Thioesterase variants with improved activity for the production of medium-chain fatty acid derivatives

Abstract

Engineered thioesterase variants are provided that have improved activity for the production of medium-chain fatty acid derivatives. [Solution] This disclosure relates to engineered plant acyl-ACP thioesterases with improved activity for the production of medium-chain fatty acid derivatives, including, for example, 8-carbon and 10-carbon fatty acids and fatty acid derivatives. This disclosure further relates to recombinant host cells containing engineered plant acyl-ACP thioesterases with improved activity for the production of medium-chain fatty acid derivatives. This disclosure also relates to methods for reducing toxicity and improving production of medium-chain fatty acids and derivatives.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. USSN 62 / 481,078, filed April 3, 2017, which is incorporated herein by reference in its entirety.

[0002] Field The present disclosure relates to molecular tools useful for the production of medium-chain fatty acids and fatty acid derivatives. Accordingly, the present disclosure relates to genes that confer tolerance to medium-chain fatty acids and fatty acid derivatives on microorganisms. The present disclosure further relates to novel engineered thioesterase variants and polynucleotides encoding them that have improved activity and / or selectivity for the production of medium-chain fatty acid derivatives, including, for example, 8-carbon and 10-carbon fatty acids and fatty acid derivatives. Accordingly, the present disclosure also relates to host cells containing the engineered thioesterase variants and the polynucleotides encoding them, as well as related cell cultures. Additionally, methods for producing medium-chain fatty acid derivatives by employing host cells expressing the engineered thioesterase variants and biologically produced medium-chain fatty acid derivative compositions are encompassed. [Background technology]

[0003] background There is considerable interest in producing medium-chain fatty acid (MCFA) derived products. Medium-chain fatty acids and medium-chain fatty acid derivatives have numerous industrial applications, such as biofuels, lubricants and greases, metalworking fluids, coatings and adhesives, cosmetics and personal care products, fragrances, food and nutrition, pharmaceuticals, plastics and rubber, and other feedstocks for the chemical industry.

[0004] In addition to their value in industry, medium-chain fatty acids have valuable uses as food supplements and nutraceuticals (see, e.g., Stig Bengmark (2013) Nutrients 5(1): 162-207). In fact, medium-chain fatty acids and their derivatives exhibit antibacterial properties (see, for example, Nobmann et al. International Journal of Food Microbiology. 2009;128(3):440-445 (Non-Patent Document 2); BW Petschow, et al. (1996) Antimicrob. Agents Chemother. 40(2):302-306 (Non-Patent Document 3)), suppress the accumulation of body fat, and prevent metabolic syndrome (see, for example, Takeuchi H., et al. (2008) Asia Pac J Clin Nutr. 17 Suppl 1:320-3 (Non-Patent Document 4); Koji Nagao (2010) Pharmacological Research 61:208-212 (Non-Patent Document 5)). Omura Y., et al. (2011) Acupuncture Electrotherm Res. 36(1-2):19-64 (see Non-Patent Document 6)) and has anticonvulsant effects at clinically relevant concentrations (see, e.g., Chang et al., (2013) Neuropharmacology 2013; 69: 105-14 (Non-Patent Document 7); Wlaz et al., (2015) Prog Neuropsychopharmacol Biol Psychiatry 2015; 57: 110-16 (Non-Patent Document 8)).

[0005] Given the numerous useful applications, it is not surprising that the demand for medium-chain fatty acids has been on the rise over the past few years. Unfortunately, the supply of medium-chain fatty acids has always been tied to the production of longer-chain free fatty acid (FFA) products from plants (palm oil) or chemical synthesis, with medium-length chains being produced as a shoulder, representing less than 20% of the total fatty acyl species (see, e.g., Kostik, V. et al. (2013) J. Hyg. Eng. Des. 4:112-116). This makes the supply of medium-chain fatty acids highly variable and unstable. Therefore, there is a need in the art for methods that can provide a reliable, stable, and sustainable supply of these compounds.

[0006] An alternative to current sources of medium-chain fatty acids is their production using biological systems, such as microbial fermentation. However, the production of free fatty acids by biological systems presents two major challenges. First, it often relies on thioesterases that act on alkyl thioester molecules produced by the host organism. Available thioesterases active on medium-chain alkyl thioesters either have suboptimal catalytic activity or are too broad in specificity, acting on a range of alkyl thioester chain lengths. Second, medium-chain fatty acids are often highly toxic to microbial cells, hindering their production at high levels. Additionally, the toxicity of medium-chain acyl compounds can make the selection and engineering of highly active thioesterases unfavorable. Therefore, for biological systems to provide alternative sources of medium-chain fatty acids, biological systems with improved thioesterases that are more active and selective for medium-chain alkyl thioesterases and that exhibit improved tolerance to medium-chain fatty acids are needed.

[0007] Fortunately, as will become apparent from the disclosure that follows, the present invention meets these and other needs. [Prior art documents] [Non-patent literature]

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Summary of the Invention

[0009] Summary One aspect of the present disclosure provides engineered thioesterase variants with improved activity for the production of medium chain fatty acid derivatives. Accordingly, in one embodiment, the present disclosure provides engineered thioesterase variants with improved activity for the production of medium chain fatty acid derivatives. In one embodiment, the engineered thioesterase variant of claim 1 has improved activity for the production of C8 fatty acid derivatives. In one embodiment, the engineered thioesterase variant has a sequence similar to SEQ ID NO: 1. and has an amino acid sequence having at least 90% sequence identity with NO:1 and at least one substitution mutation at an amino acid position selected from the group consisting of 3, 4, 6, 14, 15, 17, 22, 37, 44, 45, 50, 54, 56, 64, 67, 73, 76, 91, 99, 102, 110, 111, 114, 129, 132, 137, 158, 162, 165, 176, 178, 185, 186, 196, 197, 198, 203, 213, 217, 225, 227, 236, 244, 254, 256, 258, 278, 282, 292, 297, 298, 299, 300, 301, 302, 316, 321, and 322. In one embodiment of the engineered thioesterase, the at least one substitution mutation is (a) a lysine at amino acid position 3; (b) a methionine at amino acid position 4; (c) an arginine at amino acid position 6; (d) a glycine or arginine at amino acid position 14; (e) a leucine or tryptophan at amino acid position 15; (f) an alanine or cysteine ​​at amino acid position 17; (g) an arginine at amino acid position 22; (h) a proline at amino acid position 37; (i) a glycine or isoleucine at amino acid position 44; (j) a serine at position 45; (k) a tryptophan at amino acid position 50; (l (a) arginine at amino acid position 54; (b) lysine or cysteine ​​at amino acid position 56; (c) arginine or proline at amino acid position 64; (d) leucine at amino acid position 67; (e) valine at position 73; (f) phenylalanine or leucine or tyrosine at amino acid position 76; (g) methionine at amino acid position 91; (h) lysine or proline at amino acid position 99; (i) isoleucine at amino acid position 102; (j) leucine at amino acid position 110; (k) threonine at position 111; (k) lysine at position 114; (k) valine at amino acid position 129;(y) tryptophan at amino acid position 132; (z) cysteine ​​at amino acid position 137; (aa) glutamine at amino acid position 158; (bb) glutamic acid at amino acid position 162; (cc) valine at amino acid position 176; (dd) proline at amino acid position 178; (ee) alanine at amino acid position 185; (ff) glycine at amino acid position 186; (gg) valine at amino acid position 196; (hh) alanine at amino acid position 197 (ii) tryptophan at amino acid position 198; (jj) arginine at amino acid position 203; (kk) histidine or arginine at amino acid position 213; (ll) arginine at amino acid position 217; (mm) leucine at amino acid position 225; (nn) glycine at amino acid position 227; (oo) threonine at amino acid position 236; (pp) methionine or arginine at amino acid position 244; (qq) amino a member selected from the group consisting of: glycine at amino acid position 254; (rr) cysteine ​​or arginine at amino acid position 256; (ss) threonine or valine at amino acid position 258; (tt) lysine or valine at amino acid position 278; (uu) serine or valine at amino acid position 282; (vv) phenylalanine at amino acid position 292; (ww) threonine or aspartic acid or valine at amino acid position 297; (xx) valine or cysteine ​​at amino acid position 298; (yy) leucine at amino acid position 299; (zz) lysine or tryptophan or leucine at amino acid position 300; (aaa) cysteine ​​at amino acid position 301; (bbb) threonine at amino acid position 302; (ccc) arginine at amino acid position 316; (ddd) arginine at amino acid position 321; and (eee) lysine at amino acid position 322.

[0010] In one aspect, the engineered thioesterase variant is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO The member is selected from SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59.

[0011] In one aspect, the engineered thioesterase variants have an increased overall net positive charge compared to the thioesterase having SEQ ID NO: 1. In one aspect, the engineered thioesterase variants have an increased overall net positive charge compared to the thioesterase having variant SEQ ID NO: 4.

[0012] In one aspect, the engineered thioesterase variants are selected from the group consisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:45, and SEQ ID NO:46.

[0013] In one aspect, the engineered thioesterase variant has an increased surface positive charge compared to SEQ ID NO: 15. In one aspect, the engineered thioesterase variant is a member selected from the group consisting of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51.

[0014] In one aspect, the engineered thioesterase variants have improved solubility. In one aspect, the engineered thioesterase variants have improved solubility compared to SEQ ID NO:49.

[0015] In one aspect, the engineered thioesterase variant has a truncation mutation at amino acids 2-40 of SEQ ID NO: 49. In one aspect, the engineered thioesterase variant is a member selected from the group consisting of SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59.

[0016] In one aspect, the variant thioesterase has improved activity for the production of C10 fatty acid derivatives. In one aspect, the variant thioesterase has improved activity for the production of C8 fatty acid derivatives.

[0017] In one aspect, the present disclosure provides a recombinant host cell comprising one or more heterologous genes encoding a biochemical pathway that converts a first fatty acid derivative into a second fatty acid derivative, wherein the second fatty acid derivative has a higher minimum inhibitory concentration (MIC) than the first fatty acid derivative, and wherein the presence of the second fatty acid derivative increases the MIC of the first fatty acid derivative.

[0018] In one aspect, the biochemical pathway comprises a carboxylic acid reductase, a carboxylic acid reductase and an alcohol dehydrogenase, a carboxylic acid reductase and an alcohol-O-acetyltransferase, a carboxylic acid reductase and an alcohol dehydrogenase and an alcohol O-acetyltransferase, an ester synthase, an ester synthase and a fatty acyl-CoA synthetase, an acyl-CoA reductase, an acyl-CoA reductase and an acyl-CoA synthetase, an acyl-CoA reductase and an alcohol O-acetyltransferase, an acyl-CoA reductase, an alcohol O-acetyltransferase, and an acyl-CoA synthetase. A synthetase, O-methyltransferase, acyl-ACP reductase, acyl-ACP reductase and aldehyde decarbonylase, acyl-ACP reductase and aldehyde oxidative deformylase, acyl-ACP reductase and alcohol O-acetyltransferase, acyl-ACP reductase, alcohol-O-acetyltransferase, and alcohol dehydrogenase, OleA protein, OleA, OleC, and OleD proteins, OleA protein and fatty acyl-CoA synthetase, or OleA, OleC, and OleD proteins and fatty acyl-CoA synthetase.

[0019] In some embodiments, the first fatty acid derivative is a fatty acid, the second fatty acid derivative is a fatty acid alkyl ester, and the biochemical pathway comprises an ester synthase and a fatty acyl-CoA synthetase.

[0020] In one embodiment, the fatty acid alkyl ester is a fatty acid methyl ester or a fatty acid ethyl ester.

[0021] In one aspect, the first fatty acid derivative is a fatty alcohol, the second fatty acid derivative is a fatty alcohol acetate ester, and the biochemical pathway comprises a carboxylic acid reductase and an alcohol-O-acetyltransferase.

[0022] In one embodiment, the first fatty acid derivative and the second fatty acid derivative are medium chain fatty acid derivatives.

[0023] In one aspect, the recombinant host cell further comprises an engineered thioesterase variant.

[0024] In one aspect, the engineered thioesterase variants have at least 90% sequence identity to SEQ ID NO: 1, as well as 3, 4, 6, 14, 15, 17, 22, 37, 44, 45, 50, 54, 56, 64, 67, 73, 76, 91, 99, 102, 110, 111, 114, 129, 132, 137, 158, 162, 165, 176, 178, 185, 186, 196, 197, 198, 203, 213, 214, 215, 216, 217, 218, 220, 222, 224, 226, 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 and variant thioesterases having an amino acid sequence with at least one substitution mutation at an amino acid position selected from the group consisting of: 17, 225, 227, 236, 244, 254, 256, 258, 278, 282, 292, 297, 298, 299, 300, 301, 302, 316, 321, and 322.

[0025] In one aspect, the at least one substitution mutation is (a) a lysine at amino acid position 3; (b) a methionine at amino acid position 4; (c) an arginine at amino acid position 6; (d) a glycine or arginine at amino acid position 14; (e) a leucine or tryptophan at amino acid position 15; (f) an alanine or cysteine ​​at amino acid position 17; (g) an arginine at amino acid position 22; (h) a proline at amino acid position 37; (i) a glycine or isoleucine at amino acid position 44; (j) a serine at position 45; (k) a tryptophan at amino acid position 50; (l) an amino acid (m) arginine at amino acid position 54; (n) arginine or proline at amino acid position 64; (o) leucine at amino acid position 67; (p) valine at position 73; (q) phenylalanine, leucine, or tyrosine at amino acid position 76; (r) methionine at amino acid position 91; (s) lysine or proline at amino acid position 99; (t) isoleucine at amino acid position 102; (u) leucine at amino acid position 110; (v) threonine at position 111; (w) lysine at position 114; (x) valine at amino acid position 129 (y) tryptophan at amino acid position 132; (z) cysteine ​​at amino acid position 137; (aa) glutamine at amino acid position 158; (bb) glutamic acid at amino acid position 162; (cc) valine at amino acid position 176; (dd) proline at amino acid position 178; (ee) alanine at amino acid position 185; (ff) glycine at amino acid position 186; (gg) valine at amino acid position 196; (hh) asparagine at amino acid position 197; (ii) tryptophan at amino acid position 198; (jj) arginine at amino acid position 203; (kk) amino acid (ll) histidine or arginine at amino acid position 213; (ll) arginine at amino acid position 217; (mm) leucine at amino acid position 225; (nn) glycine at amino acid position 227; (oo) threonine at amino acid position 236; (pp) methionine or arginine at amino acid position 244; (qq) glycine at amino acid position 254; (rr) cysteine ​​or arginine at amino acid position 256; (ss) threonine or valine at amino acid position 258; (tt) lysine or valine at amino acid position 278; (uu) serine or valine at amino acid position 282;(vv) a phenylalanine at amino acid position 292; (ww) a threonine or aspartic acid or valine at amino acid position 297; (xx) a valine or cysteine ​​at amino acid position 298; (yy) a leucine at amino acid position 299; (zz) a lysine or tryptophan or leucine at amino acid position 300; (aaa) a cysteine ​​at amino acid position 301; (bbb) a threonine at amino acid position 302; (ccc) an arginine at amino acid position 316; (ddd) an arginine at amino acid position 321; and (eee) a lysine at amino acid position 322.

[0026] In one aspect, the engineered thioesterase variant is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO The member is selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59.

[0027] In one aspect, the engineered thioesterase variant has an increased surface positive charge compared to SEQ ID NO: 1. In one aspect, the engineered thioesterase variant has an increased surface positive charge compared to SEQ ID NO: 4. In one aspect, the engineered thioesterase variants are selected from the group consisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:45, and SEQ ID NO:46.

[0028] In one aspect, the engineered thioesterase variant has an increased surface positive charge compared to SEQ ID NO: 15. In one aspect, the engineered thioesterase variant is a member selected from the group consisting of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51.

[0029] In one aspect, the engineered thioesterase variant has improved solubility. In one aspect, the engineered thioesterase variant has improved solubility compared to SEQ ID NO:49. In one aspect, the engineered thioesterase variant has a truncation mutation at amino acids 2-40 of SEQ ID NO:49. In one aspect, the engineered thioesterase variant is a member selected from the group consisting of SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59.

[0030] In another aspect, the disclosure provides a method for producing medium-chain fatty acid derivatives at commercial titers, comprising culturing a recombinant host cell comprising an engineered thioesterase variant in the presence of a carbon source under conditions suitable for producing the medium-chain fatty acid derivative, wherein the recombinant host cell comprises one or more heterologous genes encoding a biochemical pathway that converts a first fatty acid derivative to a second fatty acid derivative, wherein the second fatty acid derivative has a higher minimum inhibitory concentration (MIC) than the first fatty acid derivative, and wherein the presence of the second fatty acid derivative increases the MIC of the first fatty acid derivative.

[0031] In one aspect, the first fatty acid derivative is a medium chain fatty acid, the second fatty acid derivative is a medium chain fatty acid alkyl ester, and the biochemical pathway comprises an ester synthase and a fatty acyl-CoA synthetase.

[0032] In one embodiment, the fatty acid alkyl ester is a medium chain fatty acid methyl ester or a medium chain fatty acid ethyl ester.

[0033] In one aspect, the first fatty acid derivative is a medium chain fatty alcohol, the second fatty acid derivative is a medium chain fatty alcohol acetate ester, and the biochemical pathway includes a carboxylic acid reductase and an alcohol-O-acetyltransferase.

[0034] In one aspect, the engineered thioesterase variant has SEQ ID and has an amino acid sequence having at least 90% sequence identity with NO:1 and at least one substitution mutation at an amino acid position selected from the group consisting of 3, 4, 6, 14, 15, 17, 22, 37, 44, 45, 50, 54, 56, 64, 67, 73, 76, 91, 99, 102, 110, 111, 114, 129, 132, 137, 158, 162, 165, 176, 178, 185, 186, 196, 197, 198, 203, 213, 217, 225, 227, 236, 244, 254, 256, 258, 278, 282, 292, 297, 298, 299, 300, 301, 302, 316, 321, and 322. In one aspect, the at least one substitution mutation is: (a) lysine at amino acid position 3; (b) methionine at amino acid position 4; (c) arginine at amino acid position 6; (d) glycine or arginine at amino acid position 14; (e) leucine or tryptophan at amino acid position 15; (f) alanine or cysteine ​​at amino acid position 17; (g) arginine at amino acid position 22; (h) proline at amino acid position 37; (i) glycine or isoleucine at amino acid position 44; (j) serine at position 45; (k) tryptophan at amino acid position 50; (l) arginine at amino acid position 54; (m) lysine or cysteine ​​at amino acid position 56; (n) arginine or proline at amino acid position 64; (o) leucine at amino acid position 67; (p) valine at position 73; (q) amino acid position (r) phenylalanine or leucine or tyrosine at amino acid position 76; (r) methionine at amino acid position 91; (s) lysine or proline at amino acid position 99; (t) isoleucine at amino acid position 102; (u) leucine at amino acid position 110; (v) threonine at position 111; (w) lysine at position 114; (x) valine at amino acid position 129; (y) tryptophan at amino acid position 132; (z) cysteine ​​at amino acid position 137; (aa) glutamine at amino acid position 158; (bb) glutamic acid at amino acid position 162; (cc) valine at amino acid position 176; (dd) proline at amino acid position 178; (ee) alanine at amino acid position 185; (ff) glycine at amino acid position 186; (gg) valine at amino acid position 196; (hh) asparagine at amino acid position 197;(ii) tryptophan at amino acid position 198; (jj) arginine at amino acid position 203; (kk) histidine or arginine at amino acid position 213; (ll) arginine at amino acid position 217; (mm) leucine at amino acid position 225; (nn) glycine at amino acid position 227; (oo) threonine at amino acid position 236; (pp) methionine or arginine at amino acid position 244; (qq) glycine at amino acid position 254; (rr) cysteine ​​or arginine at amino acid position 256; (ss) threonine or valine at amino acid position 258; (tt) lysine or valine at amino acid position 278; (uu) amino (vv) a serine or valine at amino acid position 282; (vv) a phenylalanine at amino acid position 292; (ww) a threonine or aspartic acid or valine at amino acid position 297; (xx) a valine or cysteine ​​at amino acid position 298; (yy) a leucine at amino acid position 299; (zz) a lysine or tryptophan or leucine at amino acid position 300; (aaa) a cysteine ​​at amino acid position 301; (bbb) a threonine at amino acid position 302; (ccc) an arginine at amino acid position 316; (ddd) an arginine at amino acid position 321; and (eee) a lysine at amino acid position 322.

