# Patent

## Title
Enzyme variants with improved ester synthase properties

## Abstract

The disclosure relates to enzyme variants with improved ester synthase properties for the production of fatty acid esters. Further contemplated are recombinant host cells that express such variants, cell cultures comprising the recombinant host cells and fatty acid ester compositions produced by such recombinant host cells.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the National Stage of International Application No. PCT/US2013/032564, filed Mar. 15, 2013, which claims the benefit of U.S. Provisional Application No. 61/708,424, filed Oct. 1, 2012, and U.S. Provisional Application No. 61/701,191, filed Sep. 14, 2012, the entire disclosures of which are hereby incorporated by reference.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy, created on Jan. 17, 2017, is named 109112-0388 SL.txt and is 167,781 bytes in size.
FIELD
The disclosure relates to enzyme variants with improved ester synthase properties for the production of fatty esters.
BACKGROUND
Petroleum is a limited, natural resource found in the earth in liquid, gaseous, or solid forms. However, petroleum products are developed at considerable costs, both financial and environmental. In its natural form, crude petroleum extracted from the Earth has few commercial uses. It is a mixture of hydrocarbons, e.g., paraffins (or alkanes), olefins (or alkenes), alkynes, napthenes (or cylcoalkanes), aliphatic compounds, aromatic compounds, etc. of varying length and complexity. In addition, crude petroleum contains other organic compounds (e.g., organic compounds containing nitrogen, oxygen, sulfur, etc.) and impurities (e.g., sulfur, salt, acid, metals, etc.). Due to its high energy density and its easy transportability, most petroleum is refined into fuels, such as transportation fuels (e.g., gasoline, diesel, aviation fuel, etc.), heating oil, liquefied petroleum gas, etc.
Petrochemicals can be used to make specialty chemicals, such as plastics, resins, fibers, elastomers, pharmaceuticals, lubricants, or gels. Examples of specialty chemicals which can be produced from petrochemical raw materials include fatty acids, hydrocarbons (e.g., long chain hydrocarbons, branched chain hydrocarbons, saturated hydrocarbons, unsaturated hydrocarbons, etc.), fatty alcohols, fatty esters, fatty aldehydes, ketones, lubricants, etc. Specialty chemicals have many commercial uses. Fatty acids are used commercially as surfactants. Surfactants can be found in detergents and soaps. Fatty acids can also be used as additives in fuels, lubricating oils, paints, lacquers, candles, shortenings, cosmetics, and emulsifiers. In addition, fatty acids are used as accelerator activators in rubber products. Fatty acids can also be used as a feedstock to produce methyl esters, amides, amines, acid chlorides, anhydrides, ketene dimers, and peroxy acids and esters.
Esters have many commercial uses. For example, biodiesel, an alternative fuel, is comprised of esters (e.g., fatty acid methyl ester, fatty acid ethyl esters, etc.). Some low molecular weight esters are volatile with a pleasant odor which makes them useful as fragrances or flavoring agents. In addition, esters are used as solvents for lacquers, paints, and varnishes. Furthermore, some naturally occurring substances, such as waxes, fats, and oils are comprised of esters. Esters are also used as softening agents in resins and plastics, plasticizers, flame retardants, and additives in gasoline and oil. In addition, esters can be used in the manufacture of polymers, films, textiles, dyes, and pharmaceuticals.
There is a need for alternative routes to create both fuels and products currently derived from petroleum. As such, microbial systems offer the potential for the biological production of numerous types of biofuels and chemicals. Renewable fuels and chemicals can be derived from genetically engineered organisms (such as bacteria, yeast and algae). Naturally occurring biosynthetic pathways can be genetically altered to enable engineered organisms to synthesize renewable fuel and chemical products. In addition, microbes can be tailored, or metabolically engineered, to utilize various carbon sources as feedstock for the production of fuel and chemical products. For example, FAME (fatty acid methyl ester) can be produced by adding methanol to a culture of a recombinant host cell expressing a wax ester synthase, for example, a wax ester synthase derived from Marinobacter hydrocarbonoclasticus (“M. hydrocarbonoclasticus”). However, the expression of the wild type wax ester synthase from M. hydrocarbonoclasticus does not produce significant amounts of FAME, thus development of an improved wax ester synthase is highly desirable. In addition, the wild-type wax ester synthase from M. hydrocarbonoclasticus produces significant amounts of beta hydroxy (“β-OH”) esters, which are not typically found in fuel or chemicals derived from plant sources. See, e.g., Patent Cooperation Treaty Application No. PCT/US12/31682, which is incorporated by reference herein. Thus, it would be desirable to engineer an ester synthase to produce higher yields of fatty ester; and an ester synthase that produces higher yields of fatty ester without producing significant amounts of β-OH ester, when expressed in a recombinant host cell. Finally, it would be desirable to engineer other enzymes that have ester synthase activity to further improve the yield of fatty ester production.
Notwithstanding the advances in the field, there remains a need for improvements in genetically modified enzymes, recombinant host cells, methods and systems in order to achieve robust and cost-effective production of fuels and chemicals of interest by fermentation of recombinant host cells. The present disclosure addresses this need by providing a number of ester synthase enzyme variants that increase the yield of fatty esters. Some of the ester synthase enzyme variants of the disclosure also alter (i.e., increase or reduce) the β-OH ester content of the fatty ester composition produced by fermentation of recombinant host cells that express the ester synthase enzyme variants. The present disclosure further addresses this need by providing a number of thioesterase variants that have been engineered to have greater ester synthase activity, thereby further enhancing fatty ester production.
SUMMARY
One aspect of the disclosure provides a variant ester synthase polypeptide having SEQ ID NO: 2 , wherein the variant ester synthase polypeptide is genetically engineered to have at least one mutation at an amino acid position including, but not limited to, amino acids 4, 5, 7, 15, 24, 30, 33, 39, 40, 41, 43, 44, 58, 73, 76, 77, 78, 80, 98, 99, 101, 102, 111, 122, 131, 146, 147, 149, 150, 155, 157, 162, 164, 166, 170, 171, 172, 173, 177, 179, 182, 184, 185, 186, 187, 188, 190, 192, 193, 195, 197, 201, 202, 203, 206, 207, 212, 216, 219, 234, 239, 242, 243, 244, 246, 255, 257, 258, 259, 260, 262, 263, 264, 265, 266, 267, 285, 288, 289, 293, 294, 301, 302, 303, 304, 306, 307, 309, 310, 311, 313, 314, 315, 316, 317, 319, 320, 323, 328, 334, 348, 349, 351, 352, 353, 356, 357, 360, 366, 375, 381, 393, 394, 395, 409, 411, 413, 420, 424, 442, 443, 447, 454, 455, 457, 458, 461, 466, 468, and 472, wherein the variant ester synthase polypeptide has improved fatty acid methyl ester activity compared to a corresponding wild type polypeptide. In a related aspect, the variant polypeptide is a Marinobacter hydrocarbonoclasticus (WS377) ester synthase polypeptide. In another related aspect, the expression of the variant ester synthase polypeptide in a recombinant host cell results in a higher titer of fatty ester compositions compared to a recombinant host cell expressing the corresponding wild type polypeptide. The titer is at least about 5 percent or greater. The fatty ester compositions include, but are not limited to, fatty acid methyl esters (FAME), fatty acid ethyl esters (FAEE), fatty acid propyl esters, fatty acid isopropyl esters, fatty acid butyl esters, monoglycerides, fatty acid isobutyl esters, fatty acid 2-butyl esters, and fatty acid tert-butyl esters. The variant ester synthase has improved ester synthase activity, particularly improved fatty acid methyl ester activity that results in improved properties including, but not limited to, increased beta hydroxy esters, decreased beta hydroxyl esters, increased chain lengths of fatty acid esters, and decreased chain lengths of fatty acid esters. With respect to the ester synthase polypeptide sequences described herein “M” (ATG) is considered to be amino acid “0”. The first amino acid after the ATG is designated amino acid “1”.
Another aspect of the disclosure provides a variant ester synthase polypeptide having SEQ ID NO: 2, wherein the variant ester synthase polypeptide is genetically engineered to have at least one mutation at an amino acid position including, but not limited to, amino acids 7, 24, 30, 41, 99, 111, 122, 146, 147, 149, 150, 171, 172, 173, 179, 187, 192, 212, 234, 239, 244, 246, 258, 264, 266, 267, 285, 301, 302, 303, 304, 306, 307, 309, 310, 311, 313, 314, 315, 316, 320, 348, 349, 352, 356, 357, 360, 375, 381, 393, 409, 411, 424, 443, 455, 457, 458, 461 and 472. The expression of the variant ester synthase polypeptide in a recombinant host cell may result in the production of an altered percentage of beta hydroxy esters as compared to a recombinant host cell expressing the corresponding wild type ester synthase polypeptide, wherein the altered percentage of beta hydroxy esters is an increased or decreased percentage of beta hydroxy esters. With respect to the ester synthase polypeptide sequences described herein “M” (ATG) is considered to be amino acid “0”. The first amino acid after the ATG is designated amino acid “1”.
Another aspect of the disclosure provides a variant ester synthase polypeptide having SEQ ID NO: 2, wherein the variant ester synthase polypeptide is genetically engineered to have at least one mutation at an amino acid position including, but not limited to, amino acids 5, 7, 15, 24, 33, 44, 58, 73, 78, 101, 111, 162, 171, 179, 184, 187, 188, 192, 201, 207, 212, 234, 243, 244, 255, 257, 267, 307, 310, 317, 320, 348, 349, 353, 357, 366, 381, 394, 409, 411, 413, 442, 443, and 461. With respect to the ester synthase polypeptide sequences described herein “M” (ATG) is considered to be amino acid “0”. The first amino acid after the ATG is designated amino acid “1”.
Another aspect of the disclosure provides a variant ester synthase polypeptide having SEQ ID NO: 2, wherein the variant ester synthase polypeptide is genetically engineered to have at least one mutation at an amino acid position including, but not limited to, amino acids G4R, T5P, T5S, D7N, S15G, T24M, T24W, L30H, G33D, G33S, L39A, L39M, L39S, R40S, D41A, D41G, D41H, D41Y, V43K, V43S, T44A, T44F, T44K, Y58C, Y58L, A73Q, V76L, D77A, K78F, K78W, 180V, R98D, E99Q, G101L, I102R, P111D, P111G, P111S, H122S, R131M, I146K, I146L, I146R, S147A, V149L, R150P, V155G, T157S, R162E, N164D, N164R, P166S, T170R, T170S, V171E, V171F, V171H, V171R, V171W, R172S, R172W, P173W, R177V, A179S, A179V, D182G, E184F, E184G, E184L, E184R, E184S, A185L, A185M, S186T, V187G, V187R, P188R, A190P, A190R, A190W, S192A, S192L, S192V, Q193R, Q193S, M195G, A197T, A197V, Q201A, Q201V, Q201W, A202L, D203R, P206F, R207A, G212A, G212L, V216I, V219L, V234C, H239G, T242K, T242R, A243R, Q244G, R246A, R246G, R246L, R246Q, R246V, R246W, D255E, L257I, K258R, N259E, N259Q, L260V, H262Q, A263V, S264D, S264V, S264W, G265N, G266A, G266S, S267G, A285L, A285R, A285V, N288D, N289E, N289G, T293A, P294G, V301A, N302G, I303G, I303R, I303W, R304W, A306G, D307F, D307G, D307L, D307N, D307R, D307V, E309A, E309G, E309S, G310H, G310R, G310V, T311S, T313S, Q314G, I315F, S316G, F317W, I319G, A320C, A323G, D328F, Q334K, Q334S, Q348A, Q348R, K349A, K349C, K349H, K349Q, P351G, K352I, K352N, S353K, S353T, T356G, T356W, Q357V, M360Q, M360R, M360S, M360W, Y366G, Y366W, G375A, G375V, G375S, V381F, E393G, E393R, E393W, G394E, T395E, V409L, L411A, A413T, I420V, S424G, S424Q, S442E, S442G, M443G, A447C, A447I, A447L, L454V, D455E, L457Y, E458W, I461G, I461L, I461V, K466N, A468G, K472T and K472. In a particular aspect of the disclosure the variant ester synthase polypeptide includes mutations D7N, A179V and V381F; D7N, A179V, Q348R and V381F; D7N, A179V, V187R, G212A, Q357V, V381F and M443G; T5S, S15G, P111S, V171R, P188R, F317W, S353T, V409L and S442G; T5S, S15G, K78F, P111S, V171R, P188R, S192V, A243R, F317W, K349H, S353T, V409L and S442G; T5S, S15G, V76L, P111S, V171R, P188R, K258R, S316G, F317W, S353T, M360R, V409L and S442G; T5S, S15G, P111S, V171R, P188R, Q244G, S267G, G310V, F317W, A320C, S353T, Y366W, V409L and S442G; S15G, P111S, V155G, P166S, V171R, P188R, F317W, Q348A, S353T, V381F, V409L and S442G; S15G, L39S, D77A, P111S, V171R, P188R, T313S, F317W, Q348A, S353T, V381F, V409L, I420V and S442G; T5S, S15G, T24W, T44F, P111S, I146L, V171R, P188R, D307N, F317W, S353T, V409L and S442G; T5S, S15G, K78F, P111S, V171R, P188R, S192V, R207A, A243R, D255E, L257I, N259Q, L260V, H262Q, G265N, A285V, N288D, N289G, F317W, Q348R, S353T, V381F, V409L and S442G; T5S, S15G, K78F, P111S, V171R, P188R, S192V, R207A, A243R, D255E, L257I, N259Q, L260V, H262Q, G265N, A285V, N288D, N289G, F317W, H349K, S353T, V381F, V409L and S442G; T5P, S15G, G33D, T44K, Y58L, P111S, R162E, V171R, P188R, R207A, V234C, Q244G, D255E, S267G, D307N, G310V, F317W, A320C, S353T, Y366W, G394E, V409L, S442G and I461V; T5P, S15G, G33D, T44K, Y58C, P111S, V171R, P188R, R207A, V234C, Q244G, L257I, S267G, D307N, G310V, F317W, A320C, S353T, Y366W, G394E, V409L, A413T, S442G and I461V; T5P, S15G, T44A, P111S, T157S, N164D, T170S, V171R, P188R, R207A, V216I, V234C, Q244G, L257I, S267G, D307N, G310V, F317W, A320C, Q334K, S353T, Y366W, G394E, V409L, S442G and I461V; or mutations T5P, S15G, Y58L, P111S, R162E, T170S, V171R, A179S, P188R, R207A, V234C, Q244G, L257I, S267G, D307N, G310V, F317W, A320C, S353K, Y366W, G394E, V409L, S442G and I461V. With respect to the ester synthase polypeptide sequences described herein “M” (ATG) is considered to be amino acid “0”. The first amino acid after the ATG is designated amino acid “1”.
Another aspect of the disclosure provides a recombinant host cell genetically engineered to express a variant ester synthase polypeptide. In a related aspect, the disclosure provides a cultured recombinant host cell having a polynucleotide sequence encoding a variant ester synthase polypeptide, wherein the recombinant host cell when expressing the variant ester synthase polypeptide produces a fatty acid ester composition with higher titer, higher yield and/or higher productivity of fatty acid esters compared to a host cell expressing the corresponding wild type polypeptide, when cultured in medium containing a carbon source under conditions effective to express the variant ester synthase polypeptide. The titer is at least about 5 percent or greater. In a related aspect, the recombinant host cell when expressing the variant ester synthase polypeptide produces a fatty ester composition with an altered percentage of beta hydroxy esters as compared to a host cell that expresses the wild type ester synthase polypeptide. Herein, the altered percentage of beta hydroxy esters is an increased or decreased percentage of beta hydroxy esters.
Still another aspect of the invention provides a cell culture including the recombinant host cell and a fatty ester composition. The fatty ester composition may include one or more of a C6, C8, C10, C12, C13, C14, C15, C16, C17 or C18 fatty ester. In addition, the fatty ester composition may include unsaturated fatty esters and/or saturated fatty esters and/or branched chain fatty esters. The fatty ester composition may further include a fatty ester having a double bond at position 7 in the carbon chain between C7 and C8 from the reduced end of the fatty ester.
The disclosure further contemplates a variant Marinobacter hydrocarbonoclasticus (WS377) ester synthase polypeptide having SEQ ID NO: 2 , wherein the variant WS377 polypeptide has improved fatty acid methyl ester activity compared to the wild type WS377 polypeptide, and wherein the variant WS377 has at least one mutation at an amino acid position including, but not limited to, amino acids 4, 5, 7, 15, 24, 30, 33, 39, 40, 41, 43, 44, 58, 73, 76, 77, 78, 80, 98, 99, 101, 102, 111, 122, 131, 146, 147, 149, 150, 155, 157, 162, 164, 166, 170, 171, 172, 173, 177, 179, 182, 184, 185, 186, 187, 188, 190, 192, 193, 195, 197, 201, 202, 203, 206, 207, 212, 216, 219, 234, 239, 242, 243, 244, 246, 255, 257, 258, 259, 260, 262, 263, 264, 265, 266, 267, 285, 288, 289, 293, 294, 301, 302, 303, 304, 306, 307, 309, 310, 311, 313, 314, 315, 316, 317, 319, 320, 323, 328, 334, 348, 349, 351, 352, 353, 356, 357, 360, 366, 375, 381, 393, 394, 395, 409, 411, 413, 420, 424, 442, 443, 447, 454, 455, 457, 458, 461, 466, 468, and 472. With respect to the ester synthase polypeptide sequences described herein “M” (ATG) is considered to be amino acid “0”. The first amino acid after the ATG is designated amino acid “1”.
Another aspect of the disclosure provides a variant thioesterase polypeptide comprising an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 51, wherein the variant ester synthase polypeptide is genetically engineered to have at least one mutation at an amino acid position including, but not limited to, amino acids 32, 33, 34, 38, 45, 46, 48, 49, 51, 52, 76, 81, 82, 84, 88, 112, 115, 116, 118, 119, 148, 149, 156, 159 and 164, wherein the variant thioesterase polypeptide has greater ester synthase activity compared to the corresponding wild type polypeptide. In a related aspect of the disclosure, the variant polypeptide is a Photobacterium profundum (Ppro) thioesterase polypeptide. The expression of the variant thioesterase polypeptide in a recombinant host cell results in a higher titer of fatty ester compositions compared to a recombinant host cell expressing the corresponding wild type polypeptide, wherein said titer is at least about 5 percent or greater.
Another aspect of the disclosure provides a variant thioesterase polypeptide having at least one mutation including, but not limited to, amino acids K32M, Q33A, Q33R, Q34R, I38Y, I45R, S46K, S46V, S46W, D48E, D48F, D48L, D48M, T49D, T49L,T49V, T49W, G51R, N52C, N52F, N52K, N52L, N52R, N76I, N76L, N76V, G81C, F82G, F82I, F82R, Q84A, Q84L, Q84V, R88H, V112L, N115F, N115W, N115Y, Y116H, Y116N, Y116P, Y116R, Y116S, Y116T, K118Q, R119C, I148Y, L149F, L149M, L149T, N156K, N156R, N156Y, L159K, L159M and D164T. A related aspect of the disclosure provides a variant thioesterase polypeptide comprising SEQ ID NO: 51 having mutations S46V and Y116H; S46V, D48M, G51R, N76V and Y116H; S46V, D48M, T49L, G51R, N76V, Y116H and I148Y; S46V, D48M, T49L, G51R, N76V, N115F, Y116H and I148Y; Q33R, Q34R, S46V, D48M, T49L, G51R, N52R, N76V, V112L, N115W, Y116H and I148Y; Q33R, S46V, D48M, T49L, G51R, N52K, N76V, V112L, N115W, Y116H, I148Y and L149M; or mutations S46V, D48M, T49L, G51R, N52R, N76V, Q84A, V112L, N115W, Y116H, I148Y and L149M.
The disclosure further encompasses a recombinant host cell genetically engineered to express a variant thioesterase polypeptide that has ester synthase activity. A cultured recombinant host cell including a polynucleotide sequence encoding a variant thioesterase polypeptide is further contemplated by the disclosure. The recombinant host cell, when expressing the variant thioesterase polypeptide, produces a fatty acid ester composition with higher titer, higher yield and/or higher productivity of fatty acid esters compared to a host cell expressing the corresponding wild type thioesterase polypeptide, when cultured in medium containing a carbon source under conditions effective to express the variant thioesterase polypeptide. The titer is at least about 5 percent or greater. The cell culture further includes the recombinant host cell and a fatty ester composition. The fatty ester composition may include one or more of a C6, C8, C10, C12, C13, C14, C15, C16, C17 or C18 fatty ester. In addition, the fatty ester composition may include unsaturated fatty esters and/or saturated fatty esters and/or branched chain fatty esters. The fatty ester composition may further include a fatty ester having a double bond at position 7 in the carbon chain between C7 and C8 from the reduced end of the fatty ester.
Another aspect of the disclosure provides a variant thioesterase polypeptide with ester synthase activity, wherein the variant thioesterase polypeptide includes amino acids 73-81 of SEQ ID NO: 51 as follows LG[AG][VIL]D[AG]LRG.
Yet, another aspect of the disclosure provides a variant Photobacterium profundum (Ppro) thioesterase polypeptide having at least about 90% sequence identity to a corresponding wild type Ppro polypeptide sequence having SEQ ID NO: 51, wherein the variant Ppro polypeptide has greater ester synthase activity compared to the wild type Ppro polypeptide, and where the variant Ppro polypeptide has at least one mutation including, but not limited to, amino acids 32, 33, 34, 38, 45, 46, 48, 49, 51, 52, 76, 81, 82, 84, 88, 112, 115, 116, 118, 119, 148, 149, 156, 159 and 164.
The disclosure still encompasses a recombinant host cell including (a) a polynucleotide encoding a variant polypeptide with increased ester synthase activity, (b) an activated fatty acid, and (c) an alcohol, wherein the recombinant host cell produces a fatty ester composition under conditions effective to express an ester synthase activity of the variant polypeptide. The activated fatty acid is an Acyl-ACP or an Acyl-CoA. The recombinant host cells may further utilize a carbon source. The fatty ester composition is produced at a titer of least about 5 percent or higher.
Another aspect of the disclosure provides a polynucleotide encoding a variant ester synthase polypeptide, wherein the polynucleotide has at least about 85% sequence identity to SEQ ID NO: 1, wherein the polynucleotide further includes SEQ ID NO: 27 prior to ATG start.
Yet, another aspect of the disclosure provides a variant ester synthase polypeptide having an amino acid sequence that has at least about 95% sequence identity to SEQ ID NO: 29; or 95% sequence identity to SEQ ID NO: 33; or 95% sequence identity to SEQ ID NO: 35; or 95% sequence identity to SEQ ID NO: 37; or 95% sequence identity to SEQ ID NO: 39; or 95% sequence identity to SEQ ID NO: 41; or 95% sequence identity to SEQ ID NO: 43. Herein, the expression of the variant ester synthase polypeptide in a recombinant host cell results in a production of a higher titer, higher yield, or increased productivity of fatty acid esters as compared to a recombinant host cell expressing a wild type Marinobacter hydrocarbonoclasticus (WS377) ester synthase polypeptide. The titer is at least about 5% or higher.



BRIEF DESCRIPTION OF THE FIGURES
The present disclosure is best understood when read in conjunction with the accompanying figures, which serve to illustrate the preferred embodiments. It is understood, however, that the disclosure is not limited to the specific embodiments disclosed in the figures.
FIG. 1 is a schematic depiction of an exemplary three gene biosynthetic pathway that may be incorporated into a recombinant host cells for production of fatty acid esters of various chain lengths. The abbreviations used include TE for thioesterase, FACS for fatty acyl CoA synthetase, and AT for acyltransferase.
FIG. 2 is a schematic depiction of a proposed futile cycle catalyzed by thioesterase.
FIG. 3 is a schematic depiction of two exemplary biosynthetic pathways for production of fatty esters starting with acyl-ACP, where the production of fatty esters is accomplished by a one enzyme system or a three enzyme system.
FIG. 4 shows the results of a GC-FID screen from a plate fermentation of combination library hits. The combination library was built using Group 0 primers and pKEV38 as a template, transformed into BD64.
FIG. 5 shows FAME titers and % β-OH FAME produced by mutant strains KEV040, KEV075 and KEV085 compared to a strain which contains wild-type '377 ester synthase.
FIG. 6 shows the titer of total fatty acid species produced by KEV040 (Wax) and becos.128 (Ppro) when methanol, ethanol, 1-propanol, isopropanol, 1-butanol or glycerol was fed to the strains.
FIGS. 7A and 7B show the titer and % FAME (FIG. 7A) or FAEE (FIG. 7B) produced when a Ppro combination library built was fed methanol to evaluate the ability of Ppro variants to use methanol to make FAME (FIG. 7A) or ethanol to make FAEE (FIG. 7B).
FIG. 8 shows the composition of fatty acid species produced by becos.128 when cultured in the presence of (fed) methanol, ethanol, 1-propanol, isopropanol, 1-butanol or glycerol. FFA refers to free fatty acid species and FAxE refers to the ester species.



