAATCCTAGGTATCCACTTATAAGTG AACTTAAGGATATCTATATAAAATCATTTTAA SEQ ID NO: 3 (mmCAR) MSPITREERLERRIQDLYANDPQFAAAKPATAITAAIERPGLPLPQIIETVMTGYADRPAL AQRSVEFVTDAGTGHTTLRLLPHFETISYGELWDRISAL DVLSTEQTVKPGDRVCLLGF NSVDYATIDMTLARLGAVAVPLQTSAAITQLQPIVAETQPTMIAASVDAL DATELALS GQTATRVLVFDHHRQVDAHRAAVESARERLAGSAVVETLAEAIARGDVPRGASAGSAP GTDVSDDSLALLIYTSGSTGAPKGAMYPRRNVATFWRKRTWFEGGYEPSITLNFMPMS HVMGRQILYGTLCNGGTAYFVAKSDLSTLFEDLALVRPTELTFVPRVWDMVFDEFQSE VDRRLVDGADRVALEAQVKAEIRNDVLGGRYTSALTGSAPISDEMKAWVEELLDMHL VEGYGSTEAGMILIDGAIRRPAVLDYKLVDVPDLGYFLTDRPHPRGELLVKTDSLFPGY YQRAEVT DVFDADGFYRTGDIMAEVGPEQFVYLDRRNNVLKLSQGEFVTVSKLEAVF GDSPLVRQIYIYGNSARAYLLAVIVPTQEALDAVPVEELKARLGDSLQEVAKAAGLQSY EIPRDFIIETTPWTLENGLLTGIRKLARPQLKKHYGELLEQIYTDLAHGQADELRSLRQSG ADAPVLVTVCRAAAALLGGSASDVQPDAHFTDLGGDSLSALSFTNLLHEIFDIEVPVGVI VSPANDLQAL DYVEAARKPGSSRPTFASVHGASNGQVTEVHAGDLSLDKFIDAATLA EAPRLPAANTQVRTVLLTGATGFLGRYLALEWLERMDLVDGKLICLVRAKSDTEARAR LDKTFDSGDPELLAHYRALAGDHLEVLAGDKGEADLGLDRQTWQRLADTVDLIVDPA ALVNHVLPYSQLFGPNALGTAELLRLALTSKIKPYSYTSTIGVADQIPPSAFTEDADIRVIS ATRAVDDSYANGYSNSKWAGEVLLREAHDLCGLPVAVFRCDMILADTTWAGQLNVPD MFTRMILSLAATGIAPGSFYELAADGARQRAHYDGLPVEFIAEAISTLGAQSQDGFHTYH VMNPYDDGIGLDEFVDWLNESGCPIQRIADYGDWLQRFETALRALPDRQRHSSLLPLLH NYRQPERPVRGSIAPTDRFRAAVQEAKIGPDKDIPHVGAPIIVKYVSDLRLLGLL SEQ ID NO: 4 (mmCAR nucleotide) ATGTCCCCAATCACCCGTGAAGAACGTTTGGAACGTCGTATTCAGGACCTTTACGCA AATGATCCCCAGTTCGCCGCGGCGAAACCTGCCACAGCAATTACAGCAGCGATCGA ACGCCCAGGCTTGCCTCTTCCGCAAATCATCGAGACGGTTATGACGGGGTATGCTGA CCGCCCTGCGCTTGCTCAGCGCTCTGTTGAGTTCGTCACGGATGCCGGTACAGGTCA CACGACTTTACGCTTATTACCTCACTTCGAGACGATTTCTTACGGTGAGCTGTGGGA CCGCATTTCCGCGCTTGCGGACGTGTTGTCAACAGAGCAGACGGTCAAGCCGGGGG ACCGTGTTTGTCTGTTGGGGTTCAACTCGGTAGACTATGCCACGATTGATATGACGT TGGCCCGCTTGGGTGCTGTTGCTGTTCCGCTTCAGACCTCAGCTGCCATTACTCAGTT ACAGCCGATCGTCGCAGAAACTCAGCCAACCATGATTGCCGCGAGTGTTGACGCGC TTGCAGACGCAACTGAGTTGGCGTTGTCAGGTCAAACAGCCACACGTGTGTTAGTCT TTGACCATCATCGCCAGGTGGACGCTCACCGCGCTGCAGTCGAATCTGCTCGTGAGC GTCTTGCGGGATCAGCGGTCGTCGAAACACTGGCCGAAGCGATCGCGCGCGGAGAC GTTCCTCGCGGCGCTTCAGCAGGATCTGCCCCTGGAACCGATGTTAGTGACGATTCA TTGGCTCTTTTGATCTACACTTCGGGCAGTACAGGTGCCCCGAAAGGTGCGATGTAC