[0035] In one aspect, the engineered thioesterase variant is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO The member is selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59.

[0036] In one aspect, the engineered thioesterase variant has an increased surface positive charge compared to SEQ ID NO:1.

[0037] In one aspect, the engineered thioesterase variant has an increased surface positive charge compared to SEQ ID NO:4. In one aspect, the engineered thioesterase variants are selected from the group consisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:45, and SEQ ID NO:46.

[0038] In one aspect, the engineered thioesterase variant has an increased surface positive charge compared to SEQ ID NO: 15. In one aspect, the engineered thioesterase variant is a member selected from the group consisting of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51.

[0039] In one aspect, the engineered thioesterase variant has improved solubility. In one aspect, the engineered thioesterase variant has improved solubility compared to SEQ ID NO:49. In one aspect, the engineered thioesterase variant has a truncation mutation at amino acids 2-40 of SEQ ID NO:49. In one aspect, the engineered thioesterase variant is a member selected from the group consisting of SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59.

[0040] In another aspect, the present disclosure provides a composition of medium-chain fatty acid derivatives having a ratio of C8 fatty acid derivatives to C10 fatty acid derivatives (C8 / C10) of at least 3.6. In one embodiment, the ratio of C8 fatty acid derivatives to C10 fatty acid derivatives is 7.7. [The present invention 1001] Engineered thioesterase variants with improved activity for the production of medium-chain fatty acid derivatives. [The present invention 1002] The engineered thioesterase variants of the present invention 1001 have improved activity for the production of C8 fatty acid derivatives. [The present invention 1003] at least 90% sequence identity with SEQ ID NO: 1, as well as 3, 4, 6, 14, 15, 17, 22, 37, 44, 45, 50, 54, 56, 64, 67, 73, 76, 91, 99, 102, 110, 111, 114, 129, 132, 137, 158, 162, 165, 176, 178, 185, 186, 196, 197, 198, 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, 26 1001. An engineered thioesterase variant of the present invention having an amino acid sequence with at least one substitution mutation at an amino acid position selected from the group consisting of 13, 217, 225, 227, 236, 244, 254, 256, 258, 278, 282, 292, 297, 298, 299, 300, 301, 302, 316, 321, and 322. [The present invention 1004] At least one substitution mutation is: (a) lysine at amino acid position 3; (b) methionine at amino acid position 4; (c) arginine at amino acid position 6; (d) glycine or arginine at amino acid position 14; (e) leucine or tryptophan at amino acid position 15; (f) alanine or cysteine ​​at amino acid position 17; (g) arginine at amino acid position 22; (h) proline at amino acid position 37; (i) glycine or isoleucine at amino acid position 44; (j) serine at position 45; (k) tryptophan at amino acid position 50; (l) alanine at amino acid position 54; (m) lysine or cysteine ​​at amino acid position 56; (n) arginine or proline at amino acid position 64; (o) leucine at amino acid position 67; (p) valine at position 73; (q) phenylalanine, leucine, or tyrosine at amino acid position 76; (r) methionine at amino acid position 91; (s) lysine or proline at amino acid position 99; (t) isoleucine at amino acid position 102; (u) leucine at amino acid position 110; (v) threonine at position 111; (w) lysine at position 114; (x) valine at amino acid position 129; (y (aa) glutamine at amino acid position 158; (bb) glutamic acid at amino acid position 162; (cc) valine at amino acid position 176; (dd) proline at amino acid position 178; (ee) alanine at amino acid position 185; (ff) glycine at amino acid position 186; (gg) valine at amino acid position 196; (hh) asparagine at amino acid position 197; (ii) tryptophan at amino acid position 198; (jj) arginine at amino acid position 203; (kk) amino acid position (ll) histidine or arginine at amino acid position 213; (ll) arginine at amino acid position 217; (mm) leucine at amino acid position 225; (nn) glycine at amino acid position 227; (oo) threonine at amino acid position 236; (pp) methionine or arginine at amino acid position 244; (qq) glycine at amino acid position 254; (rr) cysteine ​​or arginine at amino acid position 256; (ss) threonine or valine at amino acid position 258; (tt) lysine or valine at amino acid position 278; (uu) serine or valine at amino acid position 282;1003. The engineered thioesterase variant of the present invention, wherein the engineered thioesterase variant is a member selected from the group consisting of: (vv) phenylalanine at amino acid position 292; (ww) threonine or aspartic acid or valine at amino acid position 297; (xx) valine or cysteine ​​at amino acid position 298; (yy) leucine at amino acid position 299; (zz) lysine or tryptophan or leucine at amino acid position 300; (aaa) cysteine ​​at amino acid position 301; (bbb) threonine at amino acid position 302; (ccc) arginine at amino acid position 316; (ddd) arginine at amino acid position 321; and (eee) lysine at amino acid position 322. [The present invention 1005] Variant thioesterases are also disclosed in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO 1004. The engineered thioesterase variant of the present invention, wherein the variant is a member selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59. [The present invention 1006] An engineered thioesterase variant of the present invention 1005 having an increased overall net positive charge compared to the thioesterase having SEQ ID NO:1. [The present invention 1007] 1006. An engineered thioesterase variant of the present invention having an increased overall net positive charge compared to the variant thioesterase having SEQ ID NO:4. [The present invention 1008] SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and SEQ ID 1007. An engineered thioesterase variant of the present invention, which is a member selected from the group consisting of NO:46. [The present invention 1009] An engineered thioesterase variant of the present invention 1006 having an increased surface positive charge compared to SEQ ID NO:15. [The present invention 1010] 1009. An engineered thioesterase variant of the present invention, which is a member selected from the group consisting of SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51. [The present invention 1011] 1005. Engineered thioesterase variants of the present invention having improved solubility. [The present invention 1012] An engineered thioesterase variant of the present invention 1011 having improved solubility compared to SEQ ID NO:49. [The present invention 1013] An engineered thioesterase variant of the present invention 1012 having a truncation mutation at amino acids 2-40 of SEQ ID NO:49. [The present invention 1014] The engineered thioesterase variant of the present invention 1016, which is a member selected from the group consisting of SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59. [The present invention 1015] 1001. A variant thioesterase enzyme of the present invention, wherein the variant thioesterase has improved activity for the production of C10 fatty acid derivatives. [The present invention 1016] 1. A recombinant host cell comprising one or more heterologous genes encoding a biochemical pathway that converts a first fatty acid derivative into a second fatty acid derivative, the second fatty acid derivative has a higher minimum inhibitory concentration (MIC) than the first fatty acid derivative; A recombinant host cell wherein the presence of a second fatty acid derivative increases the MIC of a first fatty acid derivative. [The present invention 1017] The biochemical pathway a. carboxylic acid reductase, b. carboxylic acid reductase and alcohol dehydrogenase, c. carboxylic acid reductase and alcohol-O-acetyltransferase, d. carboxylic acid reductase, and alcohol dehydrogenase, and alcohol O-acetyltransferase; e. ester synthase, f. ester synthase and fatty acyl-CoA synthetase; g. acyl-CoA reductase, h. acyl-CoA reductase and acyl-CoA synthetase, i. acyl-CoA reductase and alcohol O-acetyltransferase, j. acyl-CoA reductase, alcohol O-acetyltransferase, and acyl-CoA synthetase; k. O-methyltransferase, l. acyl-ACP reductase, m. acyl-ACP reductase and aldehyde decarbonylase, n. acyl-ACP reductase and aldehyde oxidative deformylase, o. acyl-ACP reductase and alcohol O-acetyltransferase, p. acyl-ACP reductase, alcohol-O-acetyltransferase, and alcohol dehydrogenase, q. OleA protein, r. OleA, OleC, and OleD proteins, s. OleA protein and fatty acyl-CoA synthetase, or t. OleA, OleC, and OleD proteins and fatty acyl-CoA synthetase 1016. A recombinant host cell of the present invention, comprising one of: [The present invention 1018] the first fatty acid derivative is a fatty acid and the second fatty acid derivative is a fatty acid alkyl ester; The biochemical pathway includes an ester synthase and a fatty acyl-CoA synthetase; A recombinant host cell of the present invention. [The present invention 1019] The fatty acid alkyl ester is a fatty acid methyl ester or a fatty acid ethyl ester. A recombinant host cell of the present invention. [The present invention 1020] the first fatty acid derivative is a fatty alcohol and the second fatty acid derivative is a fatty alcohol acetate; the biochemical pathway includes carboxylic acid reductase and alcohol-O-acetyltransferase; A recombinant host cell of the present invention. [The present invention 1021] the first fatty acid derivative and the second fatty acid derivative are medium-chain fatty acid derivatives; A recombinant host cell of the present invention. [The present invention 1021] Further comprising engineered thioesterase variants, A recombinant host cell of the present invention. [The present invention 1022] The engineered thioesterase variants may have at least 90% sequence identity with SEQ ID NO: 1, as well as 3, 4, 6, 14, 15, 17, 22, 37, 44, 45, 50, 54, 56, 64, 67, 73, 76, 91, 99, 102, 110, 111, 114, 129, 132, 137, 158, 162, 165, 176, 178, 185, 186, 196, 197, 198, 203, 213, 214, 215, 216, 217, 218, 220, 222, 224, 226, 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, 2 and wherein the amino acid sequence of the variant thioesterase is at least one substitution mutation at an amino acid position selected from the group consisting of: 17, 225, 227, 236, 244, 254, 256, 258, 278, 282, 292, 297, 298, 299, 300, 301, 302, 316, 321, and 322. A recombinant host cell of the present invention. [The present invention 1023] At least one substitution mutation is: (a) lysine at amino acid position 3; (b) methionine at amino acid position 4; (c) arginine at amino acid position 6; (d) glycine or arginine at amino acid position 14; (e) leucine or tryptophan at amino acid position 15; (f) alanine or cysteine ​​at amino acid position 17; (g) arginine at amino acid position 22; (h) proline at amino acid position 37; (i) glycine or isoleucine at amino acid position 44; (j) serine at position 45; (k) tryptophan at amino acid position 50; (l) alanine at amino acid position 54; (m) lysine or cysteine ​​at amino acid position 56; (n) arginine or proline at amino acid position 64; (o) leucine at amino acid position 67; (p) valine at position 73; (q) phenylalanine, leucine, or tyrosine at amino acid position 76; (r) methionine at amino acid position 91; (s) lysine or proline at amino acid position 99; (t) isoleucine at amino acid position 102; (u) leucine at amino acid position 110; (v) threonine at position 111; (w) lysine at position 114; (x) valine at amino acid position 129; (y (aa) glutamine at amino acid position 158; (bb) glutamic acid at amino acid position 162; (cc) valine at amino acid position 176; (dd) proline at amino acid position 178; (ee) alanine at amino acid position 185; (ff) glycine at amino acid position 186; (gg) valine at amino acid position 196; (hh) asparagine at amino acid position 197; (ii) tryptophan at amino acid position 198; (jj) arginine at amino acid position 203; (kk) amino acid position (ll) histidine or arginine at amino acid position 213; (ll) arginine at amino acid position 217; (mm) leucine at amino acid position 225; (nn) glycine at amino acid position 227; (oo) threonine at amino acid position 236; (pp) methionine or arginine at amino acid position 244; (qq) glycine at amino acid position 254; (rr) cysteine ​​or arginine at amino acid position 256; (ss) threonine or valine at amino acid position 258; (tt) lysine or valine at amino acid position 278; (uu) serine or valine at amino acid position 282;1022. The recombinant cell of the present invention, wherein the amino acid sequence is a member selected from the group consisting of: (vv) phenylalanine at amino acid position 292; (ww) threonine or aspartic acid or valine at amino acid position 297; (xx) valine or cysteine ​​at amino acid position 298; (yy) leucine at amino acid position 299; (zz) lysine or tryptophan or leucine at amino acid position 300; (aaa) cysteine ​​at amino acid position 301; (bbb) threonine at amino acid position 302; (ccc) arginine at amino acid position 316; (ddd) arginine at amino acid position 321; and (eee) lysine at amino acid position 322. [The present invention 1024] The engineered thioesterase variants are shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID 1023. A recombinant host cell of the present invention, wherein the recombinant host cell is a member selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59. [The present invention 1025] 1024. A recombinant host cell of the invention, wherein the engineered thioesterase variant has an increased positive surface charge compared to SEQ ID NO:1. [The present invention 1026] 1025. A recombinant host cell of the invention, wherein the engineered thioesterase variant has an increased positive surface charge compared to SEQ ID NO:4. [The present invention 1027] Engineered thioesterase variants are shown in SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO: 1026. The recombinant host cell of the present invention, which is a member selected from the group consisting of NO:46. [The present invention 1028] 1025. A recombinant host cell of the invention, wherein the engineered thioesterase variant has an increased positive surface charge compared to SEQ ID NO:15. [The present invention 1029] 1028. The recombinant host cell of the invention, wherein the engineered thioesterase variant is a member selected from the group consisting of SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51. [The present invention 1030] The recombinant host cell of the present invention 1024, wherein the engineered thioesterase variant has improved solubility. [The present invention 1031] 1030. The recombinant host cell of the present invention, wherein the engineered thioesterase variant has improved solubility compared to SEQ ID NO:49. [The present invention 1032] 1031. The recombinant host cell of the invention, wherein the engineered thioesterase variant comprises a truncation mutation at amino acids 2-40 of SEQ ID NO:49. [The present invention 1033] 1032. The recombinant host cell of the present invention, wherein the engineered thioesterase variant is a member selected from the group consisting of SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59. [The present invention 1034] 1. A method for producing medium chain fatty acid derivatives at commercial titers, comprising culturing a recombinant host cell containing an engineered thioesterase variant in the presence of a carbon source under conditions suitable for the production of medium chain fatty acid derivatives, the recombinant host cell comprises one or more heterologous genes encoding a biochemical pathway that converts a first fatty acid derivative into a second fatty acid derivative; the second fatty acid derivative has a higher minimum inhibitory concentration (MIC) than the first fatty acid derivative; the presence of the second fatty acid derivative increases the MIC of the first fatty acid derivative; method. [This invention 1035] the first fatty acid derivative is a medium-chain fatty acid and the second fatty acid derivative is a medium-chain fatty acid alkyl ester; The biochemical pathway includes an ester synthase and a fatty acyl-CoA synthetase; The method of the present invention 1034. [The present invention 1036] The fatty acid alkyl ester is a medium-chain fatty acid methyl ester or a medium-chain fatty acid ethyl ester. The method of the present invention 1035. [This invention 1037] the first fatty acid derivative is a medium-chain fatty alcohol and the second fatty acid derivative is a medium-chain fatty alcohol acetate; the biochemical pathway includes carboxylic acid reductase and alcohol-O-acetyltransferase; The method of the present invention 1035. [The present invention 1038] The engineered thioesterase variants may have at least 90% sequence identity with SEQ ID NO: 1, as well as 3, 4, 6, 14, 15, 17, 22, 37, 44, 45, 50, 54, 56, 64, 67, 73, 76, 91, 99, 102, 110, 111, 114, 129, 132, 137, 158, 162, 165, 176, 178, 185, 186, 196, 197, 198, 1034. The method of claim 1034, wherein the polypeptide has an amino acid sequence having at least one substitution mutation at an amino acid position selected from the group consisting of 203, 213, 217, 225, 227, 236, 244, 254, 256, 258, 278, 282, 292, 297, 298, 299, 300, 301, 302, 316, 321, and 322. [This invention 1039] At least one substitution mutation is: (a) lysine at amino acid position 3; (b) methionine at amino acid position 4; (c) arginine at amino acid position 6; (d) glycine or arginine at amino acid position 14; (e) leucine or tryptophan at amino acid position 15; (f) alanine or cysteine ​​at amino acid position 17; (g) arginine at amino acid position 22; (h) proline at amino acid position 37; (i) glycine or isoleucine at amino acid position 44; (j) serine at position 45; (k) tryptophan at amino acid position 50; (l) alanine at amino acid position 54; (m) lysine or cysteine ​​at amino acid position 56; (n) arginine or proline at amino acid position 64; (o) leucine at amino acid position 67; (p) valine at position 73; (q) phenylalanine, leucine, or tyrosine at amino acid position 76; (r) methionine at amino acid position 91; (s) lysine or proline at amino acid position 99; (t) isoleucine at amino acid position 102; (u) leucine at amino acid position 110; (v) threonine at position 111; (w) lysine at position 114; (x) valine at amino acid position 129; (y (aa) glutamine at amino acid position 158; (bb) glutamic acid at amino acid position 162; (cc) valine at amino acid position 176; (dd) proline at amino acid position 178; (ee) alanine at amino acid position 185; (ff) glycine at amino acid position 186; (gg) valine at amino acid position 196; (hh) asparagine at amino acid position 197; (ii) tryptophan at amino acid position 198; (jj) arginine at amino acid position 203; (kk) amino acid position (ll) histidine or arginine at amino acid position 213; (ll) arginine at amino acid position 217; (mm) leucine at amino acid position 225; (nn) glycine at amino acid position 227; (oo) threonine at amino acid position 236; (pp) methionine or arginine at amino acid position 244; (qq) glycine at amino acid position 254; (rr) cysteine ​​or arginine at amino acid position 256; (ss) threonine or valine at amino acid position 258; (tt) lysine or valine at amino acid position 278; (uu) serine or valine at amino acid position 282;1036. The method of claim 1036, wherein the amino acid is a member selected from the group consisting of: (vv) phenylalanine at amino acid position 292; (ww) threonine or aspartic acid or valine at amino acid position 297; (xx) valine or cysteine ​​at amino acid position 298; (yy) leucine at amino acid position 299; (zz) lysine or tryptophan or leucine at amino acid position 300; (aaa) cysteine ​​at amino acid position 301; (bbb) threonine at amino acid position 302; (ccc) arginine at amino acid position 316; (ddd) arginine at amino acid position 321; and (eee) lysine at amino acid position 322. [The present invention 1038] The engineered thioesterase variants are shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59, The method of the present invention 1037. [This invention 1039] The engineered thioesterase variant has an increased surface positive charge compared to SEQ ID NO:1. The method of the present invention 1038. [The present invention 1040] The engineered thioesterase variants have increased surface positive charge compared to SEQ ID NO:4. The method of the present invention 1039. [This invention 1041] Engineered thioesterase variants are shown in SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO: The method of claim 1040, wherein the compound is a member selected from the group consisting of NO:46. [The present invention 1042] 1039. The method of claim 1039, wherein the engineered thioesterase variant has an increased surface positive charge compared to SEQ ID NO:15. [This invention 1043] 1042. The method of claim 1042, wherein the engineered thioesterase variant is a member selected from the group consisting of SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51. [This invention 1044] The method of claim 1038, wherein the engineered thioesterase variant has improved solubility. [This invention 1045] 1044. The method of claim 1044, wherein the engineered thioesterase variant has improved solubility compared to SEQ ID NO:49. [The present invention 1046] 1045. The method of claim 1045, wherein the engineered thioesterase variant comprises a truncation mutation at amino acids 2-40 of SEQ ID NO:49. [This invention 1047] 1046. The method of claim 1046, wherein the engineered thioesterase variant is a member selected from the group consisting of SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59. [This invention 1048] A composition of medium chain fatty acid derivatives having a ratio of C8 fatty acid derivatives to C10 fatty acid derivatives (C8 / C10) of at least 3.6. [This invention 1049] The composition of claim 1048, wherein the ratio of C8 fatty acid derivatives to C10 fatty acid derivatives is 7.7. [Brief explanation of the drawings]