DETAILED DESCRIPTION
General Overview
The disclosure relates to variant enzymes with improved ester synthase properties for the production of fatty esters. Herein, the disclosure relates to enzymes such as variant ester synthase enzymes having improved ester synthase properties and variant thioesterase enzymes designed to have ester synthase activity. Both types of enzymes are engineered to improve the production of fatty esters when used alone or in combination with other proteins. In order to illustrate this, the applicants have engineered an ester synthase from Marinobacter hydrocarbonoclasticus and a thioesterase from Photobacterium profundum to produce fatty esters in vivo without the need to overexpress a fatty acyl-CoA synthetase or a different thioesterase. So far, it has been thought that in order to produce higher yields of fatty esters, at least an acyl-CoA synthetase or a thioesterase enzyme has to be simultaneously overexpressed with an ester synthase because any fatty ester production requires the co-expression of thioesterase, acyl-CoA synthetase, and ester synthase. Moreover, native Marinobacter ester synthases, and native thioesterases in general are not known to utilize short chain alcohols. However, the applicants have engineered variant enzymes with ester synthase activity such that the enzymes can utilize short chain alcohols, including methanol and ethanol, allowing the enzymes to produce fatty esters. For example, the variant ester synthase and variant thioesterase with ester synthase activity can produce fatty acid methyl ester(s) (FAME) and/or fatty acid ethyl ester(s) (FAEE) at high quantities. As such, the applicants have engineered variant enzymes that each lead to a higher production of esters in vivo.
Herein, the applicants employ a convenient one-gene-system (see FIG. 3) for the production of fatty esters. A one-gene-system typically employs a gene that encodes a variant enzyme with improved ester synthase activity in combination with an activated fatty acid, such as an Acyl-ACP or Acyl-CoA, (i.e., as a substrate) and alcohol to produce fatty esters in a recombinant host cell. There are certain advantages of using a one-gene-system (i.e., rather than a multi-gene-system) for the production of fatty esters. For example, one such advantage is the simplicity of the system itself which allows it to be faster implemented and more readily monitored. Another advantage is that a one-gene-system is more energy efficient for the cell because it likely bypasses or avoids the so-called futile cycle (i.e., a substrate futile cycle, wherein two metabolic pathways run simultaneously in opposite directions and have no overall effect), thereby increasing the yield of ester production in vivo.
More specifically, fatty acid ester production in Escherichia coli typically relies on the co-expression of a thioesterase ('tesA), an acyl-CoA synthetase (fadD) and a wax or ester synthase. In order to maximize ester production the three genes are carefully balanced in order to minimize production of free fatty acids (“FFA”). The thioesterase can act on both acyl-ACP (product of fatty acid biosynthesis) and acyl-CoA (product of acyl-CoA synthetase) generating free fatty acids (substrate of acyl-CoA synthetase). The present disclosure is directed to a modified ester synthase which has improved properties relative to the wild type enzyme, cloned from Marinobacter hydrocarbonoclasticus DSM 8798 GenBank: EF219377 (referred to herein as “'377”). The modified '377 has been shown to act on both acyl-ACP and acyl-CoA to generate fatty acid esters such as fatty acid methyl ester (“FAME”).
In addition, the percentage of FAME of the total acyl species (FAME+FFA) generated varies depending on the thioesterase being used. The 'tesA of Photobacterium profundum (“P. profundum” or “Ppro”) naturally produces a small percentage of FAME. The Photobacterium profundum tesA was engineered to produce a higher percentage of FAME. Thus, the present disclosure is further directed to a modified thioesterase ('tesA) from Photobacterium profundum (referred to herein as “Ppro”) that was genetically engineered to produce a higher percentage of FAME without the need to overexpress any other gene in a host cell such as E. coli. While not wanting to be bound by theory, an ester synthase that acts directly on acyl-ACP (a product of fatty acid biosynthesis) could eliminate the futile cycle that theoretically exists in the current biodiesel pathway, thus reserving resources from the cell, which could be directed towards fatty acid biosynthesis, which should also improve the production.
Definitions
As used in this 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 recombinant host cell” includes two or more such recombinant host cells, reference to “a fatty ester” includes one or more fatty esters, or mixtures of esters, reference to “a nucleic acid coding sequence” includes one or more nucleic acid coding sequences, reference to “an enzyme” includes one or more enzymes, and the like.
Sequence Accession numbers throughout this description were obtained from databases provided by the NCBI (National Center for Biotechnology Information) maintained by the National Institutes of Health, U.S.A. (which are identified herein as “NCBI Accession Numbers” or alternatively as “GenBank Accession Numbers”), and from the UniProt Knowledgebase (UniProtKB) and Swiss-Prot databases provided by the Swiss Institute of Bioinformatics (which are identified herein as “UniProtKB Accession Numbers”).
EC numbers are established by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB), description of which is available on the IUBMB Enzyme Nomenclature website on the World Wide Web. EC numbers classify enzymes according to the reaction catalyzed.
As used herein, the term “nucleotide” refers to a monomeric unit of a polynucleotide that consists of a heterocyclic base, a sugar, and one or more phosphate groups. The naturally occurring bases (guanine, (G), adenine, (A), cytosine, (C), thymine, (T), and uracil (U)) are typically derivatives of purine or pyrimidine, though it should be understood that naturally and non-naturally occurring base analogs are also included. The naturally occurring sugar is the pentose (five-carbon sugar) deoxyribose (which forms DNA) or ribose (which forms RNA), though it should be understood that naturally and non-naturally occurring sugar analogs are also included. Nucleic acids are typically linked via phosphate bonds to form nucleic acids or polynucleotides, though many other linkages are known in the art (e.g., phosphorothioates, boranophosphates, and the like).
The term “polynucleotide” refers to a polymer of ribonucleotides (RNA) or deoxyribonucleotides (DNA), which can be single-stranded or double-stranded and which can contain non-natural or altered nucleotides. The terms “polynucleotide,” “nucleic acid sequence,” and “nucleotide sequence” are used interchangeably herein to refer to a polymeric form of nucleotides of any length, either RNA or DNA. These terms refer to the primary structure of the molecule, and thus include double- and single-stranded DNA, and double- and single-stranded RNA. The terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to methylated and/or capped polynucleotides. The polynucleotide can be in any form, including but not limited to, plasmid, viral, chromosomal, EST, cDNA, mRNA, and rRNA.
As used herein, the terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues. The term “recombinant polypeptide” refers to a polypeptide that is produced by recombinant techniques, wherein generally DNA or RNA encoding the expressed protein is inserted into a suitable expression vector that is in turn used to transform a host cell to produce the polypeptide.
As used herein, the to Ins “homolog,” and “homologous” refer to a polynucleotide or a polypeptide comprising a sequence that is at least about 50% identical to the corresponding polynucleotide or polypeptide sequence. Preferably homologous polynucleotides or polypeptides have polynucleotide sequences or amino acid sequences that have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least about 99% homology to the corresponding amino acid sequence or polynucleotide sequence. As used herein the terms sequence “homology” and sequence “identity” are used interchangeably.
One of ordinary skill in the art is well aware of methods to determine homology between two or more sequences. Briefly, calculations of “homology” between two sequences can be performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In one preferred embodiment, the length of a first sequence that is aligned for comparison purposes is at least about 30%, preferably at least about 40%, more preferably at least about 50%, even more preferably at least about 60%, and even more preferably at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or about 100% of the length of a second sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions of the first and second sequences are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent homology between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, that need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm, such as BLAST (Altschul et al., J. Mol. Biol., 215(3): 403-410 (1990)). The percent homology between two amino acid sequences also can be determined using the Needleman and Wunsch algorithm that has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 (Needleman and Wunsch, J. Mol. Biol., 48: 444-453 (1970)). The percent homology between two nucleotide sequences also can be determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. One of ordinary skill in the art can perform initial homology calculations and adjust the algorithm parameters accordingly. A preferred set of parameters (and the one that should be used if a practitioner is uncertain about which parameters should be applied to determine if a molecule is within a homology limitation of the claims) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. Additional methods of sequence alignment are known in the biotechnology arts (see, e.g., Rosenberg, BMC Bioinformatics, 6: 278 (2005); Altschul, et al., FEBS J., 272(20): 5101-5109 (2005)).
The term “hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous and non-aqueous methods are described in that reference and either method can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions—6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes can be increased to 55° C. for low stringency conditions); 2) medium stringency hybridization conditions—6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringency hybridization conditions—6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and 4) very high stringency hybridization conditions—0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) are the preferred conditions unless otherwise specified.
An “endogenous” polypeptide refers to a polypeptide encoded by the genome of the parental microbial cell (also termed “host cell”) from which the recombinant cell is engineered (or “derived”). An “exogenous” polypeptide refers to a polypeptide which is not encoded by the genome of the parental microbial cell. A variant (i.e., mutant) polypeptide is an example of an exogenous polypeptide.
The term “heterologous” as used herein typically refers to a nucleotide, polypeptide or protein sequence, not naturally present in a given organism. For example, a polynucleotide sequence endogenous to a plant can be introduced into a host cell by recombinant methods, and the plant polynucleotide is then heterologous to that recombinant host cell. The term “heterologous” may also be used with reference to a nucleotide, polypeptide, or protein sequence which is present in a recombinant host cell in a non-native state. For example, a “heterologous” nucleotide, polypeptide or protein sequence may be modified relative to the wild type sequence naturally present in the corresponding wild type host cell, e.g., a modification in the level of expression or in the sequence of a nucleotide, polypeptide or protein.
As used herein, the term “fragment” of a polypeptide refers to a shorter portion of a full-length polypeptide or protein ranging in size from four amino acid residues to the entire amino acid sequence minus one amino acid residue. In certain embodiments of the disclosure, a fragment refers to the entire amino acid sequence of a domain of a polypeptide or protein (e.g., a substrate binding domain or a catalytic domain).
The term “mutagenesis” refers to a process by which the genetic information of an organism is changed in a stable manner Mutagenesis of a protein coding nucleic acid sequence produces a mutant protein. Mutagenesis also refers to changes in non-coding nucleic acid sequences that result in modified protein activity.
A “mutation”, as used herein, refers to a permanent change in a nucleic acid position of a gene or in an amino acid position of a polypeptide or protein. Mutations include substitutions, additions, insertions, and deletions. For example, a mutation in an amino acid position can be a substitution of one type of amino acid with another type of amino acid (e.g., a serine (S) may be substituted with an alanine (A); a lysine (L) may be substituted with an T (Threonine); etc.). As such, a polypeptide or a protein can have one or more mutations wherein one amino acid is substituted with another amino acid. For example, an ester synthase polypeptide or protein can have one or more mutations in its amino acid sequence.
An “ester synthase variant” is an ester synthase polypeptide or protein that has one or more mutations in its amino acid sequence. In one example, the amino acid sequence ranges from 0 (i.e., the initial methionine (M) based on the ATG start site) to 473. Such an ester synthase variant can have one or more mutation in the amino acid position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321,322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, and/or 473. Notably, amino acid positions 139 and 140 are part of a conserved motif, and thus, are important for the activity of the enzyme. In one embodiment, the mutations include mutations of SEQ ID NO: 2 comprising G4R, T5P, T5S, D7N, S15G, T24M, T24N, T24W, Q26T, L30H, G33D, G33N, G33S, L39A, L39M, L39S, R40S, D41A, D41G, D41H, D41Y, V43K, V43S, T44A, T44F, T44K, E48A, E48Q, A49D, A49T, Y58C, Y58L, I70L, I70V, A73G, A73Q, A73S, A73T, V76L, D77A, K78F, K78W, D79H, D79K, I80V, R98D, E99Q, G101L, I102R, P111D, P111G, P111S, H122S, V123I, V123M, R131M, I146K, I146L, I146R, S147A, V149L, R150P, V155G, T157S, T158E, T158K, R162E, R162K, C163I, C163R, N164D, N164R, P166L, P166S, T170R, T170S, V171E, V171F, V171H, V171R, V171W, R172S, R172W, P173W, H174E, Q175R, Q175S, R176T, R177V, A179K, A179S, A179V, D182G, K183S, E184F, E184G, E184L, E184R, E184S, A185L, A185M, S186T, V187G, V187R, P188R, A189G, A190P, A190R, A190W, V191I, V191L, S192A, S192L, S192V, Q193R, Q193S, M195G, D196E, A197T, A197V, Q201A, Q201V, Q201W, A202L, D203R, P206F, R207A, G212A, G212L, G212M, G212S, V216I, V219L, V234C, V236A, V236K, L237I, H239G, T242K, T242R, A243G, A243R, Q244G, R246A, R246G, R246L, R246Q, R246V, R246W, D255E, L257I, L257M, K258R, N259A, N259E, N259Q, L260M, L260V, H262Q, H262R, A263V, S264D, S264V, S264W, G265N, G266A, G266S, S267G, A285L, A285R, A285V, N288D, N289E, N289G, T293A, T293I, P294G, V301A, N302G, I303G, I303R, I303W, R304W, A306G, A306S, D307F, D307G, D307L, D307N, D307R, D307V, E309A, E309G, E309S, G310H, G310R, G310V, T311S, T313S, Q314G, I315F, S316G, F317W, I319G, A320C, A323G, D328F, N331I, N331K, N331T, Q334K, Q334S, Q335C, Q335N, Q335S, T338A, T338E, T338H, Q348A, Q348R, K349A, K349C, K349H, K349Q, P351G, K352I, K352N, S353K, S353T, T356G, T356W, Q357V, M360R, M360Q, M360S, M360W, Y366G, Y366W, G375A, G375V, G375S, V381F, E393G, E393R, E393W, G394E, G394R, T395E, R402K, V409L, L411A, A413T, I420V, S424G, S424Q, S442E, S442G, M443G, A447C, A447I, A447L, L454V, D455E, L457Y, E458W, I461G, I461L, I461V, K466N, A468G, K472T and K472.
The terms “thioesterase variant with ester synthase activity” or “thioesterase variant” are used interchangeably herein and refer to a thioesterase polypeptide or protein that has ester synthase activity and has one or more mutations in its amino acid sequence. In one example, the amino acid sequence ranges from 0 (i.e., the initial methionine (M) based on the ATG start site) to 182. Such a thioesterase variant can have one or more mutation(s) in the amino acid position 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 158, 159, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 and/or 182. However, amino acid positions 10, 157 and 160 are part of the catalytic triad, and thus, are important for the activity of the enzyme. In one embodiment, the mutations include mutations to SEQ ID NO: 51 comprising R18C, K32M, Q33A, Q33R, Q34R, E37D, I38Y, I45R, I45T, S46K, S46V, S46W, D48E, D48F, D48L, D48M, T49D, T49L,T49V, T49W, G51R, N52C, N52F, N52K, N52L, N52R, K65M, N76I, N76L, N76V, G81C, F82G, F82I, F82R, Q84A, Q84L, Q84V, R88H, D102V, V112L, N115F, N115W, N115Y, Y116H, Y116N, Y116P, Y116R, Y116S, Y116T, K118Q, R119C, I148Y, L149F, L149M, L149T, N156K, N156R, N156Y, L159K, L159M and D164T.
As used herein, the term “gene” refers to nucleic acid sequences encoding either an RNA product or a protein product, as well as operably-linked nucleic acid sequences affecting the expression of the RNA or protein (e.g., such sequences include but are not limited to promoter or enhancer sequences) or operably-linked nucleic acid sequences encoding sequences that affect the expression of the RNA or protein (e.g., such sequences include but are not limited to ribosome binding sites or translational control sequences).
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. 185, Academic Press, San Diego, Calif. (1990). In the methods of the disclosure, an expression control sequence is operably linked to a polynucleotide sequence. By “operably linked” is meant that a polynucleotide sequence and an expression control sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the expression control sequence(s). Operably linked promoters are located upstream of the selected polynucleotide sequence in terms of the direction of transcription and translation. Operably linked enhancers can be located upstream, within, or downstream of the selected polynucleotide.
As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid, i.e., a polynucleotide sequence, to which it has been linked. One type of useful vector is an episome (i.e., a nucleic acid capable of extra-chromosomal replication). Useful vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors.” In general, expression vectors of utility in recombinant DNA techniques are often in the form of “plasmids,” which refer generally to circular double stranded DNA loops that, in their vector form, are not bound to the chromosome. The terms “plasmid” and “vector” are used interchangeably herein, in as much as a plasmid is the most commonly used form of vector. However, also included are such other forms of expression vectors that serve equivalent functions and that become known in the art subsequently hereto. In some embodiments, a recombinant vector further includes a promoter operably linked to the polynucleotide sequence. In some embodiments, the promoter is a developmentally-regulated, an organelle-specific, a tissue-specific, an inducible, a constitutive, or a cell-specific promoter. The recombinant vector typically comprises at least one sequence selected from an expression control sequence operatively coupled to the polynucleotide sequence; a selection marker operatively coupled to the polynucleotide sequence; a marker sequence operatively coupled to the polynucleotide sequence; a purification moiety operatively coupled to the polynucleotide sequence; a secretion sequence operatively coupled to the polynucleotide sequence; and a targeting sequence operatively coupled to the polynucleotide sequence. In certain embodiments, the nucleotide sequence is stably incorporated into the genomic DNA of the host cell, and the expression of the nucleotide sequence is under the control of a regulated promoter region. The expression vectors described herein include a polynucleotide sequence described herein in a form suitable for expression of the polynucleotide sequence in a host cell. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc. The expression vectors described herein can be introduced into host cells to produce polypeptides, including fusion polypeptides, encoded by the polynucleotide sequences as described herein.
As used herein, a recombinant or engineered “host cell” is a host cell, e.g., a microorganism used to produce one or more of fatty acid derivatives include, for example, acyl-CoA, fatty acids, fatty aldehydes, short and long chain alcohols, hydrocarbons, fatty alcohols, fatty esters (e.g., waxes, fatty acid esters, fatty esters, and/or fatty fatty esters), terminal olefins, internal olefins, and ketones. In some embodiments, the recombinant host cell comprises one or more polynucleotides, each polynucleotide encoding a polypeptide having fatty acid biosynthetic enzyme activity, wherein the recombinant host cell produces a fatty ester composition when cultured in the presence of a carbon source under conditions effective to express the polynucleotides.
As used herein “acyl-ACP” refers to an acyl thioester formed between the carbonyl carbon of alkyl chain and the sulfhydryl group of the phosphopantetheinyl moiety of an acyl carrier protein (ACP). The phosphopantetheinyl moiety is post-translationally attached to a conserved serine residue on the ACP by the action of holo-acyl carrier protein synthase (ACPS), a phosphopantetheinyl transferase. In some embodiments an acyl-ACP is an intermediate in the synthesis of fully saturated acyl-ACPs. In other embodiments an acyl-ACP is an intermediate in the synthesis of unsaturated acyl-ACPs. In some embodiments, the carbon chain will have about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 carbons. Each of these acyl-ACPs are substrates for enzymes that convert them to fatty acid derivatives.
As used herein, the term “fatty acid derivative” means a “fatty acid” or a “fatty acid derivative”, which may be referred to as a “fatty acid or derivative thereof”. The term “fatty acid” means a carboxylic acid having the formula RCOOH. R represents an aliphatic group, preferably an alkyl group. R can comprise between about 4 and about 22 carbon atoms. Fatty acids can have a branched chain or straight chain and may be saturated, monounsaturated, or polyunsaturated. A “fatty acid derivative” is a product made in part from the fatty acid biosynthetic pathway of the production host organism. “Fatty acid derivatives” include products made in part from acyl-ACP or acyl-ACP derivatives. Exemplary fatty acid derivatives include, acyl-CoA, fatty acids, fatty aldehydes, short and long chain alcohols, fatty alcohols, hydrocarbons, esters (e.g., waxes, fatty acid esters, fatty esters), terminal olefins, internal olefins, and ketones.
A “fatty acid derivative composition” as referred to herein is produced by a recombinant host cell and typically includes a mixture of fatty acid derivatives. In some cases, the mixture includes more than one type of fatty acid derivative product (e.g., fatty acids, fatty esters, fatty alcohols, fatty aldehydes, fatty ketones, hydrocarbons, etc.). In other cases, a fatty acid derivative composition may include, for example, a mixture of fatty esters (or another fatty acid derivative) with different chain lengths, saturation and/or branching characteristics. In still other cases, the fatty acid derivative composition may comprise both a mixture of more than one type of fatty acid derivative product and fatty acid derivatives with different chain lengths, saturation and/or branching characteristics. In still other cases, a fatty acid derivative composition may include, for example, a mixture of fatty esters and beta hydroxy esters.
As used herein, the term “fatty acid biosynthetic pathway” means a biosynthetic pathway that produces fatty acid derivatives. The fatty acid biosynthetic pathway may include additional enzymes to produce fatty acids derivatives having desired characteristics.
As used herein, “fatty ester” means an ester having the formula RCOOR′. A fatty ester as referred to herein can be any ester made from a fatty acid, for example a fatty acid ester. In some embodiments, the R group is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19, carbons in length. Alternatively, or in addition, the R group is 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, or 6 or less carbons in length. Thus, the R group can have an R group bounded by any two of the above endpoints. For example, the R group can be 6-16 carbons in length, 10-14 carbons in length, or 12-18 carbons in length. In some embodiments, the fatty ester composition comprises one or more of a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, and a C26 fatty ester. In other embodiments, the fatty ester composition includes one or more of a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, and a C18 fatty ester. In still other embodiments, the fatty ester composition includes C12, C14, C16 and C18 fatty esters; C12, C14 and C16 fatty esters; C14, C16 and C18 fatty esters; or C12 and C14 fatty esters.
The R group of a fatty acid derivative, for example a fatty ester, can be a straight chain or a branched chain. Branched chains may have more than one point of branching and may include cyclic branches. In some embodiments, the branched fatty acid, branched fatty aldehyde, or branched fatty ester is a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, or a C26 branched fatty acid, branched fatty aldehyde, or branched fatty ester. In particular embodiments, the branched fatty acid, branched fatty aldehyde, or branched fatty ester is a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, or C18 branched fatty acid, or branched fatty ester. A fatty ester of the present disclosure may be referred to as containing an A side and a B side. As used herein, an “A side” of an ester refers to the carbon chain attached to the carboxylate oxygen of the ester. As used herein, a “B side” of an ester refers to the carbon chain comprising the parent carboxylate of the ester. When the fatty ester is derived from the fatty acid biosynthetic pathway, the A side is typically contributed by an alcohol, and the B side is contributed by a fatty acid.
Any alcohol can be used to form the A side of the fatty esters. For example, the alcohol can be derived from the fatty acid biosynthetic pathway, such as those describe herein. Alternatively, the alcohol can be produced through non-fatty acid biosynthetic pathways. Moreover, the alcohol can be provided exogenously. For example, the alcohol can be supplied in the fermentation broth in cases where the fatty ester is produced by an organism. Alternatively, a carboxylic acid, such as a fatty acid or acetic acid, can be supplied exogenously in instances where the fatty ester is produced by an organism that can also produce alcohol.
The carbon chains comprising the A side or B side of the ester can be of any length. In one embodiment, the A side of the ester is at least about 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, or 18 carbons in length. When the fatty ester is a fatty acid methyl ester, the A side of the ester is 1 carbon in length. When the fatty ester is a fatty acid ethyl ester, the A side of the ester is 2 carbons in length. The B side of the ester can be at least about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 carbons in length. The A side and/or the B side can be straight or branched chain. The branched chains can have one or more points of branching. In addition, the branched chains can include cyclic branches. Furthermore, the A side and/or B side can be saturated or unsaturated. If unsaturated, the A side and/or B side can have one or more points of unsaturation. In addition, the alcohol group of a fatty ester produced in accordance with the present disclosure need not be in the primary (C1) position. In one embodiment, the fatty ester is produced biosynthetically. In this embodiment, first the fatty acid is “activated.” Non-limiting examples of “activated” fatty acids are acyl-CoA, acyl ACP, and acyl phosphate. Acyl-CoA can be a direct product of fatty acid biosynthesis or degradation. In addition, acyl-CoA can be synthesized from a free fatty acid, a CoA, and an adenosine nucleotide triphosphate (ATP). An example of an enzyme which produces acyl-CoA is acyl-CoA synthase.
In certain embodiments, the branched fatty acid derivative is an iso-fatty acid derivative, for example an iso-fatty ester, or an anteiso-fatty acid derivative, e.g., an anteiso-fatty ester. In exemplary embodiments, the branched fatty acid derivative is selected from iso-C7:0, iso-C8:0, iso-C9:0, iso-C10:0, iso-C11:0, iso-C12:0, iso-C13:0, iso-C14:0, iso-C15:0, iso-C16:0, iso-C17:0, iso-C18:0, iso-C19:0, anteiso-C7:0, anteiso-C8:0, anteiso-C9:0, anteiso-C10:0, anteiso-C11:0,anteiso-C12:0, anteiso-C13:0, anteiso-C14:0, anteiso-C15:0, anteiso-C16:0, anteiso-C17:0, anteiso-C18:0, and an anteiso-C19:0 branched fatty ester.
The R group of a branched or unbranched fatty acid derivative can be saturated or unsaturated. If unsaturated, the R group can have one or more than one point of unsaturation. In some embodiments, the unsaturated fatty acid derivative is a monounsaturated fatty acid derivative. In certain embodiments, the unsaturated fatty acid derivative is a C6:1, C7:1, C8:1, C9:1, C10:1, C11:1, C12:1, C13:1, C14:1, C15:1, C16:1, C17:1, C18:1, C19:1, C20:1, C21:1, C22:1, C23:1, C24:1, C25:1, or a C26:1 unsaturated fatty acid derivative. In certain embodiments, the unsaturated fatty acid derivative, is a C10:1, C12:1, C14:1, C16:1, or C18:1 unsaturated fatty acid derivative. In other embodiments, the unsaturated fatty acid derivative is unsaturated at the omega-7 position. In certain embodiments, the unsaturated fatty acid derivative comprises a cis double bond.
As used herein, the term “clone” typically refers to a cell or group of cells descended from and essentially genetically identical to a single common ancestor, for example, the bacteria of a cloned bacterial colony arose from a single bacterial cell.
As used herein, the term “culture” typical refers to a liquid media comprising viable cells. In one embodiment, a culture comprises cells reproducing in a predetermined culture media under controlled conditions, for example, a culture of recombinant host cells grown in liquid media comprising a selected carbon source and nitrogen. “Culturing” or “cultivation” refers to growing a population of recombinant host cells under suitable conditions in a liquid or solid medium. In particular embodiments, culturing refers to the fermentative bioconversion of a substrate to an end-product. Culturing media are well known and individual components of such culture media are available from commercial sources, e.g., DifcoTM media and BBLTM media. In one non-limiting example, the aqueous nutrient medium is a “rich medium” including complex sources of nitrogen, salts, and carbon, such as YP medium, comprising 10 g/L of peptone and 10 g/L yeast extract of such a medium.
As used herein, the term “under conditions effective to express said heterologous nucleotide sequence(s)” means any conditions that allow a host cell to produce a desired fatty acid derivative. Suitable conditions include, for example, fermentation conditions.
As used herein, “modified” or an “altered level of” activity of a protein, for example an enzyme, in a recombinant host cell refers to a difference in one or more characteristics in the activity determined relative to the parent or native host cell. Typically, differences in activity are determined between a recombinant host cell, having modified activity, and the corresponding wild-type host cell (e.g., comparison of a culture of a recombinant host cell relative to the corresponding wild-type host cell). Modified activities can be the result of, for example, modified amounts of protein expressed by a recombinant host cell (e.g., as the result of increased or decreased number of copies of DNA sequences encoding the protein, increased or decreased number of mRNA transcripts encoding the protein, and/or increased or decreased amounts of protein translation of the protein from mRNA); changes in the structure of the protein (e.g., changes to the primary structure, such as, changes to the protein's coding sequence that result in changes in substrate specificity, changes in observed kinetic parameters); and changes in protein stability (e.g., increased or decreased degradation of the protein). In some embodiments, the polypeptide is a mutant or a variant of any of the polypeptides described herein, e.g., WS377 (“'377”), Ppro. In certain instances, the coding sequences for the polypeptides described herein are codon optimized for expression in a particular host cell. For example, for expression in E. coli, one or more codons can be optimized as described in, e.g., Grosjean et al., Gene 18:199-209 (1982).
The term “regulatory sequences” as used herein typically refers to a sequence of bases in DNA, operably-linked to DNA sequences encoding a protein that ultimately controls the expression of the protein. Examples of regulatory sequences include, but are not limited to, RNA promoter sequences, transcription factor binding sequences, transcription termination sequences, modulators of transcription (such as enhancer elements), nucleotide sequences that affect RNA stability, and translational regulatory sequences (such as, ribosome binding sites (e.g., Shine-Dalgarno sequences in prokaryotes or Kozak sequences in eukaryotes), initiation codons, termination codons). As used herein, the phrase “the expression of said nucleotide sequence is modified relative to the wild type nucleotide sequence,” means an increase or decrease in the level of expression and/or activity of an endogenous nucleotide sequence or the expression and/or activity of a heterologous or non-native polypeptide-encoding nucleotide sequence. 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. As used herein, the term “express” with respect to a polynucleotide is to cause it to function. A polynucleotide which encodes a polypeptide (or protein) will, when expressed, be transcribed and translated to produce that polypeptide (or protein). As used herein, the term “overexpress” means to express or cause to be expressed a polynucleotide or polypeptide in a cell at a greater concentration than is normally expressed in a corresponding wild-type cell under the same conditions.
As used herein, the term “titer” refers to the quantity of fatty acid derivative produced per unit volume of host cell culture. In any aspect of the compositions and methods described herein, a fatty acid derivative is produced at a titer of about 25 mg/L, about 50 mg/L, about 75 mg/L, about 100 mg/L, about 125 mg/L, about 150 mg/L, about 175 mg/L, about 200 mg/L, about 225 mg/L, about 250 mg/L, about 275 mg/L, about 300 mg/L, about 325 mg/L, about 350 mg/L, about 375 mg/L, about 400 mg/L, about 425 mg/L, about 450 mg/L, about 475 mg/L, about 500 mg/L, about 525 mg/L, about 550 mg/L, about 575 mg/L, about 600 mg/L, about 625 mg/L, about 650 mg/L, about 675 mg/L, about 700 mg/L, about 725 mg/L, about 750 mg/L, about 775 mg/L, about 800 mg/L, about 825 mg/L, about 850 mg/L, about 875 mg/L, about 900 mg/L, about 925 mg/L, about 950 mg/L, about 975 mg/L, about 1000 mg/L, about 1050 mg/L, about 1075 mg/L, about 1100 mg/L, about 1125 mg/L, about 1150 mg/L, about 1175 mg/L, about 1200 mg/L, about 1225 mg/L, about 1250 mg/L, about 1275 mg/L, about 1300 mg/L, about 1325 mg/L, about 1350 mg/L, about 1375 mg/L, about 1400 mg/L, about 1425 mg/L, about 1450 mg/L, about 1475 mg/L, about 1500 mg/L, about 1525 mg/L, about 1550 mg/L, about 1575 mg/L, about 1600 mg/L, about 1625 mg/L, about 1650 mg/L, about 1675 mg/L, about 1700 mg/L, about 1725 mg/L, about 1750 mg/L, about 1775 mg/L, about 1800 mg/L, about 1825 mg/L, about 1850 mg/L, about 1875 mg/L, about 1900 mg/L, about 1925 mg/L, about 1950 mg/L, about 1975 mg/L, about 2000 mg/L (2 g/L), 3 g/L, 5 g/L, 10 g/L, 20 g/L, 30 g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L, 80 g/L, 90 g/L, 100 g/L or a range bounded by any two of the foregoing values. In other embodiments, a fatty acid derivative is produced at a titer of more than 100 g/L, more than 200 g/L, or more than 300 g/L. One preferred titer of fatty acid derivative produced by a recombinant host cell according to the methods of the disclosure is from 5 g/L to 200 g/L, 10 g/L to 150 g/L, 20 g/L to 120 g/L and 30 g/L to 100 g/L. The titer may refer to a particular fatty acid derivative or a combination of fatty acid derivatives produced by a given recombinant host cell culture. For example, the expression of a variant polypeptide with ester synthase activity in a recombinant host cell such as E. coli results in the production of a higher titer as compared to a recombinant host cell expressing the corresponding wild type polypeptide. In one embodiment, the higher titer ranges from at least about 5 g/L to about 200 g/L.
As used herein, the “yield of fatty acid derivative produced by a host cell” refers to the efficiency by which an input carbon source is converted to product (i.e., a fatty ester) in a host cell. Host cells engineered to produce fatty acid derivatives according to the methods of the disclosure have a yield of at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, or at least about 30% or a range bounded by any two of the foregoing values. In other embodiments, a fatty acid derivative or derivatives is produced at a yield of more than about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more. Alternatively, or in addition, the yield is about 30% or less, about 27% or less, about 25% or less, or about 22% or less. Thus, the yield can be bounded by any two of the above endpoints. For example, the yield of a fatty acid derivative or derivatives produced by the recombinant host cell according to the methods of the disclosure can be about 5% to about 15%, about 10% to about 25%, about 10% to about 22%, about 15% to about 27%, about 18% to about 22%, about 20% to about 28%, or about 20% to about 30%. The yield may refer to a particular fatty acid derivative or a combination of fatty acid derivatives produced by a given recombinant host cell culture. For example, the expression of a variant polypeptide with ester synthase activity in a recombinant host cell such as E. coli results in the production of a higher yield of fatty acid esters as compared to a recombinant host cell expressing the corresponding wild type polypeptide. In one embodiment, the higher yield ranges from about 10% to about 100% of theoretical yield.
As used herein, the term “productivity” refers to the quantity of a fatty acid derivative or derivatives produced per unit volume of host cell culture per unit time. In any aspect of the compositions and methods described herein, the productivity of a fatty acid derivative or derivatives produced by a recombinant host cell is at least 100 mg/L/hour, at least 200 mg/L/hour, at least 300 mg/L/hour, at least 400 mg/L/hour, at least 500 mg/L/hour, at least 600 mg/L/hour, at least 700 mg/L/hour, at least 800 mg/L/hour, at least 900 mg/L/hour, at least 1000 mg/L/hour, at least 1100 mg/L/hour, at least 1200 mg/L/hour, at least 1300 mg/L/hour, at least 1400 mg/L/hour, at least 1500 mg/L/hour, at least 1600 mg/L/hour, at least 1700 mg/L/hour, at least 1800 mg/L/hour, at least 1900 mg/L/hour, at least 2000 mg/L/hour, at least 2100 mg/L/hour, at least 2200 mg/L/hour, at least 2300 mg/L/hour, at least 2400 mg/L/hour, 2500 mg/L/hour, or as high as 10 g/L/hr (dependent upon cell mass). For example, the productivity of a fatty acid derivative or derivatives produced by a recombinant host cell according to the methods of the may be from 500 mg/L/hour to 2500 mg/L/hour, or from 700 mg/L/hour to 2000 mg/L/hour. The productivity may refer to a particular fatty acid derivative or a combination of fatty acid derivatives produced by a given recombinant host cell culture. For example, the expression of a variant polypeptide with ester synthase activity in a recombinant host cell such as E. coli results in the production of an increased productivity of fatty acid esters as compared to a recombinant host cell expressing the corresponding wild type polypeptide. In one embodiment, the higher productivity ranges from about 0.3 g/L/h to about 3 g/L/h.
As used herein, the term “total fatty species” and “total fatty acid product” may be used interchangeably herein with reference to the amount of fatty esters and fatty acids, as evaluated by GC-FID. The same terms may be used to mean fatty alcohols, fatty aldehydes and free fatty acids when referring to a fatty alcohol analysis.
As used herein, the term “glucose utilization rate” means the amount of glucose used by the culture per unit time, reported as grams/liter/hour (g/L/hr).
As used herein, the term “greater ester synthase activity as compared to the wild type polypeptide” is used with reference to an ester synthase or thioesterase with ester synthase activity with a higher titer, higher yield and/or higher productivity than the corresponding wild type enzyme. In one preferred embodiment, the titer, yield or productivity is at least twice that of the wild type polypeptide. In other preferred embodiments, the titer, yield or productivity is at least 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times or 10 times that of the wild type polypeptide. In one particular embodiment, the titer is at least about 5% or greater. In some cases, “greater ester synthase activity as compared to the wild type polypeptide”, also means that in addition to the higher titer, yield and/or productivity than the corresponding wild type ester synthase enzyme, the ester synthase produces a low or lower percentage (e.g., 1-5%) of beta hydroxy esters (see Examples, infra). In other cases, “greater ester synthase activity as compared to the wild type polypeptide”, also means that in addition to the higher titer, yield and/or productivity than the corresponding wild type ester synthase enzyme, the ester synthase produces a higher percentage of beta hydroxy esters (see Examples, infra).
The term “improved fatty acid methyl ester activity” means that the variant polypeptide or enzyme can produce fatty acid methyl esters, fatty acid ethyl esters, fatty acid propyl esters, fatty acid isopropyl esters, fatty acid butyl esters, monoglycerides, fatty acid isobutyl esters, fatty acid 2-butyl esters, and fatty acid tert-butyl esters, and the like. A variant polypeptide or enzyme with improved fatty acid methyl ester activity has improved properties including, but not limited to, increased beta hydroxy esters, decreased beta hydroxyl esters, increased chain lengths of fatty acid esters, and decreased chain lengths of fatty acid esters. A variant ester synthase polypeptide with improved fatty acid methyl ester activity produces a titer that is at least about 5% or greater when the variant polypeptide is expressed in a recombinant microorganism.
As used herein, the term “carbon source” refers to a substrate or compound suitable to be used as a source of carbon for prokaryotic or simple eukaryotic cell growth. Carbon sources can be in various forms, including, but not limited to polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides, and gases (e.g., CO and CO2). Exemplary carbon sources include, but are not limited to, monosaccharides, such as glucose, fructose, mannose, galactose, xylose, and arabinose; oligosaccharides, such as fructo-oligosaccharide and galacto-oligosaccharide; polysaccharides such as starch, cellulose, pectin, and xylan; disaccharides, such as sucrose, maltose, cellobiose, and turanose; cellulosic material and variants such as hemicelluloses, methyl cellulose and sodium carboxymethyl cellulose; saturated or unsaturated fatty acids, succinate, lactate, and acetate; alcohols, such as ethanol, methanol, and glycerol, or mixtures thereof. The carbon source can also be a product of photosynthesis, such as glucose. In certain preferred embodiments, the carbon source is biomass. In other preferred embodiments, the carbon source is glucose. In other preferred embodiments the carbon source is sucrose. In other embodiments the carbon source is glycerol.
As used herein, the term “biomass” refers to any biological material from which a carbon source is derived. In some embodiments, a biomass is processed into a carbon source, which is suitable for bioconversion. In other embodiments, the biomass does not require further processing into a carbon source. The carbon source can be converted into a composition comprising fatty esters. Fatty esters find utility in a number of products including, but not limited to, surfactants, polymers, films, textiles, dyes, pharmaceuticals, fragrances and flavoring agents, lacquers, paints, varnishes, softening agents in resins and plastics, plasticizers, flame retardants, and additives in gasoline and oil.
An exemplary source of biomass is plant matter or vegetation, such as corn, sugar cane, or switchgrass. Another exemplary source of biomass is metabolic waste products, such as animal matter (e.g., cow manure). Further exemplary sources of biomass include algae and other marine plants. Biomass also includes waste products from industry, agriculture, forestry, and households, including, but not limited to, glycerol, fermentation waste, ensilage, straw, lumber, sewage, garbage, cellulosic urban waste, and food leftovers (e.g., soaps, oils and fatty acids). The term “biomass” also can refer to sources of carbon, such as carbohydrates (e.g., monosaccharides, disaccharides, or polysaccharides).
As used herein, the term “isolated,” with respect to products (such as fatty acids and derivatives thereof) refers to products that are separated from cellular components, cell culture media, or chemical or synthetic precursors. The fatty acids and derivatives thereof produced by the methods described herein can be relatively immiscible in the fermentation broth, as well as in the cytoplasm. Therefore, the fatty acids and derivatives thereof can collect in an organic phase either intracellularly or extracellularly.
As used herein, the terms “purify,” “purified,” or “purification” mean the removal or isolation of a molecule from its environment by, for example, isolation or separation. “Substantially purified” molecules are at least about 60% free (e.g., at least about 70% free, at least about 75% free, at least about 85% free, at least about 90% free, at least about 95% free, at least about 97% free, at least about 99% free) from other components with which they are associated. As used herein, these terms also refer to the removal of contaminants from a sample. For example, the removal of contaminants can result in an increase in the percentage of fatty acid derivatives in a sample. For example, when a fatty acid derivative is produced in a recombinant host cell, the fatty acid derivative can be purified by the removal of host cell proteins. After purification, the percentage of fatty acid derivative in the sample is increased. The terms “purify,” “purified,” and “purification” are relative terms which do not require absolute purity. Thus, for example, when a fatty acid derivative is produced in recombinant host cells, a purified fatty acid derivative is a fatty acid derivative that is substantially separated from other cellular components (e.g., nucleic acids, polypeptides, lipids, carbohydrates, or other hydrocarbons). As used herein, the term “attenuate” means to weaken, reduce, or diminish. For example, a polypeptide can be attenuated by modifying the polypeptide to reduce its activity (e.g., by modifying a nucleotide sequence that encodes the polypeptide).
Ester Synthase Variants
The present disclosure relates to, among other things, variant ester synthase enzymes, polypeptide sequences of such variants and functional fragments thereof with improved properties; polynucleotides encoding variant ester synthase polypeptide sequences; recombinant microorganisms including a nucleic acid encoding an improved ester synthase polypeptide; microorganisms capable of expressing the improved ester synthase polypeptide; cultures of such microorganisms; processes for producing fatty acid esters; fatty acid ester compositions and other compositions derived therefrom using the improved ester synthase polypeptides; and the resultant compositions.
Particularly, ester synthase polypeptides with improved properties and microorganisms expressing these polypeptides are provided herein. Wild-type ester synthase polypeptides from Marinobacter hydrocarbonoclasticus strain DSM8798, have been described in Holtzapple et al. (see J Bacteriol. (2007) 189(10):3804-3812) and U.S. Pat. No. 7,897,369. SEQ ID NO: 2 is the amino acid sequence of the wild-type ester synthase, i.e., ES9/DSM8798 from Marinobacter hydrocarbonoclasticus (M. hydrocarbonoclasticus; GenBankAccession No. ABO21021), which was used as a template to generate the improved ester synthase enzymes in order to illustrate the disclosure (see Examples, infra). A preferred ester synthase variant has at least about 90% sequence identity to the amino acid sequence of the wild type M. hydrocarbonoclasticus ester synthase enzyme (referred to herein as “WS377” or “'377”) of SEQ ID NO: 2. Furthermore, SEQ ID NO: 1 is the polynucleotide sequence encoding SEQ ID NO: 2. With respect to the ester synthase polypeptide sequences described herein “M” (ATG) is considered to be amino acid “0”. The first amino acid after the ATG is designated amino acid “1”.
In one aspect, the disclosure provides improved ester synthase polypeptides with enhanced ester synthase activity and nucleotide sequences that encode them. In another aspect, it can be seen that substitutions introduced at numerous different amino acid positions (also referred to herein as “residues”) within the wild-type '377 polypeptide of SEQ ID NO: 2 yield improved '377 polypeptides, capable of catalyzing increased production of fatty esters relative to wild-type '377. Depending upon the position mutated, single amino acid changes at specified positions give rise to increases or decreases in fatty ester production. Single amino acid changes at specified positions may also give rise to increases or decreases in beta hydroxy OH) ester production. In one embodiment, a single amino acid change results in an increase in fatty ester production and a decrease in β-OH ester production. In another embodiment, a single amino acid change results in an increase in fatty ester production and no change in n-OH ester production. In still another embodiment, a single amino acid change results in an increase in fatty ester production and an increase inn-OH ester production. A single amino acid change may also result in a decrease in fatty ester production and an increase, decrease or no change in β-OH ester production. In other embodiments, single or multiple amino acid changes result in an increase in fatty ester production. In still other embodiments, single or multiple amino acid changes result in an increase or decrease in β-OH production.
Thus, combinations of two or more amino acid changes at specified positions may give rise to increases or decreases in fatty ester and/or free fatty acid production. The effect of each individual amino acid change on fatty ester and free fatty acid production may or may not be additive to the effect of other individual amino acid changes on fatty ester and free fatty acid production. In some preferred embodiments, a combination of two or more amino acid changes at specified positions results in an increase in fatty ester production and a decrease in free fatty acid production. Accordingly, multiple amino acid changes at specified positions give rise to increases or decreases in fatty ester production. As such, multiple amino acid changes at specified positions may also give rise to decreases in β-OH ester production. In some embodiments, multiple amino acid changes result in an increase in fatty ester production and a decrease in β-OH ester production. In other embodiments, multiple amino acid changes result in an increase in fatty ester production and no change in β-OH ester production. In still other embodiments, multiple amino acid changes result in an increase in fatty ester production and an increase in β-OH ester production. Multiple amino acid changes may also result in a decrease in fatty ester production and an increase, decrease or no change in β-OH ester production.
A full saturation library was prepared using WS377 ester synthase as a template, as described in Example 1 (infra). Over 200 beneficial mutations were identified based on increased titer of fatty ester and/or a decrease in the percentage of beta hydroxy ester produced. A combination library was prepared based on “hits” from the saturation library, as detailed in Examples 2 and 5. In one aspect, the disclosure relates to improved ester synthase polypeptides with at least about 70% sequence identity to SEQ ID NO: 2. In some embodiments, an improved ester synthase polypeptide shows at least about 75% (e.g., at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%) sequence identity to the wild-type '377 sequence of SEQ ID NO: 2 and also includes one or more substitutions which results in useful characteristics and/or properties as described herein. In one aspect of the disclosure, the improved ester synthase polypeptide has about 100% sequence identity to SEQ ID NO: 4. In another aspect of the disclosure, the improved ester synthase polypeptide has about 100% sequence identity to any one of the following SEQ ID NOS, including, but not limited to, SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 and SEQ ID NO: 43.
In a related aspect, an improved ester synthase polypeptide is encoded by a nucleotide sequence having 100% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 7. In another related aspect, an improved ester synthase polypeptide is encoded by a nucleotide sequence having about 100% sequence identity to any one of the following SEQ ID NOS including, but not limited to, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40 and SEQ ID NO: 42.
In another aspect, the disclosure relates to improved ester synthase polypeptides with at least about 70% sequence identity to SEQ ID NO: 4. In some embodiments, an improved ester synthase polypeptide has at least about 75% (e.g., at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%) sequence identity to the ester synthase sequence of SEQ ID NO: 4 and also includes one or more substitutions which results in improved characteristics and/or properties as described herein. In another aspect, the disclosure relates to improved ester synthase polypeptides with at least about 70% sequence identity to any one of the following SEQ ID NOS including, but not limited to, SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 and SEQ ID NO: 43. In some embodiments, an improved ester synthase polypeptide has at least about 75% (e.g., at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%) sequence identity to the ester synthase sequence of any one of the following SEQ ID NOS including, but not limited to, SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 and SEQ ID NO: 43, and also includes one or more substitutions which results in improved characteristics and/or properties as described herein.
In another aspect, the disclosure relates to improved ester synthase polypeptides that comprise an amino acid sequence encoded by a nucleic acid sequence that has at least about 75% (e.g., at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or and at least 99%) sequence identity to the ester synthase sequence of SEQ ID NO: 5. In some embodiments the nucleic acid sequence encodes an ester synthase variant with one or more substitutions which results in improved characteristics and/or properties as described herein. In other embodiments, the improved or variant ester synthase nucleic acid sequence is derived from a species other than Marinobacter hydrocarbonoclasticus. In another aspect, the disclosure relates to improved ester synthase polypeptides that comprise an amino acid sequence encoded by a nucleic acid sequence that has at least about 75% (e.g., at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or and at least 99%) sequence identity to the ester synthase sequence of any one of the following SEQ ID NOS including, but not limited to, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40 and SEQ ID NO: 42. In some embodiments the nucleic acid sequence encodes an ester synthase variant with one or more substitutions which results in improved characteristics and/or properties as described herein. In other embodiments, the improved or variant ester synthase nucleic acid sequence is derived from a species other than Marinobacter hydrocarbonoclasticus. 
In another aspect, the disclosure relates to ester synthase polypeptides that comprise an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions over substantially the entire length of a nucleic acid corresponding to any one of the following SEQ ID NOS including, but not limited to, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40 and SEQ ID NO: 42. In some embodiments the nucleic acid sequence encodes an improved or variant ester synthase nucleic acid sequence derived from a species other than Marinobacter hydrocarbonoclasticus. In a related aspect, the disclosure provides ester synthase encoded by a nucleotide sequence having at least about 70% (e.g., at least 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 at least 99%) sequence identity to SEQ ID NO: 1 and comprises one or more of the substitutions disclosed herein.
Improved Properties of Ester Synthase Variants
The wild type ester synthase from Marinobacter hydrocarbonoclasticus DSM 8798 (“'377”) was engineered to produce a high percentage of fatty ester without the need to overexpress any other gene using expression in E. coli as an illustrative model. In addition, when expressed in a recombinant host cell such as E. coli, variants of the wild type ester synthase enzyme that result in a higher titer, yield or productivity also produce greater than about 20%, 25%, 30%, 35%, 40%, 45%, 50% or more β-OH esters. In one aspect, the present disclosure provides an improved variant of an ester synthase that has a higher titer, yield or productivity than the wild type ester synthase, and also produces a lower percentage of β-OH esters than the wild type enzyme. In a preferred embodiment, the variant ester synthase enzymes of the disclosure produce less than about 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or less than 1% β-OH esters. As such, the ester synthase variants of the disclosure exhibit improved characteristics and/or properties as described herein, for example, an increase in titer, yield and/or productivity of fatty acid esters; and/or a decrease in titer, yield or productivity of beta hydroxyl esters. Thus, the engineering of an ester synthase to have a higher titer, yield and/or productivity essentially prevents E. coli from using resources in a futile cycle (see FIG. 2), while allowing for a high level production of fatty acid esters, e.g., for use as biodiesel or in the manufacture of surfactants.
Photobacterium profundum Variants
The present disclosure relates to, among other things, variant thioesterase enzymes with increased ester synthase activity; polypeptide sequences of such variants and functional fragments thereof with improved properties; polynucleotides encoding variant thioesterase polypeptide sequences with increased ester synthase activity; recombinant microorganisms comprising a nucleic acid encoding an improved thioesterase polypeptide with increased ester synthase activity; microorganisms capable of expressing the improved thioesterase polypeptide sequences with increased ester synthase activity; cultures of such microorganisms; processes for producing fatty acid esters; fatty acid ester compositions and other compositions derived therefrom using the improved thioesterase polypeptide sequences with increased ester synthase activity; and the resultant compositions.
Particularly, thioesterase polypeptides with improved properties such as an increased ester synthase activity and microorganisms expressing these polypeptides are provided herein. Wild-type Photobacterium profundum (Ppro) polypeptides have been described in Vezzi et al. (see Science (2005) 307:1459-1461). SEQ ID NO: 51 is the wild-type amino acid sequence for thioesterase from Photobacterium profundum. SEQ ID NO: 50 is the polynucleotide sequence encoding SEQ ID NO: 51. The leader sequence that targets the thioesterase into the periplasm was removed from SEQ ID NO: 50. Improved thioesterase polypeptides, for example, variant Ppro polypeptides, have enhanced ester synthase activity. With respect to the Ppro polypeptide sequences described herein “M” (ATG) is considered to be amino acid “0”. The first amino acid after the ATG is designated amino acid “1”.
In one aspect, the disclosure provides improved Ppro polypeptides with enhanced ester synthase activity and nucleotide sequences that encode them. Improved Ppro polypeptides are examples of variant thioesterase polypeptides with increased or enhanced ester synthase activity. In another aspect, it can be seen that substitutions introduced at numerous different amino acid positions (also referred to herein as “residue” positions) within the wild-type Ppro of SEQ ID NO: 51 yield improved Ppro polypeptides capable of catalyzing increased production of fatty esters relative to wild-type Ppro. Depending upon the position mutated, single amino acid changes at specified positions gives rise to increases or decreases in fatty ester production. Single amino acid changes at specified positions may also give rise to increases or decreases in free fatty acid production. In some preferred embodiments, a single amino acid change results in an increase in fatty ester production and a decrease in free fatty acid production. In other embodiments, a single amino acid change results in an increase in fatty ester production and no change in free fatty acid production. In still other embodiments, a single amino acid change results in an increase in fatty ester production and an increase in free fatty acid production. A single amino acid change may also result in a decrease in fatty ester production and an increase, decrease or no change in free fatty acid production. Combinations of two or more amino acid changes at specified positions may also give rise to increases or decreases in fatty ester and/or free fatty acid production. The effect of each individual amino acid change on fatty ester and free fatty acid production may or may not be additive to the effect of other individual amino acid changes on fatty ester and free fatty acid production. In some preferred embodiments, a combination of two or more amino acid changes at specified positions results in an increase in fatty ester production and a decrease in free fatty acid production.
In one aspect, the disclosure relates to Ppro polypeptides that have at least about 70% sequence identity with SEQ ID NO: 51. In some embodiments, a Ppro polypeptide shows at least about 75% (e.g., at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or and at least 99%) sequence identity to the wild-type Ppro sequence of SEQ ID NO: 51 and also includes one or more substitutions which results in improved characteristics and/or properties as described herein. In a related aspect the Ppm polypeptide has a 100% sequence identity with SEQ ID NO: 51. In other related aspects the Ppm polypeptides have 100% sequence identity with SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, or SEQ ID NO: 31.
In one aspect, the disclosure relates to Ppro polypeptides that have at least about 70% sequence identity with SEQ ID NO: 61. In some embodiments, a Ppro polypeptide has at least about 75% (e.g., at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or and at least 99%) sequence identity to the wild-type Ppro sequence of SEQ ID NO: 61 and also includes one or more substitutions which results in improved characteristics and/or properties as described herein.
In another aspect the disclosure relates to Ppro polypeptides that have at least about 70% sequence identity with SEQ ID NO: 31. In some embodiments, a Ppm polypeptide has at least about 75% (e.g., at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or and at least 99%) sequence identity to the wild-type Ppro sequence of SEQ ID NO: 31 and also includes one or more substitutions which results in improved characteristics and/or properties as described herein.
In another aspect, the disclosure relates to Ppm polypeptides that comprise an amino acid sequence encoded by a nucleic acid sequence that has at least about 75% (e.g., at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or and at least 99%) sequence identity to the wild-type Ppro sequence of SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64 or SEQ ID NO: 30. In some embodiments the nucleic acid sequence encodes a Ppro variant with one or more substitutions which results in improved characteristics and/or properties as described herein. In other embodiments, the Ppro nucleic acid sequence is derived from a species other than Photobacterium profundum. 
In another aspect, the disclosure relates to Ppro polypeptides that comprise an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions over substantially the entire length of a nucleic acid corresponding to SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64 or SEQ ID NO: 30. In some embodiments the nucleic acid sequence encodes a Ppro variant derived from a species other than Photobacterium profundum. In a related aspect, the disclosure provides Ppro encoded by a nucleotide sequence having at least about 70% (e.g., at least 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 at least 99%) sequence identity to SEQ ID NO: 50 and comprises substitutions disclosed herein (see, e.g., Table 13).
Improved Properties of Ppro Variants
In order to illustrate the disclosure, the 'tesA from P. profundum was engineered to produce a higher percentage of FAME without the need to overexpress any other gene in E. coli. An ester synthase that acts directly on acyl-ACP (product of fatty acid biosynthesis) could be directed towards increased ester production, as well as allow direct pull of the pathway from the fatty acid biosynthesis, which should also improve the production. In addition, one of the libraries was tested for production of FAEE, and it was observed that engineered 'tesA from P. profundum for FAME, also produced higher levels of FAEE than the wild type strain. These Ppro variants exhibit improved characteristics and/or properties as described herein, for example an increase in titer, yield and/or productivity of fatty acid esters; and/or a decrease in titer, yield and/or productivity of free fatty acids. As an example, the tesA of P. profundum (Ppro) was engineered to have ester synthase activity. In other words, the tesA of Ppro was engineered to act as an ester synthase. When screened in plates, the wild type tesA Ppro gene produced about 15-18% FAME of the total acyl products (FAME+free fatty acids (“FFA”)). Mutants were identified that resulted in an increase of FAME production of 42-44% when tested on plates. When these same mutants were tested in shake flasks, they produced 62-65% FAME when compared to the wild type tesA Ppro which only produced 30% FAME (see Example 7, infra).
Methods of Making Ester Synthase or Ppro Variants
In practicing the methods of the present disclosure, mutagenesis is used to prepare groups of recombinant host cells for screening. Typically, the recombinant host cells comprise one or more polynucleotide sequences that include an open reading frame for an ester synthase or Ppro polypeptide, such as a variant of ester synthase or a variant of thioesterase (e.g., Ppro) together with operably-linked regulatory sequences. Numerous examples of variant ester synthase polypeptides and variant thioesterase polypeptides useful in the practice of the methods of the present disclosure are described herein. Examples of regulatory sequences useful in the practice of the methods of the present disclosure are also described herein. Mutagenesis of such polynucleotide sequences can be performed using genetic engineering techniques, such as site directed mutagenesis, random chemical mutagenesis, exonuclease ITT deletion procedures, or standard cloning techniques. Alternatively, mutations in polynucleotide sequences can be created using chemical synthesis or modification procedures.
Mutagenesis methods are well known in the art and include, for example, the following. In error prone PCR (see, e.g., Leung et al., Technique 1:11-15, 1989; and Caldwell et al., PCR Methods Applic. 2:28-33, 1992), PCR is performed under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product. Briefly, in such procedures, polynucleotides to be mutagenized are mixed with PCR primers, reaction buffer, MgCl2, MnCl2, Taq polymerase, and an appropriate concentration of dNTPs for achieving a high rate of point mutation along the entire length of the PCR product. For example, the reaction can be performed using 20 fmoles of nucleic acid to be mutagenized, 30 pmole of each PCR primer, a reaction buffer comprising 50 mM KCl, 10 mM Tris HCl (pH 8.3), and 0.01% gelatin, 7 mM MgCl2, 0.5 mM MnCl2, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, and 1 mM dTTP. PCR can be performed for 30 cycles of 94° C. for 1 min., 45° C. for 1 min., and 72° C. for 1 min. It will be appreciated that these parameters can be varied as appropriate. The mutagenized polynucleotides are then cloned into an appropriate vector and the activities of the affected polypeptides encoded by the mutagenized polynucleotides are evaluated. Mutagenesis can also be performed using oligonucleotide directed mutagenesis (see, e.g., Reidhaar-Olson et al., Science 241:53-57, 1988) to generate site-specific mutations in any cloned DNA of interest. Briefly, in such procedures a plurality of double stranded oligonucleotides bearing one or more mutations to be introduced into the cloned DNA are synthesized and assembled into the cloned DNA to be mutagenized. Clones containing the mutagenized DNA are recovered, and the activities of affected polypeptides are assessed. Another mutagenesis method for generating polynucleotide sequence variants is assembly PCR. Assembly PCR involves the assembly of a PCR product from a mixture of small DNA fragments. A large number of different PCR reactions occur in parallel in the same vial, with the products of one reaction priming the products of another reaction. Assembly PCR is described in, for example, U.S. Pat. No. 5,965,408. Still another mutagenesis method of generating polynucleotide sequence variants is sexual PCR mutagenesis (Stemmer, PNAS, USA 91:10747-10751, 1994). In sexual PCR mutagenesis, forced homologous recombination occurs between DNA molecules of different, but highly related, DNA sequence in vitro as a result of random fragmentation of the DNA molecule based on sequence homology. This is followed by fixation of the crossover by primer extension in a PCR reaction.
Ester synthase (e.g., '377) or thioesterase (e.g., Ppro) sequence variants can also be created by in vivo mutagenesis. In some embodiments, random mutations in a nucleic acid sequence are generated by propagating the polynucleotide sequence in a bacterial strain, such as an E. coli strain, which carries mutations in one or more of the DNA repair pathways. Such “mutator” strains have a higher random mutation rate than that of a wild-type strain. Propagating a DNA sequence in one of these strains will eventually generate random mutations within the DNA. Mutator strains suitable for use for in vivo mutagenesis are described in, for example, PCT International Publication No. WO 91/16427.
Ester synthase (e.g., '377) or thioesterase (e.g., Ppro) sequence variants can also be generated using cassette mutagenesis. In cassette mutagenesis, a small region of a double stranded DNA molecule is replaced with a synthetic oligonucleotide “cassette” that differs from the starting polynucleotide sequence. The oligonucleotide often contains completely and/or partially randomized versions of the starting polynucleotide sequence. There are many applications of cassette mutagenesis; for example, preparing mutant proteins by cassette mutagenesis (see, e.g., Richards, J. H., Nature 323, 187 (1986); Ecker, D. J., et al., J. Biol. Chem. 262:3524-3527 (1987)); codon cassette mutagenesis to insert or replace individual codons (see, e.g., Kegler-Ebo, D. M., et al., Nucleic Acids Res. 22(9): 1593-1599 (1994)); preparing variant polynucleotide sequences by randomization of non-coding polynucleotide sequences comprising regulatory sequences (e.g., ribosome binding sites, see, e.g., Barrick, D., et al., Nucleic Acids Res. 22(7): 1287-1295 (1994); Wilson, B. S., et al., Biotechniques 17:944-953 (1994)).
Recursive ensemble mutagenesis (see, e.g., Arkin et al., PNAS, USA 89:7811-7815, 1992) can also be used to generate polynucleotide sequence variants. Recursive ensemble mutagenesis is an algorithm for protein engineering (i.e., protein mutagenesis) developed to produce diverse populations of phenotypically related mutants whose members differ in amino acid sequence. This method uses a feedback mechanism to control successive rounds of combinatorial cassette mutagenesis. Exponential ensemble mutagenesis (see, e.g., Delegrave et al., Biotech. Res. 11:1548-1552, 1993) can also be used to generate polynucleotide sequence variants. Exponential ensemble mutagenesis is a process for generating combinatorial libraries with a high percentage of unique and functional mutants, wherein small groups of residues are randomized in parallel to identify, at each altered position, amino acids which lead to functional proteins. Random and site-directed mutagenesis can also be used (see, e.g., Arnold, Curr. Opin. Biotech. 4:450-455, 1993).
Further, standard methods of in vivo mutagenesis can be used. For example, host cells, comprising one or more polynucleotide sequences that include an open reading frame for an ester synthase (e.g., '377) or thioesterase (e.g., Ppro) polypeptide, as well as operably-linked regulatory sequences, can be subject to mutagenesis via exposure to radiation (e.g., UV light or X-rays) or exposure to chemicals (e.g., ethylating agents, alkylating agents, or nucleic acid analogs). In some host cell types, for example, bacteria, yeast, and plants, transposable elements can also be used for in vivo mutagenesis.
The mutagenesis of one or more polynucleotide sequences that encode an ester synthase variant such as a '377 variant generally results in expression of an ester synthase polypeptide product that demonstrates a modified and improved biological function. Similarly, the mutagenesis of one or more polynucleotide sequences that encode a Ppro variant generally results in expression of a Ppro polypeptide product that demonstrates a modified and improved biological function such as enhanced ester synthase activity. For example, when preparing a group of recombinant microorganisms by mutagenesis of one or more polynucleotide sequences including the open reading frame encoding a '377 polypeptide and operably-linked regulatory sequences, the protein expressed from the resulting mutagenized polynucleotide sequences may maintain the '377 ester synthase biological function, however, an improved yield of fatty esters, a decreased yield of beta hydroxy esters and/or an improved compositions comprising a modified mixture of fatty ester products (in terms of chain length, saturation, and the like) is observed upon culture of the recombinant microorganism under conditions effective to express the mutant '377 polynucleotide. Similarly, when preparing a group of recombinant microorganisms by mutagenesis of one or more polynucleotide sequences including the open reading frame encoding a Ppro polypeptide and operably-linked regulatory sequences, the protein expressed from the resulting mutagenized polynucleotide sequences may maintain the Ppm thioesterase biological function but an ester synthase activity is observed in the recombinant microorganism. Because of the ester synthase activity, an improved yield of fatty esters, an improved compositions comprising a modified mixture of fatty ester products (in terms of chain length, saturation, and the like) is observed upon culture of the recombinant microorganism under conditions effective to express the mutant Ppm polynucleotide. In another embodiment, the mutagenesis of one or more Ppro polynucleotide sequences generally results in expression of a Ppm polypeptide product that may retain the same biological function as the wild type or parent Ppro polypeptide even though the mutant Ppro polypeptide demonstrates a modified biological function. For example, when preparing a group of recombinant microorganisms by mutagenesis of one or more polynucleotide sequences including the open reading frame encoding a Ppm polypeptide and operably-linked regulatory sequences, the protein expressed from the resulting mutagenized polynucleotide sequences may maintain the Ppro thioesterase biological function but a ester synthase activity is observed in the recombinant microorganism.
Hot Spots
The disclosure is also based, at least in part, on the identification of certain structurally conserved “hot spots” among variant ester synthase polypeptides such as '377 polypeptides. Hot spots are regions where a high number of mutations were observed that lead to a higher titer of fatty ester product and/or a lower production of beta hydroxy esters in '377 polypeptides. Notably, such regions are seen in variant ester synthase polypeptides that exhibit a greater ester synthase activity as compared to the wild type polypeptide that lacks those hot spots. Hot spots include amino acid regions 39-44; 76-80; 98-102; 146-150; 170-207, in particular 182-207 (e.g., showing the highest number of mutations); 242-246; 300-320; 348-357; and 454-458.
Motifs
The disclosure is also based, at least in part, on the identification of certain structurally conserved motifs among thioesterase polypeptides with enhanced ester synthase activity, wherein a thioesterase polypeptide, such as Ppro, that comprises one of these motifs has greater ester synthase activity as compared to the wild type thioesterase polypeptide which lacks the motif. Accordingly, the disclosure features variant thioesterase polypeptides, such as Ppm, that comprise a motif at amino acid region 73-81 of SEQ ID NO: 51, with the substitutions indicated below.