CCTCGCCGTAATGTCGCCACATTTTGGCGTAAGCGTACCTGGTTCGAGGGAGGTTAC GAGCCGAGCATTACTCTGAATTTTATGCCAATGAGCCATGTTATGGGCCGCCAAATT CTGTATGGGACATTGTGCAACGGGGGGACGGCTTATTTCGTAGCCAAGTCTGATCTG AGCACTCTTTTTGAAGATCTGGCTCTGGTTCGCCCCACCGAGCTGACCTTCGTACCGC GCGTATGGGATATGGTCTTTGACGAATTTCAGTCTGAGGTAGATCGTCGTTTAGTGG ACGGAGCAGATCGCGTCGCCTTGGAGGCGCAGGTCAAGGCAGAGATCCGCAATGAC GTCTTGGGGGGACGTTACACCAGTGCCCTGACAGGCTCAGCTCCTATTAGTGACGAA ATGAAAGCATGGGTTGAAGAGTTGTTAGACATGCATCTGGTAGAAGGATATGGGTC CACAGAGGCGGGCATGATCTTGATCGACGGGGCCATCCGCCGTCCCGCGGTTCTGG ACTATAAACTGGTGGACGTCCCGGACCTTGGTTATTTTCTGACGGACCGTCCACACC CGCGTGGTGAGTTGCTTGTCAAAACGGATAGCTTATTCCCCGGTTACTATCAACGTG CTGAGGTCACAGCAGACGTCTTCGACGCCGATGGCTTTTATCGTACCGGAGACATCA TGGCGGAAGTCGGCCCCGAGCAGTTCGTTTACTTAGACCGCCGCAATAACGTCTTGA AGTTGAGCCAGGGCGAGTTTGTGACCGTGAGCAAGCTTGAAGCAGTATTCGGTGAC TCGCCGCTTGTCCGTCAGATCTACATTTATGGGAATTCAGCCCGTGCTTATTTGCTGG CCGTAATTGTCCCCACGCAAGAAGCTTTAGATGCCGTCCCTGTTGAAGAATTGAAAG CGCGTTTAGGGGACAGCCTGCAGGAGGTTGCTAAAGCCGCCGGTCTGCAAAGCTAC GAAATTCCGCGCGACTTCATCATCGAAACGACCCCGTGGACCCTTGAAAATGGCCTG TTAACTGGAATCCGTAAACTGGCGCGCCCGCAGCTGAAAAAACACTACGGCGAATT GTTAGAACAAATTTACACCGACCTGGCCCACGGACAAGCTGATGAGCTTCGTTCACT GCGCCAATCCGGTGCTGACGCGCCGGTCCTGGTAACTGTCTGCCGTGCAGCAGCCGC CCTTTTGGGTGGGTCTGCCTCAGATGTGCAGCCAGATGCTCATTTCACAGACTTGGG CGGCGATAGTCTGTCTGCGTTATCATTCACTAATTTACTGCATGAGATCTTTGACATT GAAGTACCTGTCGGCGTGATTGTCTCCCCTGCTAACGATCTGCAAGCGCTTGCGGAC TACGTCGAAGCGGCACGTAAACCAGGTTCCTCACGTCCAACCTTTGCCAGCGTACAC GGTGCGAGTAATGGACAGGTGACCGAGGTTCATGCCGGAGATCTTAGCTTAGACAA GTTCATTGACGCCGCAACTTTGGCAGAAGCGCCCCGTTTACCTGCTGCTAACACACA GGTACGTACTGTTTTGTTAACAGGAGCAACCGGATTTCTGGGGCGCTATCTTGCACT TGAGTGGCTGGAACGTATGGACTTGGTGGATGGAAAGTTAATTTGCCTTGTACGCGC AAAAAGCGACACCGAGGCCCGTGCGCGTCTTGATAAAACCTTTGACAGCGGTGACC CAGAGTTACTTGCACACTACCGCGCATTAGCTGGGGACCATCTTGAAGTGTTGGCGG GAGATAAGGGCGAGGCGGACCTTGGGTTGGATCGTCAGACTTGGCAGCGCCTGGCA GATACAGTCGACCTTATTGTTGACCCGGCTGCCCTTGTGAATCACGTATTACCATACT CTCAATTGTTTGGGCCTAACGCATTGGGTACTGCTGAACTGTTACGCTTAGCGTTAA CCTCCAAAATTAAGCCCTATTCTTATACTTCGACAATCGGAGTAGCCGACCAAATCC CCCCTAGTGCATTCACGGAGGACGCTGACATCCGTGTTATCTCCGCAACTCGCGCCG