[0041] [Figure 1] Illustrates minimum inhibitory concentration (MIC) curves for different C8 aliphatic compounds. [Figure 2] The partition coefficients (logPwo) of different medium-chain aliphatic compounds are shown. [Figure 3] Illustrating protection from 1-octanol toxicity in the presence of octyl acetate. When exposed to 1-octanol, E. coli cell viability was completely lost after 5 hours of exposure. However, when 50 g / L of octyl acetate (a non-toxic concentration for E. coli cells) was also added, the reduction in cell viability was less than 20% in the presence of up to 10 g / L of 1-octanol. [Figure 4] Illustrates a pathway for the production of medium-chain fatty alcohols and their acetylation to fatty acetates. R: CH3(CH2)n [where n = 1, 2, 3, 4, or 5]; FFA: free fatty acid; FALD: fatty aldehyde; FALC: fatty alcohol; FACE: fatty alcohol acetate; ACP: acyl carrier protein; AAR: acyl-ACP reductase; ADH: aldehyde / alcohol dehydrogenase; TE: thioesterase; ACR: acyl-CoA reductase; CAR: carboxylate reductase; AAT: o-alcohol acetyltransferase. [Figure 5A] Figure 5 illustrates different measures showing improved tolerance and production of medium-chain fatty alcohol (FALC) compounds due to expression of alcohol acetyltransferase. Figure 5A illustrates that the FALC-producing strain (sRG.674) was unable to grow on minimal salts medium with glucose as the carbon source. In contrast, there was no growth inhibition on the same medium with expression of o-alcohol acetyltransferase (AAT) in strain sJN.209. [Figure 5B]Figure 5B illustrates the levels of total fatty acids (FAS) produced by the FALC-producing strain (sRG.674) and the AAT-expressing strain sJN.209. [Figure 5C] Figure 5C illustrates different measures showing improved tolerance and production of medium-chain fatty alcohol (FALC) compounds due to expression of alcohol acetyltransferase. Figure 5C illustrates the levels and composition of lipid species produced by the FALC-producing strain (sRG.674) compared with the AAT-expressing strain sJN.209. [Figure 6] Illustrates pathways for the esterification of free fatty acids. R: CH3(CH2)n, where n = 1, 2, 3, 4, or 5; FFA: free fatty acid; FAEE: fatty acid ethyl ester; TE: thioesterase; ES: ester synthase. [Figure 7A] Figure 1 illustrates different measures of improved viability and medium-chain fatty acid derivative production by strains expressing the medium-chain alkyl ester biosynthetic pathway compared to strains expressing only the medium-chain fatty acid biosynthetic pathway. Strain sRS.786 was engineered to express a medium-chain thioesterase (chFatB2) and produces only free fatty acids (FFAs). Strain Stpay.179 is isogenic to sRS.786 and also expresses fatty acyl-CoA synthetase and ester synthase, producing medium-chain fatty alkyl esters when short-chain alcohols (e.g., methanol, ethanol, etc.) are provided in the medium. Strains sRS.786 and Stpay.179 were grown in minimal salts medium with glucose as the carbon source. Additionally, ethanol was fed during the course of the fermentation run to maintain an alcohol concentration of approximately 2 g / L. In Figure 7A, the strain producing FFAs alone (sRS.786) stopped growing and consuming glucose approximately 10 hours after addition of IPTG to induce expression of the medium-chain acyl-ACP thioesterase. In contrast, the Stpay.179 strain, which expressed the esterification pathway, was able to continue growing after IPTG induction. [Figure 7B]In Figure 7B, strain sRS.786 stopped producing medium-chain fatty acid species (FAS) approximately 10 hours after IPTG addition to induce expression of medium-chain acyl-ACP thioesterase, ultimately producing only approximately 5 g of C8 + C10. In contrast, strain Stpay.179 continued to grow and produce FAS throughout the entire fermentation run, ultimately producing over 84 g / kg of total fatty acid species. [Figure 7C] Figure 7C. The Stpay.179 strain, which expressed the esterification pathway, was able to grow and produce titers of over 84 g / kg of total fatty acid species, with 93% of the total fatty acid species being C8-C10 FFAs. [Figure 8] 1 illustrates the plasmid pIR.108. [Figure 9] 1 illustrates the structure-based sequence alignment used to build a model of SEQ ID NO:1 disclosed in Example 6. [Figure 10] Illustrates the final full-length model for the 3D structure of SEQ ID NO: 1. Surface residues are shown as balls and sticks. [Figure 11] Western blot to assess the solubility of various FatB2 truncations (1 = total cell fraction, 2 = soluble fraction). [Figure 12] 1 illustrates the characteristic end-product compositions of medium-chain-length fatty alcohol acetate esters produced by strains sRG.825 and sDH.377 when cultured under the conditions of Example 8. [Figure 13] 1 shows the characteristic end-product composition of medium-chain fatty acid ethyl esters produced by the sAZ918 strain when cultured under the conditions of Example 11. DETAILED DESCRIPTION OF THE INVENTION

[0042] Detailed Description definition Singular articles such as "a," "an," and "the" and similar referents used in this specification and the appended claims in the context of describing elements should be construed to include both the singular and the plural unless otherwise indicated herein or clearly contradicted by context. Thus, for example, reference to "a host cell" includes two or more such host cells, reference to "a nucleic acid sequence" includes one or more nucleic acid sequences, reference to "an enzyme" includes one or more enzymes, and so forth.

[0043] As used herein, "about" is understood by those of ordinary skill in the art and can vary to some extent depending on the context in which it is used. If "about" is used in a way that would not be clear to persons of ordinary skill in the art given the context in which it is used, then "about" will mean up to plus or minus 10% of the particular term.

[0044] As will be understood by one of ordinary skill in the art, for any and all purposes, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Furthermore, as will be understood by one of ordinary skill in the art, a range includes each individual member. Thus, for example, a group having 1 to 3 atoms refers to groups having 1, 2, or 3 atoms. Similarly, a group having 1 to 5 atoms refers to groups having 1, 2, 3, 4, or 5 atoms, and so on.

[0045] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In particular, this disclosure utilizes commonly used techniques in the fields of recombinant genetics, organic chemistry, fermentation science, and biochemistry. A basic text disclosing general terms in molecular biology and genetics includes, for example, Lackie, Dictionary of Cell and Molecular Biology, Elsevier (5th ed. 2013). A basic text disclosing general methods and general terms in biochemistry includes, for example, Lehninger Principles of Biochemistry Sixth Edition, David L. Nelson and Michael M. Cox eds. W. H. Freeman (2012). A basic text disclosing general methods and general terms in fermentation science includes, for example, Principles of Fermentation Technology, 3rd Edition by Peter F. Stanbury, Allan Whitaker, and Stephen J. Hall. Butterworth-Heinemann (2016). Basic texts disclosing general methods and general terminology in organic chemistry include, for example, Favre, Henri A. and Powell, Warren H. Nomenclature of Organic Chemistry. IUPAC Recommendations and Preferred Name 2013. Cambridge, UK: The Royal Society of Chemistry, 2013; Practical Synthetic Organic Chemistry: Reactions, Principles, and Techniques, Stephane Caron ed., John Wiley and Sons Inc. (2011); Organic Chemistry, 9th Edition - Francis Carey and Robert Giuliano, McGraw Hill (2013).

[0046] Throughout this specification, sequence accession numbers are obtained from databases provided by NCBI (National Center for Biotechnology Information), maintained by the United States National Institutes of Health (identified herein as "NCBI Accession Number" or alternatively as "GenBank Accession Number" or alternatively simply as "Accession Number"), and the UniProt Knowledgebase (UniProtKB) and Swiss-Prot databases provided by the Swiss Bioinformatics Institute (identified herein as "UniProtKB Accession Number").

[0047] Enzyme Classification (EC) numbers were established by the Nomenclature Commission of the International Union of Biochemistry and Molecular Biology (IUBMB), and descriptions are available on the World Wide Web at the IUBMB Enzyme Nomenclature website. EC numbers classify enzymes according to the reaction they catalyze. For example, the enzymatic activity of thioesterases is classified as EC 3.1.2.1-3.1.2.27 and 3.1.2.-. Specific classifications are based on the activity of different thioesterases on different substrates.

[0048] For example, in some exemplary embodiments, thioesterases that catalyze the hydrolysis of the thioester bond of C6-C18 alkyl thioesters, such as acyl-acyl carrier protein thioesters (acyl-ACP) and acyl-Coenzyme A thioesters (acyl-CoA), are classified in EC 3.1.2.- to 3.1.2.14. Thioesterases are present in most prokaryotes and in the chloroplasts of most plants and algae. Thioesterase functionality is conserved across species in most prokaryotes. Thus, different microbial species can exhibit the same thioesterase enzyme activity as those classified in EC 3.1.2.1-3.1.2.27 and 3.1.2.-.

[0049] As used herein, the term "fatty acid" refers to an aliphatic carboxylic acid having the formula RCOOH, where R is an aliphatic group having at least four carbon atoms, typically from about 4 to about 28 carbon atoms. The aliphatic R group can be saturated or unsaturated, branched or unbranched. Unsaturated "fatty acids" can be monounsaturated or polyunsaturated.

[0050] As used herein, one or more " fatty acids " can be produced in cells or supplied to cells through fatty acid biosynthesis process or through reverse fatty acid beta-oxidation.As is well known in the art, fatty acid biosynthesis is generally the malonyl-CoA-dependent synthesis of acyl-ACP, while reverse beta-oxidation results in acyl-CoA.The fatty acid supplied to cells is converted into acyl-CoA.

[0051] Fatty acid biosynthesis and degradation occur in all life forms, including prokaryotes, unicellular eukaryotes, higher eukaryotes, and archaea. The tools and methods disclosed herein are useful for producing medium-chain fatty acid derivatives derived from any one or more of fatty acid synthesis, degradation, or supply in any organism that naturally produces alkyl thioesters.

[0052] As used herein, the term "medium chain fatty acid" or, by analogy, "medium chain length fatty acid" refers to a fatty acid having a carbon chain length of 6 to 10. Thus, in some exemplary embodiments, a "medium chain fatty acid" is a fatty acid having a carbon chain length of 6 carbons, 7 carbons, 8 carbons, 9 carbons, or 10 carbons.

[0053] The term "fatty acid derivative" as used herein refers to a product produced by derivatization of a fatty acid. Thus, "fatty acid derivative" includes "fatty acid" and "medium-chain fatty acid" as defined above. Generally, "fatty acid derivative" includes malonyl-CoA-derived compounds, including acyl-ACP or acyl-ACP derivatives. "Fatty acid derivative" also includes malonyl-CoA-derived compounds, such as acyl-CoA or acyl-CoA derivatives. Thus, "fatty acid derivative" includes molecules / compounds obtained from metabolic pathways that include a thioesterase reaction. Exemplary fatty acid derivatives include fatty acids, fatty acid esters (e.g., waxes, fatty acid esters, fatty acid methyl esters (FAMEs), fatty acid ethyl esters (FAEEs)), fatty alcohol acetates (FACEs), fatty amines, fatty aldehydes, fatty alcohols, hydrocarbons such as alkanes, alkenes, etc., ketones, terminal olefins, internal olefins, 3-hydroxy fatty acid derivatives, difunctional fatty acid derivatives (e.g., ω-hydroxy fatty acids, 1,3 fatty diols, α,ω-diols, α,ω-3-hydroxytriols, ω-hydroxy FAMEs, ω-OH FAEEs, etc.), and unsaturated fatty acid derivatives containing the unsaturated compounds of each of the above fatty acid derivatives.

[0054] As used herein, the phrase "fatty acid derivative composition" refers to a composition of fatty acid derivatives, e.g., a fatty acid composition produced by an organism. A "fatty acid derivative composition" may include a single fatty acid derivative species or a mixture of fatty acid derivative species. In some exemplary embodiments, the fatty acid derivative mixture includes more than one type of fatty acid derivative product (e.g., fatty acids, fatty acid esters, fatty alcohols, fatty alcohol acetate esters, fatty aldehydes, fatty amines, difunctional fatty acid derivatives, etc.). In other exemplary embodiments, the fatty acid derivative mixture includes a mixture of fatty acid esters (or other fatty acid derivatives) having different chain lengths, degrees of saturation, and / or branching characteristics. In other exemplary embodiments, the fatty acid derivative mixture primarily includes one type of fatty acid derivative, e.g., a medium-chain fatty acid derivative composition. In yet other exemplary embodiments, the fatty acid derivative mixture includes more than one type of fatty acid derivative product, e.g., a mixture of fatty acid derivatives having different chain lengths, degrees of saturation, and / or branching characteristics. In still other exemplary embodiments, the fatty acid derivative mixture includes a mixture of fatty esters and beta-hydroxy esters. In yet other exemplary embodiments, the fatty acid derivative composition comprises a mixture of fatty alcohols and fatty aldehydes. In yet other exemplary embodiments, the fatty acid derivative composition comprises a mixture of FAMEs and / or FAEEs, particularly a mixture of medium chain FAMEs and / or FAEEs. In yet other exemplary embodiments, the fatty acid derivative composition comprises a mixture of fatty alcohol acetate esters (FACEs), particularly a mixture of medium chain fatty alcohol acetate esters (FACEs).

[0055] The term "nucleotide" used herein has the conventional meaning known in the art. In addition to representing natural ribonucleotide or deoxyribonucleotide monomer, the term "nucleotide" also encompasses nucleotide analogs and modified nucleotides such as amino-modified nucleotides. In addition, "nucleotide" includes non-natural analog structures. Thus, for example, the individual units of peptide nucleic acid, each containing a base, may be referred to herein as nucleotides.

[0056] The term "polynucleotide" refers to a polymer of ribonucleotides (RNA) or deoxyribonucleotides (DNA), typically linked by phosphodiester bonds, which can be single- or double-stranded and may contain natural and / or non-natural and / or modified 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 single-stranded, double-stranded, triple-stranded, quadruple-stranded, partially double-stranded, branched, hairpinned, circular, padlocked, and other polynucleotides. These terms include, by equivalents, but are not limited to, nucleotide analogs and analogs of either RNA or DNA made from modified polynucleotides, such as methylated and / or capped polynucleotides. The polynucleotides may be in any form, including but not limited to, plasmid, viral, chromosomal, EST, cDNA, mRNA, and rRNA, and may be prepared by any known method, including synthetic, recombinant, ex vivo generated, or a combination thereof, and using any purification method known in the art.

[0057] As used herein, the terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues that are typically 12 or more amino acids in length. Polypeptides less than 12 amino acids in length are referred to herein as "peptides." The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of corresponding naturally occurring amino acids, as well as to naturally occurring and unnatural amino acid polymers. The term "recombinant polypeptide" generally refers to a polypeptide produced by recombinant techniques, in which DNA or RNA encoding the protein to be expressed is inserted into an appropriate expression vector, which in turn is used to transform a host cell to produce the polypeptide. In some exemplary embodiments, DNA or RNA encoding the expressed peptide, polypeptide, or protein is inserted into a host chromosome by homologous recombination or other means well known in the art, and then used to transform a host cell to produce the peptide or polypeptide. Similarly, the terms "recombinant polynucleotide" or "recombinant nucleic acid" or "recombinant DNA" refer to polypeptides produced by recombinant techniques well known to those of skill in the art (e.g., Sambrook et al., Molecular Cloning--A Laboratory Manual, Cold Spring Harbor Press, 2004). th Edition (Cold Spring Harbor, NY 2012) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998) and Supplements 1-115 (1987-2016).

[0058] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) as well as pyrrolysine and selenocysteine. In some exemplary embodiments, the single-letter codes shown in the table below are used to represent specific members of the 20 common naturally occurring amino acids. Single-letter amino acid codes are well known in the art (see, e.g., Lehninger, supra). TIFF0007727616000001.tif56128

[0059] When referring to two nucleotide or polypeptide sequences, the "percentage of sequence identity" between the two sequences is determined by comparing two optimally aligned sequences over a comparison window, where, due to optimal alignment of the two sequences, the portion of the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions). The "percentage of sequence identity" is calculated by determining the number of positions where the same nucleic acid base or amino acid residue is present in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity.

[0060] Thus, the terms "percent identity," or, by analogy, "percent sequence identity," in the context of two or more nucleic acid sequences or peptides or polypeptides, refer to the percent identity as determined, for example, using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters (see, e.g., Altschul et al. (1990) J. Mol. Biol. 215(3):403-410) and / or the NCBI website at ncbi.nlm.nih.gov / BLAST / ) or by manual alignment and visual inspection, represent two or more sequences or subsequences that are the same or have a specified percentage of the same nucleotides or amino acids (e.g., about 50% identity, preferably 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared over a comparison window or designated region and aligned for maximum correspondence). The percent sequence identity between two nucleic acid or amino acid sequences can also be determined using, for example, the Needleman and Wunsch algorithm 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 (1970) J. Mol. Biol. 48:444-453). The percent sequence identity between two nucleotide sequences can also 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 skill in the art can perform initial sequence identity calculations and adjust the algorithm parameters accordingly.If the practitioner is unsure which parameters to apply to determine whether a molecule is within the claimed homology limits, one set of parameters that can be used is the Blossum 62 scoring matrix, with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. Additional methods of sequence alignment are known in the art of biotechnology (see, e.g., Rosenberg (2005) BMC Bioinformatics 6:278; Altschul et al. (2005) FEBS J. 272(20):5101-5109).

[0061] Two or more nucleic acid or amino acid sequences are said to be "substantially identical" when aligned and analyzed as described above and found to share about 50% identity over a specified region, preferably 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. Two nucleic acid or polypeptide sequences are said to be "identical" if the sequence of nucleotides or amino acid residues in the two sequences, respectively, is the same when aligned for maximum correspondence as described above. This definition can also refer to or apply to the complement of a test sequence. Identity is typically calculated over a region that is at least about 25 amino acids or nucleotides in length or longer, more preferably over a region that is 50-100 amino acids or nucleotides in length, or over the entire length of a given sequence.

[0062] The phrase "hybridizing under low stringency, medium stringency, high stringency, or very high stringency conditions" describes the conditions for hybridization and washing. Guidance for performing hybridization reactions can be found, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. Aqueous and non-aqueous methods are described in the cited references, 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 at least 50° C. (for low stringency conditions, the wash temperature can be increased to 55° C.); (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—0.5 M sodium phosphate, 7% SDS at 65° C., followed by 0.2× SSC, 1% SDS at 65° C. One or more washes in SDS. Unless otherwise stated, very high stringency conditions (4) are the preferred conditions.

[0063] As used herein, the term "endogenous" refers to a substance, e.g., a nucleic acid, a protein, etc., that is produced from within a cell. Thus, an "endogenous" polynucleotide or polypeptide refers to a polynucleotide or polypeptide that is produced by a cell. In some exemplary embodiments, an "endogenous" polypeptide or polynucleotide is encoded by the genome of a parent cell (or host cell). In other exemplary embodiments, an "endogenous" polypeptide or polynucleotide is encoded by an autonomously replicating plasmid harbored by a parent cell (or host cell). In some exemplary embodiments, an "endogenous" gene is a gene that was present in the cell when the cell was originally isolated from nature, i.e., the gene is "native to the cell." In other exemplary embodiments, an "endogenous" gene has been modified by recombinant techniques, e.g., by altering the relationship of regulatory and coding sequences. Thus, a "heterologous" gene can be "endogenous" to a host cell in some exemplary embodiments.

[0064] In contrast, an "exogenous" polynucleotide or polypeptide, or other substance (e.g., a fatty acid derivative, a small molecule compound, etc.), refers to a polynucleotide or polypeptide or other substance that is not produced by the parent cell and, as a result, is added extracellularly to a cell, cell culture, or assay.

[0065] As used herein, the term "native" refers to a form of a nucleic acid, protein, polypeptide, or fragment thereof that is isolated from nature, or that does not contain intentionally introduced mutations.

[0066] 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 two amino acid residues to the entire amino acid sequence minus one amino acid residue. In certain aspects of the disclosure, the fragment represents the entire amino acid sequence of a domain (e.g., a substrate-binding domain or a catalytic domain) of a polypeptide or protein.

[0067] The term "mutagenesis" refers to a process in which the genetic information of an organism is stably altered to produce "mutants" or "variants." Mutagenesis of a protein-coding nucleic acid sequence to produce a mutant nucleic acid sequence produces a mutant protein. Mutagenesis also refers to the alteration of a non-coding nucleic acid sequence. In some exemplary embodiments, mutations in the non-coding nucleic acid sequence result in altered protein activity.