(SEQ ID NO: 66)


Leu-Gly-[Ala or Gly]-[Val or Ile or Leu]-Asp-


 


[Ala or Gly]-Leu-Arg-Gly LG[AG][VIL]D[AG]LRG






The potential substitutions include alanine (A) or glycine (G) at amino acid position 75; valine (V), isoleucine (I) or leucine (L) at amino acid position 76; and alanine (A) or glycine (G) at amino acid position 78.
Recombinant Host Cells and Recombinant Host Cell Cultures
The recombinant host cells of the disclosure comprise one or more polynucleotide sequences that comprise an open reading frame encoding a polypeptide having ester synthase activity, e.g., any polypeptide which catalyzes the conversion of an acyl-thioester to a fatty ester, together with operably-linked regulatory sequences that facilitate expression of the polypeptide having ester synthase activity in a recombinant host cell. In one preferred embodiment, the polypeptide having improved ester synthase activity is a variant or mutant of '377. In another preferred embodiment, the polypeptide having improved ester synthase activity is a variant or mutant of Ppro. In a recombinant host cell of the disclosure, the open reading frame coding sequences and/or the regulatory sequences may be modified relative to the corresponding wild-type coding sequence of the '377 or Ppro polypeptide. A fatty ester composition is produced by culturing a recombinant host cell in the presence of a carbon source under conditions effective to express the variant ester synthase polypeptide (e.g., '377) or the variant thioesterase polypeptide with ester synthase activity (e.g., Ppm). Expression of mutant or variant '377 polypeptides results in production of fatty ester compositions with increased yields of fatty esters and decreased yields of beta hydroxyl esters. Expression of mutant or variant Ppm polypeptides results in production of fatty ester compositions with increased yields of fatty esters.
In order to illustrate the disclosure, the applicants have constructed host strains that express variant ester synthase polypeptide sequences (see Examples, infra). Examples of variant ester synthase polypeptide sequences that when expressed in a recombinant host cell result in a higher titer of fatty esters include but are not limited to, sequences expressed in host strains 9B12 (SEQ ID NO: 4), pKEV022 (SEQ ID NO: 10), KASH008 (SEQ ID NO: 16), KASH280 (SEQ ID NO: 33) and KASH281 (SEQ ID NO: 35). Examples of variant ester synthase polypeptide sequences that when expressed in a recombinant host cell result in a higher titer of fatty esters and a decrease in β-OH ester production include polypeptide sequences expressed in host strains pKEV28 (SEQ ID NO: 12), and KASH008 (SEQ ID NO: 16), KASH285 (SEQ ID NO: 37), KASH286 (SEQ ID NO: 39), KASH288 (SEQ ID NO: 41) and KASH289 (SEQ ID NO: 43).
Examples of variant Ppro polypeptide sequences that when expressed in a recombinant host cell result in a higher titer of fatty esters include but are not limited to, sequences expressed in host strains PROF1 (SEQ ID NO: 53), PROF2 (SEQ ID NO: 55), P1B9 (SEQ ID NO: 58), N115F (SEQ ID NO: 61), Vc7P4H5 (SEQ ID NO: 63), Vc7P5H9 (SEQ ID NO: 65) and Vc7P6F9 (SEQ ID NO: 31).
The recombinant host cell may produce a fatty ester, such as a fatty acid methyl ester (FAME), a fatty acid ethyl ester (FAEE), a wax ester, or the like. The fatty acid esters are typically recovered from the culture medium. The fatty acid ester composition produced by a recombinant host cell can be analyzed using methods known in the art, for example, GC-FID, in order to determine the distribution of particular fatty acid esters as well as chain lengths and degree of saturation of the components of the fatty ester composition.
Methods of Making Recombinant Host Cells and Cultures
Various methods well known in the art can be used to genetically engineer host cells to produce fatty esters and/or fatty ester compositions. The methods can include the use of vectors, preferably expression vectors, comprising a nucleic acid encoding a mutant or variant '377 ester synthase or Ppro thioesterase with ester synthase activity, as described herein. Those skilled in the art will appreciate a variety of viral and non-viral vectors can be used in the methods described herein.
In some embodiments of the present disclosure, a “higher” titer of fatty esters in a particular composition is a higher titer of a particular type of fatty acid ester or a combination of fatty acid esters produced by a recombinant host cell culture relative to the titer of the same fatty acid ester or combination of fatty acid esters produced by a control culture of a corresponding wild-type host cell. In some embodiments, a mutant or variant '377 ester synthase polynucleotide (or gene) sequence or a mutant or variant Ppro polynucleotide (or gene) sequence is provided to the host cell by way of a recombinant vector, which comprises a promoter operably linked to the polynucleotide sequence. In certain embodiments, the promoter is a developmentally-regulated, an organelle-specific, a tissue-specific, an inducible, a constitutive, or a cell-specific promoter. The recombinant vector typically comprises at least one sequence selected from an expression control sequence operatively coupled to the polynucleotide sequence; a selection marker operatively coupled to the polynucleotide sequence; a marker sequence operatively coupled to the polynucleotide sequence; a purification moiety operatively coupled to the polynucleotide sequence; a secretion sequence operatively coupled to the polynucleotide sequence; and a targeting sequence operatively coupled to the polynucleotide sequence. The one or more polynucleotide sequences, comprising open reading frames encoding proteins and operably-linked regulatory sequences can be integrated into a chromosome of the recombinant host cells, incorporated in one or more plasmid expression system resident in the recombinant host cells, or both.
The expression vectors described herein include a polynucleotide sequence described herein in a form suitable for expression of the polynucleotide sequence in a host cell. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc. The expression vectors described herein can be introduced into host cells to produce polypeptides, including fusion polypeptides, encoded by the polynucleotide sequences as described herein. Expression of genes encoding polypeptides in prokaryotes, for example, E. coli, is most often carried out with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides. Suitable expression systems for both prokaryotic and eukaryotic cells are well known in the art; see, e.g., Sambrook et al., “Molecular Cloning: A Laboratory Manual,” second edition, Cold Spring Harbor Laboratory, (1989). In certain embodiments, a polynucleotide sequence of the disclosure is operably linked to a promoter derived from bacteriophage T5. In one embodiment, the host cell is a yeast cell. In this embodiment, the expression vector is a yeast expression vector. Vectors can be introduced into prokaryotic or eukaryotic cells via a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell. Suitable methods for transforming or transfecting host cells can be found in, for example, Sambrook et al. (supra).
For stable transformation of bacterial cells, it is known that, depending upon the expression vector and transformation technique used, only a small fraction of cells will take-up and replicate the expression vector. In order to identify and select these transformants, a gene that encodes a selectable marker (e.g., resistance to an antibiotic) can be introduced into the host cells along with the gene of interest. Selectable markers include those that confer resistance to drugs such as, but not limited to, ampicillin, kanamycin, chloramphenicol, or tetracycline. Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a polypeptide described herein or can be introduced on a separate vector. Cells stably transformed with the introduced nucleic acid can be identified by growth in the presence of an appropriate selection drug.
Examples of host cells that are microorganisms, include but are not limited to cells from the genus Escherichia, Bacillus, Lactobacillus, Zymomonas, Rhodococcus, Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete, Pleurotus, Trametes, Chrysosporium, Saccharomyces, Stenotrophamonas, Schizosaccharomyces, Yarrowia, or Streptomyces. In some embodiments, the host cell is a Gram-positive bacterial cell. In other embodiments, the host cell is a Gram-negative bacterial cell. In some embodiments, the host cell is an E. coli cell. In other embodiments, the host cell is a Bacillus lentus cell, a Bacillus brevis cell, a Bacillus stearothermophilus cell, a Bacillus lichenoformis cell, a Bacillus alkalophilus cell, a Bacillus coagulans cell, a Bacillus circulans cell, a Bacillus pumilis cell, a Bacillus thuringiensis cell, a Bacillus clausii cell, a Bacillus megaterium cell, a Bacillus subtilis cell, or a Bacillus amyloliquefaciens cell.
In still other embodiments, the host cell is a Trichoderma koningii cell, a Trichoderma viride cell, a Trichoderma reesei cell, a Trichoderma longibrachiatum cell, an Aspergillus awamori cell, an Aspergillus fumigates cell, an Aspergillus foetidus cell, an Aspergillus nidulans cell, an Aspergillus niger cell, an Aspergillus oryzae cell, a Humicola insolens cell, a Humicola lanuginose cell, a Rhodococcus opacus cell, a Rhizomucor miehei cell, or a Mucor michei cell. In yet other embodiments, the host cell is a Streptomyces lividans cell or a Streptomyces murinus cell. In yet other embodiments, the host cell is an Actinomycetes cell. In some embodiments, the host cell is a Saccharomyces cerevisiae cell.
In other embodiments, the host cell is a cell from a eukaryotic plant, algae, cyanobacterium, green-sulfur bacterium, green non-sulfur bacterium, purple sulfur bacterium, purple non-sulfur bacterium, extremophile, yeast, fungus, an engineered organism thereof, or a synthetic organism. In some embodiments, the host cell is light-dependent or fixes carbon. In some embodiments, the host cell has autotrophic activity.
In some embodiments, the host cell has photoautotrophic activity, such as in the presence of light. In some embodiments, the host cell is heterotrophic or mixotrophic in the absence of light. In certain embodiments, the host cell is a cell from Arabidopsis thaliana, Panicum virgatum, Miscanthus giganteus, Zea mays, Botryococcuse braunii, Chlamydomonas reinhardtii, Dunaliela salina, Synechococcus Sp. PCC 7002, Synechococcus Sp. PCC 7942, Synechocystis Sp. PCC 6803, Thermosynechococcus elongates BP-1, Chlorobium tepidum, Chlorojlexus auranticus, Chromatiumm vinosum, Rhodospirillum rubrum, Rhodobacter capsulatus, Rhodopseudomonas palusris, Clostridium ljungdahlii, Clostridium thermocellum, Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pseudomonasfluorescens, or Zymomonas mobilis. 
Culture and Fermentation of Genetically Engineered Host Cells
As used herein, the term “fermentation” broadly refers to the conversion of organic materials into target substances by host cells, for example, the conversion of a carbon source by recombinant host cells into fatty acids or derivatives thereof by propagating a culture of the recombinant host cells in a media comprising the carbon source. As used herein, the term “conditions permissive for the production” means any conditions that allow a host cell to produce a desired product, such as a fatty acid ester composition. Similarly, the term “conditions in which the polynucleotide sequence of a vector is expressed” means any conditions that allow a host cell to synthesize a polypeptide. Suitable conditions include, for example, fermentation conditions. Fermentation conditions can comprise many parameters, including but not limited to temperature ranges, levels of aeration, feed rates and media composition. Each of these conditions, individually and in combination, allows the host cell to grow. Fermentation can be aerobic, anaerobic, or variations thereof (such as micro-aerobic). Exemplary culture media include broths or gels. Generally, the medium includes a carbon source that can be metabolized by a host cell directly. In addition, enzymes can be used in the medium to facilitate the mobilization (e.g., the depolymerization of starch or cellulose to fermentable sugars) and subsequent metabolism of the carbon source.
For small scale production, the engineered host cells can be grown in batches of, for example, about 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 1 mL, 5 mL, 10 mL, 15 mL, 25 mL, 50 mL, 75 mL, 100 mL, 500 mL, 1 L, 2 L, 5 L, or 10 L; fermented; and induced to express a desired polynucleotide sequence, such as a polynucleotide sequence encoding a polypeptide having ester synthase activity. For large scale production, the engineered host cells can be grown in cultures having a volume batches of about 10 L, 100 L, 1000 L, 10,000 L, 100,000 L, 1,000,000 L or larger; fermented; and induced to express a desired polynucleotide sequence. The fatty ester compositions described herein are found in the extracellular environment of the recombinant host cell culture and can be readily isolated from the culture medium. A fatty acid derivative may be secreted by the recombinant host cell, transported into the extracellular environment or passively transferred into the extracellular environment of the recombinant host cell culture. The fatty ester composition may be isolated from a recombinant host cell culture using routine methods known in the art.
Screening Genetically Engineered Host Cells
In one embodiment of the present disclosure, the activity of a mutant or variant '377 ester synthase polypeptide is determined by culturing recombinant host cells (comprising one or more mutagenized ester synthase such as '377 polynucleotide sequences), followed by screening to identify characteristics of fatty acid ester compositions produced by the recombinant host cells; for example, titer, yield and productivity of fatty acid esters; and percent beta hydroxy esters. In another embodiment, the activity of a mutant or variant Ppro polypeptide is determined by culturing recombinant host cells (comprising one or more mutagenized thioesterase such as Ppro polynucleotide sequences), followed by screening to identify characteristics of fatty acid ester compositions produced by the recombinant host cells; for example: titer, yield and productivity of fatty acid esters; and free fatty acids. Mutant or variant '377 ester synthase polypeptides or mutant or variant Ppro polypeptides and fragments thereof can be assayed for ester synthase activity using routine methods. For example, a mutant or variant '377 ester synthase polypeptide or Ppro polypeptide or fragment thereof is contacted with a substrate (e.g., an acyl-CoA, an acyl-ACP, a free fatty acid, or an alcohol) under conditions that allow the polypeptide to function. A decrease in the level of the substrate or an increase in the level of a fatty ester or a fatty ester composition can be measured to determine the ester synthase activity.
Products Derived from Recombinant Host Cells
As used herein, “fraction of modem carbon” or fM has the same meaning as defined by National Institute of Standards and Technology (MST) Standard Reference Materials (SRMs 4990B and 4990C, known as oxalic acids standards HOxI and HOxII, respectively. The fundamental definition relates to 0.95 times the 14C/12C isotope ratio HOxI (referenced to AD 1950). This is roughly equivalent to decay-corrected pre-Industrial Revolution wood. For the current living biosphere (plant material), fM is approximately 1.1.
Bioproducts (e.g., the fatty ester compositions produced in accordance with the present disclosure) comprising biologically produced organic compounds, and in particular, the fatty ester compositions produced using the fatty acid biosynthetic pathway herein, have been produced from renewable sources and, as such, are new compositions of matter. These new bioproducts can be distinguished from organic compounds derived from petrochemical carbon on the basis of dual carbon-isotopic fingerprinting or 14C dating. Additionally, the specific source of biosourced carbon (e.g., glucose vs. glycerol) can be determined by dual carbon-isotopic fingerprinting (see, e.g., U.S. Pat. No. 7,169,588). The ability to distinguish bioproducts from petroleum based organic compounds is beneficial in tracking these materials in commerce. For example, organic compounds or chemicals comprising both biologically based and petroleum based carbon isotope profiles may be distinguished from organic compounds and chemicals made only of petroleum based materials. Hence, the bioproducts herein can be followed or tracked in commerce on the basis of their unique carbon isotope profile. Bioproducts can be distinguished from petroleum based organic compounds by comparing the stable carbon isotope ratio (13C/12C) in each sample. The 13C/12C ratio in a given bioproduct is a consequence of the 13C/12C ratio in atmospheric carbon dioxide at the time the carbon dioxide is fixed. It also reflects the precise metabolic pathway. Regional variations also occur. Petroleum, C3 plants (the broadleaf), C4 plants (the grasses), and marine carbonates all show significant differences in 13C/12C and the corresponding δ13C values. Both C4 and C3 plants exhibit a range of 13C/12C isotopic ratios, but typical values are about −7 to about −13 per mil for C4 plants and about −19 to about −27 per mil for C3 plants (see, e.g., Stuiver et al., Radiocarbon 19:355 (1977)). Coal and petroleum fall generally in this latter range.