TAGATGATTCTTACGCGAACGGTTACTCGAATAGTAAATGGGCAGGCGAAGTTTTAT TGCGTGAAGCTCACGACTTGTGTGGGCTTCCCGTGGCCGTGTTTCGTTGCGACATGA TCTTAGCCGACACAACTTGGGCGGGCCAGCTTAATGTACCGGATATGTTCACGCGCA TGATCTTATCGTTAGCAGCTACCGGTATTGCTCCTGGCTCTTTTTACGAACTGGCTGC CGATGGTGCTCGCCAACGCGCCCACTACGATGGTCTTCCAGTGGAGTTCATTGCCGA GGCCATTTCAACACTTGGCGCTCAATCCCAGGATGGCTTCCACACATACCATGTAAT GAATCCTTATGACGATGGCATCGGCCTTGATGAGTTCGTTGACTGGTTAAACGAGAG TGGCTGCCCCATCCAGCGTATCGCGGACTATGGAGATTGGCTTCAGCGTTTCGAAAC CGCGCTTCGTGCGCTTCCAGATCGTCAGCGCCATTCGTCACTGTTGCCCCTTTTGCAC AATTATCGTCAACCCGAGCGCCCCGTGCGTGGGAGCATCGCGCCTACTGACCGCTTT CGCGCGGCCGTCCAGGAGGCAAAGATTGGCCCGGACAAGGATATCCCACACGTTGG GGCTCCGATTATCGTGAAATATGTTAGTGATCTGCGCCTTCTTGGATTGCTTTAA SEQ ID NO: 5 (PTDH) MLPKLVITHRVHDEILQLLAPHCELMTNQTDSTLTREEILRRCRDAQAMMAFMPDRVD ADFLQACPELRVVGCALKGFDNFDVDACTARGVWLTFVPDLLTVPTAELAIGLAVGLG RHLRAADAFVRSGEFQGWQPQFYGTGLDNATVGILGMGAIGLAMADRLQGWGATLQY HARKALDTQTEQRLGLRQ VAC SELF AS S DF I LLA LPLN A DTQHLVN AELLA LVRPG A LL VNPCRGSVVDEAAVLAALERGQLGGYAADVFEMEDWARADRPRLIDPALLAHPNTLFT PHIGSAVRAVRLEIERCAAQNIIQVLAGARPINAANRLPKAEPAAC SEQ ID NO: 6 (PTDH nucleotide) ATGTTACCCAAGCTTGTAATTACTCACAGAGTACATGATGAAATTTTACAATTATTA GCTCCTCATTGTGAGTTAATGACTAATCAAACTGATAGTACTTTGACTCGTGAAGAA ATATTAAGAAGATGCAGAGATGCACAAGCTATGATGGCTTTTATGCCGGATAGAGTT GATGCAGATTTTTTACAAGCTTGTCCCGAATTAAGAGTTGTTGGATGCGCTTTAAAA GGCTTTGATAATTTTGATGTAGATGCTTGTACTGCAAGAGGAGTATGGTTGACTTTTG TACCTGATCTTTTGACTGTTCCAACAGCAGAATTAGCAATAGGTCTTGCAGTAGGAT TAGGAAGACATCTTAGAGCTGCTGATGCATTTGTAAGGTCAGGAGAATTTCAAGGTT GGCAACCCCAATTTTATGGAACTGGTTTAGATAATGCTACAGTAGGTATTTTGGGTA TGGGGGCAATAGGGTTAGCTATGGCTGATAGATTACAAGGCTGGGGTGCAACTTTA CAATATCATGCAAGAAAAGCTTTAGATACACAAACAGAACAAAGATTAGGGTTAAG ACAAGTAGCATGTTCTGAATTATTCGCTAGTTCTGATTTTATTTTATTAGCTCTTCCA TTAAACGCTGATACACAACACCTTGTTAATGCTGAGTTATTAGCTTTAGTAAGACCT GGTGCTTTATTAGTTAATCCATGTAGGGGTTCAGTTGTAGATGAAGCAGCAGTATTA GCTGCTTTAGAAAGGGGACAATTGGGCGGTTATGCTGCTGATGTTTTTGAGATGGAA GATTGGGCAAGAGCAGATAGACCACGTTTGATAGATCCTGCATTATTAGCTCATCCT AATACTTTATTCACACCTCACATTGGAAGCGCAGTTAGAGCAGTTCGTTTAGAAATA GAAAGATGTGCAGCTCAAAATATAATACAAGTTTTAGCTGGTGCTAGACCGATTAAT