[0068] Thus, as used herein, a "mutation" refers to a permanent change in a nucleic acid position of a gene or an amino acid position (residue) of a polypeptide or protein. Indeed, the term "mutation," in the context of a polynucleotide, refers to a modification to a polynucleotide sequence that results in a change in the polynucleotide sequence relative to a control or reference polynucleotide sequence. In some exemplary embodiments, a variant polynucleotide sequence represents an alteration, e.g., related to codon optimization for expression purposes, that does not change the encoded amino acid sequence. In other exemplary embodiments, a mutation in a polynucleotide sequence modifies a codon to result in a modification of the encoded amino acid sequence. Thus, a polynucleotide encoding an engineered thioesterase variant with improved ability to produce medium-chain fatty acid derivatives will have at least one mutation compared to a polynucleotide encoding a reference thioesterase.

[0069] Similarly, in the context of proteins, the term "mutation" or "mutated" refers to a modification to an amino acid sequence that results in a change in the protein sequence relative to a control or reference protein sequence. A mutation can refer to the substitution of one amino acid with another amino acid, or the insertion or deletion of one or more amino acid residues. In some exemplary embodiments, a "mutation" is the substitution of an amino acid with a non-natural amino acid or a chemically modified amino acid residue. In other exemplary embodiments, a "mutation" is the shortening (e.g., deletion or interruption) of a sequence or subsequence relative to a precursor sequence, or a sequence truncation by deletion from one or another end. In other exemplary embodiments, a mutation is the addition of amino acids or subsequences within the protein or at either end of the protein (e.g., two or more amino acids in a stretch inserted between two adjacent amino acids in a precursor protein sequence), thereby increasing (or extending) the length of the protein. Mutations can be introduced into a polynucleotide by numerous methods known to those of skill in the art, including, for example, random mutagenesis, site-specific mutagenesis, oligonucleotide-directed mutagenesis, gene shuffling, directed evolution techniques, combinatorial mutagenesis, chemical synthesis, site-saturation mutagenesis, and the like.

[0070] As used herein, the term "mutant" or, equivalently, "variant" refers to a polynucleotide or polypeptide sequence that contains at least one mutation. Thus, an engineered thioesterase variant with improved ability to produce medium-chain fatty acid derivatives will have at least one mutation in its polypeptide sequence compared to a reference thioesterase.

[0071] As used herein, the term "engineered thioesterase variant" refers to a mutant or variant thioesterase having at least one mutation compared to SEQ ID NO:1, wherein the thioesterase variant has improved activity for the production of medium-chain fatty acid derivatives.

[0072] As used herein, the term "gene" refers to a nucleic acid sequence, e.g., a DNA sequence, that encodes either an RNA or protein product, and operably linked nucleic acid sequences (e.g., expression control sequences, e.g., promoters, enhancers, ribosomal binding sites, translation control sequences, etc.) that affect the expression of the RNA or protein product. The term "gene product" refers to any RNA, e.g., tRNA, mRNA, and / or protein, expressed from a particular gene.

[0073] The terms "expression" or "expressed," as used herein with respect to a gene, refer to the production of one or more transcription and / or translation products of the gene. In exemplary embodiments, the level of expression of a DNA molecule in a cell is determined based on either the amount of corresponding mRNA present in the cell or the amount of protein encoded by the DNA produced by the cell. The term "expressed gene" refers to genes that are transcribed into messenger RNA (mRNA) and then translated into protein, as well as genes that are transcribed into other types of RNA, such as transfer RNA (tRNA), ribosomal RNA (rRNA), and regulatory RNA, which are not translated into protein.

[0074] The expression level of a nucleic acid molecule in a cell system or a cell-free system is influenced by an "expression control sequence" or, by the same token, a "regulatory sequence." "Expression control sequence" or "regulatory sequence" are known in the art and include, for example, promoters, enhancers, polyadenylation signals, transcription terminators, nucleotide sequences that affect RNA stability, internal ribosome entry sites (IRES), and the like, that provide for the expression of a polynucleotide sequence in a host cell. In exemplary embodiments, an "expression control sequence" specifically interacts with a cellular protein involved in transcription (see, e.g., Maniatis et al., Science, 236: 1237-1245 (1987); Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990)). In exemplary methods, an expression control sequence is operably linked to a polynucleotide sequence. By "operably linked" is meant that the polynucleotide sequence and expression control sequence are operably connected to permit expression of the polynucleotide sequence when an appropriate molecule (e.g., a transcriptional activator protein) is contacted with the expression control sequence. In exemplary embodiments, an operably linked promoter is located upstream of the selected polynucleotide sequence in terms of the direction of transcription and translation. In some exemplary embodiments, an operably linked enhancer can be located upstream, within, or downstream of the selected polynucleotide sequence.

[0075] Generally, the "minimum inhibitory concentration" (MIC) is the lowest concentration of an antimicrobial agent that inhibits visible growth of a microorganism after overnight incubation. MICs can be determined by solid growth medium plates or by the broth dilution method. For example, to identify the MIC by broth dilution, equal amounts of bacteria are cultured in wells of liquid medium containing decreasing concentrations of the drug. The minimum inhibitory concentration of an antibiotic lies between the concentration in the final well at which bacteria do not grow and the next lower amount at which bacteria do grow. As used herein, the phrase "minimum inhibitory concentration" or "MIC" refers to the concentration of a compound that results in a 50% reduction in the growth of a microbial culture during a 24-hour incubation period compared to a control. In one embodiment, the "minimum inhibitory concentration" of a potentially toxic compound, e.g., octanol, is measured by growing a culture of cells, e.g., E. coli cells, in various concentrations of the potentially toxic compound and then determining how much growth of the culture occurs in the presence of the potentially toxic compound over a 24-hour period. In an exemplary embodiment, the growth of the culture is measured by measuring total protein from the lysed culture after 24 hours of growth, as a measure of the total cell number in the culture.

[0076] As used herein, a "modified activity" or "altered level of activity" of a protein / polypeptide, e.g., an engineered thioesterase variant, refers to a difference in one or more characteristics of the activity of the protein / polypeptide compared to the characteristics of an appropriate control protein, e.g., a corresponding parent protein or a corresponding wild-type protein. Thus, in exemplary embodiments, the difference in activity of a protein having a "modified activity" compared to a corresponding control protein is determined by measuring the activity of the modified protein in a recombinant host cell and comparing it to a measure of the same activity of the corresponding control protein in an otherwise isogenic host cell. Modified activity can be the result of, for example, a structural change in the protein (e.g., a change in primary structure, such as an alteration to the nucleotide coding sequence of the protein, resulting in, e.g., altered substrate specificity, altered observed kinetic parameters, altered solubility, etc.); an altered stability of the protein (e.g., increased or decreased proteolysis), etc. In some exemplary embodiments, a polypeptide having a "modified activity" is a mutant or engineered TE variant disclosed herein.

[0077] In exemplary embodiments, the polypeptides disclosed herein have a "modified activity," e.g., an "improved level of activity." As used herein, the phrase "improved level of activity" refers to a polypeptide having a higher level of biochemical or biological function (e.g., DNA binding activity or enzymatic activity) compared to the level of biochemical and / or biological function of a corresponding control polypeptide under the same conditions. The degree of improved activity can be about 10% or more, about 20% or more, about 50% or more, about 75% or more, about 100% or more, about 200% or more, about 500% or more, about 1000% or more, or any range therein.

[0078] Thus, "improved activity" can refer to improved catalytic activity or improved catalytic efficiency of a polypeptide, where catalytic efficiency refers to, for example, an increase in the reaction rate of a reaction catalyzed by such an enzyme of the polypeptide. Catalytic activity / catalytic efficiency can be improved, for example, by improving one or more rate parameters (measures or calculations) of the reaction, such as V (the maximum rate at which the reaction can proceed), K (Michaelis constant), k (the number of substrate molecules turned over per second per molecule of enzyme), or any ratio between these parameters, such as k / K (a measure of enzyme efficiency). Thus, "improved catalytic activity" or "improved catalytic efficiency" of a polypeptide can be measured in a number of ways. For example, "improved activity" may be measured as an increase in titer (concentration: g / L, or mg / L, or g / Kg), a change in composition (amount of specific fatty acid species / total fatty acid derivatives (FAS) produced), an improved ratio of molecular components (e.g., C8 / C10 content or C10 / C12 content), or an increase in FOC (fold over control, see below) of the product produced by a recombinant cell expressing an enzyme with improved activity.

[0079] Thus, as used herein, the phrases "having improved activity for the production of medium-chain fatty acid derivatives" or "having improved activity for the production of medium-chain length fatty acid derivative compounds" or "having improved activity for the production of medium-chain aliphatic compounds" or "having improved ability to produce medium-chain length fatty acid derivatives" or "having improved ability to produce medium-chain fatty acid derivatives" refer to an "improved activity", e.g., "improved catalytic activity", of a polypeptide / protein that leads to increased production of medium-chain fatty acid derivative species (fatty acids and fatty acid derivatives having alkyl chains of 6 to 10 carbons in length) when compared under the same conditions with a suitable control polypeptide / protein.

[0080] In some exemplary embodiments, a polypeptide / protein "having improved activity for the production of medium-chain fatty acid derivatives" or, by analogy, "having improved ability to produce medium-chain fatty acid derivatives" has improved activity for the production of medium-chain fatty acid derivatives of a particular chain length. Thus, for example, the phrase "having improved activity for the production of C8 fatty acid derivatives" as used herein refers to a polypeptide / protein with "improved catalytic activity" or "improved activity" that leads to increased production of 8-carbon fatty acid derivatives (e.g., as measured as % C8 FAS, increased C8 / C10 ratio, etc.).

[0081] Similarly, in some exemplary embodiments, a polypeptide / protein that "has improved activity for the production of medium-chain fatty acid derivatives" or, by analogy, "has improved ability to produce medium-chain fatty acid derivatives" has "improved activity for the production of C10 fatty acid derivatives." Thus, such a polypeptide / protein has "improved activity" that leads to increased production of 10-carbon fatty acid derivatives (e.g., as measured as % C10 FAS, increased C10 / C12 ratio, etc.).

[0082] As used herein, the phrase "fold over control," or by analogy, "FOC," refers to the ratio of a particular metric measured in cells containing an engineered thioesterase variant to the same metric measured in an appropriate control cell, e.g., an isogenic host cell containing a control thioesterase that does not have the engineered mutation. Thus, generally, FOC equals metric A of variant / metric A of control. (In some exemplary embodiments, FOC of %C8 refers to the %C8 produced by cells containing an engineered thioesterase variant compared to the %C8 of an appropriate control, e.g., an isogenic control containing a thioesterase that has not been engineered to contain a particular mutation.) Thus, in exemplary embodiments, recombinant cells containing an engineered thioesterase variant with an FOC of %C8 of 1.1 exhibit a 10% improvement (increase) in the percent of 8-carbon fatty acid derivatives produced by cells containing the engineered thioesterase variant compared to the %C8 of an isogenic control containing the control thioesterase.

[0083] A "control" sample, e.g., a "control" nucleotide sequence, a "control" polypeptide sequence, a "control" cell, etc., or value, refers to a sample that serves as a reference, typically a known reference, for comparison with a test sample. For example, in an exemplary embodiment, the test sample comprises a fatty acid derivative composition made by an engineered thioesterase variant, while the control sample comprises a fatty acid derivative composition made by a corresponding or designated unmodified / unvariant thioesterase (e.g., SEQ ID NO:1). Practitioners will recognize that controls can be designed for evaluation of any number of parameters. Furthermore, those skilled in the art will understand which controls are valuable in a given situation and how data can be analyzed based on comparison to the control value.

[0084] As used herein, the term "recombinant" refers to a genetically modified polynucleotide, polypeptide, cell, tissue, or organism. The term "recombinant" applies equally to the first generation genetically modified polynucleotide, polypeptide, cell, tissue, or organism, and to progeny of the genetically modified polynucleotide, polypeptide, cell, tissue, or organism bearing the genetic modification.

[0085] The term "recombinant" when used with respect to a cell indicates that the cell has been modified by the introduction of a heterologous nucleic acid or protein, or by alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified and the derived cell contains the modification. Thus, for example, a "recombinant cell" or, by analogy, a "recombinant host cell" can be modified to express a gene not found in the native (non-recombinant) form of the cell, or to aberrantly express a native gene, e.g., the native gene can be overexpressed, underexpressed, or not expressed at all. In exemplary embodiments, a "recombinant cell" or "recombinant host cell" has been engineered to express a heterologous thioesterase, such as an engineered thioesterase variant with improved activity for the production of medium-chain fatty acid derivatives. Recombinant cells can be derived from microorganisms such as bacteria, viruses, or fungi. Additionally, recombinant cells can be derived from plant or animal cells. In exemplary embodiments, a "recombinant host cell" or "recombinant cell" is used to produce one or more fatty acid derivatives, including, but not limited to, fatty acids, fatty esters (e.g., waxes, fatty acid esters, fatty esters, fatty acid methyl esters (FAMEs), fatty acid ethyl esters (FAEEs)), fatty alcohol acetates (FAces), fatty alcohols, fatty aldehydes, hydrocarbons, fatty amines, terminal olefins, internal olefins, ketones, difunctional fatty acid derivatives (e.g., omega-hydroxy fatty acids, omega-hydroxy diols, omega-hydroxy FAMEs, omega-hydroxy FAEEs), and the like. Thus, in some exemplary embodiments, a "recombinant host cell" is a "production host" or, equivalently, a "production host cell." In some exemplary embodiments, the recombinant cell comprises one or more polynucleotides, each polynucleotide encoding a polypeptide having fatty acid biosynthetic enzyme activity, wherein the recombinant cell produces a fatty acid derivative composition when cultured in the presence of a carbon source under conditions effective for expression of the polynucleotides.

[0086] The term "recombinant," or by analogy, "heterologous," when used in reference to a polynucleotide, indicates that the polynucleotide has been modified relative to a native or naturally occurring form of the polynucleotide, or relative to a naturally occurring variant polynucleotide. In exemplary embodiments, a recombinant polynucleotide (or a copy or complement of a recombinant polynucleotide) is a polynucleotide that has been manipulated by the hand of man to differ from its naturally occurring form. Thus, in exemplary embodiments, a recombinant polynucleotide is a mutant form of a native gene or a mutant form of a naturally occurring variant of a native gene, where the mutations are created by deliberate human manipulation, such as by saturation mutagenesis using mutagenic oligonucleotides, UV radiation, or the use of mutagenic chemicals. Such recombinant polynucleotides may contain one or more point mutations, deletions, and / or insertions relative to the native or naturally occurring variant form of the gene. Similarly, a polynucleotide comprising a promoter operably linked to a second polynucleotide (e.g., a coding sequence) is a "recombinant" polynucleotide. Thus, recombinant polynucleotides include combinations of polynucleotides not found in nature. Recombinant proteins (supra) are typically those expressed from recombinant polynucleotides, and recombinant cells, tissues, and organisms are those that contain recombinant sequences (polynucleotides and / or polypeptides).

[0087] As used herein, the term "microorganism" generally refers to a microscopic organism. Microorganisms can be prokaryotic or eukaryotic. Exemplary prokaryotic microorganisms include, for example, bacteria, archaea, cyanobacteria, etc. An exemplary bacterium is Escherichia coli. Exemplary eukaryotic microorganisms include, for example, yeast, protozoa, algae, etc. In exemplary embodiments, a "recombinant microorganism" is a microorganism that has been genetically modified to thereby express or include heterologous nucleic acid sequences and / or heterologous proteins.

[0088] A "production host" or, equivalently, a "production host cell" is a cell used to produce a product. A "production host" disclosed herein is typically modified to express or overexpress a selected gene or to have attenuated expression of a selected gene. Thus, a "production host" or "production host cell" is a "recombinant host" or, equivalently, a "recombinant host cell." Non-limiting examples of production hosts include plant, animal, human, bacterial, yeast, cyanobacterial, algae, and / or filamentous fungal cells. An exemplary "production host" is a recombinant E. coli cell.

[0089] As used herein, "acyl-ACP" refers to an acyl thioester formed between the carbonyl carbon of an acyl chain and the sulfhydryl group of a phosphopantetheinyl moiety of an acyl carrier protein (ACP). In some exemplary embodiments, the acyl-ACP is a synthetic intermediate of a fully saturated acyl-ACP. In other exemplary embodiments, the acyl-ACP is a synthetic intermediate of an unsaturated acyl-ACP. In some exemplary embodiments, the carbon chain of the acyl group of the acyl-ACP has 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 carbons. In other exemplary embodiments, the carbon chain of the acyl group of the acyl-ACP is a medium-length chain and has 6, 7, 8, 9, 10, 11, or 12 carbons. In other exemplary embodiments, the carbon chain of the acyl group of the acyl-ACP is 8 carbons in length. In yet other exemplary embodiments, the carbon chain of the acyl group of the acyl-ACP is 10 carbons in length. Each of these acyl-ACPs is a substrate for an enzyme, such as, for example, a thioesterase, e.g., an engineered thioesterase variant, that converts the acyl-ACP into a fatty acid derivative.

[0090] As used herein, the phrase "fatty acid derivative biosynthetic pathway" refers to a biochemical pathway that produces fatty acid derivatives. Enzymes comprising a "fatty acid derivative biosynthetic pathway" are therefore referred to herein as "fatty acid derivative biosynthetic polypeptides" or, equivalently, "fatty acid derivative enzymes." As noted above, the term "fatty acid derivative" includes molecules / compounds derived from a biochemical pathway that includes a thioesterase reaction. Thus, a thioesterase enzyme (e.g., an enzyme having thioesterase activity EC 3.1.1.14) is a "fatty acid derivative biosynthetic peptide" or, equivalently, a "fatty acid derivative enzyme." In addition to thioesterases, a fatty acid derivative biosynthetic pathway may include additional enzymes for producing fatty acid derivatives with desired characteristics. Thus, the terms "fatty acid derivative enzymes" or, equivalently, "fatty acid derivative biosynthetic polypeptides" refer to enzymes that, collectively and individually, can be expressed or overexpressed to produce fatty acid derivatives. Non-limiting examples of "fatty acid derivative enzymes" or, similarly, "fatty acid derivative biosynthetic polypeptides" include, for example, fatty acid synthetases, thioesterases, acyl-CoA synthetases, acyl-CoA reductases, acyl-ACP reductases, alcohol dehydrogenases, alcohol O-acyltransferases, fatty alcohol-forming acyl-CoA reductases, fatty acid decarboxylases, fatty aldehyde decarbonylases and / or oxidative deformylases, carboxylic acid reductases, fatty alcohol O-acetyltransferases, ester synthases, and the like. A "fatty acid derivative enzyme" or, similarly, a "fatty acid derivative biosynthetic polypeptide" converts a substrate into a fatty acid derivative. In exemplary embodiments, a suitable substrate for a fatty acid derivative enzyme can be a first fatty acid derivative that is converted by the fatty acid derivative enzyme to a different second fatty acid derivative.

[0091] As used herein, the term "culture" refers to a liquid medium containing viable cells. In one embodiment, a culture includes cells growing under controlled conditions in a predetermined medium, e.g., a recombinant host cell culture grown in a liquid medium containing a selected carbon source and nitrogen. "Culturing" or "culturing" refers to growing a population of host cells (e.g., recombinant host cells) in liquid or solid medium under appropriate conditions. In certain embodiments, culturing refers to the bioconversion of a substrate to an end-product. Culture media are well known, and the individual components of such culture media are available from commercial sources, e.g., Difco™ medium and BBL™ medium. In one non-limiting example, a liquid nutrient medium is a "rich medium" containing a complex nitrogen, salt, and carbon source, such as YP medium containing 10 g / L peptone and 10 g / L yeast extract.

[0092] The term "titer" as used herein refers to the amount of fatty acid derivative, e.g., medium-chain fatty acid derivative, produced per unit volume of host cell culture. Titer can refer to the amount of a particular fatty acid derivative, e.g., medium-chain fatty acid derivative, or a combination of fatty acid derivatives of different chain lengths or different functionalities, such as, for example, a mixture of saturated and unsaturated medium-chain fatty acid derivatives or a fatty acid derivative composition, produced by a given recombinant host cell culture.