δ13C (‰)=[(13C/12C) sample−(13C/12C) standard]/(13C/12C) standard×1000

A series of alternative RMs have been developed in cooperation with the IAEA, USGS, NIST, and other selected international isotope laboratories. Notations for the per mil deviations from PDB is δ13C. Measurements are made on CO2 by high precision stable ratio mass spectrometry (IRMS) on molecular ions of masses 44, 45, and 46. The compositions described herein include fatty ester compositions and products produced by any of the methods described herein. Specifically, fatty ester composition or product can have a δ13C of about −28 or greater, about −27 or greater, −20 or greater, −18 or greater, −15 or greater, −13 or greater, −10 or greater, or −8 or greater. For example, the fatty ester composition or product can have a δ13C of about −30 to about −15, about −27 to about −19, about −25 to about −21, about −15 to about −5, about −13 to about −7, or about −13 to about −10. In other instances, the fatty ester composition or product t can have a δ13C of about −10, −11, −12, or −12.3. Fatty ester compositions and products produced in accordance with the disclosure herein can also be distinguished from petroleum based organic compounds by comparing the amount of 14C in each compound. Because 14C has a nuclear half-life of 5730 years, petroleum based fuels containing “older” carbon can be distinguished from fatty ester compositions and bioproducts which contain “newer” carbon (see, e.g., Currie, “Source Apportionment of Atmospheric Particles”, Characterization of Environmental Particles, J. Buffle and H. P. van Leeuwen, Eds., 1 of Vol. I of the IUPAC Environmental Analytical Chemistry Series (Lewis Publishers, Inc.) 3-74, (1992)).
The basic assumption in radiocarbon dating is that the constancy of 14C concentration in the atmosphere leads to the constancy of 14C in living organisms. However, because of atmospheric nuclear testing since 1950 and the burning of fossil fuel since 1850, 14C has acquired a second, geochemical time characteristic. Its concentration in atmospheric CO2, and hence in the living biosphere, approximately doubled at the peak of nuclear testing, in the mid-1960s. It has since been gradually returning to the steady-state cosmogenic (atmospheric) baseline isotope rate (14C/12C) of about 1.2×10-12, with an approximate relaxation “half-life” of 7-10 years. (This latter half-life must not be taken literally; rather, one must use the detailed atmospheric nuclear input/decay function to trace the variation of atmospheric and biospheric14C since the onset of the nuclear age.) It is this latter biospheric14C time characteristic that holds out the promise of annual dating of recent biospheric carbon. 14C can be measured by accelerator mass spectrometry (AMS), with results given in units of “fraction of modern carbon” (fM). The fatty ester compositions and products described herein include bioproducts that can have an fM 14C of at least about 1. For example, the bioproduct of the disclosure can have an fM 14C of at least about 1.01, an fM 14C of about 1 to about 1.5, an fM 14C of about 1.04 to about 1.18, or an fM 14C of about 1.111 to about 1.124.
Another measurement of 14C is known as the percent of modern carbon (pMC). For an archaeologist or geologist using 14C dates, AD 1950 equals “zero years old”. This also represents 100 pMC. “Bomb carbon” in the atmosphere reached almost twice the normal level in 1963 at the peak of thermo-nuclear weapons. Its distribution within the atmosphere has been approximated since its appearance, showing values that are greater than 100 pMC for plants and animals living since AD 1950. It has gradually decreased over time with today's value being near 107.5 pMC. This means that a fresh biomass material, such as corn, would give a 14C signature near 107.5 pMC. Petroleum based compounds will have a pMC value of zero. Combining fossil carbon with present day carbon will result in a dilution of the present day pMC content. By presuming 107.5 pMC represents the 14C content of present day biomass materials and 0 pMC represents the 14C content of petroleum based products, the measured pMC value for that material will reflect the proportions of the two component types. For example, a material derived 100% from present day soybeans would give a radiocarbon signature near 107.5 pMC. If that material was diluted 50% with petroleum based products, it would give a radiocarbon signature of approximately 54 pMC. A biologically based carbon content is derived by assigning “100%” equal to 107.5 pMC and “0%” equal to 0 pMC. For example, a sample measuring 99 pMC will give an equivalent biologically based carbon content of 93%. This value is referred to as the mean biologically based carbon result and assumes all the components within the analyzed material originated either from present day biological material or petroleum based material. A bioproduct comprising one or more fatty esters as described herein can have a pMC of at least about 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100. In other instances, a fatty ester composition described herein can have a pMC of between about 50 and about 100; about 60 and about 100; about 70 and about 100; about 80 and about 100; about 85 and about 100; about 87 and about 98; or about 90 and about 95. In yet other instances, a fatty ester composition described herein can have a pMC of about 90, 91, 92, 93, 94, or 94.2.
Fatty Ester Compositions
Examples of fatty esters include fatty acid esters, such as those derived from short-chain alcohols, including FAEE and FAME, and those derived from longer chain fatty alcohols. The fatty esters and/or fatty ester compositions that are produced can be used, individually or in suitable combinations, as a biofuel (e.g., a biodiesel), an industrial chemical, or a component of, or feedstock for, a biofuel or an industrial chemical. In some aspects, the disclosure pertains to a method of producing a fatty ester composition comprising one or more fatty acid esters, including, for example, FAEE, FAME and/or other fatty acid ester derivatives of longer chain alcohols. In related aspects, the method comprises a genetically engineered production host suitable for making fatty esters and fatty ester compositions including, but not limited to, FAME, FAEE, fatty acid propyl esters, fatty acid isopropyl esters, fatty acid butyl esters, monoglycerides, fatty acid isobutyl esters, fatty acid 2-butyl esters, and fatty acid tert-butyl esters, and the like.
Accordingly, in one aspect, the disclosure features a method of making a fatty ester composition that may comprise a decreased percentage of beta hydroxy esters and free fatty acids relative to a fatty ester compositions produced by a wild type ester synthase enzyme, such as '377. The variant '377 polypeptide or enzyme with improved fatty acid methyl ester activity has improved properties including, but not limited to, increased beta hydroxy esters, decreased beta hydroxyl esters, increased chain lengths of fatty acid esters, and decreased chain lengths of fatty acid esters. The method includes expressing in a host cell a gene encoding a polypeptide having ester synthase activity. In another aspect, the disclosure features a method of making a fatty ester composition that may or may not comprise free fatty acids, wherein the method includes expressing in a host cell a gene encoding a Ppro polypeptide having ester synthase activity. In some embodiments, the gene encoding the ester synthase polypeptide or thioesterase polypeptide with ester synthase activity is selected from the enzymes classified as EC 2.3.1.75 or EC 3.1.2, respectively, and any other polypeptides capable of catalyzing the conversion of an acyl thioester to fatty esters, including, without limitation, thioesterases, ester synthases, acyl-CoA:alcohol transacylases, alcohol O-fatty acid-acyl-transferase, acyltransferases, and fatty acyl-coA:fatty alcohol acyltransferases, or a suitable variant thereof.
In certain embodiments, an endogenous thioesterase of the host cell, if present, is unmodified. In certain other embodiments, the host cell expresses an attenuated level of a thioesterase activity or the thioesterase is functionally deleted. In some embodiments, the host cell has no detectable thioesterase activity. As used herein the term “detectable” means capable of having an existence or presence ascertained. For example, production of a product from a reactant (e.g., production of a certain type of fatty acid esters) is detectable using the methods known in the art or provided herein. In certain embodiments, the host cell expresses an attenuated level of a fatty acid degradation enzyme, such as, for example, an acyl-CoA synthase, or the fatty acid degradation enzyme is functionally deleted. In some embodiments, the host cell has no detectable fatty acid degradation enzyme activity. In particular embodiments, the host cell expresses an attenuated level of a thioesterease, a fatty acid degradation enzyme, or both. In other embodiments, the thioesterase, the fatty acid degradation enzyme, or both, are functionally deleted. In some embodiments, the host cell can convert an acyl-ACP or acyl-CoA into fatty acids and/or derivatives thereof such as esters, in the absence of a thioesterase, a fatty acid derivative enzyme, or both. Alternatively, the host cell can convert a free fatty acid to a fatty ester in the absence of a thioesterase, a fatty acid derivative enzyme, or both. In certain embodiments, the method further includes isolating a fatty ester composition, a fatty ester or a free fatty acid from the host cell or from the host cell culture. In preferred embodiments of the disclosure, the fatty acid derivative composition comprises a high percentage of fatty esters. In certain embodiments, the fatty ester or fatty ester composition is derived from a suitable alcohol substrate such as a short- or long-chain alcohol.
In general, the fatty ester or fatty ester composition is isolated from the extracellular environment of the host cell. In some embodiments, the fatty ester or fatty ester composition is spontaneously secreted, partially or completely, from the host cell. In alternative embodiments, the fatty ester or fatty ester composition is transported into the extracellular environment, optionally with the aid of one or more transport proteins. In still other embodiments, the fatty ester or fatty ester composition is passively transported into the extracellular environment.
In some embodiments, the disclosure comprises a method of producing a fatty acid ester or a fatty acid ester composition which composition may comprise a decreased percentage of beta hydroxy esters and/or free fatty acids by culturing a genetically engineered host cell under conditions that allow expression or overexpression of a mutant or variant '377 polypeptide. In an alternative embodiment, the disclosure comprises a method of producing a fatty acid ester or a fatty acid ester composition which composition may comprise an increased percentage of beta hydroxy esters and/or free fatty acids by culturing a genetically engineered host cell under conditions that allow expression or overexpression of a mutant or variant '377 polypeptide. In some embodiments, the method further comprises culturing the genetically engineered host cell in medium comprising carbon source (such as a carbohydrate) under conditions that permit production of a fatty acid ester or a fatty acid ester composition which may comprise an increased or decreased percentage of beta hydroxy esters and/or free fatty acids. In other embodiments, the disclosure comprises a method of producing a fatty acid ester or a fatty acid ester composition which composition may comprise free fatty acids by culturing a genetically engineered host cell under conditions that allow expression or overexpression of a Ppro polypeptide having ester synthase activity. In some embodiments, the method further comprises culturing the genetically engineered host cell in medium comprising carbon source such as a carbohydrate under conditions that permit production of a fatty acid ester or a fatty acid ester composition which may comprise free fatty acids.
In some embodiments, the disclosure provides an ester synthase polypeptide comprising the amino acid sequence of SEQ ID NO: 2, with one or more amino acid substitutions, additions, insertions, or deletions wherein the polypeptide has ester synthase activity. A mutant or variant '377 polypeptide has greater ester synthase activity than the corresponding wild type '377 polypeptide. For example, a mutant or variant '377 polypeptide is capable, or has an improved capacity, of catalyzing the conversion of thioesters, for example, fatty acyl-CoAs or fatty acyl-ACPs, to fatty acids and/or fatty acid derivatives. In particular embodiments, the mutant or variant '377 polypeptide is capable, or has an improved capacity, of catalyzing the conversion of thioester substrates to fatty acids and/or derivatives thereof, such as fatty esters, in the absence of a thioesterase activity, a fatty acid degradation enzyme activity, or both. For example, a mutant or variant '377 polypeptide can convert fatty acyl-ACP and/or fatty acyl-CoA into fatty esters in vivo, in the absence of a thioesterase or an acyl-CoA synthase activity.
In other embodiments, the disclosure provides a Ppro polypeptide comprising the amino acid sequence of SEQ ID NO: 51, with one or more amino acid substitutions, additions, insertions, or deletions wherein the polypeptide has ester synthase activity. In certain embodiments, a Ppm polypeptide of the disclosure has greater ester synthase activity than the corresponding wild type Ppro polypeptide. For example, a Ppro polypeptide of the disclosure having ester synthase activity is capable, or has an improved capacity, of catalyzing the conversion of thioesters, for example, fatty acyl-CoAs or fatty acyl-ACPs, to fatty acids and/or fatty acid derivatives. In particular embodiments, the Ppm polypeptide having ester synthase activity is capable, or has an improved capacity, of catalyzing the conversion of thioester substrates to fatty acids and/or derivatives thereof, such as fatty esters, in the absence of a thioesterase activity, a fatty acid degradation enzyme activity, or both. For example, a Ppro polypeptide having ester synthase activity can convert fatty acyl-ACP and/or fatty acyl-CoA into fatty esters in vivo, in the absence of a thioesterase or an acyl-CoA synthase activity.
In some embodiments, the mutant or variant '377 polypeptide or mutant or variant Ppm polypeptide comprises one or more of the following conserved amino acid substitutions: replacement of an aliphatic amino acid, such as alanine, valine, leucine, and isoleucine, with another aliphatic amino acid; replacement of a serine with a threonine; replacement of a threonine with a serine; replacement of an acidic residue, such as aspartic acid and glutamic acid, with another acidic residue; replacement of residue bearing an amide group; exchange of a basic residue, such as lysine and arginine, with another basic residue; and replacement of an aromatic residue, such as phenylalanine and tyrosine, with another aromatic residue. In some embodiments, the mutant or variant '377 polypeptide or mutant or variant Ppro polypeptide having ester synthase activity has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acid substitutions, additions, insertions, or deletions. In some embodiments, the polypeptide variant has ester synthase and/or acyltransferase activity. For example, the mutant or variant '377 polypeptide polypeptide or mutant or variant Ppro polypeptide is capable of catalyzing the conversion of thioesters to fatty acids and/or fatty acid derivatives, using alcohols as substrates. In a non-limiting example, the mutant or variant '377 polypeptide polypeptide or mutant or variant Ppro polypeptide is capable of catalyzing the conversion of a fatty acyl-CoA and/or a fatty acyl-ACP to a fatty acid and/or a fatty acid ester, using a suitable alcohol substrate, such as, for instance, a methanol, ethanol, propanol, isopropanol, butanol, pentanol, hexanol, heptanol, octanol, decanol, dodecanol, tetradecanol, or hexadecanol.
EXAMPLES
The following specific examples are intended to illustrate the disclosure and should not be construed as limiting the scope of the claims. The applicants have employed a one-gene-system for production of fatty acid esters in recombinant host cells. This includes expressing in suitable host cells mutant or variant '377 polypeptides that have been engineered to have improved ester synthase activity in order to produce fatty ester compositions at increased titer and yield. The fatty ester compositions obtained include FAME as well as increased and decreased amounts of beta hydroxy esters. The same strategy was used for making Ppro polypeptides with enhanced ester synthase activity for the production of fatty esters at increased titer and yield.
Protocols:
All protocols use 96 well plates, master block, 2 mL (Greiner Bio-One, Monroe, N.C. or Corning, Amsterdam, The Netherlands) for growing cultures and Costar plates for extracting fatty acid species from the culture broth. The protocols below include the fermentation conditions and can be used to evaluate fatty acid species production. Alternative protocols can also be used to evaluate fatty acid species production.
Protocol 1 (FA4P, 32° C.):
From an LB culture growing in 96 well plate: 30 μL LB culture was used to inoculate 270 μL FA2P (Table 4), which was then incubated for approximately 16 hours at 32° C. shaker. 30 μL of the overnight seed was then used to inoculate 300 μL FA4P+2% MeOH+1 mM IPTG (Table 4). The cultures were then incubated at 32° C. shaker for 24 hours, when they were extracted following the standard extraction protocol detailed below (Protocol 5).
Protocol 2 (BP3G2P, 32° C.):
Library glycerol stocks were thawed and 10 μL transferred to 150 μL LB+100 μg/mL spectinomycin in 96-shallow-well plates, which were incubated at 32° C. for 20 hours. 20 μL of this culture was used to inoculate 280 μL BS1G4Pmedia (Table 4), which was incubated at 32° C. shaking at 250 rpm for 20 hours. 20 μl of this culture was used to inoculate 380 μL BP3G2P media (Table 4). Cultures were incubated at 32° C. shaking for 24 hours, when they were either subjected to the nile red assay following the standard protocol detailed below or extracted following the standard extraction protocol detailed below (Protocol 5).
Protocol 3 (FA2, 32° C.):
From an LB culture growing in a 96 well plate: 304 of LB culture was used to inoculate 270 μL FA2, which was then incubated for approximately 16 hours at 32° C. on a shaker. 30 μL of the overnight seed was then used to inoculate 300 μL FA2+2% MeOH+1 mM IPTG. The cultures were then incubated at 32° C. on a shaker for 24 hours, when they were extracted following the standard extraction protocol detailed below (Protocol 5).
Protocol 4 (FA2P, 32° C.):
From an LB culture growing in 96 well plate: 30 μL LB culture was used to inoculate 270 μL FA2P, which was then incubated for approximately 16 hours at 32° C. shaker. 30 μL of the overnight seed was then used to inoculate 300 μL FA2P+2% MeOH+1 mM IPTG. The cultures were then incubated at 32° C. shaker for 24 hours, when they were extracted following the standard extraction protocol detailed below (Protocol 5).
Protocol 5 (Fatty Acid Species Standard Extraction):
To each well to be extracted 40 μL 1M HCl, then 300 μL butyl acetate with 500 mg/L C11-FAME as internal standard was added. The 96 well plate was then heat-sealed using a plate sealer (ALPS-300; Abgene, ThermoScientific, Rockford, Ill.), and shaken for 15 minutes at 2000 rpm using MixMate(Eppendorf, Hamburg, Germany). After shaking, the plate was centrifuged for 10 minutes at 4500 rpm at room temperature (Allegra X-15R, rotor SX4750A, Beckman Coulter, Brea, Calif.) to separate the aqueous and organic layers. 50 μL of the organic layer was transferred to a 96 well plate (96-well plate, polypropylene, Corning, Amsterdam, The Netherlands). The plate was heat sealed then stored at −20° C. until it was evaluated by GC-FID by employing standard automated methods.
Protocol 6 (Fatty Acid Species Standard Nile Red Assay):
After 24 hour fermentation, Nile Red assay was performed by adding 70 μL of fermentation broth to 130 μL of 1.54 μg/mL Nile Red in 84.6% water and 15.4% acetonitrile solution (for a final assay concentration of 1 μg/mL Nile Red) in a Greiner MicrolonFluotrac 200 plate and mixed by pipetting up and down. Relative fluorescence units were measured at excitation of 540 nm and emission of 630 nm using the SpectraMax M2.
Protocol 7 (Building a Saturation Library):
Standard techniques known to those of skill in the art were used to prepare saturation libraries. For example, the vector backbone may be prepared by using restriction endonucleases in the vector, while the creation of diversity in the DNA insert can be generated using degenerate primers. For example, the cloning of the vector backbone and a DNA insert with diversity can be performed using InFusion Cloning System (Clontech Laboratories, Inc., Mountain View, Calif.) according to the manufacturer's protocol.
Protocol 8 (Building a Combination Library):
Mutations identified as beneficial were combined to provide Ppro or 377 variants with further improvements in the production of fatty acid species or FAME. Standard techniques known to those of skill in the art were used to prepare the combination libraries. For example, the vector backbone can be prepared by using restriction endonucleases in the vector, while the creation of diversity in the DNA insert can be generated using primers to introduce the desired mutations. For example, the cloning of the vector backbone and a DNA insert with diversity can be performed using InFusion Cloning System (Clontech Laboratories, Inc., Mountain View, Calif.), according to manufacturer's protocol. For example, combination libraries can be generated using the transfer PCR (tPCR) protocol (Erijman et al., 2011. J. Structural Bio. 175, 171-177).
Protocol 9 (Library Screening):
Once the library diversity was generated in a saturation library or combination library, it was screened using one of the methods described above (Protocols 1-4). When screening ester synthase variants, three types of hits were identified: (1) increased fatty acid ester species titer (“FAS” titer); (2) increased amount of FAME produced; e.g., C14-FAME; and/or (3) decreased amount of beta hydroxyl ester produced. The mutations in the '377 variants within each hit were identified by sequencing using standard techniques routinely employed by those of skill in the art. The mutations in the '377 variants within each hit were identified by sequencing using standard techniques routinely employed by those of skill in the art. Tables 1 and 2 list the mutations (“hits”) identified as beneficial in saturation libraries and Table 5 lists mutations (“hits”) identified as beneficial in combination libraries. When screening Ppro variants, two types of hits were identified (1) increased fatty acid ester species; (2) increase in the amount of FAME produced. The mutations in the Ppro variants within each hit were identified by sequencing and using standard techniques routinely employed by those of skill in the relevant field. Tables 8 through 11 list the mutations (“hits”) identified as beneficial in saturation libraries and Tables 12 and 14 list mutations (“hits”) identified as beneficial in combination libraries.
Example 1
Saturation Library Prepared Using WS377 as a Template
A full saturation library of the ester synthase from Marinobacter hydrocarbonoclasticus (“377”), was built and screened for variants that showed improvement over the wild type WS377 (Protocol 7). The plasmid used to make the full saturation library was designated pKEV027 which expressed SEQ ID NO: 1 (i.e., the wild type nucleic acid sequence of WS377). The full saturation library was screened in an E. coli strain (BD64) that was engineered to block beta oxidation (via deletion of FadE) and overexpress a number of fatty acid biosynthetic pathway enzymes (e.g., FabA, FabB, FabF, FabG, FabI, FabH, FabV, FabZ, accABCD) through standard manipulation techniques. However, any suitable host strain can be used herein. The libraries were screened using one of the standard protocols (Protocols 1-2) described above. The improvements were originally classified as either improving titer of FAME or reducing the fraction of beta hydroxy esters (referred to herein as “β-OH esters” or “β-OH FAME” or “beta-OH” or “beta-hydroxy”) without necessarily affecting FAME titer. The results from screening saturation libraries are shown in Tables 1 and 2 below. Table 1 presents results for mutations that all showed increased fatty ester titers when using '377 as the template (SEQ ID NO: 1). Table 2 presents results for mutations that led to a decrease in % β-OH FAME using '377 as the template. Notably, some mutants showed both, an improved titer of FAME as well as a decrease in β-OH FAME, which is beneficial if the final desired product is mostly FAME. Some mutants showed an improved titer of FAME as well as an increase in β-OH FAME. In addition, some mutants resulted in an increase or decrease of short-chain esters.
As can be seen in Table 1 below, mutations from the '377 saturation libraries correlated with improved fatty ester titer in all mutants, while the production of β-OH esters and shorter chain length ester varied from mutant to mutant. For example, a substitution of the amino acid at position 15 (Serine to Glycine) changed the total FAME titer from baseline 1 to 2.402. Thus, the titer increased by 2.4 fold compared to baseline (i.e., wild type). In this particular mutant, (β-OH esters increased by about 2.66 fold. The production of esters with a carbon chain length of 14 over esters with a carbon chain length of 16 increased by 1.48 fold over baseline (i.e., wild type). On the other hand, a substitution of the amino acid position 393 (Glutamate to Glycine) increased the FAME titer by 2.3 fold and decreased the β-OH esters to 70% of control while C14/C16 ester production stayed about the same. The mutations of Table 1 correspond to the indicated residues of SEQ ID NO: 2.
With respect to the '377 sequences described herein “M” (ATG) is considered to be amino acid “0”. The first amino acid after the ATG is designated amino acid “1”.










TABLE 1










Average













Total 
% β-




Mutation 
FAME
OH
C14/C16
















S15G
2.402
2.664
1.476



Q26T
1.356
2.233
1.163



L39M
1.64
2.82
0.936



L39S
2.228
2.368
1.127



L39A
1.876
2.412
0.94



R40S
1.328
2.587
1.259



D41H
2.225
2.344
1.312



D41G
2.031 
3.474
1.217



D41A
1.792
2.38
2.074



V43K
2.109 
1.617
0.421



V43S
1.768
2.283 
1.053



T44F
2.007 
1.577 
0.872



A73Q
2.195
1.877
0.915



V76L
1.553
1.255 
0.76



D77A
1.952 
1.645
0.887



K78W
1.758
1.469 
0.826



K78F
1.794
1.984 
0.767



I80V
2.961
2.903
1.028



R93T
1.649
1.587 
0.734



G101L
1.787
1.294 
1.288



I102R
1.718
1.418
0.928



N110R
1.582
2.038 
0.957



P111S
2.05
1.09
1.176



P111G
1.855
1.228
1.154



G126D
1.963
1.065
0.751



R131M
2.139 
1.857 
1.115



V155G
2.267
0.865
1.127



N164R
1.692
1.114
0.806



V171R
2.657 
2.152
0.815



V171E
1.472
1.065
1.042



R172W
1.499
6.244
0.922



D182G
1.89
1.18
1.147



E184L
2.352 
1.055
1.157



E184F
1.589
1.026
1.108



E184S
1.566
0.937
1.147



E184R
1.733
1.185
1.242



E184G
1.647
1.463
1.138



A185M
1.518
1.134
1.131



A185L
1.754
2.777
1.176



P188R
2.519
1.138
1.108



A190R
1.86
1.204
1.314



A190W 
1.559
1.645
1.196



S192V
3.748
2.086
1.148



S192A
1.518
0.998
0.902



Q193R
1.735
1.2
1.344



Q201W
1.748
1.357
1.176



Q201V
1.79
1.289
1.32



Q201A
1.929
1.154
1.191



A202L
2.571
1.224
1.192



D203R
1.998
1.042
1.552



P206F
1.466
1.726
1.043



G212L
1.919
1.179
1.167



V219L
2.017
1.396
0.737



T242R
1.879
0.894
1.072



T242K
1.678
2.272
1.115



A243R
2.56 
1.232
0.912



R246Q
3.571
0.692
0.412



R246L
2.083
0.746
0.711



V272A
1.984
1.511
0.78



Q287S
1.723
1.275
0.646



D292F
1.827
1.162
0.814



P294G
1.649
1.692
0.874



G298G
1.511
1.383
0.796



I303G
3.36
0.388
0.21



I303W
2.075
0.578
0.58



R304W
1.578
0.425
0.354



F317W
2.208
2.076
1.529



I319G
2.515
0.89 
0.721



A323G
1.766
1.188
0.751



D328F
1.816
1.055
0.671



Q334S
1.573
0.935
0.821



Q348A
1.442
3.247
2.463



P351G
1.875
1.211
1.312



S353T
2.123
2.007
0.957



M360W
2.303
0.634
0.582



M360S
1.502
0.397
0.394



Y366W
1.506
0.969
1.175



Y366G
1.407
1.378
0.91



G375A
1.96
1.821
1.24



M378A
1.877
1.616
0.834



E393G
2.305
0.723
0.932



T395E
1.655
1.224
0.719



V409L
2.314
0.796
0.295



I420V
1.474
1.119
0.828



S442G
1.947
1.166
1.226



A447C
1.775
1.819
1.132



A447L
1.545
1.341
1.021



A447I
1.867
1.459
1.117



L454V
1.804
1.137
0.672



R2S, Q348A
1.628
3.976
2.475



R2R, S235S
1.178
1.949
1.213



G4R, N331G
2.262
2.431
1.324



T5P, S186T
1.6
2.837
1.198



T5S, R98D
2.287
2.01
1.277



T5P, G310H
1.711
2.141
1.217



L6Q, Q1935
1.529
2.069
2.878



L6Q, A279V
1.414
3.573
1.003



D7N, T170R
3.163
1.079
0.853



G33S, S442E
2.28
1.453
1.199



R150P, A279G
1.702
1.434
0.769



E184G, K472T
2.619
1.022
1.269



V187G, *474Y
1.678
1.481
1.028



P188R, A197V
2.944
1.078
1.146



M195G, A197T
1.291
4.127
1.223



R207A, P366P
1.877
1.164
0.685



D307V, T470P
1.858
1.094
1.026



24 by repeat
1.996
2.34
0.944



24bp repeat, R177V
2.553
2.516
1.518



24bp repeat, V171R
2.724
2.338
0.821










Some mutations in the tables have stars, for example, *474Y, which means that the position has a stop codon.
The applicants discovered a 24 bp repeat in one of the variant mutant nucleic acid sequences (i.e., SEQ ID NO: 28). The 24 bp repeat is a duplication of nucleic acid region ATGAAACGTCTCGGAACCCTGGAC (SEQ ID NO: 27) and located at the start of the gene prior to the ATG start codon. Thus, SEQ ID NO: 28 is a variant '377 nucleic acid sequence (this sequence is the result of SEQ ID NO: 27 and SEQ ID NO: 1 combined with no additional mutations) that codes for the amino acid sequence of SEQ ID NO: 29. SEQ ID NO: 29 gave surprisingly good titer as shown in Table 1. However, two additional mutants were detected that included SEQ ID NO: 29 as a template sequence with additional mutations in it, wherein the mutations increased the titer even further (see Table 1). (The numbering of mutations does not include the 24 bp repeat.) Notably, the 24 bp repeat led to an increase in titer of FAS and % beta-OH FAME, as seen in Table 1 which depicts the titer obtained when expressing SEQ ID NO: 29 in a host strain.
As can be seen in Table 2 below, some of the mutations from the '377 saturation libraries correlated with a decrease in % β-OH FAME while still increasing the total FAME tighter to a lesser degree compared in Table 1 (supra). However, some mutants significantly decreased % β-OH FAME while still significantly increasing total FAME titer. With respect to the '377 sequences described herein “M” (ATG) is considered to be amino acid “0”. The first amino acid after the ATG is designated amino acid “1”. The mutations of Table 2 correspond to the indicated residues of SEQ ID NO: 2.