GCAGCTAATAGATTACCTAAAGCAGAACCAGCAGCATGTTAA SEQ ID NO: 7 (sfp) MKIYGIYMDRPLSQEENERFMTFISPEKREKCRRFYHKEDAHRTLLGDVLVRSVISRQYQ LDKSDIRFSTQEYGKPCIPDLPDAHFNISHSGRWVIGAFDSQPIGIDIEKTKPISLEIAKRFFS KTEYSDLLAKDKDEQTDYFYHLWSMKESFIKQEGKGLSLPLDSFSVRLHQDGQVSIELP DSHSPCYIKTYEVDPGYKMAVCAAHPDFPEDITMVSYEELL SEQ ID NO: 8 (sfp nucleotide) ATGAAAATCTATGGCATTTACATGGATCGTCCGCTGAGTCAGGAAGAAAACGAACG CTTTATGACCTTCATCAGCCCGGAAAAACGTGAAAAATGCCGTCGCTTTTATCATAA AGAAGATGCACACCGCACGCTGCTGGGCGATGTGCTGGTTCGTAGCGTGATCTCTCG CCAGTATCAGCTGGATAAATCTGATATTCGTTTCAGTACCCAGGAATACGGTAAACC GTGTATTCCGGATCTGCCGGATGCACATTTTAATATCAGCCACTCTGGCCGCTGGGT TATTGGTGCGTTCGATTCTCAGCCGATTGGTATCGATATTGAAAAAACGAAACCGAT CAGTCTGGAAATTGCCAAACGTTTCTTTAGCAAAACCGAATATTCTGATCTGCTGGC AAAAGATAAAGATGAACAGACGGATTACTTTTACCATCTGTGGAGTATGAAAGAAT CTTTTATCAAACAGGAAGGCAAAGGTCTGAGCCTGCCGCTGGATAGTTTTAGCGTGC GCCTGCATCAGGATGGCCAGGTTTCTATCGAACTGCCGGATTCTCACAGTCCGTGCT ATATTAAAACCTACGAAGTTGATCCGGGCTATAAAATGGCCGTTTGTGCGGCCCACC CGGATTTCCCGGAAGATATTACGATGGTGAGCTACGAAGAACTGCTGTAA SEQ ID NO: 9 (CAT1 promoter) AGAGCCCAAATCTTTGAAAATAATGTTCTTTCTTTGTATAGAAAGGACATTATTTTTT TATAGTTGTTTTGTAAACCATAGCATTGTTAAGTTATTTTCAGCTACAGCTATTATTT TAATAATAACATTGATGTAATTATGTTATTTTAACCAAAAGAAAATCATATTAATTTT GAATAAATGGATATATTATAATATAATATTAAAAGGAAGTTCAGGTTGTATATTATA CAACATCTATTTTTTACTCATAATTGTAGTTTTTTTAACAATCATAATGGAAGTTAAT TATTAAATTTTATATAATTTATGAAAGGGTGGTTTTT SEQ ID NO: 10 (tRNA inserts) ATTAAAAACTTTAAAAAATTTTGAAATAACTATTGACGAATTACATTATTTCGTGTA GAATAATCTATGTCGTTGAGTGCTTGAGAGTTTTCAAAGAAAACTTGGGTACAGACA ATAGATTTATTGATTTTAATTAAATACTGTATGGGGCATTAGCGCAGTTGGGAGCGC ATCACACTGGCAGTGTGGGGGTCAGGGGTTCAAGTCCCCTATGCTCCACCATTATGA CGGATTAGCTCAGTTGGTTAGAGTACTACCTTGACATGGTAGGGGTCACTGGTTCGA GCCCAGTATTCGTCACCAGAGTGCTTGGCGTCCCGGACAGGAATCGAACCCGTATCA GTGGAACCGGAATCCACCGTCTTATCCATTGGACTACCGAGACATATTGATAAGTCC ATGATTGCGCTAGTAGCTCAGTTGGATAGAGCAGTTGGCTACGAACCAGCGTGCCG GGGGTTCGAATCCTCTCTGGCGCACCATTTTGTTTATAAGGAAATTTTATATTTTGAG CACCCATAGCTCAGCTGGATAGAGTGATGGACTTCGAATCCAGAGGCCGTGGGTTC GAATCCCACTGGGTGCACCATAAAACATTGATAACACTGACTTTCAGTGTTTTTTATT TTCCTCTAAATTTGATATTCCCG SEQ ID NO: 11 (P619 REV_adhE2)