[0093] As used herein, the phrase "commercial titer" or "commercial titers" refers to the amount of fatty acid derivatives, e.g., medium-chain fatty acid derivatives, produced per unit volume of host cell culture that makes commercial production economically viable. Typically, commercial titers are in the range of about 10 g / L (or equivalently, 10 g / Kg) to about 200 g / L or more. Thus, commercial potencies are ≧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, ≧110 g / L, ≧120 g / L, ≧130 g / L, ≧140 g / L, ≧150 g / L, ≧160 g / L, ≧170 g / L, ≧180 g / L, ≧190 g / L, ≧200 g / L.

[0094] As used herein, the "fatty acid derivative yield" produced by a "host cell," e.g., the yield of a medium-chain fatty acid derivative or other compound, refers to the efficiency with which an input carbon source is converted into a product (i.e., a medium-chain fatty acid derivative) in the host cell. Thus, the expression "fatty acid derivative yield" refers to the amount of product produced from a given amount of carbon substrate. The percent yield is the percent of the theoretical yield (the product synthesized under ideal conditions without carbon or energy loss). Thus, percent yield = (mass of product / mass of theoretical yield) × 100. The yield may refer to a specific medium-chain fatty acid derivative or a combination of fatty acid derivatives.

[0095] As used herein, the term "productivity" refers to the amount of medium-chain fatty acid derivatives, e.g., 6-carbon fatty acid derivatives, 8-carbon fatty acid derivatives, 10-carbon fatty acid derivatives, etc., produced per unit volume of host cell culture per unit time. Productivity can refer to the specific 8- and / or 10-carbon fatty acid derivatives, or combinations of fatty acid derivatives or other compounds, produced by a given host cell culture. Thus, in exemplary embodiments, expression of an engineered thioesterase variant in a recombinant host cell, e.g., E. coli, results in increased productivity of 8- and / or 10-carbon fatty acid derivatives and / or other compounds compared to a recombinant host cell expressing a corresponding control thioesterase or other suitable control. As used herein, the terms "total fatty species," "total fatty acid product," and "total fatty acid derivatives" may be used interchangeably herein in reference to the amount (titer) of fatty acid derivatives produced by a host cell, e.g., a host cell expressing an engineered thioesterase variant. Total fatty species, etc., can be assessed by gas chromatography with a flame ionization detector (GC-FID). When referring to total fatty acid derivative analysis, the same term may be used to refer to, for example, total fatty esters, total fatty alcohols, total fatty aldehydes, total fatty amines, and total free fatty acids. In particular, the same term may be used to refer to total fatty acid methyl esters, fatty acid ethyl esters, or fatty alcohol acetate esters.

[0096] The term "carbon source" as used herein refers to a substrate or compound suitable for use as a carbon source for the growth of prokaryotic or simple eukaryotic cells. Carbon sources can be in a variety of forms, including, but not limited to, polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides, and gases (e.g., CO and CO). Exemplary carbon sources include, but are not limited to, monosaccharides such as glucose, fructose, mannose, galactose, xylose, and arabinose; oligosaccharides such as fructooligosaccharides and galactooligosaccharides; polysaccharides such as starch, cellulose, pectin, and xylan; disaccharides such as sucrose, maltose, cellobiose, and turanose; cellulosic materials and variants such as hemicellulose, methylcellulose, and sodium carboxymethylcellulose; saturated or unsaturated fatty acids, succinates, lactates, and acetates; alcohols such as ethanol, methanol, and glycerol, or mixtures thereof. The carbon source can also be a photosynthetic product such as glucose. In certain embodiments, the carbon source is biomass. In other aspects, the carbon source is glucose. In other aspects, the carbon source is sucrose. In other aspects, the carbon source is glycerol. In other aspects, the carbon source is a simple carbon source. In other aspects, the carbon source is a renewable carbon source. In other examples, the carbon source is natural gas or a natural gas component such as methane, ethane, or propane.

[0097] As used herein, the term "biomass" refers to any biological material from which a carbon source is derived. In some aspects, the biomass is processed into a carbon source suitable for bioconversion. In other aspects, the biomass does not require further processing into a carbon source. The carbon source can be converted into a composition comprising medium-chain fatty acid derivatives.

[0098] An exemplary source of biomass is plant material or vegetation, such as plant material or vegetation derived from corn, sugarcane, switchgrass, rice, wheat, hardwoods, softwoods, palms, hemp, etc. Another exemplary source of biomass is metabolic waste, such as animal material (e.g., cow manure). Further exemplary sources of biomass include algae and marine plants, such as macroalgae and kelp. Biomass also includes industrial, agricultural, forestry, and household waste, including, but not limited to, glycerol, fermentation waste, ensilage, straw, wood, pulp, sewage, garbage, cellulose waste, municipal solid waste, oleochemical waste, and food waste (e.g., soap, oil, and fatty acids). The term "biomass" can also refer to a carbon source, such as a carbohydrate (e.g., monosaccharide, disaccharide, or polysaccharide).

[0099] The term "isolated" as used herein with respect to a product (such as a medium-chain fatty acid derivative) refers to a product that is separated from cellular components, cell culture medium, or chemical or synthetic precursors. The medium-chain fatty acid derivatives produced by the methods disclosed herein can be relatively immiscible in the fermentation broth and cytoplasm. Thus, in exemplary embodiments, the medium-chain fatty acid derivatives collect extracellularly in an organic phase, thereby being "isolated."

[0100] As used herein, the terms "purify," "purified," or "purification" refer to the removal or isolation of a molecule from its environment, e.g., by isolation or separation. A "substantially purified" molecule is at least about 60% free (e.g., at least about 65% free, at least about 70% free, at least about 75% free, at least about 80% free, at least about 85% free, at least about 90% free, at least about 95% free, at least about 96% free, at least about 97% free, at least about 98% free, at least about 99% free) from other components with which it is 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 medium-chain fatty acid derivatives or other compounds in a sample. For example, when medium-chain fatty acid derivatives or other compounds are produced in recombinant host cells, the medium-chain fatty acid derivatives or other compounds can be purified by removing the host cell biomass or components thereof, such as proteins, nucleic acids, and other cellular components, once the host cells are lysed. After purification, the percentage of malonyl-CoA-derived compounds, including medium-chain fatty acid derivatives or other compounds, in the sample increases.The terms "purify", "purified" and "purification" are relative terms that do not require absolute purity.Thus, for example, when medium-chain fatty acid derivatives are produced in recombinant host cells, the medium-chain fatty acid derivatives are substantially separated from other cellular components (e.g., nucleic acids, polypeptides, lipids, carbohydrates, or other carbohydrates).

[0101] As used herein, the term "attenuate" means to weaken, reduce, or diminish. For example, the activity of a polypeptide can be attenuated, for example, by modifying the polypeptide structure to reduce its activity (e.g., by modifying the nucleotide sequence encoding the polypeptide).

[0102] I. Introduction As mentioned above, there has been great interest in medium-chain fatty acid (MCFA) derivatives and products derived from medium-chain fatty acids (MCFA).MCFA has been valued for its many advantageous properties.In fact, MCFA is used as a renewable and biodegradable component, such as surfactants, adhesives, emulsifiers, edible oils, flavors, fragrances, monomers, polymers, natural product pesticides and antibacterial agents.

[0103] Due to their numerous uses, the demand for medium-chain fatty acid derivative compounds in industrial and nutraceutical applications has been on the rise over the past few years and continues to increase. Unfortunately, however, the supply of medium-chain fatty acid derivatives is highly tied to the production of other longer-chain free fatty acid products from plants or chemical synthesis, and therefore the supply is highly variable and unstable.

[0104] Therefore, what is needed in the art are materials and methods that can provide a robust and stable supply chain for MCFAs and their derivatives. Fortunately, the present disclosure provides the tools and methods necessary to support a robust, selective, and stable supply chain for medium-chain fatty acid derivatives, thus addressing this and other needs.

[0105] II. Engineered Thioesterase Variants with Improved Activity for the Production of Medium-Chain Fatty Acid Derivatives A. General Method This disclosure utilizes conventional techniques in the field of recombinant genetics. Examples of basic texts disclosing common methods and terminology in molecular biology and genetics include, for example, Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Press 4th edition (Cold Spring Harbor, NY 2012); Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998) and Supplements 1-115 (1987-2016). This disclosure also utilizes conventional techniques in the field of biochemistry. Examples of basic texts disclosing common methods and terminology in biochemistry include, for example, Lehninger Principles of Biochemistry sixth edition, David L. Nelson and Michael M. Cox eds. W. H. Freeman (2012). This disclosure also utilizes conventional techniques in industrial fermentation. Basic texts disclosing common methods and terminology in fermentation include, for example, "Principles of Fermentation Technology, 3rd Edition by Peter F. Stanbury, Allan Whitaker and Stephen J. Hall. Butterworth-Heinemann (2016)"; "Fermentation Microbiology and Biotechnology, 2nd Edition, EMT El-Mansi, CFA Bryce, Arnold L. Demain and AR Allman eds. CRC Press (2007)." The present disclosure also makes use of conventional techniques in the field of organic chemistry.Basic texts disclosing common methods and terminology in organic chemistry include, for example, Practical Synthetic Organic Chemistry: Reactions, Principles, and Techniques, Stephane Caron ed., John Wiley and Sons Inc. (2011); The Synthetic Organic Chemist's Companion, Michael C. Pirrung, John Wiley and Sons Inc. (2007); Organic Chemistry, 9th Edition - Francis Carey and Robert Giuliano, McGraw Hill (2013).

[0106] For nucleic acids, sizes are given in either kilobases (kb) or base pairs (bp). Estimates are typically obtained from agarose or acrylamide gel electrophoresis, sequenced nucleic acids, or published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or amino acid residue numbers. Protein sizes are estimated from gel electrophoresis, sequenced proteins, derived amino acid sequences, or published protein sequences.

[0107] Non-commercially available oligonucleotides can be chemically synthesized, for example, by the solid-phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automated synthesizer as described in Van Devanter et al., Nucleic Acids Res. 12:6159-6168 (1984). Oligonucleotide purification can be performed, for example, by either native acrylamide gel electrophoresis or anion-exchange HPLC as described in Pearson & Reanier, J. Chrom. 255:137-149 (1983).

[0108] The sequences of cloned genes and synthetic oligonucleotides can be verified after cloning using, for example, the chain termination method for sequencing double-stranded templates of Wallace et al., Gene 16:21-26 (1981).

[0109] B. Thioesterase 1. General Thioesterases or thiol ester hydrolases catalyze the hydrolysis of thioesters into acids and thiols. Thioesterases (TEs) are classified into EC 3.1.2.1 to EC 3.1.2.27 based on their activity on different substrates, with many remaining unclassified (EC 3.1.2.-) (see, for example, Cantu, DC, et al. (2010) Protein Science 19:1281-1295). TEs can be obtained from a variety of sources. Exemplary TEs include plant TEs (see, e.g., Voelker and Davies, J. Bact., Vol., 176, No. 23, pp. 7320-27, 1994; U.S. Patent Nos. 5,667,997 and 5,455,167), bacterial TEs (see, e.g., U.S. Patent No. 9,175,234); cyanobacterial TEs, as well as TEs from algal, mammalian, insect, and fungal sources.

[0110] In particular, acyl-acyl carrier protein (ACP) thioesterases (TEs), classified under EC number 3.1.2.14, selectively hydrolyze the thioester bond of acyl-ACPs to release free fatty acids (FFAs) and ACPs. Therefore, acyl-ACP thioesterases play an important role in determining the carbon chain length of the fatty acid derivatives resulting from the hydrolysis of alkyl thioesters.

[0111] FatB2 thioesterase (ChFatB2) from Cuphea hookeriana is an exemplary acyl-ACP thioesterase. ChFatB2 naturally has a high selectivity for medium-chain fatty acid derivatives. However, this plant enzyme has low activity when expressed in microorganisms such as the industrial microorganism Escherichia coli. As disclosed in detail herein, the poor ability to produce medium-chain fatty acids in microorganisms is a result of their low activity, poor selectivity for C8 and C10, and poor solubility.

[0112] The polypeptide / protein sequence of wild-type ChFatB2 from Cuphea hookeriana has GenBank accession number AAC49269 (see, e.g., Dehesh, K., et al. (1996) The Plant Journal 9(2):167-72). The amino acid sequence of the ChFatB2 polypeptide disclosed herein comprises a wild-type sequence in which the first 88 amino acids, including the plant-translocated leader sequence at the N-terminus of the wild-type protein, have been removed and replaced with methionine (M) to facilitate production of the active enzyme in the bacterial cytoplasm. Thus, the amino acid sequence of the wild-type ChFatB2 thioesterase disclosed herein, known as wild-type (wt) ChFatB2, is set forth below as SEQ ID NO:1. TIFF0007727616000002.tif38149

[0113] The activity of SEQ ID NO:1 is known to be specific for saturated 8-carbon (8:0) and saturated 10-carbon (10:0) ACP substrates (see, e.g., Dehesh, K., et al. (1996), supra). Unfortunately, however, the medium-chain fatty acid derivative production capacity possessed by SEQ ID NO:1 is insufficient for large-scale production of medium-chain fatty acid derivatives. Therefore, in response to the need for a stable and reliable supply of medium-chain fatty acid derivatives, in exemplary embodiments, SEQ ID NO:1 is modified to produce engineered TE variants with improved activity for the production of medium-chain fatty acid derivatives.

[0114] Thus, in exemplary aspects, the disclosure provides engineered TE variant polypeptides with improved activity for the production of medium-chain fatty acid derivatives, such as medium-chain fatty esters, such as medium-chain fatty acid methyl esters (FAMEs) and medium-chain fatty acid ethyl esters (FAEEs), medium-chain fatty alcohol acetates (FACEs), medium-chain fatty amines, medium-chain fatty aldehydes, medium-chain fatty alcohols, medium-chain hydrocarbons, medium-chain fatty ketones, medium-chain alkanes, medium-chain terminal olefins, medium-chain internal olefins, medium-chain hydroxy fatty acid derivatives, medium-chain difunctional fatty acid derivatives, e.g., medium-chain fatty diacids, medium-chain fatty diols, unsaturated medium-chain fatty acid derivatives, etc., compared to the enzyme having SEQ ID NO:1.

[0115] In some exemplary embodiments, engineered TE variants of SEQ ID NO:1 (e.g., SEQ ID NO:16 to SEQ ID NO:46) with improved activity for the production of medium-chain fatty acid derivatives have an increased net positive surface charge compared to a non-variant / non-engineered control thioesterase, e.g., SEQ ID NO:1.

[0116] 2. Assays for engineered thioesterase variants with improved activity for the production of medium-chain fatty acid derivatives In exemplary embodiments, engineered TE variants with improved activity for the production of medium-chain fatty acid derivatives are identified by measuring the medium-chain fatty acid derivatives (e.g., free fatty acids (FFA), fatty acid ethyl esters, FAEE, fatty alcohols (FALC), fatty alcohol acetate esters (FACe), etc.) produced by a bacterial strain (i.e., a test strain) containing the engineered TE variant, and comparing these medium-chain fatty acid derivatives to measurements of medium-chain fatty acid derivatives (e.g., FFA, FAEE, FALC, FACE, etc.) produced by a suitable control test strain that is isogenic to the test strain except for containing the control TE.

[0117] In some exemplary embodiments, the total titer of medium-chain fatty acid derivatives is measured and compared between the test strain and the control strain. In some exemplary embodiments, the percentage of the total titer of medium-chain fatty acid derivatives, including a particular medium-chain fatty acid derivative (e.g., a C8 fatty acid derivative), produced by the test strain is measured and compared to the percentage of the total titer of medium-chain fatty acid derivatives, including a particular medium-chain fatty acid derivative, produced by a suitable control strain that is isogenic to the test strain except for containing a control TE (e.g., SEQ ID NO:1).

[0118] In an exemplary embodiment, gas chromatography with a flame ionization detector (GC-FID) is used to assay medium-chain fatty acid derivatives. GC-FID is known in the art (see, for example, Adlard, ER; Handley, Alan J. (2001). Gas chromatographic techniques and applications. London: Sheffield Academic). However, any method suitable for quantification and analysis, such as mass spectrometry (MS), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), thin-layer chromatography (TLC), etc., can be used.

[0119] C. Methods for Producing Engineered Thioesterase Variants Engineered TE variants can be prepared by any method known in the art (see, e.g., Current Protocols in Molecular Biology, supra). Thus, in an exemplary embodiment, mutagenesis is used to prepare polynucleotide sequences encoding engineered TE variants, which can then be screened for improved activity for the production of medium-chain fatty acid derivatives. In another exemplary embodiment, polynucleotide sequences encoding engineered TE variants, which can then be screened for improved activity for the production of medium-chain fatty acid derivatives, are prepared by chemical synthesis of the polynucleotide sequence (see, e.g., M. H. Caruthers et al. (1987) Methods in Enzymology Volume 154, Pages 287-313; Beaucage, S. L. and Iyer, R. P. (1992) Tetrahedron 48(12):2223-2311).

[0120] Mutagenesis methods are well known in the art. Exemplary mutagenesis techniques for preparing engineered TE variants with improved activity for the production of medium-chain fatty acid derivatives include, for example, site-saturation mutagenesis (see, for example, Chronopoulou EG1, Labrou NE. Curr. Protoc. Protein Sci. 2011 Feb; Chapter 26:Unit 26.6, John Wiley and Sons, Inc; Steffens, DL and Williams., JGK (2007) J Biomol Tech. 18(3): 147-149; Siloto, RMP and Weselake, RJ (2012) Biocatalysis and Agricultural Biotechnology 1(3):181-189).

[0121] Another exemplary mutagenesis technique for preparing engineered TE variants with improved activity for the production of medium-chain fatty acid derivatives includes transfer PCR (tPCR). See, e.g., Erijman A., et al. (2011) J. Struct. Biol. 175(2):171-7.

[0122] Other exemplary mutagenesis techniques include, for example, error-prone polymerase chain reaction (PCR) (see, e.g., Leung et al. (1989) Technique 1:11-15; and Caldwell et al. (1992) PCR Methods Applic. 2:28-33).

[0123] Another exemplary mutagenesis technique for preparing engineered TE variants with improved activity for the production of medium-chain fatty acid derivatives includes using oligonucleotide-directed mutagenesis to create site-specific mutations in any cloned DNA of interest (see, e.g., Reidhaar-Olson et al. (1988) Science 241:53-57).

[0124] The mutagenized polynucleotide resulting from any method of synthesis or mutagenesis, such as those described above, is then cloned into an appropriate vector and the activity of the affected polypeptide encoded by the mutagenized polynucleotide is assessed as disclosed above.

[0125] Those skilled in the art will recognize that the protocols and procedures disclosed herein can be modified, and that such modifications are in accordance with the variations of the present disclosure. For example, when method steps are described in a certain order, the order of these steps can be modified and / or performed in parallel or sequentially.

[0126] III. Host Cells and Host Cell Cultures In light of the present disclosure, one of skill in the art will recognize that any of the embodiments contemplated herein can be practiced using any host cell or microorganism that can be genetically modified through the introduction of one or more nucleic acid sequences encoding the disclosed engineered TE variants. Thus, the recombinant microorganisms disclosed herein function as host cells and include one or more polynucleotide sequences that include an open reading frame encoding an engineered TE variant polypeptide having improved activity for the production of medium-chain fatty acid derivatives, together with operably linked regulatory sequences that facilitate expression of the engineered TE variant polypeptide in the host cell.

[0127] Exemplary microorganisms that provide suitable host cells include, but are not limited to, species of the genera Escherichia, Bacillus, Lactobacillus, Zymomonas, Rhodococcus, Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, and the like. ), Mucor, Myceliophtora, Marinobacter, Penicillium, Phanerochaete, Pleurotus, Trametes, Chrysosporium, Saccharomyces, Stenotrophomonas, Schizosaccharomyces, Yarrowia, or Streptomyces. In some exemplary embodiments, the host cell is a Gram-positive bacterial cell. In other exemplary embodiments, the host cell is a Gram-negative bacterial cell. In some embodiments, the host cell is an Escherichia coli cell.In other exemplary 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 cells, or Bacillus amyloliquefaciens cells.