TABLE 2








Average











Total 
% β-



Mutations 
FAME
OH
C14/C16





T24W
1.141 
0.533 
1.024


V69W
1.154
0.661
0.657


S105G
1.146
0.875
1.109


P111D
1.271 
0.802
0.822


D113V
1.242
0.687
0.629


D113A
1.279 
0.740
0.580


S115R
1.118
0.880
0.925


C121S
1.088 
0.756
0.969


H122S
1.186
0.905
0.780


L127G
1.455
0.644
0.507


L134T
1.350
0.495
0.611


T136G
1.090
0.591
0.472


I146K
1.523
0.381
0.342


I146R
1.328
0.199
0.220


S147A
1.233
0.796
0.945


T158R
1.694
0.749
0.855


M165K
1.260
0.889
0.569


V171H
1.168
0.677
0.902


V171W
1.168
0.091
0.812


R172S
1.590
0.505
0.897


P173W
1.189
0.647
0.652


S192L
1.215
0.898
1.176


A228G
1.147
0.787
0.503


V234C
1.059
0.185
0.775


H239G
1.318
0.833
0.874


Q244G
1.397
0.601
0.923


R246W
1.191
0.312
0.323


R246Q
3.571
0.692
0.412


R246G
1.660
0.669
0.431


R246V
1.130
0.454
0.838


R246L
2.083
0.746
0.711


R246A
1.470
0.757
0.559


Q250W
1.061
0.850
0.860


Q253G
1.186
0.772
0.877


L254R
1.409
0.647
0.900


L254T
1.089
0.704
0.868


K258R
1.098
0.657
0.759


S264D
1.220
0.876
0.549


S264V
1.411
0.730
0.489


S264W
1.772
0.646
0.491


G266S
1.121
0.846
0.613


S267G
1.214
0.262
0.263


Y274G
1.115
0.789
0.556


A285R
1.273
0.864
0.893


V301A
1.500
0.755
0.440


N302G
1.355
0.254
0.182


I303G
3.360
0.388
0.210


I303W
2.075
0.578
0.580


I303R
1.257
0.373
0.857


R304W
1.578
0.425
0.354


A306G
1.103
0.808
1.164


D307G
1.079
0.645
1.095


D307L
1.197
0.817
1.279


D307R 
1.537
0.679
0.848


D307V 
1.273
0.585
0.984


E309A 
1.190
0.544
0.775


E309G 
1.392
0.735
0.667


E309S
1.249
0.730
0.768


G310R
1.801
0.886
1.146


G310V
1.098
0.732
0.879


T311S
1.163
0.634
0.297


T313S
1.208
0.587
0.903


Q314G
1.140
0.347
0.694


I315F
1.345
0.178
0.182


S316G
1.079
0.000
0.413


A320C
1.387
0.777
0.595


A327I
1.057
0.373
0.889


K349C 
1.145
0.781
0.481


K349Q
1.105
0.637
0.693


K349H
1.243
0.663
0.458


K349A
1.200
0.484
0.472


K352I 
1.207
0.576
0.418


K352N 
1.084
0.783
0.679


T356W
1.153
0.345
0.727


T356G
1.135
0.688
1.019


M360R
1.238
0.375
0.487


M360S
1.502
0.397
0.394


M360W
2.303
0.634
0.582


M360Q
1.062
0.735
0.865


M363W
1.218
0.000
0.497


P365G
1.102
0.611
0.376


I367M 
1.274
0.704
0.768


M371R
1.297
0.073
0.554


G373R
1.440
0.720
0.436


G375S
1.146
0.609
0.816


G375V
1.429
0.770
0.833


T385G
1.215
0.875
0.609


E393R
1.290
0.234
0.478


E393W
1.768
0.817
0.852


E393G
2.305
0.723
0.932


E404K
1.350
0.762
0.603


E404R
1.089
0.793
0.529


V409L
2.314
0.796
0.295


S410G
1.576
0.786
0.653


L411A
1.274
0.735
1.060


C422G
1.067
0.581
0.572


S424G
1.755
0.538
0.657


S424Q
1.268
0.395
0.629


D455E
1.096
0.737
0.749


E458W
1.428
0.654
0.503


I461G
1.259
0.927
0.584


K1T, L1_355_L3 
1.183
0.874
0.732


K1N, I146L
1.269
0.695
0.692


L30H, K472*
1.054
0.872
1.139


D41Y, D307F
1.604
0.664
0.633


E99Q, D307N
1.610
0.801
0.969


V149L, N430G
1.232
0.569
0.900


R150P, A190P, E286H
1.186
0.614
0.338


V171F, R469R
1.085
0.619
0.713


L457Y, R467T
1.111
0.544
0.594









Example 2
Combination Library Prepared Using WS377 as a Template
Standard techniques were employed to prepare combination libraries (Protocol 8). The mutations tested in combination libraries (Tables 3A and 3B) were originally identified in the full saturation library of '377 (as described in Example 1). The plasmid used to make the combination libraries was pKEV38, which was constructed by removing the accABCD_birA from pKEV027. Without wanting to be bound by theory, the removal of the accABCD_birA is believed to reduce the intermediate levels of the fatty acids in the biosynthetic pathway such that the affinity of the mutant enzyme for the substrate is more easily detectable. Cells were plated on 2NBT+Nile Red agar (Table 4). Colonies were observed using the Dark Reader DR88X Transilluminator at an excitation range of 400 nm-500 nm and an amber filter permissive at 580-620 nm, handpicked into LB+100 μg/mL spectinomycin, grown overnight, and screened using one of the standard protocols (Protocols 1-2) described above. As shown in FIG. 4, one of the variants from this library showed a dramatic increase in FAS over the control (BD64/pKEV038, SEQ ID NO: 2). FIG. 4 shows the results of a plate fermentation of combination library hits. Several library hits outperformed the control (i.e., BD64/pKEV038) showing that '377 variants can support high production of FAS. The plasmid of the highest-producing variant, named pSHU10 (expressing variant '377 of SEQ ID NO: 14), was isolated and used as template for subsequent combination libraries.
The combination libraries were screened in strain BD64 (supra) by using one of the standard protocols (Protocols 1-2; supra). The results from screening '377 combination libraries are shown in Table 5.
Table 3A below shows the amino acid substitutions that were introduced in the first round of combination libraries. The mutations of Table 3A correspond to the indicated residues of SEQ ID NO: 2.








TABLE 3A







Mutations
T5S; S15G; G33S; L39S; L39A; D41A; V43K; I102R; 



P111S; R131M; V171R; P188R; S192V; Q201V; R246Q; 



R246L; I303W; I303G; F317W; I319G; S353T; E393G; 



V409L; S442G









Table 3B below shows the amino acid substitutions that were introduced in the second round of combination libraries. The mutations of Table 3B correspond to the indicated residues of SEQ ID NO: 2.













TABLE 3B







K1T
G101L
R172S
D203R
A285R
A327I
T385G


K1N
I102R
R172W
P206F
E286H
D328F
E393G


G4R
S105G
P173W
R207A
Q287S
N331G
E393R


S5P
N110R
R177V
G212L
D292F
Q334S
E393W


S5T
S111P
A179V
G212A
P294G
Q348A
T395E


L6Q
S111G
D182G
V219L
V301A
Q348R
E404K


D7N
S111D
E184L
A228G
N302G
K349C
E404R


G15S
D113V
E184G
V234C
I303W
K349Q
L409V


T24W
D113A
E184R
H239G
I303G
K349H
S410G


Q26T
S115R
E184F
T242R
I303R
K349A
L411A


L30H
C121S
E184S
T242K
R304W
P351G
I420V


G33S
H122S
A185L
A243R
A306G
K352I
C422G


L39A
G126D
A185M
Q244G
D307F
K352N
S424G


L39M
L127G
S186T
R246Q
D307N
T353S
S424Q


L39S
R131M
V187G
R246L
D307G
T356G
N430G


R40S
L134T
V187R
R246G
D307L
T356W
G442S


D41A
T136G
R188P
R246W
D307R
Q357V
G442E


D41H
I146K
A190R
R246V
D307V
M360R
M443G


D41G
I146R
A190P
R246A
E309A
M360W
A447C


D41Y
I146L
A190W
Q250W
E309G
M360S
A447L


V43K
S147A
S192V
Q253G
E309S
M360Q
A447I


V43S
V149L
S192L
L254R
G310R
M363W
L454V


T44F
R150P
S192A
L254T
G310H
P365G
D455E


V69W
V155G
Q193S
K258R
G310V
Y366W
L457Y


A73Q
T158R
Q193R
S264D
T311S
Y366G
E458W


V76L
N164R
M195G
S264V
T313S
I367M
I461G


D77A
M165K
A197V
S264W
Q314G
M371R
R467T


K78W
T170R
A197T
G266S
I315F
G373R
K472T


K78F
R171V
L200R
S267G
S316G
G375S
K472*


I80V
R171H
Q201V
V272A
W317F
G375V


R93T
R171E
Q201W
Y274G
I319G
G375A


R98D
R171F
Q201A
A279G
A320C
M378A


E99Q
R171W
A202L
A279V
A323G
V381F









Table 4 below shows the media names and formulations that were used.








TABLE 4





Media Name
Formulation


















2NBT + Nile Red
1
X
P-lim Salt Soln w/(NH4)2SO4


agar
1
mg/ml
Thiamine



1
mM
MgSO4



1
mM
CaCl2



30
g/L
glucose



1
X
TM2



5
g/L
casaminoacids



15
g/L
Agarose



2%

Methanol



0.5
mg/L
Nile Red



10
mg/L
Fe Citrate



100
mg/L
spectinomycin



100
mM
BisTris (pH 7.0)


BS1G4P
1
X
P-lim Salt Soln w/(NH4)2SO4



1
mg/ml
Thiamine



1
mM
MgSO4



0.1
mM
CaCl2



10
g/L
glucose



1
X
TM2



10
mg/L
Fe Citrate



100
μg/mL
spectinomycin



100
mM
BisTris (pH 7.0)


BP3G2P
0.5
X
P-lim Salt Soln w/(NH4)2SO4



1
mg/ml
Thiamine



1
mM
MgSO4



0.1
mM
CaCl2



50
g/L
glucose



1
X
TM2



10
mg/L
Fe Citrate



100
μg/mL
spectinomycin



100
mM
BisTris (pH 7.0)



2.5
g/L
(NH4)2SO4



2%

MeOH



1
mM
IPTG


FA2P
1
X
P-lim Salt Soln



2
g/L
NH4Cl



1
mg/ml
Thiamine



1
mM
MgSO4



0.1
mM
CaCl2



30
g/L
glucose



1
X
TM2



10
mg/L
Fe Citrate



100
mM
BisTris (pH 7.0)


FA4P
0.5
X
P-lim Salt Soln



2
g/L
NH4C1



1
mg/ml
Thiamine



1
mM
MgSO4



0.1
mM
CaCl2



50
g/L
glucose



1
X
TM2



10
mg/L
Fe Citrate



100
mM
BisTris (pH 7.0)


FA2 (2 g/L NH4C1) 
1
X
Salt Soln


for 96 well plate
1
g/L
NH4Cl


screen
1
mg/ml
Thiamine



1
mM
MgSO4



0.1
mM
CaCl2



30
g/L
glucose



1
X
TM2



10
mg/L
Fe Citrate



100
mM
BisTris (pH 7.0)


FA2 (2 g/L NH4C1)
1
X
Salt Soln


with Triton for shake
1
g/L
NH4Cl


flask validation
1
mg/ml
Thiamine



1
mM
MgSO4



0.1
mM
CaCl2



30
g/L
glucose



1
X
TM2



10
mg/L
Fe Citrate



0.05%

Triton X-100



100
mM
BisTris (pH 7.0)









Table 5 below depict the secondary screen, showing the mutations over pSHU10 (which contains mutations T5S, S15G, P111S, V171R, P188R, F317W, S353T, V409L, S442G) and the normalized GC-FID results. The results depicted are relative to control (i.e., pSHU10, SEQ ID NO: 14). Each line on the table represents a different combination mutant that was made. All mutants were measured to have distinct properties in that they either affected the total FAME produced, the total FAS produced, the percent FAME produced, or the ester species in the final composition (with C12/C14 or C14/C16 chain length), or a combination of these properties. As can be seen in Table 5, some mutants behaved similar to other mutants in terms of their properties. The mutations of Table 5 correspond to the indicated residues of SEQ ID NO: 2.
For example, as shown on the first line in Table 5 below, the mutant containing mutations A190P, N302G, T313S, and Q348R produced a higher percent FAME (and reduction of β-0H FAME), and also increased the production of C14/C16 chain length esters, meaning it produced more of C14 esters than C16 esters in the final product. This is desirable since shorter chain esters can be used for many products including biodiesel. The results also show that the applicants were able to alter the final chain length of ester species in the final composition. The mutations of Table 5 correspond to the indicated residues of SEQ ID NO: 2.
The following combinations of mutations relate to the results shown herein; their performance is relative to the control (pSHU10; SEQ ID NO: 14). KASH008 (SEQ ID NO: 16) increased the % FAME by 1.4 fold, while increasing titer by 1.06 fold. KASH032 (SEQ ID NO: 18) increased % FAME by 1.29 fold, while affecting titer. KASH040 (SEQ ID NO: 20) increased % FAME by 1.16 fold, while the titer decreased to 0.91 times the control. KASH078 (SEQ ID NO: 26) increased the % FAME by 1.25 fold, while decreasing titers to 0.76 of control. KASH060 (SEQ ID NO: 22) and KASH061 (SEQ ID NO: 24) are of special interest because they affected not only the % FAME, but they also produced significantly shorter ester chain products, as was shown by an increase in C12/C14 (6.8 fold for KASH060 and 3.1 fold for KASH061) and C14/C16 (4.0 fold for KASH060 and 3.2 fold for KASH061).