[0296] GTATTTCATTCCTGTTTCATCAT SEQ ID NO: 12 (P619I FWD adhE2) ATGAAACAGGAATGAAATACGGATTAACTTCTTATGAATTGACCCCAA SEQ ID NO: 13 (V608S_REV_adhE2)

[0297] TGCAAAAGGTGTTGCCTCTG SEQ ID NO: 14 (V608S_FWD adhE2) CAGAGGCAACACCTTTTGCATCAATAACTAATGATGAAACAGGAATG SEQ ID NO: 15 (D485G_FWD adhE2) GAGCCTTTATAGTAACAGGTAAAGATCTTTTTAAACTTGGATATG SEQ ID NO: 16 (D485G_REV_adhE2) TGTTACTATAAAGGCTCTTTTCTTATTC

[0298] SEQ ID NO: 17 (L488A FWD adhE2) TTTAAACTTGGATATGTTAATAAAATAAC SEQ ID NO: 18 (L488A REV_adhE2)

[0299] ACATATCCAAGTTTAAATGCATCTTTATCTGTTACTATAAAGGC

Claims

CLAIMSWhat is claimed is:

1. A recombinant microorganism (e.g., Clostridium tyrobutyricum), wherein the recombinant microorganism is engineered cell to express or overexpress a heterologous carboxylic acid reductase (CAR) gene and a heterologous aldehyde / alcohol dehydrogenase E2 (adhE2) gene.

2. The recombinant microorganism of claim 1, wherein the heterologous aldehyde / alcohol dehydrogenase E2 (adhE2) gene comprises one or mutations that increase cofactor selectivity, butyraldehyde selectivity, or a combination thereof.