[0128] In still other exemplary 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 In still other exemplary embodiments, the host cell is a Humicola lanuginose cell, a Rhodococcus opacus cell, a Rhizomucor miehei cell, or a Mucor michei cell. In still other exemplary embodiments, the host cell is a Streptomyces lividans cell or a Streptomyces murinus cell. In still other embodiments, the host cell is an Actinomycetes cell. In some exemplary embodiments, the host cell is a Saccharomyces cerevisiae cell.

[0129] In still other exemplary embodiments, the host cell is a cell of a eukaryotic plant, algae, cyanobacteria, green sulfur bacteria, green non-sulfur bacteria, purple sulfur bacteria, purple non-sulfur bacteria, extremophile, yeast, fungus, engineered organisms thereof, or synthetic organisms. In some exemplary embodiments, the host cell is selected from the group consisting of Arabidopsis thaliana, Panicum virgatums, Miscanthus giganteus, Zea mays, Botryococcus braunii, Chalamydomonas reinhardtii, Dunaliela salina, Thermosynechococcus elongatus, Synechococcus elongatus, Synechococcus sp., Synechocystis sp., Chlorobium tepidum, and the like. tepidum, Chloroflexus auranticus, Chromatium vinosum, Rhodospirillum rubrum, Rhodobacter capsulatus, Rhodopseudomonas palusris, Clostridium ljungdahlii, Clostridiuthermocellum, or Pencillium chrysogenum.In some other exemplary embodiments, the host cell is a cell derived from Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica, Schizosaccharomyces pombe, Pseudomonas fluorescens, Pseudomonas putida, or Zymomonas mobilis. In still further exemplary embodiments, the host cell is a cell of Synechococcus species PCC 7002, Synechococcus species PCC 7942, or Synechocystis species PCC 6803. In some exemplary embodiments, the host cell is a CHO cell, a COS cell, a VERO cell, a BHK cell, a HeLa cell, a Cv1 cell, an MDCK cell, a 293 cell, a 3T3 cell, or a PC12 cell. In some exemplary embodiments, the host cell is an E. coli cell. In some exemplary embodiments, the E. coli cell is a B, C, K, or W strain E. coli cell.

[0130] In some exemplary embodiments, the host cells include any genetic manipulations and modifications that can be used interchangeably for each host cell, depending on which other heterologous enzymes and native enzyme pathways are present in the host cell. In one exemplary embodiment, the host cells optionally include a fadE and / or fhuA deletion. In other exemplary embodiments, the host cells are optionally engineered to have the ability to produce more than 200 mg / L of fatty acid derivatives, more than 1000 mg / L of fatty acid derivatives, more than 1200 mg / L of fatty acid derivatives, more than 1700 mg / L of fatty acid derivatives, more than 2000 mg / L of fatty acid derivatives, or more than 3000 mg / L of fatty acid derivatives. The above-mentioned optionally engineered strains are useful for identifying and characterizing useful engineered TE variants with improved ability to produce medium-chain fatty acid derivatives, and for selectively producing medium-chain fatty acid derivatives when expressing engineered TE variants with improved ability to produce medium-chain fatty acid derivatives.

[0131] As discussed in detail herein below, in some exemplary embodiments, the host cell or host microorganism used to express the engineered TE variant polypeptide further expresses genes having enzymatic activities that can increase the production of one or more specific fatty acid derivatives, such as, for example, fatty esters, fatty alcohols, fatty alcohol acetate esters, fatty acid methyl esters, fatty acid ethyl esters, fatty amines, fatty aldehydes, difunctional fatty acid derivatives, diacids, alkanes, alkenes or olefins, ketones, etc.

[0132] For example, the entD gene encodes a phosphopantetheinyl transferase. Overexpression of native E. coli entD, a phosphopantetheinyl transferase, is an optional genetic modification to cells expressing a carboxylic acid reductase, such as CarB, because it allows for improved activation of CarB from apo-CarB to holo-CarB, thereby allowing for improved conversion of free fatty acids by holo-CarB to fatty aldehydes, which can then be converted to fatty alcohols by fatty aldehyde reductase. See, e.g., U.S. Patent No. 9,340,801.

[0133] In exemplary embodiments, the host cell or host microorganism used to express the engineered TE variant polypeptide further expresses ester synthase activity (EC 2.3.1.75) for the production of fatty esters. In another exemplary embodiment, the host cell has acyl-ACP reductase (AAR) (EC 1.2.1.80) activity and / or alcohol dehydrogenase activity (EC 1.1.1.1.) and / or fatty alcohol acyl-CoA reductase (FAR) (EC 1.1.1.*) activity and / or carboxylic acid reductase (CAR) (EC 1.2.99.6) activity for the production of fatty alcohols. In another exemplary embodiment, the host cell has acyl-ACP reductase (AAR) (EC 1.2.1.80) activity for the production of fatty aldehydes. In another exemplary embodiment, the host cell has acyl-ACP reductase (AAR) (EC 1.2.1.80) activity and decarbonylase or fatty aldehyde oxidative deformylation activity for the production of alkanes and alkenes. In another exemplary embodiment, the host cell has acyl-CoA reductase (EC 1.2.1.50) activity and acyl-CoA synthetase (FadD) (EC 2.3.1.86) activity for the production of fatty alcohols. In another exemplary embodiment, the host cell has ester synthase activity (EC 2.3.1.75) and acyl-CoA synthetase (FadD) (EC 2.3.1.86) activity for the production of fatty esters. In another exemplary embodiment, the host cell has OleA activity for the production of ketones. In another exemplary embodiment, the host cell has OleBCD activity for the production of internal olefins. In another exemplary embodiment, the host cell has acyl-ACP reductase (AAR) (EC 1.2.1.80) activity and alcohol dehydrogenase activity (EC 1.1.1.1.) for the production of fatty alcohols. In another exemplary embodiment, the host cell has decarboxylase activity for the production of terminal olefins.Expression of enzyme activity in microorganisms and microbial cells is described, for example, in the following U.S. Patents: 9,133,406; 9,340,801; 9,200,299; 9,068,201; 8,999,686; 8,658,404; 8,597,922; 8,535,916; 8,658,404; ,530,221; 8,372,610; 8,323,924; 8,313,934; 8,283,143; 8,268,599; 8,183,028; 8,110,670; 8,110,093; and 8,097,439.

[0134] In some exemplary embodiments, the host cell or microorganism used to express the engineered TE variant polypeptide comprises certain native enzyme activities that are upregulated or overexpressed to produce one or more specific fatty acid derivatives, such as, for example, fatty esters, fatty acid methyl esters, fatty acid ethyl esters, fatty alcohols, fatty alcohol acetate esters, fatty amines, fatty amides, fatty aldehydes, difunctional fatty acid derivatives, diacids, etc.

[0135] In some exemplary embodiments, the recombinant host cells produce medium-chain fatty esters, such as medium-chain fatty acid methyl esters (FAMEs) or medium-chain fatty acid ethyl esters (FAEEs), medium-chain fatty alcohol acetates (FACEs), medium-chain fatty alcohols (FALCs), medium-chain fatty amines, medium-chain fatty aldehydes, medium-chain difunctional fatty acid derivatives, medium-chain diacids, medium-chain alkanes, medium-chain olefins, and the like.

[0136] The medium-chain fatty acid derivatives are typically recovered from the culture medium and / or isolated from the host cells. In one exemplary embodiment, the fatty acid derivatives are recovered from the culture medium (extracellular). In another exemplary embodiment, the fatty acid derivatives are isolated from the host cells (intracellular). In another exemplary embodiment, the fatty acid derivatives or non-fatty acid compounds are recovered from the culture medium and isolated from the host cells.

[0137] To determine the distribution of specific fatty acid derivatives and the chain length and saturation of the components of the fatty acid derivative composition, the fatty acid derivative composition produced by the host cell can be analyzed using methods known in the art, such as gas chromatography with a flame ionization detector (GC FID). Similarly, other compounds can be analyzed by methods known in the art.

[0138] IV. Methods of Producing Recombinant Host Cells and Cultures Any method known in the art can be used to engineer host cells to produce fatty acid derivatives and / or fatty acid derivative compositions or other compounds. Exemplary methods include the use of vectors, e.g., expression vectors, containing polynucleotide sequences encoding mutant or engineered TE variants and / or polynucleotide sequences encoding other fatty acid derivative biosynthetic pathway polypeptides disclosed herein. Those skilled in the art will recognize that a wide variety of viral and non-viral vectors can be used in the methods disclosed herein.

[0139] In some exemplary embodiments, the polynucleotide (or gene) sequence encoding the mutant or engineered TE variant is provided to the host cell by a recombinant vector comprising a promoter operably linked to the polynucleotide sequence encoding the mutant or engineered TE variant. In some exemplary embodiments, the promoter is developmentally regulated, organelle-specific, tissue-specific, inducible, constitutive, or cell-specific. In some exemplary embodiments, the promoter is inducible by the addition of lactose or isopropylthiogalactoside (IPTG).

[0140] After polynucleotide sequences encoding mutant or engineered TE variants and / or other fatty acid derivative biosynthetic pathway polypeptides have been prepared and isolated, various methods can be used to construct expression cassettes, vectors, and other DNA constructs. Expression cassettes containing polynucleotide sequences encoding mutant or engineered TE variants and / or other fatty acid biosynthetic pathway polypeptides can be constructed in a variety of ways. Experts are well aware of the genetic elements that must be present on an expression construct / vector for successful transformation, selection, and propagation of the expression construct in a host cell. Techniques for manipulating polynucleotide sequences, such as polynucleotide sequences encoding mutant or engineered TE variants, such as subcloning nucleic acid sequences into expression vectors, labeled probes, DNA hybridization, and the like, are generally described in, for example, Sambrook, et al., supra; Current Protocols in Molecular Biology, supra.

[0141] Heterologous DNA sequences, such as polynucleotide sequences encoding mutant or engineered TE variants (e.g., SEQ ID NO:3, SEQ ID NO:16-SEQ NO:46, etc.) and / or polynucleotide sequences encoding other fatty acid biosynthetic pathway polypeptides, linked to a promoter sequence, can be inserted into a wide variety of vectors. In some exemplary embodiments, the vector selected is an expression vector useful for transforming bacteria, such as E. coli. Expression vectors can be plasmids, viruses, cosmids, artificial chromosomes, nucleic acid fragments, etc. Such vectors are readily constructed through the use of recombinant DNA techniques well known to those of skill in the art (see, e.g., Sambrook et al., supra). The expression vector containing the polynucleotide sequence encoding the mutant or engineered TE variant may then be transfected / transformed into a target host cell. Successfully transformed cells are then selected based on the presence of an appropriate marker gene by methods well known in the art.

[0142] Several recombinant vectors are available to those skilled in the art for use in stable transformation / transfection of bacteria and other microorganisms (see, e.g., Sambrook, et al., supra). An appropriate vector can be readily selected by one skilled in the art. In an exemplary embodiment, a known vector is used to generate an expression construct containing a polynucleotide sequence encoding a mutant or engineered TE variant.

[0143] Typically, a transformation vector contains one or more polynucleotide sequences encoding one or more mutant or engineered TE variants and / or other fatty acid derivative biosynthetic pathway polypeptides operably linked, for example, to a promoter sequence and a selectable marker. Such transformation vectors also typically contain transcription initiation sites, ribosome binding sites, RNA processing signals, transcription termination sites, and / or polyadenylation signals as needed.

[0144] Thus, in addition to polynucleotide sequences encoding mutant or engineered TE variants and / or other fatty acid derivative biosynthetic pathway polypeptides, expression constructs prepared as disclosed herein may contain additional elements. In exemplary embodiments, expression constructs comprising polynucleotide sequences encoding mutant or engineered TE variants and / or other fatty acid derivative biosynthetic pathway polypeptides also contain enhancer sequences such that expression of the heterologous protein can be increased. As is known in the art, enhancers are typically found 5' to the transcription start site, and they can often be inserted either 5' or 3' to the coding sequence, in a forward or reverse orientation.

[0145] As mentioned above, transformation / expression vectors typically contain a selectable and / or screenable marker gene to allow easy identification of transformants. Exemplary selectable marker genes include, but are not limited to, genes encoding antibiotic resistance (e.g., resistance to kanamycin, ampicillin, etc.). Exemplary screenable markers include a six amino acid histidine tag introduced at the C-terminus of the recombinant protein.

[0146] In exemplary embodiments, a selectable or screenable marker gene is employed as, or in addition to, the specific gene of interest to provide or enhance the ability to identify transformants. Numerous selectable marker genes are known in the art (see, e.g., Sambrook et al., supra).

[0147] In some exemplary embodiments, the expression vector further comprises sequences linked to the coding sequence of the heterologous nucleic acid to be expressed, which are post-translationally removed from the initial translation product. In one exemplary embodiment, the post-translationally removed sequences facilitate transport of the protein into or across an intracellular or extracellular membrane, thereby facilitating transport of the protein to intracellular and / or extracellular compartments. In one exemplary embodiment, the post-translationally removed sequences protect the nascent protein from intracellular proteolysis. In one exemplary embodiment, a nucleic acid segment encoding a leader peptide sequence upstream and in reading frame with a selected coding sequence is used for recombinant expression of the coding sequence in a host cell.

[0148] In another exemplary embodiment, the expression construct comprises a bacterial origin of replication, e.g., the ColE1 origin. In yet another exemplary embodiment, the expression construct / vector comprises a bacterial selectable marker, e.g., an ampicillin, tetracycline, hygromycin, neomycin, or chloramphenicol resistance gene.

[0149] As is well known in the art, expression constructs typically contain restriction endonuclease sites to facilitate vector construction. Exemplary restriction endonuclease recognition sites include, but are not limited to, the recognition sites for the restriction endonucleases NotI, AatII, SacII, PmeI HindIII, PstI, EcoRI, and BamHI.

[0150] DNA constructs, polynucleotide sequences functionally encoding mutant or engineered TE variants and / or heterologous DNA sequences, e.g., promoter sequences, polynucleotide sequences encoding other fatty acid derivative biosynthetic pathway polypeptides linked to marker sequences; purification moieties; secretion sequences operably coupled to polynucleotide sequences; targeting sequences, etc., are used to transform cells and produce recombinant host cells with improved activity for the production of medium-chain fatty acid derivatives. Exemplary host cells for transformation with expression constructs containing polynucleotide sequences encoding mutant or engineered TE variants are discussed in detail in Section III above.

[0151] An appropriate transformation technique can be easily selected by a skilled artisan. Exemplary transformation / transfection methods available to those skilled in the art include, for example, electroporation, calcium chloride transformation, etc., and such methods are well known to those skilled in the art (see, e.g., Sambrook, supra). Thus, a polynucleotide sequence comprising a protein-encoding open reading frame and an operably linked regulatory sequence can be integrated into the chromosome of a recombinant host cell, integrated into one or more plasmid expression systems resident in the recombinant host cell, or both.

[0152] The expression vectors disclosed herein typically contain a polynucleotide sequence encoding a mutant or engineered TE variant and / or a polynucleotide sequence encoding a fatty acid derivative biosynthetic pathway polypeptide in a form suitable for expression of the polynucleotide sequence in a host cell. As will be recognized by those skilled in the art, the design of the expression vector can depend on factors such as the choice of host cell to be transformed, the expression level of the desired polypeptide, and the like.

[0153] V. Evaluation of Recombinant Host Cells In exemplary embodiments, the activity of an engineered TE variant polypeptide is determined by culturing the recombinant host cell and measuring characteristics of, for example, fatty acid derivative composition (e.g., medium-chain fatty esters, medium-chain fatty alcohols, medium-chain fatty aldehydes, etc.) or other compounds produced by the recombinant host cell. In exemplary embodiments, the composition, titer, yield, and / or productivity of the fatty acid derivative or other compound are analyzed.

[0154] The engineered TE variant polypeptides and fragments thereof can be assayed using conventional methods for improved activity for the production of medium-chain fatty acid derivatives (see, e.g., Example 4 herein below).

[0155] IV. Products Obtained from Recombinant Host Cells Strategies for increasing production of medium-chain fatty acid derivatives by recombinant host cells include increasing the flux through the fatty acid biosynthetic pathway, for example, by overexpression of native fatty acid biosynthetic genes in the production host and / or expression of heterologous fatty acid biosynthetic genes from the same or a different organism.

[0156] Thus, in exemplary embodiments, recombinant host cells with improved activity for medium-chain fatty acid derivative production are engineered to contain, in addition to the engineered TE variant, one or more polynucleotide sequences encoding one or more "fatty acid derivative biosynthetic polypeptides" or, equivalently, "fatty acid derivative enzymes." Metabolic engineering of fatty acid derivative biosynthetic pathways to produce fatty acid derivative compounds (e.g., fatty acid esters, alkanes, olefins, fatty ketones, fatty alcohols, fatty alcohol acetates, etc.) using microorganisms to convert biomass-derived sugars into desired products is known in the art. See, for example, U.S. Patent Nos. 9,133,406; 9,340,801; 9,200,299; 9,068,201; 8,999,686; 8,658,404; 8,597,922; 8,535,916; 8,530,221; 8,372,610; 8,323,924; 8,313,934; 8,283,143; 8,268,599; 8,183,028; 8,110,670; 8,110,093; and 8,097,439. The metabolically engineered strains can be cultured in industrial-scale bioreactors and the resulting products can be purified using conventional chemical and biochemical engineering techniques.

[0157] As is well known in the art, thioesterases catalyze the hydrolysis of alkyl thioesters to free fatty acids (FFAs). Thus, thioesterases play a role in determining the acyl chain length distribution of fatty acids and fatty acid derivatives (see, e.g., Dehesh (1996) supra; PNAS (1995) 92(23):10639-10643). Thus, recombinant host cells with improved activity for the production of medium-chain fatty acid derivatives typically contain engineered TE variants with improved activity for the production of medium-chain fatty acid derivatives (e.g., SEQ ID NO:3, and the variants shown in Table 7). In some exemplary embodiments, such recombinant host cells provide increased amounts of medium-chain fatty acid derivatives, e.g., medium-chain fatty alcohols, medium-chain fatty acids, FAEEs, FAMEs, FACEs, etc., compared to appropriate control host cells that do not contain the engineered TE variant, e.g., isogenic control host cells that have a control thioesterase (e.g., SEQ ID NO:1) instead of the engineered TE variant.

[0158] Thus, in some embodiments, a fatty acid derivative composition comprising a fatty acid is produced by culturing a recombinant host cell comprising an engineered TE variant in the presence of a carbon source under conditions effective to express a thioesterase.

[0159] In some embodiments, substantially all of the fatty acid derivatives produced by culturing recombinant host cells containing engineered TE variants with improved activity for the production of medium-chain fatty acid derivatives under conditions effective for expressing the TE are produced extracellularly. Thus, in some exemplary embodiments, the fatty acid derivatives produced are recovered from the culture medium. In some exemplary embodiments, the recovered fatty acid derivative composition is analyzed using any suitable method known in the art, such as GC FID, to determine and quantify the distribution of specific fatty acid derivatives and the chain length and saturation degree of the components of the fatty acid derivative composition.

[0160] In other embodiments, the recombinant host cell comprises a polynucleotide sequence encoding a mutant or engineered TE variant with improved activity for the production of a medium-chain fatty acid derivative, and one or more additional polynucleotides encoding polypeptides with other fatty acid derivative biosynthetic enzyme activity. Thus, in some embodiments, a first medium-chain fatty acid derivative (e.g., a medium-chain fatty acid, a medium-chain fatty alcohol, etc.) produced by the action of the engineered TE variant is converted by one or more fatty acid derivative biosynthetic enzymes to a second fatty acid derivative, e.g., a medium-chain fatty acid ester, a medium-chain fatty aldehyde, a medium-chain fatty alcohol acetate ester, a hydrocarbon, e.g., a straight-chain alkane, a straight-chain alkene, etc.

[0161] Table 1 provides a list of exemplary fatty acid derivative biosynthetic polypeptides that can be expressed in recombinant host cells to facilitate the production of medium-chain fatty acid derivatives, in addition to engineered TE variants with improved activity for the production of medium-chain fatty acid derivatives.

[0162] (Table 1) Gene names of fatty acid derivative enzymes TIFF0007727616000003.tif127152TIFF0007727616000004.tif251152TIFF0007727616 000005.tif201152TIFF0007727616000006.tif88152TIFF0007727616000007.tif219152

[0163] Production of medium-chain fatty acid derivatives As described above, recombinant host cells containing engineered TE variants with improved activity for the production of medium-chain fatty acid derivatives produce increased amounts of medium-chain fatty acids compared to suitable control host cells that do not contain the engineered TE variant, for example, isogenic control host cells that contain a control TE (such as SEQ ID NO:1).