TABLE 5






Total
Total





Mutation
FAME
FAS
% FAME
C12/C14
C14/C16




















A190P, N302G, T313S, Q348R
0.545
0.475
1.146
0.945
1.486


A190P, S264V, A327I, Q348A
0.444
0.807
0.556
2.441
3.171


A197T, V219L, R467L
0.409
0.473
0.865
1.491
1.821


A197V, Q250W, G442S
0.663
0.647
1.024
1.130
1.610


A228G, D328F, L409V
0.922
1.385
0.701
1.254
1.918


A279V, K472T
0.904
1.286
0.732
0.643
1.733


A327I
0.682
0.923
0.738
1.225
2.259


A327I, Q348R, V381F, I420V
0.473
0.974
0.485
2.536
3.363


A73Q, G101L, H122S, R172S, Q201W, Q357V
0.947
0.881
1.103
0.448
1.539


A73Q, G101L, H122S, V187G, Q201V, N331G,
0.530
0.525
1.009
0.838
1.651


A447L


A73Q, H122S, V187G, Q201A, V234C, G375S,
0.484
0.390
1.242
0.647
1.231


A447L


A73Q, Q201W, I315F, G375V, A447I
0.437
0.350
1.242
0.081
0.951


C121S, A228G, A279V, K472*
0.912
0.989
0.962
0.799
1.389


C121S, H122Q, S147A, L200R
0.654
0.608
1.122
0.244
1.164


C121S, R171H, I303R
0.901
0.805
1.199
2.550
1.812


C121S, S147A
1.016
1.172
0.945
2.072
1.144


D113V, E184F, G212L, H239G, V272A, T395E,
0.528
0.506
1.044
1.278
1.557


N430G


D113V, E184F, N430G, I461G
0.818
0.784
1.042
0.923
1.411


D113V, E184R, D203E, D307N, N430G, I461G
0.918
0.957
0.965
0.867
1.652


D307F
0.991
1.122
0.886
0.835
1.563


D307N
0.379
0.393
0.973
1.675
1.700


D328F, K472T
1.068
1.580
0.742
2.931
1.740


D328F, T356G, L409V, A468T, K472T
0.971
1.303
0.772
0.786
1.645


D41G, Q193R, Q244G, G310V, Y366G, S424G
0.669
0.569
1.176
0.000
0.576


D41H, I80V, D182G, Q193S, R207A, S267G,
0.535
0.414
1.296
1.056
0.598


G310V, S424Q


D41Y, I80V, A320C
0.579
0.558
1.043
1.109
1.219


D41Y, N110R, Q193S, Y366W
0.641
1.214
0.528
1.179
2.665


D455E
0.671
0.842
0.800
1.188
2.183


D77A, A190W, T242K, N302G, T313S
0.560
0.436
1.285
2.048
0.554


D77A, L127G, V155G, A190R, D455E
0.304
0.237
1.292
0.670
1.216


D77A, L127G, V155G, V381F
0.699
0.646
1.084
1.291
1.415


D77A, N302G, T313S, Q348R, M363W, V381F,
0.357
0.300
1.192
7.037
1.598


I420V


D77A, S105G, A190P
0.593
0.657
0.925
1.077
1.678


D77A, T170M, R177V, T242R, S264D
0.832
1.206
0.689
1.095
1.851


D77A, V155G, A190P, T242K
0.640
0.589
1.088
1.105
1.726


D7N, D41Y, R207A, G310V, Y366G
0.762
0.649
1.174
0.886
0.767


D7N, N164R, D182G, R207A, G310R, P351G,
0.509
0.405
1.258
0.000
0.391


S424Q, T470A


D7N, Q193R, Y366W, E393G, S424Q, E458G
0.655
0.603
1.086
1.653
0.719


D7N, Q193R, Y366W, E393G, S424Q, E458W
0.658
0.561
1.172
0.637
0.538


E184F, A323G, T395E, I461G
0.960
1.009
0.952
0.868
1.404


E184G, I461G
0.994
1.164
0.854
0.829
1.631


E184G, M195G, G212A, T311S, N430G, I461G
0.870
0.933
0.932
0.809
1.705


E184L, M195G, G212A, H239G, T311S, K352I
0.769
0.654
1.178
0.943
1.229


E184R, I461G
0.375
0.438
0.857
0.930
2.234


E184S, G212A, V272A, I367M, I461G
0.707
0.635
1.118
1.175
1.660


E184S, G212L, H239G, K352N, I461G
0.734
0.763
0.964
1.112
1.735


E458W
0.894
1.126
0.794
1.125
1.692


E99Q, A228G, A279G, I303R, L409V, K472T
0.788
0.709
1.193
2.434
1.496


E99Q, C121S, T356G, K472T
1.360
1.552
0.946
2.043
1.292


E99Q, I303W, T356G, K472T
1.286
1.260
1.096
1.966
1.049


E99Q, R171V, M443G
0.734
0.790
0.971
0.805
1.602


E99Q, S147A, R171W, L200R, K472*
1.019
1.540
0.696
1.224
1.863


G101L, G375A, S410G
0.642
0.577
1.113
0.604
0.973


G101L, H122S, V187R, G375S, A447I
0.280
0.237
1.187
0.520
1.544


G101L, Q201V, I315F, G375V, A447L
0.481
0.400
1.199
0.730
1.359


G126D, A202L, A306G, S316G, M360S
0.528
0.410
1.286
0.248
0.549


G126D, A306G, M360W
0.443
0.353
1.258
0.000
0.972


G126D, M378A
0.489
0.456
1.076
0.364
1.146


G126D, R188P, A306G, M360W
0.526
0.439
1.207
0.000
1.109


G15S, D113A, E184G, T395E, N430G, I461G
0.921
0.865
1.065
0.867
1.348


G15S, E184G, G212A, K352N, I461G
1.006
1.045
0.962
0.856
1.465


G15S, E184L, H239G, V272A, I461G
0.788
0.701
1.124
1.109
1.713


G15S, E184L, T311S, K352N, I461G
0.769
0.646
1.193
1.202
1.150


G15S, H239G, I461G
0.465
0.468
0.995
1.504
1.768


G15S, I461G
1.159
1.314
0.883
0.819
1.431


G15S, M165K, E184F, G212A
0.649
0.767
0.846
1.151
1.541


G15S, M165K, E184S, A323G, I367M, N430G,
0.554
0.484
1.145
1.915
1.062


I461G


G15S, M195G, H239G, I461G
0.783
0.698
1.120
1.166
1.523


G15S, R93T, T136G, G212L, H239G, I461G
0.594
0.528
1.125
0.788
1.466


G15S, V272A, I461G
0.536
0.468
1.144
1.304
1.875


G212A, D307F, K352N, I461G
0.794
0.693
1.146
1.172
1.432


G33S, I102R
0.777
1.150
0.675
0.742
1.702


G33S, I102R, M360W, M378A
0.577
0.528
1.101
0.298
1.542


G33S, L454V
0.459
0.577
0.797
0.844
2.063


G33S, P173R, R188P, R246P, A306G
0.414
0.354
1.168
0.449
2.310


G33S, R188P, M360S, L454V
0.466
0.378
1.232
0.139
1.564


G33S, R246V, A306G, M360R
0.597
0.457
1.306
0.000
0.599


G33S, T470S
0.680
1.044
0.654
0.792
1.749


G373R
1.159
1.332
0.943
2.117
1.109


G375S, A447I
0.765
0.863
0.886
0.753
1.631


G442E
0.566
0.688
0.824
0.944
1.738


G4R, G33S, G126D, P173S, R188P, R246V,
0.524
0.400
1.311
0.000
0.382


Q334S, M360W, L411P


G4R, P173W, Q175E, R246Q, K258R, M360S
0.171
0.133
1.286
0.000
0.434


G4R, R188P, R246Q
0.603
0.500
1.209
0.000
1.093


H122S, G375S
0.482
0.405
1.193
0.717
1.483


H122S, I315F
0.461
0.423
1.091
0.724
1.575


H122S, L254T, N331G
0.436
0.371
1.173
0.441
1.113


H122S, Q201V
0.522
0.898
0.590
0.741
4.476


H122S, R172S, Q201W, A447I
0.567
0.496
1.140
1.294
2.362


H122S, V187R, L254T, N331G
0.389
0.329
1.176
0.357
1.554


H122S, V234C, L254R, I315F
0.456
0.365
1.242
0.101
1.069


H239G, K352I, T395E, I461G
0.795
0.703
1.131
1.024
1.636


H239G, T311S, K352N, I461G
0.913
0.782
1.168
1.066
1.125


I102L, R246W, K258R, A306G, M360R,
0.559
0.464
1.205
0.000
0.493


L411A, L454V


I102R, P173W, A202L, Q334S, M360R
0.495
0.405
1.225
0.175
0.795


I102R, R246A, Q334S, M360R
0.580
0.451
1.286
0.000
0.519


I102R, R246L, K258R, A306G, M360R, L411A,
0.664
0.517
1.286
0.000
0.360


L454V


I102R, R246Q, K258R, M360S
0.709
0.448
1.502
0.052
0.194


I102R, R246W, L454V
0.772
0.668
1.157
0.000
0.940


I146K, V219L, D307N
0.357
0.305
1.170
2.470
1.551


I146K, V219L, D307N, G442S
0.356
0.286
1.248
3.212
0.932


I146L, A185L, D307R, G442S
0.358
0.276
1.294
2.393
0.529


I146L, V219L, D307R, G442S
0.361
0.284
1.272
4.822
0.565


I146R, A197T, D307G
0.281
0.221
1.274
4.844
0.902


I146R, A197T, D307N, G442E
0.315
0.255
1.234
3.768
1.213


I303R, D328F
0.619
0.531
1.212
1.228
1.882


I303W
1.192
1.188
1.088
1.898
1.235


I315F
0.529
0.483
1.099
0.715
1.406


I315F, N331G, G375S, A447C
0.606
0.532
1.135
0.389
1.283


I420V
0.567
0.864
0.656
1.033
2.097


I461G
0.914
1.151
0.794
0.770
1.821


K472T
0.974
1.088
0.894
1.215
1.442


K78F, R131M, S192V, G266S, K349H
0.754
0.540
1.327
0.660
0.537


K78F, S192V, A243R, K349H
1.501
1.055
1.417
0.695
0.629


K78W, S111G, C422G
0.788
0.538
1.387
0.839
0.495


L127G, A190W, D203R, A327I, I420V, D455E
0.547
0.565
0.968
1.198
1.590


L127G, A190W, S264D, V381F
0.292
0.260
1.124
0.994
1.950


L134T
0.745
1.165
0.639
1.364
2.156


L200R, A228G, M443G
0.537
0.569
0.980
0.610
1.696


L200R, A228G, T356W, G373R
1.224
1.167
1.126
1.326
0.904


L200R, I303R, Q314G, T356G
1.280
1.092
1.163
0.651
0.703


L30H, Q201V, V234C, G265D, R304W, G375V
0.271
0.217
1.242
0.784
0.696


L30H, R304W, I315F
0.507
0.397
1.282
0.626
0.650


L30H, V187G, Q201V, L254T, I315F, Q357V,
0.456
0.353
1.294
0.746
0.710


G375V


L30H, V234C, S410G, A447L
0.862
1.035
0.833
1.022
1.401


L39A, D77A, S105G, V155G, T242K, V381F,
0.595
0.557
1.071
4.219
1.897


T470S


L39A, D77A, T242R, S264D, A327I, M363W,
0.392
0.307
1.276
0.332
0.710


D455E


L39A, L127G, A190W, T242R, S264V, V381F,
0.382
0.313
1.221
0.000
1.181


D455E


L39A, T313S, Q348R, V381F
0.434
0.547
0.794
2.921
1.846


L39M, D77A, L127G, A190W, T313S
0.437
0.366
1.200
0.158
1.553


L39M, D77A, S105G, V155G, S264V, A327I,
0.542
0.628
0.862
2.186
2.129


Q348R, D455E


L39M, L127G, A190R, D203R, Q348R,
0.462
0.469
0.987
1.428
1.732


M363W, I420V


L39M, L127G, A190W, V381F, D455E
0.490
0.591
0.836
1.222
2.070


L39M, R177V, A190R, I420V
0.428
0.565
0.768
1.079
1.827


L39M, R177V, A190W, S264D, N302G, A327I
0.544
0.424
1.285
0.000
0.902


L39M, S105G, A190P, T242R, T313S, M363W
0.954
0.819
1.163
0.549
0.790


L39M, S105G, L127G, A190W, D203R, Q348R
0.480
0.525
0.917
8.297
2.700


L39S, A190P, T242R, N302G, T313S, A327I,
0.500
0.389
1.276
0.000
0.787


Q348R


L39S, R177V, D203R, T242R, S264D, A327I,
0.485
0.411
1.181
0.000
1.013


M363W


L39S, S105G, R177V, N302G, A327I, V381F
0.324
0.255
1.269
0.000
0.980


L39S, V155G, A190P, A327I, Q348R, V381F
0.239
0.287
0.837
1.552
2.340


L39S, V155G, A190R, T242R, S264D, A327I,
0.419
0.431
0.976
1.159
1.695


Q348R


L411A
0.828
1.016
0.764
1.327
0.522


L454V
0.799
1.000
0.798
0.572
1.528


L6Q, K78W, S111G, T158R, S192L, E309S,
0.167
0.104
1.526
0.000
0.282


L457Y


L6Q, K78W, S111P, R131M, A243R, G266S,
1.111
0.728
1.445
0.458
0.253


C422G


L6Q, R40S, S192A, K349Q, T385G, C422G
0.786
0.496
1.500
0.027
0.171


M165K, E184L, I461G
0.672
0.594
1.133
1.490
1.269


M165K, I367M, I461G
0.544
0.471
1.155
1.572
1.386


M360Q
0.844
0.961
0.880
0.227
1.282


M360Q, L454V
0.259
0.219
1.236
0.000
11.652


M360S
0.596
0.548
1.090
0.159
0.956


M360S, M378A, L411A
0.596
0.514
1.160
0.000
1.039


M363W, V381F, D455E
0.711
1.021
0.697
14.861
2.109


M378A
0.525
0.554
0.947
0.553
1.676


M378A, L411A
0.626
0.727
0.860
0.482
1.566


N110R, L134T, N164R, Q193R
0.526
0.805
0.655
1.535
1.835


N164R, G310V, P351G, S424Q, A468T
0.615
0.519
1.186
1.215
0.870


N331G
0.630
0.667
0.942
0.811
1.503


P173W, R246V
0.515
0.393
1.311
0.000
0.880


P294G, G310H, A320C, P351G, E393G, S424G
0.585
0.454
1.289
1.376
0.633


Q193R, G310H, E458W
0.778
0.909
0.864
1.161
1.435


Q193R, P351G, E393W, S424Q, E458W
0.552
0.449
1.228
0.000
0.873


Q193S, Q244G, G310H, P351G, E458W
0.789
0.727
1.085
1.045
1.192


Q193S, Q244G, P294G, G310R, S424G
0.764
0.637
1.199
1.015
0.859


Q201A, G375S, S410G, A447L
0.930
0.964
0.959
0.792
1.251


Q201A, I315F, G375A, A447L
0.527
0.464
1.139
1.059
1.250


Q201A, R304W, S410G
0.508
0.402
1.265
0.708
0.831


Q201V, I315F, A447I
0.622
0.591
1.047
1.053
1.398


Q201V, I315F, G375S, A447C
0.652
0.568
1.149
0.739
0.981


Q201W, R304W, S410G
0.643
0.524
1.227
0.669
0.837


Q244G, G310R, A320C, P351G, S424Q
0.664
0.534
1.245
1.226
0.610


Q244G, G310V, P351G, Y366W, E458W
0.849
0.892
0.951
1.220
1.673


Q244G, P294G, G310H, P351G, Y366W,
0.507
0.457
1.108
1.205
1.023


E393W, S424G


Q244G, S267G, G310V, A320C, Y366W
1.052
0.905
1.161
0.820
0.649


Q244G, Y366G, S424Q
0.601
0.491
1.224
0.895
0.724


Q26P, R171E, A228G, L268F
0.540
0.964
0.635
4.918
3.527


Q26T, C121S
0.740
0.886
0.837
0.599
1.366


Q26T, C121S, R171F, A228G, K472*
0.892
0.973
0.996
1.552
1.839


Q26T, C121S, R171W, A228G, K472*
0.542
0.553
0.983
1.047
2.189


Q26T, E99Q, R171W, I303W
0.618
0.563
1.184
0.529
1.472


Q26T, E99Q, S147T, R171V, S186T, I303G,
0.285
0.316
0.958
2.050
2.607


K472T


Q26T, K472*
0.826
1.343
0.618
0.880
2.071


Q26T, L200R, I303R, K472*
0.909
1.148
0.825
0.997
1.672


Q26T, Q314G
1.151
1.386
0.830
0.921
1.448


Q26T, R171E, I303W, K472T
0.618
0.602
1.054
0.527
1.840


Q26T, R171F, Q253G, T356G
0.890
1.075
0.831
1.079
1.930


Q26T, R171H, S186T, I303G, K472T
0.709
0.697
1.064
0.780
1.558


Q26T, R171V, A228G, I303R, K472T
0.792
0.995
0.807
1.668
2.166


Q26T, S147A, R171E, I303R
0.632
0.614
1.027
0.844
1.988


Q26T, V69L, E99Q, C121S, R171L, A228G,
0.479
0.460
1.130
0.000
1.484


I303W, Q314R, D328F


Q26T, V69W, C121S
0.671
0.608
1.097
0.383
1.046


Q26T, V69W, E99Q, A279G, T356G, K472*
0.771
0.712
1.077
0.454
0.984


Q26T, V69W, E99Q, K472*
0.685
0.694
1.019
0.565
1.141


Q26T, V69W, E99Q, R171F, D328F
0.650
0.549
1.174
0.071
1.241


Q26T, V69W, S186T, L200R, I303W
1.307
1.374
1.030
1.840
0.943


Q348R
0.447
0.961
0.467
2.033
3.548


Q348R, M363W
0.647
1.004
0.646
1.613
2.385


R171F, I303W
0.883
0.741
1.236
0.407
1.274


R171F, M443G
0.557
0.450
1.228
0.494
1.719


R171F, Q314G, G373R
0.710
0.573
1.251
0.424
1.208


R171H, S186T, A228G, I303G, D328F
0.594
0.479
1.230
0.432
1.791


R171V, L200R, Q253G, I303W, M443G, K472T
0.961
0.804
1.186
0.339
1.358


R171V, L200R, T356W, G373R, K472T
0.800
0.930
0.957
2.087
1.442


R171W, I303W, K472*
0.864
0.765
1.175
1.049
1.629


R171W, K472T
0.897
1.313
0.712
0.975
2.490


R172S, V187G
0.557
0.609
0.945
1.058
1.874


R172S, V187G, Q201W, R304W
0.639
0.529
1.211
0.691
1.149


R172W, V234C, A447L
0.544
0.848
0.646
1.245
2.495


R188P
0.583
0.648
0.897
0.491
1.794


R188P, A202L, R246G, K258R, Q334S, M360R
0.416
0.317
1.311
0.000
0.512


R188P, A202L, S316G, M360Q
0.513
0.401
1.286
0.000
1.088


R188P, L411A
0.526
0.539
0.979
0.672
1.468


R207A, Q244G, P294G, Y366W, E393W,
0.873
0.751
1.161
0.932
0.952


S424G


R246L, K258R, M360R, L454V, A468S
0.521
0.397
1.311
0.000
0.390


R246V, A306G, M360R, L411V, L454V
0.743
0.566
1.311
0.000
0.419


R246V, K258R, M360R
0.590
0.520
1.143
0.000
0.778


R304W, I315F
0.422
0.330
1.280
0.977
0.648


R467T
0.440
0.526
0.848
1.399
1.791


R87W, K349Q, C422G
0.349
0.217
1.522
0.333
0.281


R93T, E184F, I461G
0.474
0.491
0.974
1.611
1.770


R93T, G212A, K352I, I461G
0.869
0.917
0.955
0.999
1.782


R93T, I461G
1.124
1.474
0.762
0.791
1.740


R98D, I146K, T170R, A185L, D307L, R467T
0.354
0.292
1.208
3.175
1.166


R98D, I146L, A185M, A197V, Q250W, D307G
0.312
0.253
1.236
3.559
1.030


R98D, I146R, T170R, D307F, T353S
0.331
0.271
1.224
3.074
1.073


S105G, A190P, Q348R
0.415
0.616
0.831
1.917
2.888


S105G, A190P, S264D, M363W
0.403
0.311
1.295
0.000
1.122


S105G, L127G
0.413
0.358
1.155
0.438
1.411


S105G, L127G, D203R, T242R, A327I, Q348A,
0.364
0.446
0.818
2.635
2.283


V381F


S105G, L127G, T242K, Q348A, I420V
0.320
0.313
1.024
1.097
1.840


S105G, V155G, A190P, T242K
0.555
0.446
1.244
1.520
1.507


S105G, V155G, R177V, A190W, T242R,
0.464
0.488
0.950
3.447
1.568


Q348A


S111G, E309A, C422G
1.407
0.951
1.402
0.605
0.299


S111P, S192L, E309S, K349Q
1.111
0.858
1.230
0.681
0.399


S115R, I146R, D307F, R467T
0.325
0.255
1.272
5.998
0.542


S147A, A228G, A279V, K472*
0.920
1.143
0.806
0.887
1.905


S147A, D328F, G373R, K472T
0.883
0.822
1.120
0.245
1.198


S147A, I303R, D328F, K472*
0.400
0.349
1.232
2.937
2.254


S147A, R171E, A279G, I303W
0.624
0.546
1.187
1.400
1.972


S147A, R171E, I303R, T356G
0.980
1.052
1.008
4.987
2.253


S147A, S186T, I303R, D328F
0.504
0.428
1.226
0.819
1.923


S147A, T356G
0.669
0.751
0.901
0.660
1.404


S186T, A228G, T231S
0.662
0.914
0.760
0.610
2.169


S192A, L457Y
0.612
0.654
0.916
0.962
1.490


S264D, A327I
0.612
0.729
0.840
1.113
1.916


S264D, Q348R, M363W, V381F, D455E
0.483
0.905
0.536
2.198
3.734


S264V, A327I, D455E
0.768
0.960
0.800
1.008
1.472


S264W
0.690
0.804
0.855
0.584
1.686


S316G, L411A, L454V
0.606
0.525
1.155
0.000
0.842


S5P, D77A, S105G, A190P, S264W, N302G,
0.329
0.259
1.267
0.669
1.892


Q348A, I420V


S5P, D77A, V155G, A190R, T242K, N302G,
0.435
0.328
1.326
0.000
0.976


A327I, A426V


S5P, D77A, V155G, S264D, A327I, I420V
0.501
0.474
1.078
0.695
1.865


S5P, D77A, V155G, S264V, A327I
0.885
1.117
0.788
1.552
1.805


S5P, G15D, L39S, A190R, S264D, Q348R,
0.482
1.085
0.444
2.533
3.606


V381F


S5P, L39A, D77A, A190W, D203R, S264W,
0.752
0.721
1.039
1.437
1.514


N302G, A327I, Q348A, I420V


S5P, L39A, D77A, V155G, A190P, T242K,
0.652
0.770
0.849
1.549
1.840


L268M


S5P, L39A, V155G, A190W, T242R, N302G,
0.591
0.580
1.019
2.749
1.365


T313S, A320C, Q348A


S5P, L39A, V155G, Q348R, I420V
0.496
1.177
0.421
2.953
3.910


S5P, L39M, D77A, A190P, T242K, S264D,
0.610
0.602
1.013
0.874
1.485


Q348R, M363W, I420V


S5P, L39M, D77A, S105G, A190P, S264W,
0.359
0.568
0.799
2.419
2.998


Q348A


S5P, L39M, G50S, D77A, V155G, A190P,
0.600
0.599
0.996
1.610
1.898


T242R, S264D, A327I, V381F, D455E


S5P, L39M, S264D, I420V, D455E
0.604
0.643
0.939
1.494
3.014


S5P, L39M, V155G, A190W, D203R, T242R,
0.549
0.490
1.172
3.399
1.640


S264W, N302G, T313S, Q348A, R469L


S5P, L39S, D77A, V155G, A190P, T242K,
0.486
0.393
1.238
0.000
1.277


M363W, V381F


S5P, L39S, S105G, A190R, N302G, M363W
0.441
0.339
1.295
0.000
0.472


S5P, R177V, D203R, T242R, S264D, N302G,
0.592
0.498
1.182
1.497
1.203


A327I, Q348A, M363W, I420V


S5P, S264V, Q348A, I420V
0.261
0.432
0.605
2.409
2.984


S5P, S264W, N302G, T313S
0.469
0.365
1.285
0.000
0.524


S5P, V155G, S264V, G265D
0.622
0.525
1.183
0.827
1.540


S5P, V381F
0.503
1.148
0.437
15.293
3.054


S5T
0.876
1.151
0.761
2.978
1.548


S5T, A190W, T242K, V381F
0.476
0.555
0.859
2.342
1.950


S5T, D77A
0.722
0.804
0.899
0.508
1.186


S5T, D77A, S105G, V155G, A190R, S264D
0.640
0.508
1.261
9.479
1.589


S5T, D77A, V155G, N302G, A327I
0.399
0.306
1.303
1.174
0.961


S5T, L127G, A190R, V381F
0.405
0.382
1.080
3.787
2.108


S5T, L127G, A327I, Q348A, D455E
0.485
0.736
0.656
2.040
2.847


S5T, L39A, A190P, S264D, A327I, Q348A,
0.486
0.750
0.647
2.195
2.743


V381F, D455E


S5T, L39A, L127G, A190P, D203R, Q348R
0.347
0.352
0.986
0.000
1.415


S5T, L39A, L127G, D203R
0.704
0.665
1.055
0.283
1.068


S5T, L39M, A190P, D203R, S264W, N302G,
0.463
0.356
1.295
0.000
0.425


I420V


S5T, L39M, D77A, S105G, S264W, N302G,
0.614
0.508
1.211
0.000
0.794


T313S, Q348R, V381F


S5T, L39M, L127G, A190R, T242K, A327I,
0.442
0.477
0.922
2.084
1.958


Q348R, V381F


S5T, L39M, L127G, D203R, T242R, Q348R,
0.592
0.832
0.708
1.814
2.556


V381F


S5T, L39M, R177V, A190P, T242R
0.511
0.522
0.978
2.163
3.546


S5T, L39S, D77A, A190P
0.620
0.634
0.976
0.000
1.243


S5T, L39S, D77A, S264V, Q348R, V381F,
0.476
0.483
0.978
0.000
1.463


I420V


S5T, L39S, D77A, T313S, Q348A, V381F,
0.377
0.733
0.518
3.052
3.172


I420V


S5T, L39S, D77A, V155G, A190P, N302G,
0.394
0.306
1.284
0.886
1.282


Q348R, V381F


S5T, N302G
0.621
0.490
1.269
0.166
0.692


S5T, S105G, A190P, S264D, Q348A
0.445
0.603
0.741
2.077
2.668


S5T, T242K, N302G, V381F
0.486
0.383
1.269
0.000
0.836


S5T, V155G, N302G, A327I, Q348A, D455E
0.470
0.372
1.255
3.417
1.789


S5T, V155G, N302G, Q348R
0.497
0.391
1.269
2.115
1.577


S5T, V155G, P166S, Q348A, V381F
0.314
0.651
0.479
6.776
3.952


T170R, T353S, E404R
0.733
0.992
0.744
0.913
2.173


T24W, D307N, G442E
0.353
0.331
1.067
2.003
1.690


T24W, D307N, T353S, E404K, G442S
0.746
0.659
1.132
1.526
1.666


T24W, R98D, A185L, A197V, D307V
0.404
0.336
1.201
2.744
1.495


T24W, R98D, A185M, A197T, D307L
0.363
0.329
1.102
2.617
1.579


T24W, R98D, A197T, D307G
0.426
0.363
1.172
2.189
1.579


T24W, R98D, S115R, I146L, A197V, V219L,
0.904
0.732
1.235
2.231
1.040


T353S, G442E, R467T


T24W, T170R, A197T, D307L, T311S, G442S
0.913
0.745
1.227
1.561
1.357


T24W, T44F, I146L, D307N
0.947
0.755
1.254
2.211
1.086


T24W, T44F, R98D, A185L, R467T
0.376
0.335
1.128
2.119
1.709


T24W, T44F, R98D, T170R, A185L, V219L,
0.400
0.392
1.023
2.218
1.768


R467T


T311S, A323G, I367M
0.505
0.446
1.133
1.633
1.220


T311S, K352N, T395E, N430G, I461G
0.807
0.736
1.096
1.059
1.506


T313S, I420V
0.800
1.036
0.772
0.877
1.459


T44F, D307F
0.496
0.529
0.948
1.440
1.705


T44F, I146R, A197T, G442E, R467T
0.356
0.316
1.127
2.923
1.128


T44F, I146R, Q287S, D307R, G442S
0.826
0.649
1.274
1.907
0.450


T44F, R98D, A185M, G442E
0.604
0.717
0.844
1.168
1.904


T44F, R98D, I146R, A197T, D307G, T311S,
0.350
0.274
1.272
4.569
0.678


T353S, G442E


T44F, R98D, S115R, A197T
0.717
0.715
1.003
1.045
1.417


T44F, S115R, A185M, A197V, D307N, E404R
0.609
0.593
1.040
1.139
1.301


T44F, S115R, I146R, A197T, V219L, D307F
0.266
0.205
1.294
3.144
0.518


V149L, I315F, A447C
0.533
0.422
1.263
0.929
1.104


V149L, Q201W, A285R, Q357V, A447C
0.505
0.406
1.245
0.962
0.731


V155G, A190P
0.517
0.471
1.097
1.254
1.680


V155G, A190P, S264V, Q348R, I420V
0.371
0.376
0.989
17.841
2.093


V155G, A190W, S264D, I420V, D455E
0.802
0.851
0.943
3.273
1.703


V155G, D203R, N302G, I420V
0.792
0.608
1.295
1.038
0.969


V155G, M363W
0.628
0.517
1.216
10.265
1.556


V155G, V381F, I420V, D455E
0.408
0.554
0.880
1.907
2.389


V187G, I315F, G375A, A447L
0.606
0.539
1.126
0.960
1.331


V187G, Q201R, V234C, I315F, Q357V, A447I
0.556
0.465
1.190
0.270
1.186


V187G, Q201V, L254R, A285R, S410G, A447L
0.535
0.538
1.006
0.510
1.467


V187G, Q201W, I315F, Q357V, A447I
0.545
0.492
1.114
0.484
1.801


V187R, Q201V, I315F, G375A
0.377
0.305
1.229
0.161
0.809


V187R, Q201V, L254R, R304W, N331G,
0.516
0.413
1.242
0.376
0.768


G375A


V219L
0.327
0.347
0.952
1.786
1.657


V234C, I315F, Q357V, G375V
0.542
0.441
1.225
0.090
1.181


V381F, I420V
0.617
1.147
0.538
1.503
2.596


V43S, M165K, E184S, G212A
0.957
1.228
0.779
0.809
1.414


V43S, N430G
0.888
1.429
0.623
0.800
2.242


V43S, R93T, E184S, T395V, I461G
0.809
0.785
1.029
1.010
1.450


V69W
0.702
0.709
0.990
0.304
1.214


V69W, C121S, R171E, K472*
0.719
0.742
1.015
1.734
2.123


V69W, C121S, S147A
1.407
1.333
1.131
0.448
0.887


V69W, C121S, S186T, A279G, K472*
1.286
1.225
1.126
0.959
1.033


V69W, E99Q, Q253G, A279G, K472T
1.161
1.142
1.034
0.453
1.051


V69W, Q314G
0.684
0.593
1.200
0.298
0.847


V69W, R171E, S186T, I303W
0.915
0.763
1.190
0.691
1.652


V69W, R171E, S186T, I303W, K472*
0.933
0.838
1.193
1.431
1.585


V69W, R171W
1.147
1.263
0.983
3.002
1.738


V76L, K258R, S316G, M360R
0.713
0.555
1.286
0.000
0.657


V76L, L454V
0.736
1.056
0.698
0.814
1.770


V76L, R246L, Q334S, M360R
0.511
0.407
1.258
0.393
0.485


Y366G
0.720
0.907
0.793
1.015
1.657









Example 3
Ester Synthase Mutant Strains with Beneficial Properties
As described in Examples 1 and 2, a number of variant or mutant forms of '377 were identified with beneficial properties. In this experiment, a particular '377 mutant designated herein as 9B12 (SEQ ID NO: 4), which contains three different mutations over wild-type 377 (D7N, A179V, V381F), was cloned into pKEV027, replacing the wild-type '377 gene (SEQ ID NO: 1) coding for the wild type '377 polypeptide (SEQ ID NO: 2) and creating pKEV018. When transformed into strain BD64 (supra), strain KEV040 was created. The plasmid pKEV018 was further mutated using saturation mutagenesis at codon 348 of the gene (start codon=0) to produce the plasmid pKEV022, which contains four different mutations over wild-type '377 (D7N, A179V, Q348R, V381F). Separately, several previously identified mutations were combined with the pKEV018 plasmid to produce plasmid pKEV028 (which contains eight mutations over wild-type '377 (D7N, R87R “silent mutation”, A179V, V187R, G212A, Q357V, V381F, M443G). pKEV022 and pKEV028 were transformed into strain BD64 which resulted in the strains designated KEV075 (SEQ ID NO: 10), and KEV085 (SEQ ID NO: 12), respectively. These mutant strains were observed to produce either a significant increase in FAME titer or a reduction in the percentage of “β-OH” FAME or both (e.g., mutant strains 9B 12 and KEV085 achieved both).
Strains KEV080 (BD64/pKEV027); KEV040; KEV075 and KEV085 were screened using one of the standard protocols (Protocols 1-2) described above. FIG. 5 shows the relative FAME titers and % β-OH FAME of mutant strains KEV040, KEV075 and KEV085 compared to a strain which contains wild-type '377 ester synthase. Herein, FIG. 5 illustrates that those three variants were observed to have significantly higher titer FAME than the control (wild type); that the % β-OH FAME varied from one variant to another; and that strains 9B12 (KEV040) and KEV085 produced lower % β-OH FAME than the control (wild type).
Example 4
Ester Production by Recombinant Host Cells Engineered to Express a Variant '377 Ester Synthase and a Variant Ppro
As demonstrated in Examples 1, 2 and 3, an ester synthase from Marinobacter hydrocarbonoclasticus (WS377) was engineered to produce higher levels of fatty acid esters and a lower percentage of β-OH esters. In carrying out the present disclosure, wild type WS377 (SEQ ID NO: 1) was engineered resulting in new strains, such as 9B12, that produce higher levels of FAME directly from acyl-ACP.
The 'tesA of Photobacterium Profundum (Ppro) was engineered to have ester synthase activity. One variant of Ppro Vc7P6F9 (SEQ ID NO: 31) in plasmid pBD207 was tested in BD64 (designated herein as becos.128). Similarly, the 9B12 variant of '377 (SEQ ID NO: 4) in pKEV18 was tested in BD64 (designated herein as KEV040). Cultures of becos.128 (BD64/pBD207) and KEV040 (BD64/pKEV018) were tested in minimal media with 2% alcohol. After production for about 24 hours, they were extracted and analyzed by GC/FID or GC/MS for identification of the fatty acyl ester (following the protocols listed above).
FIG. 6 shows the production of fatty acyl esters from KEV040 (ester synthase from M. hydrocarbonoclasticus) (shown as Wax in FIG. 6) and fatty acid species from becos.128 ('tesA from P. profundum) (shown as Ppro in FIG. 6) when fed methanol, ethanol, 1-propanol, isopropanol, 1-butanol or glycerol. As negative control, water was added (lack of exogenous alcohol means that any ester production would rely on endogenous production of alcohol by E. coli). While no free fatty acid was produced by KEV040, becos.128 produced a mixture of fatty acid esters and fatty acids. FIG. 8 shows the amounts of fatty acid esters and fatty acid produced by becos.128. It should be noted that the in vitro results presented in Holtzapple and Schmidt-Dannert (see Journal of Bacteriology (2007) 189(10):3804-3812) showed that ester synthase from M. hydrocarbonoclasticus ('377) has a preference for longer chain alcohols producing primarily waxes. Holtzapple's data suggests that '377 would not be capable of using shorter chain alcohols. In contrast, the applicants work was conducted in vivo and established that the variant '377 enzyme can utilize shorter chain alcohols (C1, C2, C3 and C4), leading to shorter chain esters that can be used for a variety of valuable products (e.g., biodiesel, surfactants, etc.). The applicants used acyl-ACP as a substrate, which is believed to bring about a reduction of the futile cycle involvement (i.e., making the cell more energy efficient). In addition, the applicants showed that not all alcohols needed to have the OH group at position 1, for example, the OH group for propanol could be at position 1 or 2. FIG. 6 shows the production of fatty acyl esters with various chain lengths using a recombinant host cell genetically modified to express a single variant '377 ester synthase. This result was observed for engineered forms of both, a variant of tesA of P. profundum (Ppro) and a variant of the '377 ester synthase. It is noteworthy that the engineered mutants can convert not only primary alcohols but also secondary alcohols into a variety of different products, including FAME, FAEE, fatty acyl monoglycerides, and others. Recombinant bacteria, yeast, algae or other host cells can be used for the production of fatty esters.
Example 5
Ester Synthase Mutant Strains Expressing Combination Mutants
Standard techniques known to those of skill in the art were used to prepare combination libraries (Protocol 8). The mutations tested in combination libraries (Tables 3A and 3B) were originally identified in the full saturation library of '377 (as described in Example 1).
The combination libraries were screened in strain GLPH077 which is similar to BD64 but expressed slightly different levels of biosynthetic pathway enzymes (supra) and using one of the standard protocols (Protocols 1-2) (supra). The results from screening pKASH008 (SEQ ID NO: 16) combination libraries are shown in Table 6. As can be seen in Table 6, the resultant '377 ester synthase variants had 24 mutations compared to wild type 377. Two strains, KASH280 (SEQ ID NO: 33) and KASH281 (SEQ ID NO: 35) were used to test the variant '377. However, KASH280 expressed a '377 variant that differed from the one expressed in KASH280 by one substitution mutation. KASH280 expressed a '377 variant where a glutamine at position 348 was exchanged with an arginine (Q348R), allowing the strain to produce a higher titer FAME and a greater number of short chain esters. Conversely, KASH281 expressed a '377 variant where a lysine at position 349 was exchanged with a histidine (K349H), leading the strain to produce a lower titer FAME compared to KASH008. In addition KASH281 produced a lower titer of FAME and lesser number of short chain esters compared to KASH280. Overall, the comparison is to KASH008, which is better than pSHU10, which is better than the wild type (see examples above). The mutations of Table 6 correspond to the indicated residues of SEQ ID NO: 2.
Example 6














TABLE 6






Mutations
# Mutations
Total
Total





Strain
over wild type
over wild type
FAME
FAS
% FAME
C12/C14
C14/C16







KASH280
T5S, S15G, K78F,
24
1.59
0.66
0.42
1.65
2.36



P111S, V171R,



P188R, S192V,



R207A, A243R,



D255E, L257I,



N259Q, L260V,



H262Q, G265N,



A285V, N288D,



N289G, F317W,



Q348R, S353T,



V381F, V409L, S442G


KASH281
T5S, S15G, K78F,
24
0.80
0.87
0.92
1.38
1.22



P111S, V171R,



P188R, S192V,



R207A, A243R,



D255E, L257I,



N259Q, L260V,



H262Q, G265N,



A285V, N288D,



N289G, F317W,



K349H, S353T,



V381F, V409L, S442G









Ester Synthase Mutant Strains with Beneficial Properties from Natural Diversity
As described in Examples 1 and 2, a number of variant or mutant forms of '377 were identified with beneficial properties. An alignment of the ester synthase polypeptide from different Marinobacter organisms, such as Marinobacter adhaerens HP15 (YP_005886638.1, SEQ ID NO: 45), Marinobacter algicola DG893 (ZP_01893763.1, SEQ ID NO: 47), Marinobacter sp. ELB17 (ZP_01736818.1, SEQ ID NO: 49) with the one from Marinobacter hydrocarbonoclasticus DSM 8798 (SEQ ID NO: 2) using standard alignment procedures was used to identify mutations that already exist in nature, as this would reveal specific mutations as well as amino acid positions that naturally have diversity and thus could be used to stabilize the variant protein. The alignment was carried out and the analysis identified natural diversity within the ester synthase homologues polypeptides. Table 7 below illustrates a summary of such a group of mutations. When those mutations are combined with an engineered ester synthase from Marinobacter hydrocarbonoclasticus, beneficial variants can be observed if they are screened using one of the standard protocols (Protocols 1-2) described above. Examples of such variants are described as SEQ ID NOS: 37, 39, 41 and 43.
Table 7 below shows a list of natural diversity observed by aligning Marinobacter hydrocarbonoclasticus, M. adhaerens, M algicola and M. sp. ELB17. The mutations of Table 7 correspond to the indicated residues of SEQ ID NO: 2.











TABLE 7





AA Position
Mutation
M. adhaerens
M. algicola
M. sp. ELB17



















24
T24N
X
X
X


33
G33D


X


33
G33N

X


44
T44K


X


44
T44A

X


48
E48Q


X


48
E48A

X


49
A49D

X


49
A49T
X


58
Y58C


X


58
Y58L
X


70
I70V

X


70
I70L
X

X


73
A73S

X


73
A73T


X


73
A73G
X


79
D79K
X
X


79
D79H


X


123
V123I

X


123
V123M


X


157
T157S

X
X


158
T158E

X


158
T158K


X


161
E161G

X


161
E161D
X

X


162
R162E

X
X


162
R162K
X


163
C163I

X


163
C163R
X

X


164
N164D
X

X


166
P166L

X
X


170
T170S
X
X
X


174
H174E
X
X
X


175
Q175R
X
X


175
Q175S


X


176
R176T

X
X


179
A179S
X
X


179
A179K


X


183
K183S
X
X
X


189
A189G
X

X


191
V191L

X


191
V191I


X


196
D196E
X
X
X


212
G212M

X


212
G212S


X


216
V216I

X
X


236
V236A

X


236
V236K
X

X


237
L237I
X

X


243
A243G
X
X
X


255
D255E

X
X


257
L257I
X
X


257
L257M


X


259
N259Q

X


259
N259A


X


259
N259E
X


260
L260V

X


260
L260M


X


262
H262Q

X


262
H262R


X


263
A263V
X


265
G265N

X


266
G266S


X


266
G266A
X


285
A285V

X


285
A285L
X

X


288
N288D

X
X


289
N289G

X


289
N289E
X


293
T293I


X


293
T293A
X


306
A306S
X
X


331
N331K

X


331
N331T
X

X


334
Q334K

X
X


335
Q335S

X


335
Q335C


X


335
Q335N
X


338
T338H

X


338
T338E


X


338
T338A
X


353
S353K

X
X


393
E393G

X


393
E393T


X


393
E393Q
X


394
G394E

X
X


394
G394R
X


402
R402K
X

X


413
A413T
X

X


461
I461L


X


461
I461V

X









Example 7
Engineering of 'tesA of P. profundum (Ppro)
The 'tesA of P. profundum (Ppro) was engineered to act as an ester synthase. When screened in 96 well plates, the wild type Ppro produced about 15-18% FAME of the total acyl products (FAME+free fatty acids). Mutations were identified that increased the FAME content to 42-44% when the variants were tested on 96 well plates. When the same mutants were tested in a shake flask, they produced 62-65% FAME as compared to 30% FAME produced by the wild type enzyme. Mutations were identified based on standard techniques, such as error prone PCR or saturation libraries of Ppro (Protocol 7). The identified mutations were combined generating combination libraries, which were also tested. The libraries were then screened using one of the standard protocols (Protocols 1-4) described above.
Based on the screening of combination libraries, mutations were selected to be either (1) fixed for the next round of combination library screening; (2) re-tested in the next round of combination library screening; or (3) not tested further at that time. Mutations that were identified as beneficial were fixed for future libraries. The process can be carried out by screening using 96 well plates, validation in 96 well plates, and follow-up validation in shake flasks.
The best performers in shake flasks can then be tested in fermenters to verify that there is improvement of the engineered Ppro that is consistent with scale-up. A few of the top hits from the combination libraries can be tested in shake flasks to confirm that the Ppro variants have improved performance at a larger scale than that of 96 well plates. A shake flask validation can be carried out as follows. Desired mutants are streaked on LB+spectinomycin agar to obtain single colonies and incubated overnight at 37° C. Three single colonies from each mutant are picked into 4 mL LB+spectinomycin and incubated at 37° C. shaker for approximately 6 hours. 1.5 mL of the LB culture is used to inoculate 15 mL FA2 (2 g/L NH4Cl)+0.05% Triton X-100+spectinomycin in a baffled 125 mL flask and incubated overnight at 32° C. 3 mL of the overnight culture is used to inoculate 15 mL FA2 (2 g/L NH4C1)+0.05% Triton X-100+1 mM IPTG, 2% Methanol+spectinomycin in a baffled 125 mL flask and incubated for approximately 24 hours at 32° C. At time of extraction 300 μL of culture is sampled from the flask into a 96 well plate and extracted using a standard extraction protocol (Protocol 5).
A standard extraction protocol was carried out as described above (Protocol 5). The results from screening saturation libraries are shown in Tables 8 through 11. Table 8 presents results for single beneficial mutations found using the wild type as template, i.e, SEQ ID NO: 51. Table 9 presents results for combination mutants found using the wild type as template. Table 10 presents results for single beneficial mutations found using SEQ ID NO: 53 (referred to as PROF 1) as template (SEQ ID NO: 53 already contained two beneficial mutations in its sequence including S46V and Y116H mutations). For PROF1, the S46V and Y116H mutations were “fixed” into the template, such that future rounds of saturation/combination would be starting from that variant rather than from the wild type sequence. Table 11 presents results for double mutations found as beneficial using PROF1 as template. Table 12 presents results for combination mutants found, using the wild type as template. Table 13 presents results for combination mutants found using P1B9v2.0 (SEQ ID NO: 58) as template in BD64.
Table 8 below shows a list of single beneficial mutations found using Ppm wild type (SEQ ID NO: 51) as template.













TABLE 8








Norm
Norm
Norm
Norm



Mutation
FAME
FFA
Titer
% FAME






















T3L
0.911
0.882
0.889
1.023



P29A
1.015
1.088
1.071
0.947



P29F
0.984
0.984
0.984
1.000



P29G
1.008
1.009
1.009
0.999



P29S
1.018
1.030
1.027
0.992



I38Y
0.765
0.592
0.632
1.211



I45A
0.420
0.304
0.320
1.317



I45G
0.311
0.109
0.137
2.242



I45K
1.205
1.145
1.154
1.046



I45R
1.313
1.257
1.265
1.038



I45S
0.398
0.242
0.264
1.509



I45V
1.162
1.096
1.105
1.050



I45W
0.281
0.202
0.213
1.322



S46K
0.387
0.224
0.247
1.556



S46V
1.166
0.899
0.937
1.250



S46W
0.346
0.211
0.230
1.517



D48A
1.297
1.417
1.396
0.929



D48E
1.267
1.278
1.276
0.993



D48F
0.892
0.773
0.794
1.123



D48G
0.921
0.835
0.851
1.083



D48H
1.081
0.955
0.977
1.106



D48L
1.774
1.816
1.808
0.980



D48M
1.880
2.159
2.109
0.891



D48V
0.626
0.535
0.551
1.136



D48W
1.263
1.252
1.254
1.010



T49D
0.556
0.378
0.408
1.364



T49G
0.273
0.263
0.265
1.024



T49L
0.595
0.425
0.453
1.311



T49M
0.461
0.334
0.356
1.292



T49R
0.402
0.214
0.244
1.651



T49S
0.447
0.360
0.374
1.194



T49V
0.503
0.336
0.362
1.389



T49W
0.678
0.542
0.565
1.203



G51A
1.079
1.068
1.070
1.009



G51E
1.025
0.938
0.952
1.076



G51F
0.921
0.830
0.844
1.091



G51H
1.083
1.087
1.086
0.997



G51L
0.956
0.871
0.887
1.076



G51M
1.013
0.952
0.962
1.053



G51P
0.460
0.308
0.332
1.385



G51Q
1.047
1.082
1.075
0.972



G51R
1.377
1.452
1.440
0.955



G51S
1.088
1.046
1.053
1.033



G51T
1.062
1.115
1.107
0.959



G51V
1.003
1.036
1.031
0.973



G51Y
0.852
0.833
0.837
1.016



N52A
0.970
0.856
0.883
1.099



N52C
0.825
0.676
0.710
1.161



N52F
0.797
0.668
0.697
1.141



N52G
1.026
1.093
1.078
0.951



N52H
0.959
1.035
1.017
0.943



N52L
0.974
0.848
0.877
1.110



N52M
1.040
1.045
1.044
0.996



N52R
1.405
1.641
1.587
0.885



N52S
0.903
0.810
0.832
1.084



N52T
0.874
0.785
0.806
1.084



N52V
0.812
0.699
0.725
1.118



N76I
0.518
0.285
0.326
1.584



N76L
0.366
0.160
0.200
1.834



N76V
0.642
0.336
0.395
1.624



S92A
1.001
1.054
1.042
0.960



S92H
1.026
1.106
1.087
0.943



S92S
1.002
0.972
0.979
1.022



S92Y
1.022
1.199
1.158
0.881



I95V
0.910
0.876
0.884
1.028



Y116A
0.923
0.889
0.895
1.032



Y116F
1.077
1.035
1.042
1.034



Y116I
1.392
1.303
1.318
1.055



Y116L
1.203
1.246
1.239
0.971



Y116N
1.430
1.424
1.425
1.003



Y116P
0.628
0.460
0.488
1.284



Y116R
1.442
1.701
1.657
0.872



Y116S
0.876
0.757
0.777
1.127



Y116T
1.079
0.939
0.963
1.121



Y120R
0.490
0.399
0.420
1.164



N156A
1.048
1.090
1.082
0.969



N156C
0.903
0.858
0.866
1.042



N156G
0.984
1.070
1.055
0.933



N156L
0.841
0.821
0.825
1.018



N156R
1.477
1.942
1.856
0.796



N156W
0.824
0.678
0.705
1.168



L159G
0.399
0.245
0.273
1.455



L159M
1.254
1.651
1.577
0.795



L159N
0.513
0.323
0.358
1.433



L159R
0.554
0.540
0.542
1.021



L159S
0.390
0.237
0.265
1.466



L159Y
0.476
0.318
0.347
1.368










Table 9 below shows a list of combination mutants found using Ppro wild type (SEQ ID NO: 51) as template.












TABLE 9







Norm
Norm
Norm
Norm


Mutation
Mutation
FAME
FFA
Titer
% FAME







E37D
I45R
1.189
1.255
1.246
0.953


P29A
Stop_182I
1.014
1.058
1.047
0.967


I38L
V41I
0.843
0.760
0.779
1.082


Y116I
Q146H
1.554
1.906
1.846
0.841


Y116P
E175D
0.341
0.231
0.252
1.350


Y116Q
G117R
0.438
0.350
0.365
1.202


G8V
T49S
0.405
0.277
0.299
1.358


A30T
G51C
0.566
0.483
0.499
1.134


T49Q
Y116H
0.383
0.247
0.269
1.417


T49R
Y116H
0.441
0.303
0.324
1.362


N156K
L159K
1.456
1.955
1.862
0.782


G158D
L159V
0.334
0.224
0.244
1.362









Table 10 below shows a list of single beneficial mutations found using PROF1 (SEQ ID NO: 53) as template.













TABLE 10








Norm
Norm
Norm
Norm



Mutation
FAME
FFA
Titer
% FAME









V41D
0.394
0.310
0.330
1.195



V41E
0.891
0.855
0.863
1.033



V41K
0.999
1.016
1.012
0.989



V41Q
1.015
1.048
1.040
0.976



V41R
0.996
1.019
1.014
0.983



V41S
0.577
0.495
0.515
1.122



V41T
0.732
0.669
0.684
1.070



V41W
0.856
0.778
0.797
1.075



F82A
1.036
0.940
0.963
1.077



F82E
0.861
0.747
0.775
1.111



F82G
0.770
0.577
0.625
1.233



F82K
1.167
1.220
1.207
0.967



F82Q
0.996
0.927
0.944
1.054



F82R
1.426
1.552
1.521
0.938



F82V
0.975
0.883
0.906
1.076



F82W
1.089
1.141
1.128
0.966



Q84A
0.989
0.936
0.949
1.043



Q84E
0.990
1.050
1.036
0.957



Q84G
0.822
0.774
0.785
1.047










Table 11 shows a list of double mutations found as beneficial using PROF1 (SEQ ID NO: 53) as template.












TABLE 11







Norm

Norm
Norm


Mutation
Mutation
FAME
Norm FFA
Titer
% FAME







V41L
D48G
0.684
0.601
0.621
1.103


F82G
T86P
0.589
0.399
0.446
1.323









Discussed herein are the results of feeding ethanol to one of the Ppro libraries. One of the combination libraries was fed either methanol or ethanol, in order to test if genes that have improved ester synthase activity using methanol, also show improved ester synthase activity in the presence of ethanol. As can be seen in FIGS. 7A and 7B, the improved ester synthase activity was observed with either methanol or ethanol feeding, though higher percentage esters were obtained using methanol. Table 12 shows a list of combination mutants found using Ppro wild type (SEQ ID NO: 51) as template.