3. The recombinant microorganism of claim 2, wherein the heterologous adhE2 gene encodes a heterologous adhE2 enzyme that is at least 90% identical to SEQ ID NO: 1, wherein the heterologous adhE2 enzyme comprises one or more mutations selected from D485G, L488A, S601A, V608S, P619G, and combinations thereof.

4. The recombinant microorganism of any one of claims 1-3, wherein the heterologous CAR gene is derived from Mycobacterium marinum.

5. The recombinant microorganism of claim 4, wherein the heterologous CAR gene encodes a heterologous CAR enzyme that is at least 90% identical to SEQ ID NO: 3.

6. The recombinant microorganism of any one of claims 1-5, wherein the heterologous CAR gene encodes a heterologous CAR enzyme comprising a mutation that increases cofactor selectivity.

7. The recombinant microorganism of any one of claims 1-6, wherein the heterologous CAR gene encodes an enzyme that is at least 90% identical to SEQ ID NO: 3 and comprises one or more mutations selected from R507F, N508G, and combinations thereof.

8. The recombinant microorganism of any one of claims 1-7, wherein a heterologous sfp gene is overexpressed.

9. The recombinant microorganism of claim 8, wherein the sfp gene is derived from Bacillus subtilis.

10. The recombinant microorganism of claim 9, wherein the sfp gene encodes an enzyme that is at least 90% identical to SEQ ID NO: 7.

11. The recombinant microorganism of any one of claims 1-10, wherein an additional tRNA construct is expressed.

12. The recombinant microorganism of claim 11, wherein the additional tRNA construct expressed has at least 90% identity to SEQ ID NO: 10.

13. The recombinant microorganism of any one of claims 1-12, wherein phosphite dehydrogenase (PTDH) is overexpressed.

14. The recombinant microorganism of claim 13, wherein the PTDH is at least 90% identical to SEQ ID NO: 5.

15. The recombinant microorganism of any one of claims 13-14, wherein the PTDH comprises a mutation that increases NADPH regeneration.

16. The recombinant microorganism of any one of claims 14-15, wherein the PTDH comprises an amino acid substitution selected from G175A, A176R, and combinations thereof.

17. The recombinant microorganism of any one of claims 1-16, wherein overexpression of the heterologous CAR gene, the heterologous adhE2 gene, and / or the PTDH gene is driven by a promoter endogenous to C. tyrobutyricum or C. acetobutylicum.

18. The recombinant microorganism of claim 17, wherein the promoter is a THL, a CAT1, or an FLA promoter.

19. A method of biosynthesizing butanol, the method comprising fermenting the recombinant microorganism of any one of claims 1-18 with a growth medium and a carbohydrate- containing source under conditions effective to obtain a butanol containing product.

20. The method of claim 19, wherein the growth medium is clostridial growth medium.

21. The method of any one of claims 19-20, wherein the carbohydrate-containing source comprises a sugar selected glucose, xylose, mannitol, and combinations thereof.

22. The method of any one of claims 19-21, wherein the growth medium and carbohydrate- containing source are supplemented with at least one additional organic compound.

23. The method of claim 22, wherein the at least one additional organic compound is methyl viologen (MV).

24. The method of claim 23, wherein the MV is supplemented after 12 hours.

25. The method of claim 22, wherein the at least one additional organic compound is butyrate.

26. The method of claim 25, wherein the butyrate is supplemented after 24 hours.

27. The method of claim 19, wherein the growth medium and carbohydrate-containing source are supplemented with both MV and butyrate.

28. The method of any one of claims 19-27, wherein the butanol yield is equal to or greater than 0.23 g / g sugar.

29. A system useful for the biosynthesis of butanol, the system comprising:a) a container suitable for fermentation;b) the recombinant microorganism of any one of claims 1-18;c) growth medium; andd) a carbohydrate-containing source.

30. The system of claim 29 further comprising methyl viologen and butyrate.

31. A system configured to perform the method of any one of claims 19-27.

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