[0164] In other exemplary embodiments described in detail below, in addition to engineered TE variants with improved activity for the production of medium-chain fatty acid derivatives, the recombinant host cells further comprise additional fatty acid derivative biosynthetic polypeptides that facilitate the production of specific types of fatty acid derivatives.

[0165] Fatty aldehyde production In some exemplary embodiments, in addition to the engineered TE variants with improved activity for the production of medium-chain fatty acid derivatives, the recombinant host cells further comprise carboxylic acid reductase ("CAR") activity, such that the recombinant host cells synthesize fatty aldehydes and fatty alcohols. See, e.g., 9,340,801.

[0166] Thus, in some exemplary embodiments, fatty aldehydes are produced by expressing or overexpressing in a recombinant host cell a polynucleotide encoding a polypeptide having fatty aldehyde biosynthetic activity, such as carboxylic acid reductase (CAR) activity. Exemplary carboxylic acid reductase (CAR) polypeptides and their encoding polynucleotides include, for example, FadD9 (EC 6.2.1.-, UniProtKB Q50631, GenBank NP_217106), CarA (GenBank ABK75684), CarB (GenBank YP889972), and related polypeptides disclosed, for example, in U.S. Patent Nos. 8,097,439 and 9,340,801.

[0167] In some exemplary embodiments, the fatty aldehydes produced by the recombinant host cells are then converted to fatty alcohols or hydrocarbons. Thus, in some exemplary embodiments, in addition to the engineered TE variants with improved activity for the production of medium-chain fatty acid derivatives, the recombinant host cells further comprise acyl-CoA reductase ("FAR" or "ACR") activity, such that the recombinant host cells synthesize fatty aldehydes and fatty alcohols (see, e.g., U.S. Pat. Nos. 8,658,404, 8,268,599, and U.S. Patent Application Publication No. 2015 / 0361454).

[0168] In some embodiments, fatty aldehydes produced by a recombinant host cell are converted to fatty alcohols via the activity of a native or heterologous fatty alcohol biosynthetic polypeptide, such as an aldehyde reductase or alcohol dehydrogenase (see, e.g., U.S. Patent Application Publication No. 2011 / 0250663). Thus, in some exemplary embodiments, in addition to an engineered TE variant with improved activity for the production of medium-chain fatty acid derivatives, the recombinant host cell further comprises an aldehyde reductase activity, or equivalently, an alcohol dehydrogenase activity (EC 1.1.1.1), such that the recombinant host cell synthesizes fatty alcohols. Exemplary fatty alcohol biosynthetic genes include, but are not limited to, alcohol dehydrogenases, such as AlrA or AlrA homologs of Acentitobacter sp. M-1; and endogenous E. coli alcohol dehydrogenases, such as DkgA (NP.sub.--417485), DkgB (NP.sub.--414743), YjgB, (AAC77226), YdjL (AAC74846), YdjJ (NP.sub.--416288), AdhP (NP.sub.--415995), YhdH (NP.sub.--417719), YahK (NP.sub.--414859), YphC (AAC75598), and YqhD (Q46856).

[0169] Fatty amine production In some exemplary embodiments, recombinant host cells (e.g., those disclosed hereinabove) that contain engineered TE variants with improved activity for the production of medium-chain fatty acid derivatives and produce fatty aldehydes are further modified to contain heterologous biosynthetic enzymes with aminotransferase or amine dehydrogenase activity that convert the fatty aldehyde to a fatty amine (see, e.g., PCT Publication No. WO2015 / 085271).

[0170] Fatty alcohol production In some exemplary embodiments, in addition to the engineered TE variant with improved activity for the production of medium-chain fatty acid derivatives, the recombinant host cell further comprises a polynucleotide encoding a polypeptide with fatty alcohol biosynthetic activity, such that fatty alcohols are produced by the recombinant host cell. Thus, in exemplary embodiments, a composition comprising a medium-chain fatty alcohol, e.g., octanol, is produced by culturing the recombinant host cell under conditions effective to express the engineered TE variant with improved activity for the production of medium-chain fatty acid derivatives and the fatty alcohol biosynthetic enzymes in the presence of a carbon source.

[0171] Thus, in some exemplary embodiments, in addition to the engineered TE variants with improved activity for the production of medium-chain fatty acid derivatives, the recombinant host cells further comprise carboxylic acid reductase (CAR) activity and alcohol dehydrogenase activity, such that the recombinant host cells synthesize medium-chain fatty alcohols, e.g., octanol (see, e.g., U.S. Patent No. 9,340,801).

[0172] In some exemplary embodiments, a native fatty aldehyde biosynthetic polypeptide, such as an aldehyde reductase / alcohol dehydrogenase, present in a host cell converts a medium-chain fatty aldehyde to a medium-chain fatty alcohol. In other exemplary embodiments, the native fatty aldehyde reductase / alcohol dehydrogenase is overexpressed to convert a medium-chain fatty aldehyde to a medium-chain fatty alcohol. In other exemplary embodiments, a heterologous aldehyde reductase / alcohol dehydrogenase is introduced into a recombinant host cell and expressed or overexpressed to convert a medium-chain fatty aldehyde to a medium-chain fatty alcohol. Exemplary aldehyde reductase / alcohol dehydrogenase polypeptides useful for converting a medium-chain fatty aldehyde to a medium-chain fatty alcohol are disclosed herein above and in WO 2007 / 136762; WO 2010 / 062480; U.S. Pat. Nos. 8,110,670; and 9,068,201.

[0173] In some exemplary embodiments, in addition to the engineered TE variant with improved activity for the production of medium-chain fatty acid derivatives, the recombinant host cell further comprises a heterologous polynucleotide encoding a polypeptide having carboxylic acid reductase (EC 6.2.1.3 or EC 1.2.1.42) activity, whereby the recombinant host cell produces 1,3 fatty diols when grown in a fermentation broth having a simple carbon source. In other exemplary embodiments, in addition to the engineered TE variant with improved activity for the production of medium-chain fatty acid derivatives, the recombinant host cell further comprises a heterologous polynucleotide encoding a polypeptide having carboxylic acid reductase (EC 6.2.1.3 or EC 1.2.1.42) activity and a heterologous polynucleotide encoding a polypeptide having alcohol dehydrogenase (EC 1.1.1) activity, whereby the recombinant host cell produces 1,3 fatty diols, such as medium-chain 1,3 fatty diols, when grown in a fermentation broth having a simple carbon source (see, e.g., WO 2016 / 011430).

[0174] Production of fatty alcohol acetate esters In some embodiments, the fatty alcohol produced in the cell or in some embodiments provided to the cell is further processed by the recombinant cell to provide fatty alcohol acetate ester (FACE). In exemplary embodiments, an alcohol O-acetyltransferase (EC 2.8.1.14) enzyme processes the fatty alcohol into fatty alcohol acetate ester (FACE). See, for example, Gabriel M Rodriguez, et al. (2014) Nature Chemical Biology 10, 259-265; Jyun-Liang Lin and Ian Wheeldon (2014) PLoS One. 2014; 9(8): PMCID: PMC4122449.

[0175] An exemplary alcohol O-acetyltransferase is yeast Aft1, e.g., GenBank Accession No. AY242062; GenBank Accession No. AY242063. See, e.g., Kevin J. Verstrepen KJ, et al (2003) Appl Environ Microbiol. 2003 Sep; 69(9): 5228-5237.

[0176] In exemplary embodiments, the recombinant host cell comprising the engineered TE variant with improved ability to produce medium chain fatty acid derivatives further comprises sufficient carboxylic acid reductase activity (EC 1.2.99.6) to produce fatty aldehydes and fatty alcohols, and further comprises fatty alcohol O-acetyltransferase activity to convert the fatty alcohol to a fatty alcohol acetate ester.

[0177] In a further exemplary embodiment, a recombinant host cell comprising an engineered TE variant with improved ability to produce medium chain fatty acid derivatives further comprises a carboxylic acid reductase activity (EC 1.2.99.6) that results in the production of a first fatty acid derivative, and further comprises a fatty alcohol O-acetyltransferase activity that converts the first fatty acid derivative to a second fatty acid derivative, wherein the second fatty acid derivative has a higher MIC than the first fatty acid derivative.

[0178] In a further exemplary embodiment, a recombinant host cell comprising an engineered TE variant with improved ability to produce medium chain fatty acid derivatives further comprises a carboxylic acid reductase activity (EC 1.2.99.6) that results in the production of a first fatty acid derivative, and further comprises a fatty alcohol O-acetyltransferase activity that converts the first fatty acid derivative to a second fatty acid derivative, wherein the second fatty acid derivative has a higher LogP than the first fatty acid derivative.

[0179] In a further exemplary embodiment, a recombinant host cell comprising an engineered TE variant with improved ability to produce medium chain fatty acid derivatives further comprises a carboxylic acid reductase activity (EC 1.2.99.6) that results in the production of a first fatty acid derivative, and further comprises a fatty alcohol O-acetyltransferase activity that converts the first fatty acid derivative to a second fatty acid derivative, wherein the presence of the second fatty acid derivative results in an increase in the MIC of the first fatty acid derivative.

[0180] In a further exemplary embodiment, the recombinant host cell comprising the engineered TE variant with improved ability to produce medium chain fatty acid derivatives further comprises a carboxylic acid reductase activity (EC 1.2.99.6) that results in the production of a first fatty acid derivative, and further comprises a fatty alcohol O-acetyltransferase activity that converts the first fatty acid derivative to a second fatty acid derivative, wherein the second fatty acid derivative is less toxic than the first fatty acid derivative.

[0181] Fatty ester production In some embodiments, in addition to the engineered TE variant with improved activity for the production of medium-chain fatty acid derivatives, the recombinant host cell further comprises a polynucleotide encoding a polypeptide with fatty ester biosynthetic activity, such that medium-chain fatty esters are produced by the recombinant host cell.

[0182] As used herein, the term "fatty ester" or, by analogy, "fatty acid ester" refers to any ester made from a fatty acid. In exemplary embodiments, the fatty ester contains an "A side" and a "B side." As used herein, the "A side" of an ester refers to the carbon chain attached to the carboxylate oxygen of the ester. As used herein, the "B side" of an ester refers to the carbon chain comprising the parent carboxylate of the ester. In embodiments where the fatty ester is obtained from a fatty acid derivative biosynthetic pathway, the A side is contributed by an alcohol and the B side is contributed by a fatty acid or alkylthioester.

[0183] Any alcohol can be used to form the A side of the fatty ester. In an exemplary embodiment, the alcohol is obtained from a fatty acid derivative biosynthetic pathway. In other exemplary embodiments, the alcohol is produced via a non-fatty acid derivative biosynthetic pathway, for example, the alcohol is provided exogenously, for example, the alcohol is provided in the fermentation broth.

[0184] The carbon chain comprising the A-side or B-side can be any length. However, in exemplary embodiments in which a fatty acid derivative biosynthetic pathway comprising an engineered TE variant with improved activity for the production of medium-chain fatty acid derivatives provides either the A-side and / or the B-side of a fatty acid ester, the A-side and / or the B-side are medium-chain fatty acid derivatives and, therefore, have carbon chain lengths of 6, 7, 8, 9, or 10 carbons. Thus, in exemplary embodiments, a fatty acid derivative biosynthetic pathway comprising an engineered TE variant with improved activity for the production of medium-chain fatty acid derivatives provides the A-side of an ester, and, therefore, the A-side of the fatty ester is 6, 7, 8, 9, or 10 carbons in length. In other exemplary embodiments, a fatty acid biosynthetic pathway comprising an engineered TE variant with improved activity for the production of medium-chain fatty acid derivatives provides the B-side of an ester, and, therefore, the B-side of the fatty ester is 6, 7, 8, 9, or 10 carbons in length.

[0185] In one exemplary embodiment, the fatty ester is a fatty acid methyl ester, e.g., methyl octanoate, where the B-side is provided by a fatty acid biosynthetic pathway including an engineered TE variant with improved activity for the production of medium-chain fatty acid derivatives, and the A-side of the ester is one carbon in length. Thus, in an exemplary embodiment, the fatty acid ester is methyl octanoate. In one exemplary embodiment, the A-side is provided by the action of a fatty acid O-methyltransferase (FAMT) (EC 2.1.1.15) enzyme (see, e.g., Applied and Environmental Microbiology 77(22): 8052-8061).

[0186] In another exemplary embodiment, the fatty ester is a fatty acid ethyl ester, wherein the B side is provided by a fatty acid biosynthetic pathway that includes an engineered TE variant with improved activity for the production of medium-chain fatty acid derivatives, and the A side of the ester is two carbons in length.

[0187] In one exemplary embodiment, the A side is linear. In another exemplary embodiment, the A side is branched. In one exemplary embodiment, the B side is linear. In another exemplary embodiment, the B side is branched. A branched chain can have one or more branch points. In one exemplary embodiment, the A side is saturated. In another exemplary embodiment, the A side is unsaturated. In one exemplary embodiment, the B side is saturated. In another exemplary embodiment, the B side is unsaturated.

[0188] In an exemplary embodiment, in addition to the engineered TE variant with improved activity for the production of medium-chain fatty acid derivatives, the recombinant host cell comprises a polynucleotide encoding a polypeptide having ester synthase activity (EC 3.1.1.67). Ester synthases are known in the art. See, e.g., WO 2011 / 038134.

[0189] In some exemplary embodiments, fatty acid esters are produced by recombinant host cells comprising engineered TE variants with improved activity for the production of medium-chain fatty acid derivatives, as well as acyl-CoA synthetase (fadD) and ester synthase enzymes (see, e.g., WO 2011 / 038134; WO 2007 / 136762; U.S. Pat. No. 8,110,670).

[0190] In exemplary embodiments, the recombinant host cell comprising the engineered TE variant with improved ability to produce medium chain fatty acid derivatives further comprises sufficient ester synthase activity (EC 3.1.1.67) to produce fatty esters (such as FAMEs or FAEEs).

[0191] In a further aspect, the recombinant host cell comprising an engineered TE variant with improved activity that results in the production of a first fatty acid derivative further comprises an ester synthase activity that converts the first fatty acid derivative to a second fatty acid derivative.

[0192] In a further aspect, the recombinant host cell comprising the engineered TE variant with improved activity that results in the production of a first fatty acid derivative further comprises an ester synthase activity that converts the first fatty acid derivative to a second fatty acid derivative, wherein the second fatty acid derivative has a higher MIC than the first fatty acid derivative.

[0193] In a further aspect, the recombinant host cell comprising the engineered TE variant with improved activity that results in the production of a first fatty acid derivative further comprises an ester synthase activity that converts the first fatty acid derivative to a second fatty acid derivative, wherein the second fatty acid derivative has a higher partition coefficient (LogP) than the first fatty acid derivative.

[0194] In a further aspect, the recombinant host cell comprising an engineered TE variant with improved activity that results in the production of a first fatty acid derivative further comprises an ester synthase activity that converts the first fatty acid derivative to a second fatty acid derivative, wherein the presence of the second fatty acid derivative results in an increase in the MIC of the first fatty acid derivative.

[0195] In a further aspect, the recombinant host cell comprising the engineered TE variant with improved activity that results in the production of a first fatty acid derivative further comprises an ester synthase activity that converts the first fatty acid derivative to a second fatty acid derivative, wherein the second fatty acid derivative is less toxic than the first fatty acid derivative.

[0196] Hydrocarbon Production In some embodiments, in addition to the engineered TE variant with improved activity for the production of medium-chain fatty acid derivatives, the recombinant host cell further comprises a polynucleotide encoding a polypeptide having fatty aldehyde biosynthetic activity, e.g., an acyl-ACP reductase polypeptide (EC 6.4.1.2), and a polynucleotide encoding a polypeptide having hydrocarbon biosynthetic activity, e.g., a decarbonylase (EC 4.1.99.5), an oxidative deformylase, or a fatty acid decarboxylase; thus, the recombinant host cell exhibits enhanced production of hydrocarbons (see, e.g., U.S. Patent Application Publication No. 2011 / 0124071). Thus, in exemplary embodiments, the recombinant host cell comprising the engineered TE variant with improved activity for the production of medium-chain fatty acid derivatives produces hydrocarbons, e.g., alkanes or alkenes (e.g., terminal or internal olefins), or ketones.

[0197] In some exemplary embodiments, fatty aldehydes produced by recombinant host cells comprising engineered TE variants with improved activity for the production of medium-chain fatty acid derivatives are converted by decarbonylation, removing one carbon atom, to form hydrocarbons (see, e.g., U.S. Pat. No. 8,110,670 and WO 2009 / 140695).

[0198] In other exemplary embodiments, the fatty acids produced by the recombinant host cells are converted by decarboxylation, removing one carbon atom, to form terminal olefins. Thus, in some exemplary embodiments, in addition to expressing an engineered TE variant with improved activity for the production of medium-chain fatty acid derivatives, the recombinant cells further express or overexpress a polynucleotide encoding a hydrocarbon biosynthetic polypeptide, such as, for example, a polypeptide with decarboxylase activity disclosed in U.S. Pat. No. 8,597,922.

[0199] In other exemplary embodiments, the alkylthioester intermediate is converted by enzymatic decarboxylative condensation to form an internal olefin or ketone. Thus, in some exemplary embodiments, in addition to expressing an engineered TE variant with improved activity for the production of medium-chain fatty acid derivatives, the recombinant cell further expresses or overexpresses a polynucleotide encoding a hydrocarbon biosynthetic polypeptide, e.g., a polypeptide with OleA activity, thereby producing a ketone (see, e.g., U.S. Pat. No. 9,200,299). In other exemplary embodiments, in addition to expressing an engineered TE variant with improved activity for the production of medium-chain fatty acid derivatives, the recombinant cell further expresses or overexpresses a polynucleotide encoding a hydrocarbon biosynthetic polypeptide, e.g., OleCD or OleBCD, together with a polypeptide with OleA activity, thereby producing an internal olefin (see, e.g., U.S. Pat. No. 9,200,299).

[0200] Some exemplary hydrocarbon biosynthetic polypeptides are shown in Table 2 below.

[0201] Table 2: Exemplary hydrocarbon biosynthetic polynucleotides and polypeptides TIFF0007727616000008.tif52128

[0202] Production of omega(ω)-hydroxylated fatty acid derivatives In some embodiments, in addition to the engineered TE variant with improved activity for the production of medium-chain fatty acid derivatives, the recombinant host cell further comprises a polynucleotide encoding a polypeptide having ω-hydroxylase activity (EC 1.14.15.3). In exemplary embodiments, the modified ω-hydroxylase has modified cytochrome P450 monooxygenase (P450) enzyme activity and efficiently catalyzes the hydroxylation of the w-position of the hydrocarbon chain in vivo. Thus, the recombinant microorganism produces medium-chain omega-hydroxylated (ω-hydroxylated) fatty acid derivatives in vivo when grown in a fermentation broth in the presence of a carbon source derived from a renewable feedstock (see, e.g., PCT Application Publication WO 2014 / 201474).

[0203] In other exemplary embodiments, in addition to the engineered TE variant with improved activity for the production of medium-chain fatty acid derivatives, the recombinant host cell further comprises a polynucleotide encoding an alkane hydroxylase, such as AlkA, a CYP153A-reductase, or a CYP153A-reductase hybrid fusion polypeptide variant (see, e.g., WO 2015 / 195697), such that the recombinant host cell, when cultured in a medium containing a carbon source under conditions effective to express the alkane hydroxylase, such as AlkA, CYP153, or a CYP153A-reductase hybrid fusion polypeptide variant and the engineered TE variant with improved activity for the production of medium-chain fatty acid derivatives, produces omega-hydroxylated (ω-hydroxylated) fatty acid derivatives and bifunctional fatty acid derivatives, including ω-hydroxylated fatty esters, α,ω-diacids, α,ω-diesters, α,ω-diols, and chemicals derived therefrom, such as macrolactones and macrocyclic ketones, and compositions thereof.