TABLE 12






Norm


Norm


Mutations
FAME
Norm FFA
Norm Titer
% FAME



















D48E, R119C
1.119
1.116
1.117
1.001


D48E, R88H, Y116H
1.265
1.559
1.506
0.839


G51R, D102V
1.012
1.026
1.023
0.988


G51R, R88H, R119C, L159M
1.597
1.818
1.778
0.897


I45T, D48E, S92R, Y116H
1.060
0.991
1.004
1.055


I45T, D48E, Y116H
1.027
1.019
1.020
1.007


I45T, D48E, Y116H, R119C
0.812
0.766
0.774
1.046


I45T, R88H
0.643
0.490
0.518
1.237


I45T, S46V, R119C
0.635
0.565
0.578
1.099


I45T, S46V, Y116H
1.004
0.891
0.912
1.100


I45T, Y116H
1.158
1.223
1.211
0.957


K32M, L159M
1.400
1.717
1.659
0.843


K32M, N156Y, L159M
1.345
1.278
1.290
1.042


R18C, G51R, N52T, D102V, N115I,
1.377
1.524
1.497
0.918


P129L, L159M


R88H, Y116H
1.131
1.258
1.235
0.915


S46V, D48E, Y116H
1.209
1.052
1.081
1.117


S46V, D48E, Y116H, R119C
1.118
0.993
1.016
1.101


S46V, R88H
1.086
0.820
0.868
1.249


S46V, T86S, Y116H
1.401
1.092
1.149
1.219


S46V, Y116H
1.321
1.203
1.224
1.079


T2_50_T4, R88H, R119C
0.833
0.806
0.811
1.026


V41I, I45T, Y116H
1.057
1.129
1.116
0.945


V41L, S46V, R119C
0.897
0.631
0.680
1.318


Y116H, R119C
1.107
1.277
1.246
0.888


D48E, G51R, R88H, R119C, L159M
1.403
1.389
1.391
1.010


D48E, Y116H, R119C
1.493
1.570
1.558
0.958


G51R, N156Y
1.097
1.133
1.127
0.972


G51R, R119C, N156Y, L159M
1.364
1.566
1.534
0.888


G51R, R88H, Y116H, S121R, L159M
1.562
1.631
1.621
0.962


G51R, V112A, N115I, N156Y
0.317
0.216
0.230
1.339


G51R, Y116H, R119C
1.609
1.856
1.818
0.884


H36Q, S46V, R88H, Y116H, R119C,
0.843
0.652
0.682
1.236


L159M


I45N, S46V, D48E, G51R, Y116H,
1.016
0.848
0.875
1.162


L159M


K32M, D48E, R88H, Y116H, N156Y
0.920
0.811
0.828
1.110


K32M, D48E, Y116H
1.992
1.929
1.938
1.027


K32M, G51R, Y116H
1.722
1.943
1.908
0.902


K32M, H36Q, G51R, Y116H, L159M
1.629
1.651
1.648
0.988


K32M, S46V, D48E, A55T, R119C
0.710
0.518
0.547
1.294


K32M, S46V, D48E, G51R, Y116H,
2.067
1.562
1.634
1.264


L159M


K32M, S46V, D48E, Y116H, N156Y,
1.211
1.007
1.039
1.165


L159M


K32M, S46V, G51R
1.305
1.144
1.169
1.115


K32M, S46V, G51R, F82I, Q84L,
2.570
2.098
2.165
1.187


Y116H, L159M


K32M, S46V, G51R, L159M
1.646
1.512
1.533
1.073


K32M, S46V, G51R, R88H, L159M
1.730
1.552
1.579
1.097


K32M, S46V, G51R, R88H, Y116H,
1.516
1.397
1.415
1.072


R119C


K32M, S46V, G51R, R88H, Y116H,
0.601
0.532
0.543
1.116


R119C, S121R


K32M, S46V, G51R, Y116H
2.066
1.634
1.695
1.219


K32M, S46V, G51R, Y116H, N156Y,
1.992
1.859
1.880
1.060


L159M


K32M, S46V, G51R, Y116H, R119C,
1.866
1.578
1.619
1.153


N156Y, L159M


K32M, S46V, G51R, Y116H, S121R,
0.774
0.664
0.681
1.137


N156Y, L159M


K32M, S46V, Y116H
1.408
1.193
1.223
1.152


K32M, S46V, Y116H, E152*, L159M
0.441
0.385
0.394
1.136


K32M, S46V, Y116H, L159M
1.845
1.695
1.718
1.073


K32M, S46V, Y116H, N156Y
1.261
1.061
1.093
1.156


K32M, Y116H, R119C, L159M
1.693
1.928
1.891
0.895


P24Q, K32M, S46V, G51R, R88H,
0.615
0.491
0.511
1.189


L159M


R88H, L159M
1.414
1.525
1.507
0.939


R88H, N89S, Y116H
1.263
1.368
1.352
0.934


S46V, D48E
0.975
0.755
0.790
1.234


S46V, D48E, G51R, R119C
1.160
0.946
0.979
1.181


S46V, D48E, G51R, R119C, L159*
0.343
0.199
0.221
1.556


S46V, D48E, G51R, R88H, Y116H,
1.527
1.329
1.360
1.123


L159M


S46V, D48E, G51R, R88H, Y116H,
0.818
0.655
0.681
1.201


R119C, N156Y


S46V, D48E, G51R, Y116H
1.645
1.355
1.399
1.175


S46V, D48E, G51R, Y116H, L159M
1.669
1.453
1.487
1.122


S46V, D48E, G51R, Y116H, N156Y,
1.517
1.195
1.244
1.220


L159M


S46V, D48E, G51R, Y116H, S121N
0.532
0.485
0.492
1.078


S46V, D48E, N156K
1.411
1.112
1.159
1.217


S46V, D48E, N90K, Y116H, L159M
0.366
0.248
0.266
1.373


S46V, D48E, R119C
0.946
0.725
0.759
1.245


S46V, D48E, R88H, Y116H
1.199
1.016
1.044
1.148


S46V, D48E, Y116H, L159M
1.534
1.232
1.279
1.198


S46V, D48E, Y116H, N156Y, L159M
1.265
1.007
1.048
1.206


S46V, G51R
1.315
1.219
1.234
1.065


S46V, G51R, L79I, R88H, Y116H,
1.013
0.845
0.871
1.161


N156Y, L159M


S46V, G51R, N156Y
1.285
1.073
1.106
1.161


S46V, G51R, R88H, Y116H
1.561
1.389
1.416
1.103


S46V, G51R, R88H, Y116H, M154K
0.520
0.413
0.430
1.210


S46V, G51R, R88H, Y116H, N156Y,
1.677
1.571
1.588
1.055


L159M


S46V, G51R, R88H, Y116H, R119C
1.454
1.277
1.305
1.114


S46V, G51R, R88H, Y116H, R119C,
0.554
0.392
0.417
1.327


S163F


S46V, G51R, Y116H
1.656
1.486
1.513
1.095


S46V, G51R, Y116H, L159M
1.989
1.875
1.893
1.050


S46V, G51R, Y116H, N156Y
1.788
1.461
1.508
1.186


S46V, G51R, Y116H, R119C
1.869
1.480
1.535
1.216


S46V, G51R, Y116H, R119C, L159M
2.256
2.015
2.050
1.101


S46V, L159M
1.487
1.300
1.329
1.118


S46V, N156Y, L159M
1.360
1.113
1.148
1.184


S46V, R88H, L159M
1.684
1.434
1.469
1.146


S46V, R88H, Y116H
1.219
1.024
1.054
1.157


S46V, R88H, Y116H, L159M
1.647
1.439
1.471
1.119


S46V, R88H, Y116H, N156Y
1.213
0.951
0.988
1.228


S46V, R88H, Y116H, S125I
0.685
0.501
0.530
1.290


S46V, R88Y, Y116H, R119C, L159M
1.254
1.119
1.140
1.101


S46V, Y116H, L159M
1.671
1.510
1.534
1.089


S46V, Y116H, N156Y
1.320
1.070
1.105
1.193


S46V, Y116H, R119C
1.191
0.964
0.999
1.192


S46V, Y116H, R119C, L159M
1.588
1.397
1.427
1.113


S46V, Y116H, R119C, N156Y
1.045
0.851
0.881
1.186


S46V, Y116H, R119C, N156Y, L159M
1.295
1.086
1.118
1.158


V41E, S46V, D48E, R119C
0.570
0.396
0.423
1.347


V41L, S46V, R88H, L159M
1.138
0.858
0.898
1.268


Y116H, N156Y, L159M
1.455
1.644
1.615
0.901


S46V, D48F, G51R, Y116H, E152D,
1.100
0.904
0.936
1.176


L159M


S46V, D48F, G51R, Y116H, G117D,
0.695
0.439
0.482
1.444


L159M


S46V, D48F, G51R, Y116H, L159M
1.297
1.263
1.269
1.021


S46V, D48F, G51R, N52C, Y116N
0.304
0.163
0.187
1.632


S46V, D48F, G51R, N52C, N76V,
0.218
0.197
0.201
1.091


Y116S, N156K


S46V, D48F, G51R, N76V, Y116N
0.656
0.291
0.350
1.882


S46V, D48F, G51R, N76V, Y116H,
0.838
0.360
0.434
1.941


N156K, L159M


S46V, D48F, G51R, P114R, Y116H
0.636
0.518
0.536
1.194


S46V, D48L, G51R, Y116S, N156K
0.854
0.766
0.780
1.096


S46V, D48L, G51R, Y116H, M155I,
0.606
0.461
0.483
1.254


N156S


S46V, D48L, G51R, N52C, N76V,
1.033
0.397
0.495
2.089


Y116H


S46V, D48L, G51R, N52F, Y116H,
1.438
1.285
1.310
1.099


N156K


S46V, D48L, G51R, N52F, N76I,
0.446
0.272
0.298
1.492


Y116N


S46V, D48L, G51R, N52F, N76I,
0.536
0.279
0.321
1.667


Y116N, L149V


S46V, D48L, G51R, N52F, N76V,
1.023
0.319
0.428
2.389


Y116H, L159M


S46V, D48L, G51R, N76I, Y116P,
0.545
0.243
0.293
1.857


L159M


S46V, D48L, G51R, N76I, Y116S
0.398
0.238
0.265
1.501


S46V, D48M, G51R, Y116P
1.353
1.209
1.233
1.098


S46V, D48M, G51R, N52F, Y116H
1.951
1.559
1.620
1.204


S46V, D48M, G51R, N52F, Y116P
0.864
0.659
0.692
1.247


S46V, D48M, G51R, N52F, Y116H,
1.432
1.225
1.260
1.136


L159M


S46V, D48M, G51R, N76I, Y116N,
0.913
0.362
0.447
2.042


N156K


S46V, D48M, G51R, N76I, Y116S,
0.971
0.387
0.481
2.012


L159M


S46V, D48M, G51R, N76V, Y116P
1.353
0.418
0.562
2.405


S46V, D48M, G51R, N76V, Y116P,
1.179
0.377
0.501
2.356


K162R


S46V, D48M, G51R, P114R, Y116H,
1.481
1.318
1.345
1.102


Q151R


S46V, G47S, D48L, G51R, N52F,
0.558
0.351
0.385
1.448


Y116H, N156K


S46V, G51R, Y116N, L159M
2.259
1.823
1.890
1.196


S46V, G51R, Y116N, N156K
1.798
1.818
1.814
0.992


S46V, G51R, Y116P, N156K
1.257
1.000
1.039
1.211


S46V, G51R, Y116S, N156K
1.833
1.363
1.435
1.277


S46V, G51R, N52C, Y116H, L159M
2.011
1.595
1.659
1.212


S46V, G51R, N52C, Y116H, N156K
2.141
1.869
1.913
1.119


S46V, G51R, N52C, N76I, Y116H
0.287
0.141
0.163
1.759


S46V, G51R, N52C, N76V, Y116P
0.535
0.187
0.241
2.151


S46V, G51R, N52F, Y116H
1.296
1.006
1.051
1.234


S46V, G51R, N52F, Y116P, N156K,
0.263
0.216
0.224
1.145


L159M


S46V, G51R, N52F, Y116S, L159M
0.271
0.215
0.224
1.210


S46V, G51R, N76I, Y116H
0.210
0.188
0.191
1.098


S46V, G51R, N76V, Y116H
0.740
0.331
0.397
1.865


S46V, G51R, N76V, Y116S
0.653
0.252
0.317
2.065


S46V, G51R, V112M, Y116N, N156K
1.384
0.949
1.016
1.363


I38Y, S46V, D48F, G51R, Y116N,
0.258
0.115
0.139
1.867


L159M


I38Y, S46V, D48L, G51R, Y116S
0.218
0.127
0.142
1.510


I38Y, S46V, D48L, G51R, N76I,
0.954
0.379
0.468
2.041


Y116H, N156K, L159M


I38Y, S46V, D48L, G51R, N76I,
0.497
0.245
0.284
1.714


R80G, Y116H, N156K, L159M


I38Y, S46V, D48M, G51R, N52C,
0.441
0.305
0.327
1.348


Y116S


I38Y, S46V, D48M, G51R, N52F,
1.638
0.417
0.604
2.711


N76V, Y116H


I38Y, S46V, D48M, G51R, N52F,
1.836
0.485
0.692
2.653


N76V, Y116H, L181S, *182Y


I38Y, S46V, D48M, G51R, N76V,
1.627
0.468
0.648
2.513


Y116N, N156K, L159M


I38Y, S46V, G51R, Y116N
0.764
0.551
0.585
1.305


I38Y, S46V, G51R, Y116H, L159M
2.024
1.801
1.835
1.103


I38Y, S46V, G51R, N52C, N76I,
0.170
0.138
0.144
1.190


Y116H, N156K


S46V, D48L, G51R, F82I, Q84L,
1.663
1.293
1.373
1.211


Y116H, L159M


S46V, D48L, G51R, N76V, Y116H
0.678
0.340
0.413
1.639


S46V, D48L, G51R, N76V, F82I,
0.846
0.375
0.484
1.745


Q84L, Y116N, L159M


S46V, D48L, G51R, N76V, Y116N
0.899
0.395
0.513
1.754


S46V, D48L, G51R, N76V, Y116N,
0.897
0.403
0.517
1.731


L159M


S46V, D48L, G51R, N76V, Y116N,
0.721
0.342
0.430
1.675


L159M, K162E


S46V, D48L, G51R, N76V, Y116N,
0.888
0.382
0.492
1.805


N156K


S46V, D48L, G51R, N76V, Y116N,
0.809
0.369
0.471
1.714


N156K, L159M


S46V, D48L, G51R, N76V, Y116H,
1.073
0.414
0.557
1.924


L159M


S46V, D48L, G51R, N76V, Y116H,
0.874
0.401
0.511
1.712


N156K


S46V, D48L, G51R, N76V, Y116H,
0.949
0.425
0.547
1.735


N156K, L159M


S46V, D48L, G51R, N76V, V112M,
0.696
0.347
0.429
1.623


Y116N


S46V, D48M, G51R, N52Y, N76V,
1.153
0.424
0.594
1.941


Y116N, L159M


S46V, D48M, G51R, N52Y, N76V,
1.157
0.437
0.605
1.914


Y116H, L159M


S46V, D48M, G51R, N76V, Y116H
1.225
0.445
0.614
1.994


S46V, D48M, G51R, N76V, Y116N
1.178
0.446
0.605
1.947


S46V, D48M, G51R, N76V, Y116N,
1.267
0.480
0.663
1.908


L159M


S46V, D48M, G51R, N76V, Y116N,
1.058
0.406
0.547
1.931


N156K


S46V, D48M, G51R, N76V, Y116N,
1.176
0.490
0.650
1.811


N156K, L159M


S46V, D48M, G51R, N76V, Y116H,
1.312
0.529
0.711
1.845


L159M


S46V, D48M, G51R, N76V, Y116H,
0.799
0.352
0.449
1.779


M155R


S46V, D48M, G51R, N76V, Y116H,
1.254
0.532
0.700
1.791


N156K


S46V, D48M, G51R, N76V, Y116H,
1.257
0.524
0.695
1.810


N156K, L159M


S46V, D48M, G51R, R56P, N76V,
0.406
0.165
0.221
1.836


Y116H, L159M


S46V, G51R, N76V, Y116N, L159M
0.822
0.386
0.487
1.687


S46V, G51R, N76V, Y116N, N156K,
0.942
0.386
0.515
1.826


L159M


S46V, G51R, N76V, Y116N, R119H,
0.916
0.386
0.509
1.796


N156K, L159M


S46V, G51R, N76V, Y116H, L159M
0.935
0.373
0.504
1.854


S46V, G51R, N76V, Y116H, N156K
0.834
0.340
0.447
1.864


S46V, G51R, N76V, Y116H, N156K,
0.965
0.381
0.508
1.900


L159M


S46V, G51R, N76V, Y116H, R119L,
0.761
0.326
0.420
1.809


N156K


K32M, S46V, D48L, G51R, F82I,
1.724
1.281
1.377
1.251


Q84L, Y116H, L159M


K32M, S46V, G51R, F82I, Y116H,
1.956
2.094
2.064
0.947


L159M,


K32M, S46V, G51R, F82I, Q84L,
2.066
2.023
2.032
1.016


Y116H, L159M


K32M, S46V, G51R, F82I, Q84L,
2.050
2.348
2.284
0.897


Y116H, N156K, L159M


S46V, T49S, G51R, N76V, Q84I,
0.674
0.298
0.379
1.774


Y116H, N156K, L159M


A30E, S46V, D48M, G51R, N76V,
0.446
0.198
0.235
1.846


F82R, Y116H, I148Y


E37A, I45R, S46V, D48M, T49W,
0.625
0.318
0.367
1.704


G51R, N76V, F82G, Y116H, N156K


S46V, D48M, G51R, N76V, F82G,
2.627
0.613
0.913
2.878


Y116H, A136D, I148Y


S46V, D48M, G51R, N76V, F82G,
2.416
0.621
0.908
2.661


Y116H, I148Y


S46V, D48M, G51R, N76V, F82G,
0.339
0.239
0.254
1.331


Y116H, I148Y, W168C


S46V, D48M, G51R, N76V, F82G,
1.381
0.438
0.579
2.387


Y116H, N156R, L159M


S46V, D48M, G51R, N76V, F82G,
1.602
0.483
0.650
2.467


Q84L, Y116H


S46V, D48M, G51R, N76V, F82I,
1.164
0.465
0.569
2.047


Y116H, N156K, L159K


S46V, D48M, G51R, N76V, F82I,
1.419
0.466
0.618
2.289


Q84L, Y116H, N156R


S46V, D48M, G51R, N76V, F82R,
2.760
0.734
1.035
2.661


Y116H, L159K


S46V, D48M, G51R, N76V, F82R,
1.874
0.582
0.788
2.375


N115A, Y116H, N156K, L159K


S46V, D48M, G51R, N76V, F82R,
1.907
0.647
0.848
2.229


Y116H, N156K, L159K


S46V, D48M, G51R, N76V, Y116H,
2.793
0.796
1.114
2.505


I148Y


I45R, S46V, D48M, G51R, N76V,
1.335
0.491
0.626
2.125


F82I, Y116H, N156K, L159K


I45R, S46V, D48M, G51R, N76V,
0.448
0.238
0.269
1.660


Q84L, Y116H, N156R, L159K


I45R, S46V, D48M, T49L, G51R,
0.476
0.319
0.345
1.382


A67S, N76V, F82G, Y116H, L159K


I45R, S46V, D48M, T49L, G51R,
0.532
0.335
0.366
1.451


N76V, F82G, Y116H, L159K


I45R, S46V, D48M, T49L, G51R,
0.318
0.201
0.220
1.444


N76V, Q84L, Y116H, N156R, L159M


I45R, S46V, D48M, T49R, G51R,
1.056
0.383
0.490
2.152


N52D, N76V, F82I, Q84L, Y116H,


I148Y, N156Y


I45R, S46V, D48M, T49W, G51R,
0.204
0.174
0.179
1.147


N76V, F82I, Q84L, Y116H, I148Y


K32M, S46V, D48M, G51R, N76V,
1.289
0.436
0.574
2.246


F82G, Y116H, N156R, L159M


K32M, S46V, D48M, G51R, N76V,
1.565
0.493
0.664
2.353


F82G, Q84L, Y116H


K32M, S46V, D48M, G51R, N76V,
1.743
0.534
0.729
2.382


F82I, Y116H, L159M


K32M, S46V, D48M, G51R, N76V,
1.926
0.538
0.745
2.583


F82I, Q84L, Y116H, L159M


K32M, I45R, S46V, D48M, T49D,
0.927
0.405
0.489
1.896


G51R, N76V, F82G, Y116H, L159Q


K32M, I45R, S46V, D48M, T49D,
1.884
0.599
0.806
2.335


G51R, N76V, F82I, Y116H, I148Y


K32M, S46V, D48M, G51R, N76V,
1.289
0.472
0.603
2.138


Y116H, L159K


K32M, S46V, D48M, N76V, F82G,
0.902
0.353
0.441
2.045


Q84L, Y116H


K32M, S46V, D48M, T49D, G51R,
0.537
0.229
0.275
1.956


N76V, F82I, Q84L, Y116H


K32M, S46V, D48M, T49D, G51R,
0.841
0.343
0.423
1.988


N76V, Y116H, I148Y


K32M, S46V, D48M, T49L, G51R,
2.118
0.623
0.861
2.456


N76V, F82G, Q84L, Y116H, N156K,


L159M


K32M, S46V, D48M, T49L, G51R,
0.099
0.089
0.091
1.056


N76V, F82R, Y116H, L159K


K32M, S46V, D48M, T49W, G51R,
0.323
0.236
0.250
1.299


P58L, L73F, N76V, Q84L, Y116H,


N156K, L159K


K32M, S46V, D48M, T49W, G51R,
1.310
0.494
0.624
2.096


N76V, Q84L, Y116H, N156K, L159K


S46V, D48M, G51R, N76V, Y116H,
2.019
0.595
0.824
2.450


N156K, L159M


Q34R, I45R, S46V, D48M, T49D,
1.421
0.492
0.642
2.214


G51R, N76V, F82R, Y116H


S46V, D48M, G51R, N76V, Q84L,
1.631
0.522
0.701
2.325


Y116H, N156K


S46V, D48M, T49D, G51R, N76V,
0.302
0.220
0.233
1.284


F82G, Y116H, L159K


S46V, D48M, T49D, G51R, N76V,
0.596
0.267
0.319
1.871


F82G, Y116H, N156R


S46V, D48M, T49D, G51R, N76V,
0.554
0.284
0.327
1.691


F82I, Y116H, L159M


S46V, D48M, T49D, G51R, N76V,
0.746
0.326
0.388
1.923


F82R, Y116H, N156R


S46V, D48M, T49D, G51R, N76V,
0.679
0.305
0.361
1.882


F82R, Y116H, N156R, L159I


S46V, D48M, T49D, G51R, N76V,
0.822
0.358
0.427
1.927


F82R, Q84L, Y116H, N156K


S46V, D48M, T49D, G51R, N76V,
0.694
0.313
0.369
1.878


G81C, F82R, Q84L, Y116H, N156K


S46V, D48M, T49D, G51R, N76V,
0.676
0.284
0.343
1.973


Y116H, N156K, L159M


S46V, D48M, T49D, G51R, N76V,
0.563
0.284
0.329
1.711


Y116H, N156R, L159K


S46V, D48M, T49D, G51R, N76V,
0.545
0.289
0.330
1.653


Q84L, Y116H, L159M


S46V, D48M, T49L, G51R, N76V,
1.486
0.505
0.662
2.242


F82I, N156K


S46V, D48M, T49L, G51R, N76V,
1.729
0.569
0.754
2.292


F82I, Y116H, N156K


S46V, D48M, T49L, G51R, N76V,
0.719
0.301
0.363
1.978


F82I, Y116H, N156R, L159K


S46V, D48M, T49L, G51R, N76V,
0.854
0.345
0.427
1.998


F82R, Y116H


S46V, D48M, T49L, G51R, N76V,
0.357
0.209
0.231
1.528


F82R, Q84L, Y116H, I148Y


S46V, D48M, T49L, G51R, N76V,
3.085
0.824
1.186
2.599


Y116H, I148Y


S46V, D48M, T49L, G51R, N76V,
2.230
0.621
0.877
2.543


Y116H, N156K, L159M


S46V, D48M, T49L, G51R, N76V,
1.554
0.493
0.664
2.343


Y116H, N156R


S46V, D48M, T49L, G51R, N76V,
2.723
0.748
1.063
2.563


Q84L, Y116H


S46V, D48M, T49L, G51R, N76V,
2.411
0.699
0.972
2.480


Q84L, Y116H, K150I


S46V, D48M, T49W, G51R, N76V,
1.432
0.483
0.634
2.259


F82G, Q84L, Y116H, I148Y


S46V, D48M, T49W, G51R, N76V,
0.364
0.287
0.300
1.215


F82G, Q84L, Y116H, N156K, L159K


S46V, D48M, T49W, G51R, N76V,
0.495
0.288
0.321
1.537


F82I, Y116H


S46V, D48M, T49W, G51R, N76V,
0.466
0.270
0.302
1.543


F82I, Y116H, N156R, L159M


S46V, D48M, T49W, G51R, N76V,
0.282
0.261
0.265
1.063


F82R, Y116H, L159M


S46V, D48M, T49W, G51R, N76V,
1.323
0.491
0.624
2.118


Q84L, Y116H, N156R, L159K









Table 13 shows a list of combination mutants found using P1B9v2.0 (SEQ ID NO: 58) as Template in BD64.











TABLE 13






Norm
Norm
Norm
Norm


Mutations
FAME
FFA
Titer
% FAME



















E37D, S46V, D48M, T49L, G51R, N52K, N76V,
3.143
2.067
2.668
1.175


F82I, V112L, N115W, Y116H, I148Y, L159M


E37D, S46V, D48M, T49L, G51R, N52K, N76V,
3.859
1.827
2.962
1.305


N115Y, Y116H, I148Y


S46V, D48M, T49L, G51R, N76V, F82I, Y116H,
1.088
1.211
1.143
0.953


I148Y, L159M


S46V, D48M, T49L, G51R, N76V, F82I, Y116H,
1.535
1.202
1.370
1.119


I148Y, L159M, D164T


S46V, D48M, T49L, G51R, N76V, F82I, M94I,
1.422
1.177
1.303
1.091


N115C, Y116H, I148Y


S46V, D48M, T49L, G51R, N76V, F82I, N115F,
2.010
1.099
1.568
1.281


Y116H, I148Y


S46V, D48M, T49L, G51R, N76V, F82I, V112L,
1.216
1.030
1.128
1.079


N115F, Y116H, I148Y


S46V, D48M, T49L, G51R, N76V, G85S, N115W,
1.137
1.173
1.154
0.986


Y116H, I148Y, L159M


S46V, D48M, T49L, G51R, H64N, N76V, N115F,
1.116
0.907
1.013
1.102


Y116H, I148Y, D164T


S46V, D48M, T49L, G51R, N76V, I87V, V112L,
1.776
1.022
1.405
1.266


N115W, Y116H, I148Y


K32I, S46V, D48M, T49L, G51R, N52K, N76V,
1.591
1.126
1.366
1.166


N115F, Y116H, I148Y, L159M


S46V, D48M, T49L, G51R, K65M, N76V, F82I,
1.119
0.967
1.047
1.067


N115W, Y116H, I148Y, N156H


S46V, D48M, T49L, G51R, K65M, N76V, F82I,
1.552
1.072
1.316
1.181


V112L, N115F, Y116H, I148Y


S46V, D48M, T49L, G51R, K65M, N76V, G85S,
0.755
0.722
0.739
1.017


V112L, N115W, Y116H, I148Y, M155K


S46V, D48M, T49L, G51R, K65M, N76V, Y116H,
1.025
1.035
1.030
0.995


I148Y, L159M


S46V, D48M, T49L, G51R, K65M, N76V, N115F,
1.831
1.103
1.487
1.229


Y116H, I148Y


S46V, D48M, T49L, G51R, K65M, N76V, N89K,
1.215
1.045
1.132
1.077


V112L, N115Y, Y116H, I148Y


S46V, D48M, T49L, G51R, K65M, N76V, V112L,
1.650
0.957
1.325
1.244


N115W, Y116H, I148Y


S46V, D48M, T49L, G51R, K65M, N76V, V112L,
1.113
0.890
1.003
1.112


N115Y, Y116H, I148Y


S46V, D48M, T49L, G51R, N76V, Y116H, I148Y,
1.498
1.249
1.375
1.088


L159M, D164T


L31M, Q33R, S46V, D48M, T49L, G51R, N52K,
1.184
1.370
1.276
0.927


N76V, Y116H, I148Y


S46V, D48M, T49M, G51R, N52K, N76V, F82I,
2.161
1.375
1.780
1.217


V112L, N115Y, Y116H, I148Y, D164T


S46V, D48M, T49L, G51R, N76V, N115F, Y116H,
1.358
1.100
1.230
1.103


I148Y, L159M


S46V, D48M, T49L, G51R, N76V, N115W, Y116H,
1.337
1.013
1.184
1.126


I148Y, D164T


S46V, D48M, T49L, G51R, N76V, N115W, Y116H,
1.906
1.118
1.537
1.240


I148Y, L159M


S46V, D48M, T49L, G51R, N76V, N115Y, Y116H,
1.267
1.013
1.155
1.096


I148Y, L159M


S46V, D48M, T49L, G51R, N52K, N76V, Y116H,
1.790
1.385
1.599
1.122


I148Y, D164T


S46V, D48M, T49L, G51R, N52K, N76V, F82I,
4.051
2.136
3.154
1.287


Y116H, I148Y


S46V, D48M, T49L, G51R, N52K, N76V, F82I,
4.148
1.636
3.039
1.359


V112L, N115W, Y116H, I148Y, L159M


S46V, D48M, T49L, G51R, N52K, N76V, F82I,
2.175
1.471
1.834
1.189


V112L, N115Y, Y116H, I148Y, D164T


S46V, D48M, T49L, G51R, N52K, K65M, N76V,
1.733
1.303
1.525
1.136


Y116H, I148Y


S46V, D48M, T49L, G51R, N52K, K65M, N76V,
4.254
1.591
2.944
1.441


N115W, Y116H, I148Y


S46V, D48M, T49L, G51R, N52K, K65M, N76V,
1.068
0.794
0.933
1.146


N89I, N115W, Y116H, I148Y


S46V, D48M, T49L, G51R, N52K, K65M, N76V,
1.721
1.094
1.426
1.205


V112L, N115W, Y116H, I148Y, L159M, D164T


S46V, D48M, T49L, G51R, N52K, K65M, N76V,
1.112
1.085
1.099
1.011


V112M, Y116H, I148Y, D164V


S46V, D48M, T49L, G51R, N52K, N76V, N115F,
2.353
1.381
1.882
1.252


Y116H, I148Y, L159M


S46V, D48M, T49L, G51R, N52K, N76V, N115W,
4.639
1.722
3.264
1.424


Y116H, I148Y


S46V, D48M, T49L, G51R, N52K, N76V, N115Y,
4.403
2.051
3.365
1.311


Y116H, I148Y


S46V, D48M, T49L, G51R, N52K, N76V, N115Y,
4.704
1.945
3.411
1.381


Y116H, I148Y, D164T


S46V, D48M, T49L, G51R, N52K, N76V, T59A,
1.898
1.171
1.577
1.200


F82I, V112L, N115W, Y116H, I148Y, L159M


S46V, D48M, T49L, G51R, N52K, N76V, V112L,
3.675
2.069
2.966
1.245


Y116H, I148Y


S46V, D48M, T49L, G51R, N52K, N76V, V112L,
1.366
1.092
1.230
1.109


N115F, Y116H, I148Y, A165V


S46V, D48M, T49L, G51R, N52K, N76V, V112L,
3.776
2.131
2.962
1.256


N115F, Y116H, I148Y, L159M


S46V, D48M, T49L, G51R, N52S, N76V, V112L,
1.737
1.039
1.394
1.250


N115Y, Y116H, I148Y


P29R, Q33R, S46V, D48M, T49L, G51R, N52K,
3.873
2.125
3.054
1.271


N76V, F82I, N115F, Y116H, I148Y, D164T


Q28K, Q33R, S46V, D48M, T49L, G51R, N52K,
4.385
2.210
3.366
1.303


N76V, N115F, Y116H, I148Y, D164T


Q28K, Q34R, S46V, D48M, T49L, G51R, N52K,
4.201
2.708
3.497
1.207


N76V, F82I, V112L, N115W, Y116H, I148Y,


L159M


Q33A, S46V, D48M, T49L, G51R, N76V, F82I,
1.639
1.072
1.360
1.207


N115W, Y116H, I148Y, D164T


Q33A, S46V, D48M, T49L, G51R, K65M, N76V,
0.987
0.985
0.986
1.003


Y116H, K118R, I148Y


Q33A, S46V, D48M, T49L, G51R, K65M, N76V,
1.168
0.979
1.084
1.077


V112L, N115Y, Y116H, I148Y


Q33A, S46V, D48M, T49V, G51R, K65M, N76V,
1.046
0.900
0.976
1.071


N115Y, Y116H, I148Y, L159M


Q33A, S46V, D48M, T49L, G51R, N76V, N115Y
1.565
0.994
1.313
1.191


Q33A, S46V, D48M, T49L, G51R, N76V, N115Y,
1.423
1.010
1.219
1.166


Y116H, I148Y, L159M


Q33A, S46V, D48M, T49L, G51R, N52K, N76V,
0.885
0.791
0.844
1.047


D77E, F82I, N115F, Y116H, I148Y


Q33A, S46V, D48M, T49L, G51R, N52K, N76V,
2.639
1.713
2.230
1.187


F82I, N115F, Y116H, I148Y, D164Y


Q33A, S46V, D48M, T49L, G51R, N52K, N76V,
4.148
2.157
3.269
1.270


F82I, R88C, N115F, Y116H, I148Y


Q33A, S46V, D48M, T49L, G51R, N52K, K65M,
1.807
1.632
1.730
1.047


N76V, F82I, Y116H, I148Y


Q33A, S46V, D48M, T49L, G51R, N52K, K65M,
4.314
2.663
3.585
1.204


N76V, F82I, Y116H, K118Q, I148Y


Q33A, S46V, D48M, T49L, G51R, N52K, N76V,
4.458
1.534
3.079
1.447


N115F, Y116H, I148Y


Q33A, S46V, D48M, T49L, G51R, N52K, T59S,
4.215
2.129
3.295
1.280


N76V, F82I, N115F, Y116H, I148Y, K162R, D164E


Q33A, S46V, D48M, T49L, G51R, T59A, N76V,
1.108
1.129
1.118
0.992


V112L, Y116H, I148Y, D164T


Q33A, S46V, D48M, T49L, G51R, N76V, V112L,
1.265
1.119
1.196
1.059


Y116H, I148Y, D164T


Q33A, S46V, D48M, T49L, G51R, N76V, V112L,
1.823
1.108
1.486
1.229


N115F, Y116H, I148Y, D164T


Q33A, S46V, D48M, T49L, G51R, N76V, V112L,
1.559
0.990
1.291
1.207


N115F, Y116H, I148Y, K162N, D164T


Q33A, S46V, D48M, T49L, G51R, N76V, V112L,
1.802
1.052
1.430
1.258


N115W, Y116H, I148Y


Q33H, S46V, D48M, T49L, G51R, N52K, K65M,
2.564
1.315
1.949
1.318


N76V, N115W, Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, N76V, F82I,
1.587
1.198
1.404
1.131


N115W, Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, N76V, F82I,
1.432
1.351
1.396
1.017


V112L, N115Y, Y116H, I148Y, L159M


Q33R, S46V, D48M, T49L, G51R, N76V, I110S,
0.983
0.920
0.952
1.030


N115W, Y116H, I148Y, L159M


Q33R, S46V, D48M, T49L, G51R, K65M, N76V,
0.986
1.054
1.019
0.965


Y116H, I148Y, L159M


Q33R, S46V, D48M, T49L, G51R, K65M, N76V,
1.051
1.122
1.085
0.969


F82I, N115W, Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, K65M, N76V,
1.027
0.746
0.894
1.142


G81C, N115F, Y116H, I148Y, L159M, D164T


Q33R, S46V, D48M, T49L, G51R, K65M, N76V,
1.846
1.048
1.469
1.254


N115F, Y116H, I148Y, L159M, D164T


Q33R, S46V, D48M, T49L, G51R, N76V, L79M,
0.761
0.725
0.743
1.024


V112L, N115Y, Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, N76V, N115W,
1.960
1.238
1.603
1.222


Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, N76V, N115W,
1.396
1.048
1.225
1.139


Y116H, I148Y, L159M


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
3.939
2.752
3.352
1.175


Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
3.131
2.540
2.854
1.099


F82I, N115F, Y116H, I148Y, D164T


Q33R, S46V, D48M, T49L, G51R, N52K, G53C,
1.593
1.380
1.493
1.069


N76V, F82I, N115F, Y116H, I148Y, D164T


Q33R, S46V, D48M, T49L, G51R, N52K, K65M,
3.795
2.176
2.998
1.270


N76V, V112L, Y116H, I148Y, L159M


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
5.164
2.018
3.639
1.421


N115F, Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
4.774
1.710
3.289
1.454


N115Y, Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
2.326
1.258
1.809
1.285


N115Y, Y116H, I148Y, L159M, D164T


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
4.278
2.041
3.230
1.330


V112L, Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
2.435
1.530
2.011
1.216


V112L, Y116H, I148Y, D164Y


Q33R, Q34R, S46V, D48M, T49L, G51R, N52K,
5.192
1.873
3.583
1.451


N76V, Q63K, Y68C, N115F, Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, R51S, N52K,
1.149
1.087
1.118
1.027


K65M, N76V, V112L, Y116H, I148Y, L159M


Q33R, S46V, D48M, T49L, G51R, N76V, V112L,
2.316
1.030
1.714
1.351


N115F, Y116H, I148Y, D164T


Q33R, S46V, D48M, T49L, G51R, N76V, V112L,
1.243
0.982
1.114
1.115


N115Y, Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, Y68S, N76V,
0.938
0.912
0.925
1.011


F82I, N115W, Y116H, I148Y, D164A


Q34L, S46V, D48M, T49L, G51R, N52K, K65M,
2.948
1.885
2.425
1.206


N76V, N115Y, Y116H, I148Y, D164T


Q34R, S46V, D48M, T49L, G51R, N76V, F82I,
1.292
1.083
1.191
1.084


Y116H, I148Y, D164T


Q34R, S46V, D48M, T49L, G51R, N52K, K65M,
4.561
1.912
3.277
1.391


Y116H, I148Y


S46V, D48M, T49L, G51R, N76V, V112L, Y116H,
1.384
1.257
1.328
1.043


I148Y, L159M


S46V, D48M, T49L, G51R, N76V, V112L, N115W,
2.333
1.105
1.729
1.351


Y116H, I148Y


A30T, S46V, D48M, T49L, G51R, N76V, T86P,
1.158
0.762
0.983
1.178


V112L, N115F, Y116H, I148Y


K32I, S46V, D48M, T49L, G51R, N52K, N76V,
4.584
2.512
3.624
1.264


V112L, N115F, Y116H, I148Y


K32N, S46V, D48R, T49L, G51R, N52K, N76V,
3.361
2.327
2.888
1.164


Q84V, N115W, Y116H, I148Y, L149F


L5V, S22R, Q33A, S46V, D48M, T49L, G51R,
3.457
1.962
2.765
1.250


N52K, N76V, I87V, N115F, Y116H, I148Y, L149M


N2T, Q34R, S46V, D48M, T49L, G51R, N76V,
1.784
1.178
1.503
1.186


Q84T, V112L, N115F, Y116H, K118Q, I148Y,


L149M


S46V, D48M, T49L, G51R, N52K, N76V, G81C,
3.148
1.848
2.546
1.236


N115W, Y116H, I148Y


S46V, D48M, T49L, G51R, N52K, N76V, N115F,
4.305
3.298
3.839
1.125


Y116H, I148Y


S46V, D48M, T49L, G51R, N52K, N76V, N115F,
4.547
2.847
3.743
1.214


Y116H, K118Q, I148Y


S46V, D48M, T49L, G51R, N52K, N76V, Q84A,
4.872
2.567
3.767
1.291


V112L, N115F, Y116H, I148Y


S46V, D48M, T49L, G51R, N52K, N76V, Q84T,
4.840
2.692
3.809
1.270


N115W, Y116H, I148Y, L149F


S46V, D48M, T49L, G51R, N52K, N76V, Q84T,
3.710
2.244
3.054
1.213


V112L, N115F, Y116H, I148Y


S46V, D48M, T49L, G51R, N52K, N76V, Q84V,
2.744
1.925
2.369
1.159


N115W, Y116H, I148Y, L149F


S46V, D48M, T49L, G51R, N52K, N76V, Q84V,
3.042
1.847
2.518
1.208


V112L, N115F, Y116H, I148Y


S46V, D48M, T49L, G51R, N52K, N76V, Q84V,
1.944
1.219
1.608
1.208


V112L, N115W, Y116H, K118Q, I148Y


S46V, D48M, T49L, G51R, N52K, N76V, V112L,
4.686
2.599
3.719
1.259


N115F, Y116H, I148Y


S46V, D48M, T49L, G51R, N52K, N76V, V112L,
5.314
2.799
4.163
1.276


N115F, Y116H, K118Q, I148Y


S46V, D48M, T49L, G51R, N52K, N76V, V112L,
4.977
2.155
3.714
1.344


N115W, Y116H, I148Y


S46V, D48M, T49L, G51R, N52R, N76V, Q84A,
5.972
2.734
4.541
1.315


V112L, N115W, Y116H, I148Y, L149M


P29L, S46V, D48M, T49L, G51R, N52K, N76V,
2.850
1.792
2.377
1.200


Q84T, V112L, N115F, Y116H, I148Y


P29L, Q33A, Q34R, I45M, S46V, D48M, T49L,
1.803
1.458
1.652
1.091


G51R, N52K, N76V, F82L, R88H, V112L, N115W,


Y116H, I148Y


Q28H, Q33R, Q34R, S46V, D48M, T49L, G51R,
3.820
1.882
2.889
1.322


N52K, N76V, Q84T, V112L, N115W, Y116H, I148Y


Q33A, S46V, D48M, T49L, G51R, L54M, N76V,
1.590
1.032
1.326
1.199


Q84V, V112L, N115F


Q33A, S46V, D48I, T49L, G51R, N52K, N76V,
1.458
1.126
1.306
1.116


N115W, Y116H, I148Y, L149M


Q33A, S46V, D48M, T49L, G51R, N52K, N76V,
4.373
2.843
3.664
1.193


N115F, Y116H, I148Y, L149M


Q33A, S46V, D48M, T49L, G51R, N52K, N76V,
4.515
3.220
3.947
1.144


N115F, Y116H, I148Y, L149T


Q33A, S46V, D48M, T49L, G51R, N52K, N76V,
4.499
2.757
3.702
1.215


N115W, Y116H, I148Y, L149M


Q33A, S46V, D48M, T49L, G51R, N52K, N76V,
2.859
1.522
2.226
1.284


Q84A, V112L, N115W, Y116H, K118Q, I148Y,


L149T


Q33A, S46V, D48M, T49L, G51R, N52K, N76V,
3.673
2.095
2.982
1.232


Q84A, V112L, N115W, Y116H, I148Y, L149M


Q33A, S46V, D48M, T49L, G51R, N52K, N76V,
3.231
1.937
2.610
1.235


Q84T, N115W, Y116H, I148Y


Q33A, S46V, D48M, T49L, G51R, N52K, N76V,
3.489
1.854
2.741
1.273


Q84V, V112L, N115W, Y116H, I148Y, L149F


Q33A, S46V, D48M, T49L, G51R, N52K, N76V,
4.389
2.814
3.699
1.187


V112L, N115F, Y116H, I148Y


Q33A, S46V, D48M, T49L, G51R, N52R, N76V,
3.791
3.302
3.557
1.069


Y116H, I148Y, L149T


Q33A, S46V, D48M, T49L, G51R, N52S, N76V,
1.751
0.996
1.393
1.256


Q84V, V112L, N115F, Y116H, I148Y


Q33A, Q34R, S46V, D48M, T49L, G51R, N52K,
4.215
4.136
4.181
1.008


N76V


Q33A, Q34R, S46V, D48M, T49L, G51R, N52K,
3.306
2.121
2.782
1.189


N76V, P83R, Q84A, V112L, N115F, Y116H,


K118Q, I148Y


Q33A, Q34R, S46V, D48M, T49L, G51R, N52K,
4.736
3.094
4.010
1.181


N76V, Q84A, V112L, N115F, Y116H, K118Q,


I148Y


Q33A, Q34R, S46V, D48M, T49L, G51R, N52K,
5.554
3.159
4.504
1.234


N76V, V112L, N115W, Y116H, I148Y


Q33A, Q34R, S46V, D48M, T49L, G51R, N52R,
3.728
3.273
3.510
1.062


N76V, Y116H, I148Y, L149F


Q33A, Q34R, S46V, D48M, T49L, G51R, N52R,
5.915
2.816
4.427
1.335


N76V, Q84V, V112L, N115W, Y116H, I148Y,


L149T


Q33A, Q34R, S46V, D48M, T49L, G51R, N52R,
4.973
2.606
3.839
1.295


N76V, V112L, N115F, Y116H, I148Y, L149F


Q33A, Q34R, S46V, D48M, T49L, G51R, N76V,
1.535
0.921
1.254
1.224


Q84A, N115F, Y116H, I148Y


Q33A, Q34R, S46V, D48M, T49L, G51R, N76V,
1.508
1.013
1.270
1.187


Q84V, V112L, N115W, Y116H, I148Y, L149T


Q33A, S46V, D48M, T49L, G51R, N76V, Q84A,
1.676
1.063
1.407
1.192


V112L, N115W, Y116H, I148Y


Q33A, S46V, D48M, T49L, G51R, N76V, Q84T,
1.373
0.956
1.186
1.158


V112L, N115W, Y116H, I148Y


Q33A, S46V, D48M, T49L, G51R, N76V, V112L,
2.007
1.269
1.653
1.222


N115F, Y116H, K118Q, I148Y, L149T


Q33P, S46V, D48M, T49L, G51R, N52R, N76V,
2.207
1.365
1.804
1.238


V112L, N115F, Y116H, I148Y


Q33R, E37D, S46V, D48M, T49L, G51R, N52K,
3.331
1.850
2.645
1.259


N76V, G81C, Q84A, V112L, N115W, Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, N76V, N115F,
1.776
1.148
1.485
1.195


Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, N76V, N115F,
1.966
1.146
1.599
1.230


Y116H, K118Q, I148Y, L149F


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
3.586
2.808
3.242
1.106


F82V, V112L, N115F, Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
3.470
1.980
2.780
1.248


G81C, N115F, Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
3.143
1.816
2.535
1.240


G81C, Q84A, V112L, N115W, Y116H, I148Y,


L149M


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
2.892
1.774
2.392
1.209


L79M, V112L, N115W, Y116H, I148Y, L149T


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
3.279
2.324
2.852
1.149


N115W, Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
3.751
2.345
3.134
1.197


Q84A, V112L, N115F, Y116H, I148Y, L149F


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
1.426
1.098
1.282
1.113


Q84A, V112L, N115F, Y116H, I148Y, L149F,


M154I


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
6.010
2.759
4.521
1.329


Q84A, V112L, N115W, Y116H, I148Y, L149M


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
1.968
1.221
1.622
1.212


Q84A, V112L, N115W, Y116H, I148H


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
2.521
1.712
2.164
1.166


Q84K, V112L, N115F, Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
3.052
2.022
2.591
1.178


Q84T, N115F, Y116H, I148Y, L149M


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
3.220
1.947
2.630
1.224


Q84T, V112L, N115F, Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
4.980
2.989
4.068
1.224


Q84T, V112L, N115F, Y116H, K118Q, I148Y,


L149T


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
3.012
1.832
2.490
1.210


Q84T, V112L, N115W, Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
4.921
2.656
3.834
1.283


Q84V, V112L, N115F, Y116H, K118Q, I148Y,


L149F


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
2.999
1.686
2.398
1.249


Q84V, V112L, N115W, Y116H, K118Q, I148Y


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
3.750
1.961
2.931
1.279


Q84V, V112L, N115W, Y116H, I148Y, L149F


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
4.702
2.750
3.839
1.225


V112L, N115F, Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
5.424
2.806
4.253
1.276


V112L, N115W, Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
6.026
2.682
4.477
1.345


V112L, N115W, Y116H, I148Y, L149M


Q33R, S46V, D48M, T49L, G51R, N52K, N76V,
4.563
2.572
3.672
1.242


V112L, N115W, Y116H, I148Y, L149T


Q33R, S46V, D48M, T49L, G51R, N52R, N76V,
4.017
3.432
3.736
1.075


Y116H, I148Y, L149F


Q33R, S46V, D48M, T49L, G51R, N52R, N76V,
5.463
2.558
4.068
1.343


N115W, Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, N52R, N76V,
4.830
2.877
3.893
1.241


Q84A, N115F, Y116H, I148Y, L149F


Q33R, S46V, D48M, T49L, G51R, N52R, N76V,
2.939
1.666
2.381
1.235


Q84A, V112L, N115F, Y116H, K118Q, I148Y,


L149M


Q33R, S46V, D48M, T49L, G51R, N52R, N76V,
3.105
1.992
2.607
1.191


Q84T, V112L, N115F, Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, N52R, N76V,
5.372
2.835
4.251
1.265


T86P, V112L, N115W, Y116H, I148Y, L149T


Q33R, S46V, D48M, T49L, G51R, N52R, N76V,
4.105
2.503
3.388
1.211


V112L, N115F, Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, N52R, N76V,
3.385
2.212
2.871
1.175


V112L, N115F, Y116H, K118Q, I148Y, L149M


Q33R, S46V, D48M, T49L, G51R, N52R, N76V,
6.042
2.468
4.462
1.354


V112L, N115W, Y116H, I148Y, L149T


Q33R, Q34K, S46V, D48M, T49L, G51R, N76V,
1.484
0.957
1.231
1.205


Q84V, V112L, N115W, Y116H, I148Y, L149T


Q33R, Q34R, S46V, D48I, T49L, G51R, N52K,
1.665
1.313
1.497
1.112


N76V, N115W


Q33R, Q34R, S46V, D48M, T49L, G51R, N52K,
1.891
1.298
1.610
1.174


A67T, N76V, V112L, N115F, Y116H, I148Y,


L149M, M155I


Q33R, Q34R, S46V, D48M, T49L, G51R, N52K,
4.801
2.910
3.895
1.231


N76V, N115W, Y116H, K118Q, I148Y


Q33R, Q34R, S46V, D48M, T49L, G51R, N52K,
5.024
2.719
4.005
1.254


N76V, Q84A, V112L, N115F, Y116H, I148Y, L149F


Q33R, Q34R, S46V, D48M, T49L, G51R, N52K,
5.007
2.275
3.695
1.354


N76V, Q84T, V112L, N115W, Y116H, I148Y


Q33R, Q34R, S46V, D48M, T49L, G51R, N52K,
3.340
2.027
2.711
1.231


N76V, Q84V, N115F, Y116H, I148Y, K133R, L149T


Q33R, Q34R, S46V, D48M, T49L, G51R, N52K,
3.305
2.106
2.730
1.210


N76V, Q84V, N115F, Y116H, I148Y, L149T


Q33R, Q34R, S46V, D48M, T49L, G51R, N52K,
4.894
2.294
3.645
1.343


N76V, V112L, N115F, Y116H, I148Y


Q33R, Q34R, S46V, D48M, T49L, G51R, N52K,
4.942
2.527
3.799
1.300


N76V, V112L, N115F, Y116H, I148Y, L149M


Q33R, Q34R, S46V, D48M, T49L, G51R, N52K,
6.145
2.696
4.566
1.346


N76V, V112L, N115W, Y116H, I148Y, L149F


Q33R, Q34R, S46V, D48M, T49L, G51R, N52R,
5.301
2.946
4.223
1.255


N76V, Q84V, N115W, Y116H, K118Q, I148Y,


L149M


Q33R, Q34R, S46V, D48M, T49L, G51R, N52R,
5.818
2.358
4.270
1.363


N76V, V112L, N115W, Y116H, I148Y


Q33R, Q34R, S46V, D48M, T49L, G51R, N76V,
1.854
1.113
1.527
1.215


Q84A, V112L, N115F, Y116H, I148Y, L149F,


G158C


Q33R, Q34R, S46V, D48M, T49L, G51H, N52K,
2.840
1.559
2.225
1.275


N76V, V112L, N115F, Y116H, I148Y


Q33R, Q34R, S46V, D48M, T49L, G51R, N76V,
1.685
0.926
1.346
1.253


V112L, N115W, Y116H, I148Y


Q33R, Q34R, S46V, D48M, T49L, G51R, N76V,
1.501
1.097
1.314
1.142


V112L, N115W, Y116H, K118Q, I148Y


Q33R, S46V, D48M, T49L, G51R, N76V, Q84A,
1.737
1.143
1.452
1.196


N115F, Y116H, I148Y, L149F


Q33R, S46V, D48M, T49L, G51R, N76V, Q84A,
1.967
1.441
1.726
1.140


N115W, Y116H, K118Q, I148Y


Q33R, S46V, D48M, T49L, G51R, N76V, Q84A,
1.225
1.050
1.145
1.070


N89H, N115W, Y116H, K118Q, I148Y


Q33R, S46V, D48M, T49L, G51R, N76V, Q84T,
1.695
1.019
1.382
1.226


V112L, N115F, Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, N76V, Q84V,
1.640
1.024
1.348
1.216


N115F, Y116H, I148Y, L149T


Q33R, S46V, D48M, T49L, G51R, N76V, Q84V,
2.225
1.168
1.717
1.295


T86P, V112L, N115F, Y116H, I148Y, L149M


Q33R, S46V, D48M, T49L, G51R, R56H, N76V,
0.986
0.818
0.911
1.083


N115F, Y116H, K118Q, I148Y, L149F


Q33R, S46V, D48M, T49L, G51R, N76V, V112L,
4.155
2.220
3.289
1.262


N115F, Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, N76V, V112L,
2.305
1.130
1.742
1.322


N115F, Y116H, I148Y, L149M


Q33R, S46V, D48M, T49L, G51R, N76V, V112L,
1.756
1.057
1.447
1.213


N115F, Y116H, I148Y, L149T


Q33R, S46V, D48M, T49L, G51R, N76V, V112L,
1.825
1.178
1.529
1.194


N115W, Y116H, I148Y


Q33R, S46V, D48M, T49L, G51R, N76V, V112L,
1.821
1.164
1.530
1.190


N115W, Y116H, I148Y, L149M


Q34K, S46V, D48M, T49L, G51R, N52K, N76V,
1.571
1.126
1.376
1.142


Q84V, V112L, N115F, Y116H, I148Y


Q34K, S46V, D48M, T49L, G51R, N52R, N76V,
2.881
1.734
2.374
1.214


V112L, N115F, Y116H, I148Y


Q34L, S46V, D48M, T49L, G51R, N52K, N76V,
5.058
2.819
3.998
1.264


N115F, Y116H, K118Q, I148Y


Q34R, G35A, S46V, D48M, T49L, G51R, N76V,
1.792
1.116
1.493
1.200


Q84A, V112L, N115F, Y116H, I148Y, L149T


Q34R, S46V, D48M, T49L, G51R, N76V, N115W,
2.409
1.476
1.961
1.205


Y116H, I148Y


Q34R, S46V, D48M, T49L, G51R, N52K, N76V,
5.576
2.923
4.304
1.294


Q84A, V112L, N115F, Y116H, I148Y, L149T


Q34R, S46V, D48M, T49L, G51R, N52R, N76V,
5.448
2.512
4.039
1.348


N115W, Y116H, I148Y


Q34R, S46V, D48M, T49L, G51R, N52R, N76V,
3.948
1.967
3.080
1.282


V112L, N115W, Y116H, I148Y, L149F


Q34R, S46V, D48M, T49L, G51R, N76V, Q84A,
1.061
0.669
0.888
1.195


V112L, N115F, Y116H, I147L, I148Y, L149T


Q34R, S46V, D48M, T49L, G51R, N76V, Q84A,
1.961
1.090
1.576
1.244


V112L, N115F, Y116H, I148Y, L149T


Q34R, S46V, D48M, T49L, G51R, N76V, Q84T,
1.823
1.156
1.531
1.191


V112L, N115W, Y116H, I148Y, L149F


Q34R, S46V, D48M, T49L, G51R, N76V, Q84V,
1.684
1.102
1.429
1.179


V112L, N115W, Y116H, I148Y, L149M


S46V, D48M, T49L, G51R, N76V, Q84T, N115W,
1.146
0.904
1.034
1.108


Y116H, I148Y, L149T


S46V, D48M, T49L, G51R, N76V, Q84V, V112L,
1.599
0.886
1.257
1.271


N115F, Y116H, K118Q, I148Y


S46V, D48M, T49L, G51R, N76V, V112L, N115F,
2.233
1.193
1.735
1.287


Y116H, I148Y


S46V, D48M, T49L, G51R, N76V, V112L, N115W,
1.649
1.026
1.370
1.204


Y116H, K118Q, I148Y, L149F


S46V, D48M, T49L, G51R, N76V, V112L, N115W,
2.150
1.111
1.652
1.301


Y116H, I148Y, L149F


S46V, D48M, T49L, G51R, N76V, V112L, N115W,
1.810
1.166
1.525
1.187


Y116H, I148Y, L149M


S46V, D48M, T49L, G51R, N76V, V112L, N115W,
1.657
1.059
1.395
1.188


Y116H, I148Y, L149T









As is apparent to one with skill in the art, various modifications and variations of the above aspects and embodiments can be made without departing from the spirit and scope of this disclosure. Such modifications and variations are within the scope of this disclosure.

## Claims

1. A variant ester synthase polypeptide comprising a variant of SEQ ID NO: 67, wherein the variant of SEQ ID NO: 67 is genetically engineered to have an amino acid substitution at position 15 of SEQ ID NO: 67, and wherein said polypeptide comprises at least one additional mutation selected from the group consisting of G4R, T5P, T5S, D7N, T24M,T24W, L3OH, G33D, G33S, L39A, L39M, L39S, R40S, D41A, D41G, D41H, D41Y, V43K, V435, T44A, T44F, T44K, Y58C, Y58L, A73Q, V76L, D77A, K78F, K78W, 180V, R98D, E99Q, G101L, I102R, P111D, P111G, P111S, H122S, R131M, I146K, I146E, I146R, S147A, V149L, R150P, V155G, T157S, R162E, N164D, N164R, P166S, T170R, T1705, V171E, V171F, V171H, V171R, V171W, R172S, R172W, P173W, R177V, A179S, A179V, D182G, E184F, E184G, E184L, E184R, E184S, A185L, A185M, S186T, V187G, V187R, P188R, A190P, A190R, A190W, S192A, S192L, S192V, Q193R, Q193S, M195G, A197T, A197V, Q201A, Q201V, Q201W, A202L, D203R, P206F, R207A, G212A, G212L, V216I, V219L, V234C, H239G, T242K, T242R, A243R, Q244G, R246A, R246G, R246L, R246Q, R246V, R246W, D255E, L257I, K258R, N259E, N259Q, L260V, H262Q, A263V, S264D, S264V, S264W, G265N, G266A, G266S, S267G, A285L, A285R, A285V, N288D, N289E, N289G, T293A, P294G, V301A, N302G, I303G, I303R, I303W, R304W, A306G, D307F, D307G, D307L, D307N, D307R, D307V, E309A, E309G, E309S, G310H, G310R, G310V, T311S, T313S, Q314G, I315F, S316G, F317W, I319G, A320C, A323G, D328F, Q334K, Q334S, Q348A, Q348R, K349A, K349C, K349H, K349Q, P351G, K352I, K352N, S353K, S353T, T356G, T356W, Q357V, M360Q, M360R, M360S, M360W, Y366G, Y366W, G375A, G375V, G375S, V381F, E393G, E393R, E393W, G394E, T395E, V409L, L411A, A413T, I420V, S424G, S424Q, S442E, S442G, M443G, A447C, A447I, A447L, L454V, D455E, L457Y, E458W, I461G, I461L, I461V, K466N, A468G, K472T and K472*, wherein said variant ester synthase polypeptide has improved fatty acid methyl ester synthase activity compared to a corresponding wild type ester synthase polypeptide.

2. The variant ester synthase polypeptide of claim 1, wherein said polypeptide is from a Marinobacter hydrocarbonoclasticus (WS377) ester synthase polypeptide.

3. The variant ester synthase polypeptide of claim 1, wherein expression of the variant ester synthase polypeptide in a recombinant host cell results in a higher titer of fatty ester compositions compared to a recombinant host cell expressing the corresponding wild type polypeptide.

4. The variant ester synthase of claim 3, wherein said titer is at least about 5% or greater.

5. The variant ester synthase of claim 3, wherein said fatty ester compositions are selected from the group consisting of fatty acid methyl esters, fatty acid ethyl esters, fatty acid propyl esters, fatty acid isopropyl esters, fatty acid butyl esters, monoglycerides, fatty acid isobutyl esters, fatty acid 2-butyl esters, and fatty acid tert-butyl esters.

6. The variant ester synthase of claim 1, wherein said improved fatty acid methyl ester activity results in properties selected from the group consisting of increased beta hydroxy esters, decreased beta hydroxyl esters, increased chain lengths of fatty acid esters, and decreased chain lengths of fatty acid esters.

7. The variant ester synthase polypeptide of claim 1, wherein expression of the variant ester synthase polypeptide in a recombinant host cell results in a production of an altered percentage of beta hydroxy esters as compared to a recombinant host cell expressing the corresponding wild type ester synthase polypeptide.

8. The variant ester synthase polypeptide of claim 7, wherein said altered percentage of beta hydroxy esters is an increased or decreased percentage of beta hydroxy esters.

9. The variant ester synthase polypeptide of claim 1, wherein the polypeptide comprises the mutation S15G.

10. The variant ester synthase polypeptide of claim 9, wherein the ester synthase polypeptide comprises mutations T5S, P111S, V171R, P188R, F317W, S353T, V409L and S442G.

11. The variant ester synthase polypeptide of claim 9, wherein the ester synthase polypeptide comprises mutations T5S, K78F, P111S, V171R, P188R, S192V, A243R, F317W, K349H, S353T, V409L and S442G.

12. The variant ester synthase polypeptide of claim 9, wherein the ester synthase polypeptide comprises mutations T5S, V76L, P111S, V171R, P188R, K258R, S316G, F317W, S353T, M360R, V409L and S442G.

13. The variant ester synthase polypeptide of claim 9, wherein the ester synthase polypeptide comprises mutations T5S, P111S, V171R, P188R, Q244G, S267G, G310V, F317W, A320C, S353T, Y366W, V409L and S442G.

14. The variant ester synthase polypeptide of claim 9, wherein the ester synthase polypeptide comprises mutations P111S, V155G, P166S, V171R, P188R, F317W, Q348A, S353T, V381F, V409L and S442G.

15. The variant ester synthase polypeptide of claim 9, wherein the ester synthase polypeptide comprises mutations L39S, D77A, P111S, V171R, P188R, T313S, F317W, Q348A, S353T, V381F, V409L, 1420V and S442G.

16. The variant ester synthase polypeptide of claim 9, wherein the ester synthase polypeptide comprises mutations TSS, T24W, T44F, P111S, I146L, V171R, P188R, D307N, F317W, S353T, V409L and S442G.

17. The variant ester synthase polypeptide of claim 9, wherein the ester synthase polypeptide comprises mutations TSS, K78F, P111S, V171R, P188R, S192V, R207A, A243R, D255E, L257I, N259Q, L260V, H262Q, G265N, A285V, N288D, N289G, F317W, Q348R, S353T, V381F, V409L and S442G.

18. The variant ester synthase polypeptide of claim 9, wherein the ester synthase polypeptide comprises mutations T5S, K78F, P111S, V171R, P188R, S192V, R207A, A243R, D255E, L257I, N259Q, L260V, H262Q, G265N, A285V, N288D, N289G, F317W, H349K, S353T, V381F, V409L and S442G.

19. The variant ester synthase polypeptide of claim 9, wherein the ester synthase polypeptide comprises mutations T5P, G33D, T44K, Y58L, P111S, R162E, V171R, P188R, R207A, V234C, Q244G, D255E, S267G, D307N, G310V, F317W, A320C, S353T, Y366W, G394E, V409L, S442G and I461V.

20. The variant ester synthase polypeptide of claim 9, wherein the ester synthase polypeptide comprises mutations T5P, G33D, T44K, Y58C, P111S, V171R, P188R, R207A, V234C, Q244G, L257I, S267G, D307N, G310V, F317W, A320C, S353T, Y366W, G394E, V409L, A413T, S442G and I461V.

21. The variant ester synthase polypeptide of claim 9, wherein the ester synthase polypeptide comprises mutations T5P, T44A, P111S, T157S, N164D, T170S, V171R, P188R, R207A, V216I, V234C, Q244G, L257I, S267G, D307N, G310V, F317W, A320C, Q334K, S353T, Y366W, G394E, V409L, S442G and I461V.

22. The variant ester synthase polypeptide of claim 9, wherein the ester synthase polypeptide comprises mutations T5P, Y58L, P111S, R162E, T170S, V171R, A179S, P188R, R207A, V234C, Q244G, L257I, S267G, D307N, G310V, F317W, A320C, S353K, Y366W, G394E, V409L, S442G and I461V.

23. A recombinant host cell genetically engineered to express the variant ester synthase polypeptide of claim 1.

24. A cell culture comprising the recombinant host cell of claim 23 and a fatty ester composition.

25. The recombinant host cell of claim 23, wherein the recombinant host cell produces a fatty ester composition with an altered percentage of beta hydroxy esters as compared to a host cell that expresses the wild type ester synthase polypeptide.

26. The recombinant host cell of claim 25, wherein the altered percentage of beta hydroxy esters is an increased percentage of beta hydroxy esters.

27. The recombinant host cell of claim 25, wherein the altered percentage of beta hydroxy esters is a decreased percentage of beta hydroxy esters.

28. The cell culture of claim 24, wherein the fatty ester composition comprises one or more of a C6, C8, C10, C12, C13, C14, C15, C16, C17 or C18 fatty ester.

29. The cell culture of claim 24, wherein the fatty ester composition comprises unsaturated fatty esters.

30. The cell culture of claim 24, wherein the fatty ester composition comprises a fatty ester having a double bond at position 7 in the carbon chain between C7 and C8 from the reduced end of the fatty ester.

31. The cell culture of claim 24, wherein the fatty ester composition comprises saturated fatty esters.

32. The cell culture of claim 24, wherein the fatty ester composition comprises branched chain fatty esters.

33. A recombinant host cell comprising:
(a) a polynucleotide encoding the variant ester synthase polypeptide of claim 1;
(b) an activated fatty acid; and
(c) an alcohol, wherein said recombinant host cell produces a fatty ester composition.

34. The recombinant host cell of claim 33, wherein said activated fatty acid is an Acyl-ACP or an Acyl-CoA.

35. The recombinant host cell of claim 33, further comprising a carbon source.

36. The recombinant host cell of claim 33, wherein the fatty ester composition is produced at a titer of least about 5% or higher.

37. A polynucleotide encoding a variant ester synthase polypeptide, wherein said ester synthase polypeptide comprises a variant of SEQ ID NO: 67 having an amino acid substitution at the residue corresponding to residue 15 of SEQ ID NO: 67, and wherein said ester synthase polypeptide comprises at least one additional mutation selected from the group consisting of G4R, T5P, T5S, D7N, T24M,T24W, L30H, G33D, G33S, L39A, L39M, L39S, R40S, D41A, D41G, D41H, D41Y, V43K, V43S, T44A, T44F, T44K, Y58C, Y58L, A73Q, V76L, D77A, K78F, K78W, 180V, R98D, E99Q, G101L, I102R, P111D, P111G, P111S, H122S, R131M, 1146K, I146L, I146R, S147A, V149L, R150P, V155G, T157S, R162E, N164D, N164R, P166S, T170R, T170S, V171E, V171F, V171H, V171R, V171W, R172S, R172W, P173W, R177V, A179S, A179V, D182G, E184F, E184G, E184L, E184R, E184S, A185L, A185M, S186T, V187G, V187R, P188R, A190P, A190R, A190W, S192A, S192L, S192V, Q193R, Q193S, M195G, A197T, A197V, Q201A, Q201V, Q201W, A202L, D203R, P206F, R207A, G212A, G212L, V216I, V219L, V234C, H239G, T242K, T242R, A243R, Q244G, R246A, R246G, R246L, R246Q, R246V, R246W, D255E, L257I, K258R, N259E, N259Q, L260V, H262Q, A263V, S264D, S264V, S264W, G265N, G266A, G266S, S267G, A285L, A285R, A285V, N288D, N289E, N289G, T293A, P294G, V301A, N302G, I303G, I303R, I303W, R304W, A306G, D307F, D307G, D307L, D307N, D307R, D307V, E309A, E309G, E309S, G310H, G310R, G310V, T311S, T313S, Q314G, I315F, S316G, F317W, I319G, A320C, A323G, D328F, Q334K, Q334S, Q348A, Q348R, K349A, K349C, K349H, K349Q, P351G, K352I, K352N, S353K, S353T, T356G, T356W, Q357V, M360Q, M360R, M360S, M360W, Y366G, Y366W, G375A, G375V, G375S, V381F, E393G, E393R, E393W, G394E, T395E, V409L, L411A, A413T, I420V, S424G, S424Q, S442E, S442G, M443G, A447C, A447I, A447L, L454V, D455E, L457Y, E458W, I461G, I461L, I461V, K466N, A468G, K472T and K472*.

38. A variant ester synthase polypeptide comprising an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 35.

39. A variant ester synthase polypeptide comprising an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 37.

40. A variant ester synthase polypeptide comprising an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 39.

41. A variant ester synthase polypeptide comprising an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 41.

42. A variant ester synthase polypeptide comprising an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 43.

43. The polynucleotide of claim 37, further comprising SEQ ID NO: 27 5′ to the ATG start codon.