[0204] V. Cultivation and Fermentation of Recombinant Host Cells As used herein, fermentation broadly refers to the conversion of organic matter into a target substance by recombinant host cells. For example, this includes the conversion of a carbon source by recombinant host cells into fatty acid derivatives, such as medium-chain fatty acids, medium-chain fatty acid esters, medium-chain fatty alcohols, and medium-chain fatty alcohol acetate esters, by growing a culture of the recombinant host cells in a medium containing a carbon source. Conditions permissive for the production of target substances, such as fatty acids, fatty esters, fatty alcohols, and fatty alcohol acetate esters, are any conditions that allow the host cells to produce a desired product, such as a fatty acid derivative composition. Suitable conditions include, for example, typical fermentation conditions. See, for example, Principles of Fermentation Technology, 3rd Edition (2016), supra; Fermentation Microbiology and Biotechnology, 2nd Edition (2007), supra.

[0205] Fermentation conditions can include numerous parameters well known in the art, including, but not limited to, temperature range, pH level, aeration level, feed rate, and medium composition. Each of these conditions, individually and in combination, allows host cells to grow. Fermentation can be aerobic, anaerobic, or a variation thereof (such as microaerobic). Exemplary media include broth (liquid) or gel (solid). Generally, the medium contains a carbon source (e.g., a simple carbon source derived from a renewable feedstock) that can be directly metabolized by the host cells. Additionally, enzymes can be used in the medium to facilitate metabolism of the carbon source following mobilization (e.g., depolymerization of starch or cellulose into fermentable sugars) to produce medium-chain fatty acid derivatives.

[0206] For small-scale production, host cells engineered to produce medium-chain fatty acid derivative compositions can be grown in, 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 batches; fermented; and induced to express a desired polynucleotide sequence, such as a polynucleotide encoding a polypeptide having a specific enzymatic activity (e.g., thioesterase (TE), carboxylic acid reductase (CAR), alcohol dehydrogenase (ADH), fatty acyl-CoA / ACP reductase (FAR), acyl-CoA reductase (ACR), acetyl-CoA carboxylase (ACC), and / or acyl-ACP / CoA reductase (AAR) enzymatic activity). For large-scale production, engineered host cells can be grown in cultures having batch volumes of about 10 L, 100 L, 1000 L, 10,000 L, 100,000 L, 1,000,000 L, or more; fermented; and induced to express any desired polynucleotide sequence.

[0207] The fatty acid derivative compositions disclosed herein are often found in the extracellular environment of recombinant host cell cultures and can be easily isolated from the culture medium. Medium-chain fatty acid derivatives, such as medium-chain fatty acids, medium-chain fatty acid esters, medium-chain fatty aldehydes, medium-chain fatty ketones, medium-chain fatty alcohols, and medium-chain fatty alcohol acetate esters, may be secreted by recombinant host cells, transported into the extracellular environment of recombinant host cell cultures, or passively transported into the extracellular environment. Medium-chain fatty acid derivative compositions may be isolated from recombinant host cell cultures using conventional methods known in the art, including but not limited to centrifugation.

[0208] Exemplary microorganisms suitable for use as production host cells include, for example, bacteria, cyanobacteria, yeast, algae, filamentous fungi, etc. To produce fatty acid derivative compositions, the production host cell (or equivalently, host cell) is engineered to contain a fatty acid biosynthetic pathway that is modified compared to a non-engineered or native host cell, for example, engineered as described above and disclosed, for example, in U.S. Patent Application Publication No. 2015 / 0064782. A production host engineered to contain a modified fatty acid biosynthetic pathway can efficiently convert glucose or other renewable feedstocks into fatty acid derivatives. Protocols and procedures for high-density fermentation for the production of a variety of compounds have been established (see, e.g., U.S. Patent Nos. 8,372,610; 8,323,924; 8,313,934; 8,283,143; 8,268,599; 8,183,028; 8,110,670; 8,110,093; and 8,097,439).

[0209] In some exemplary embodiments, the production host cells are cultured in a culture medium (e.g., a fermentation medium) comprising an initial concentration of a carbon source (e.g., a simple carbon source) of about 20 g / L to about 900 g / L. In other embodiments, the culture medium comprises an initial carbon source concentration of about 2 g / L to about 10 g / L; about 10 g / L to about 20 g / L; about 20 g / L to about 30 g / L; about 30 g / L to about 40 g / L; or about 40 g / L to about 50 g / L. In some embodiments, the level of available carbon source in the culture medium can be monitored during the course of the fermentation. In some embodiments, the method further comprises adding additional carbon source to the culture medium if the initial level of carbon source in the medium is less than about 0.5 g / L.

[0210] In some exemplary embodiments, an additional carbon source is added to the culture medium when the carbon source level in the medium is less than about 0.4 g / L, less than about 0.3 g / L, less than about 0.2 g / L, or less than about 0.1 g / L. In some embodiments, an additional carbon source is added to maintain a carbon source level of about 1 g / L to about 25 g / L. In some embodiments, an additional carbon source is added to maintain a carbon source level of about 2 g / L or greater (e.g., about 2 g / L or greater, about 3 g / L or greater, about 4 g / L or greater). In certain embodiments, an additional carbon source is added to maintain a carbon source level of about 5 g / L or less (e.g., about 5 g / L or less, about 4 g / L or less, about 3 g / L or less). In some embodiments, an additional carbon source is added to maintain a carbon source level of about 2 g / L to about 5 g / L, about 5 g / L to about 10 g / L, or about 10 g / L to about 25 g / L.

[0211] In an exemplary embodiment, the carbon source for fermentation is derived from a renewable feedstock. In some embodiments, the carbon source is glucose. In other embodiments, the carbon source is glycerol. Other possible carbon sources include, but are not limited to, fructose, mannose, galactose, xylose, arabinose, starch, cellulose, hemicellulose, pectin, xylan, sucrose, maltose, cellobiose, turanose, acetate, ethane, ethanol, methane, methanol, formic acid, and carbon monoxide; cellulose materials and variants such as hemicellulose, methylcellulose, and sodium carboxymethylcellulose; saturated or unsaturated fatty acids, succinates, lactates, and acetates; alcohols such as ethanol, methanol, and glycerol, or mixtures thereof. In one embodiment, the carbon source is derived from corn, sugarcane, sorghum, sugar beet, switchgrass, ensilage, straw, wood, pulp, sewage, garbage, cellulose municipal solid waste, flue gas, synthesis gas, or carbon dioxide. The simple carbon source can also be a photosynthetic product, such as glucose or sucrose. In one embodiment, the carbon source is derived from waste materials such as glycerol, flue gas, or syngas; or from the reorganization of organic matter such as biomass; or from natural gas or methane, or from the reorganization of these materials into syngas; or from photosynthetically fixed carbon dioxide. For example, medium-chain fatty acid derivatives can be produced by photosynthetically growing recombinant cyanobacteria or algae using CO as a carbon source. In some exemplary embodiments, the carbon source is derived from biomass. An exemplary source of biomass is plant material or vegetation such as corn, sugarcane, or switchgrass. Another exemplary source of biomass is metabolic waste, such as animal matter (e.g., cow manure). Further exemplary sources of biomass include algae and other marine plants. Biomass also includes waste from industrial, agricultural, forestry, and household sources, including, but not limited to, fermentation waste, ensilage, straw, wood, sewage, garbage, cellulosic municipal waste, municipal solid waste, and food scraps.

[0212] In some exemplary embodiments, fatty acid derivatives, such as medium-chain fatty acids, medium-chain fatty esters, and medium-chain fatty alcohols, are produced at a concentration of about 0.5 g / L to about 40 g / L. In some embodiments, fatty acid derivatives are produced at a concentration of about 1 g / L or greater (e.g., about 1 g / L or greater, about 10 g / L or greater, about 20 g / L or greater, about 50 g / L or greater, or about 100 g / L or greater). In some embodiments, fatty acid derivatives are produced at a concentration of about 1 g / L to about 170 g / L, about 1 g / L to about 10 g / L, about 40 g / L to about 170 g / L, about 100 g / L to about 170 g / L, about 10 g / L to about 100 g / L, about 1 g / L to about 40 g / L, about 40 g / L to about 100 g / L, or about 1 g / L to about 100 g / L.

[0213] In other exemplary embodiments, the fatty acid derivatives, e.g., medium chain fatty acid derivatives, are at concentrations 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 560 mg / L, about 570 mg / L, about 580 mg / L, about 590 mg / L, about 600 mg / L, about 610 mg / L, about 620 mg / L, about 630 mg / L, about 640 mg / L, about 650 mg / L, about 660 mg / L, about 670 mg / L, about 680 mg / L, about 690 mg / L, about 700 mg / L, about 710 mg / L, about 720 mg / L, about 730 mg / L, about 740 mg / L, about 750 mg / L, about 760 mg / L, about 770 mg / L, about 780 mg / L, about 790 mg / L, about 800 mg / L, about 825 mg / L, about 850 mg / L, about 860 mg / L, about 870 mg / L, about 880 mg / L, about 890 mg / L, about 900 mg / L, about 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 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 117 5mg / L, about 1200mg / L, about 1225mg / L, about 1250mg / L, about 1275mg / L, about 1300mg / L, about 1325mg / L, about 1350mg / L, about 1375mg / L, about 1400mg / L, about 1425mg / L, about 1450 mg / L, approximately 1475mg / L, approximately 1500mg / L, approximately 1525mg / L, approximately 1550mg / L, approximately 1575mg / L, approximately 1600mg / L, approximately 1625mg / L, approximately 1650mg / L, approximately 1675mg / L, approximately 1700mg / L, approximately 1725m In other embodiments, the fatty acid derivatives or other compounds are produced at titers of about 100 g / L, about 200 g / L, about 300 g / L, about 400 mg / L, about 500 mg / L, about 600 mg / L, about 700 mg / L, about 800 g / L, about 900 mg / L, about 1000 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, the fatty acid derivatives or other compounds are produced at titers of greater than 100 g / L, greater than 200 g / L, or greater than 300 g / L.In exemplary embodiments, the titers of fatty acid derivatives or other compounds produced by recombinant host cells according to the methods disclosed herein are between 5 g / L and 200 g / L, between 10 g / L and 150 g / L, between 20 g / L and 120 g / L, and between 30 g / L and 100 g / L. The titers may represent the specific fatty acid derivative or combination of fatty acid derivatives or another compound or combinations of other compounds produced by a given recombinant host cell culture. In exemplary embodiments, expression of an engineered TE variant in a recombinant host cell, such as E. coli, results in the production of a higher titer 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.

[0214] In other exemplary embodiments, host cells engineered to produce fatty acid derivatives, e.g., medium chain fatty acid derivatives, according to the methods of the present disclosure have a yield of at least 1%, at least 2%, 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, one or more fatty acid derivatives or other compounds are produced in a yield of greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, or greater than about 90%. Alternatively or additionally, the yield is about 30% or less, about 27% or less, about 25% or less, or about 22% or less. In another embodiment, the yield is about 50% or less, about 45% or less, or about 35% or less. In another embodiment, the yield is about 95% or less, or 90% or less, or 85% or less, or 80% or less, or 75% or less, or 70% or less, or 65% or less, or 60% or less, or 55% or less, or 50% or less. Thus, the yield can be linked by any two of the above endpoints.For example, the yield of medium-chain fatty acid derivatives, e.g., 8- and / or 10-carbon fatty acid derivatives, produced by recombinant host cells according to the methods disclosed herein may 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%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 100%, about 100% to about 120%, about 120% to about 140%, about 140% to about 150%, about 15% to about 22%, about 1 The yield can be about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 100%, about 100% to about 200%, about 200% to about 300%, about 300% to about 400%, about 400% to about 500%, about 500% to about 600%, about 600% to about 700%, or about 700% to about 800%. The yield may represent a specific medium-chain fatty acid derivative or combination of fatty acid derivatives. In one embodiment, a higher yield is in the range of about 10% to about 800% of the theoretical yield. In addition, the yield also depends on the feedstock used.

[0215] In some exemplary embodiments, the productivity of host cells engineered to produce fatty acid derivatives, e.g., medium chain fatty acid derivatives, according to the methods of the present disclosure is at least 100 mg / L / hr, at least 200 mg / L / hr, at least 300 mg / L / hr, at least 400 mg / L / hr, at least 500 mg / L / hr, at least 600 mg / L / hr, at least 700 mg / L / hr, at least 800 mg / L / hr, at least 900 mg / L / hr, at least 1000 mg / L / hr, at least 1100 mg / L / hr, at least 1200 mg / L / hr, at least 1400 mg / L / hr, at least 1600 mg / L / hr, at least 1800 mg / L / hr, at least 1900 mg / L / hr, at least 2100 mg / L / hr, at least 2200 mg / L / hr, at least 2300 mg / L / hr, at least 2400 mg / L / hr, at least 2500 mg / L / hr, at least 2600 mg / L / hr, at least 2700 mg / L / hr, at least 2800 mg / L / hr, at least 2900 mg / L / hr, at least 3000 mg / L / hr, at least 3100 mg / L / hr, at least 3200 mg / L / hr, at least 3300 mg / L / hr, at least 3400 mg / L / hr, at least 3500 mg / L / hr, at least 3600 mg / L / hr, at least 3700 mg / L / hr, at least 3800 mg / L / hr, at least 3900 mg / L / hr, at least 4000 mg / L / hr, at least 4100 mg / L / hr The productivity of malonyl-CoA-derived compounds, including one or more fatty acid derivatives or other compounds, produced by recombinant host cells according to the disclosed methods can be as high as 500 mg / L / h to 2500 mg / L / h, or 700 mg / L / h to 2000 mg / L / h (depending on cell mass). Productivity can refer to the specific 8-carbon and / or 10-carbon fatty acid derivative or combination of fatty acid derivatives or other compounds produced by a given host cell culture. For example, expression of an engineered TE variant in a recombinant host cell, such as E. coli, results in increased productivity of 8-carbon and / or 10-carbon fatty acid derivatives or other compounds compared to a recombinant host cell expressing the corresponding wild-type polypeptide. In exemplary embodiments, the higher productivity ranges from about 0.3 g / L / h to about 3 g / L / h, about 10 g / L / h, about 100 g / L / h, or about 1000 g / L / h.

[0216] VI. Isolation Biological products, e.g., compositions, comprising the medium-chain fatty acid derivatives disclosed herein, produced using recombinant host cells as described above, are typically isolated from the fermentation broth by methods known in the art. In an exemplary embodiment, compositions comprising the medium-chain fatty acid derivatives disclosed herein, produced using recombinant host cells, are isolated from the fermentation broth by gravity settling, centrifugation, or decantation, as described above.

[0217] VII. Medium Chain Fatty Acid Derivative Compositions and Formulations The bioproducts produced utilizing the recombinant host cells described in detail above, e.g., compositions containing medium-chain fatty acids and medium-chain fatty acid derivatives, are produced from renewable sources (e.g., simple carbon sources obtained from renewable feedstocks) and, as such, are novel compositions of matter. These novel bioproducts can be identified by dual carbon-isotope fingerprinting or 14 C dating can be used to distinguish between organic compounds derived from petrochemical carbon. Additionally, the specific origin of biogenic carbon (e.g., glucose vs. glycerol) can be determined by dual carbon-isotope fingerprinting, as known in the art (see, e.g., U.S. Pat. No. 7,169,588, WO2016 / 011430A1, etc.).

[0218] Furthermore, as shown below, the composition of the bioproduct defines a unique composition of natural fatty acid derivatives produced from the organism. These unique compositions, which are unusually high in medium chain fatty acid derivatives, provide a novel and unique source of these valuable medium chain length products.

[0219] The following examples are offered to illustrate, but not to limit, the present invention. [Example]

[0220] The following specific examples are intended to illustrate the disclosure and should not be construed as limiting the scope of the claims.

[0221] Example 1 The following examples demonstrate that chemical modifications to medium-chain length fatty acid derivative compounds reduce the toxicity experienced by microorganisms to the medium-chain length fatty acid derivative compounds compared to the toxicity experienced by the microorganisms when grown in the presence of unmodified medium-chain length fatty acid derivative compounds.

[0222] As discussed hereinabove, the production of medium-chain fatty acid derivative compounds using biological systems (e.g., fermentation of microbial cells) is a desirable route for the selective production of medium-chain fatty acids / aliphatic compounds. Unfortunately, medium-chain fatty acid derivative compounds can be highly toxic to microbial cells, and this toxicity is a barrier to the commercial-scale production of medium-chain fatty acid derivative compounds via fermentation.

[0223] In this example, related compounds that differ only in whether they are modified or unmodified were evaluated for toxicity by determining the minimum inhibitory concentration (MIC) of each compound (the concentration of compound sufficient to kill 50% of the culture). Compounds with lower toxicity (i.e., higher MICs) are more amenable to production by fermentation.

[0224] E. coli cell cultures were grown with various concentrations of these compounds, and their growth was determined as a measure of the total number of cells in the culture by measuring the total protein from the lysed culture after 24 hours of growth.

[0225] In particular, E. coli cell cultures were grown in the presence of octanol, octanoic acid, methyl octanoate, and octyl acetate. The results are shown in Figure 1.

[0226] As can be seen in Figure 1, the medium-chain acid octanoic acid and the medium-chain alcohol octanol have MICs of 1-5 g / L. On the other hand, the esters of these medium-chain alcohols and acids, octyl acetate, methyl octanoate, and ethyl octanoate (not shown), have MICs 10-100 times higher than the corresponding unmodified alcohols and acids. Therefore, E. coli can tolerate 10-100 times higher concentrations of chemically modified compounds compared to unmodified compounds.

[0227] Thus, the above examples demonstrate that esters of medium-chain fatty alcohols and acids can be produced and tolerated at high concentrations by industrial fermentation processes. Furthermore, the above examples demonstrate that the toxicity of a fatty compound of a given chain length can be significantly reduced by modifying the functional groups associated with the toxic molecule or by slightly increasing its molecular weight.

[0228] Example 2 The following examples demonstrate that the toxicity of medium-chain fatty acid derivative compounds correlates with the partition coefficient (LogP).

[0229] As shown in Example 1, esters of medium-chain fatty alcohols and esters of medium-chain fatty acids are less toxic (have higher MICs) than the corresponding medium-chain fatty alcohols and medium-chain fatty acids.

[0230] Many water-soluble compounds have low partition coefficients (LogP). LogP is a measure of a compound's partition between water and octanol (see, e.g., that compounds with low LogP, such as acetic acid, lactic acid, pyruvate, 1,3 propanediol, and amino acids, can be produced and tolerated at high concentrations by microorganisms, e.g., Escherichia coli). Therefore, it can be concluded that compounds that are less hydrophobic (or equivalently more hydrophilic) and therefore have a lower LogP will be less toxic. To assess whether this is true, we measured the logP of the compounds disclosed in Figure 1 (i.e., octanol, octanoic acid, octyl acetate, and methyl octanoate).

[0231] Surprisingly, as Figure 2 shows, toxicity as a function of LogP for medium-chain aliphatic compounds is contrary to expectations: compounds with low logP, octanol and octanoic acid, have high toxicity (i.e., low MICs). Compounds with high logP, octyl acetate, methyl octanoate, and ethyl ocanoate, have even lower toxicity (high MICs).

[0232] Thus, this example demonstrates the ability to differentiate toxic medium chain aliphatic compounds with low LogP from compounds with higher LogP wo These results demonstrate that modification of compounds with the formula (I) is a useful method for reducing the toxicity of medium-chain aliphatic compounds that are toxic to industrial microorganisms such as E. coli.

[0233] Example 3 The following examples demonstrate that expression of novel biochemical pathways that catalyze the conversion of toxic medium-chain aliphatic compounds to their less toxic derivatives can enable microorganisms to tolerate pathways leading to toxic compounds and produce the derivatives at high levels.

[0234] As discussed in Examples 1 and 2 above, medium-chain fatty acid derivative compounds, such as fatty alcohols and fatty acids, are toxic to host cells, but their slightly higher molecular weight and high logP derivatives are not. Therefore, the present inventors have determined that more toxic compounds can be produced in microorganisms without killing cells by biochemically converting the more toxic compounds into less toxic compounds in vivo. The less toxic compounds can then be produced and tolerated at high levels. Once produced, these less toxic compounds can be isolated and used directly, or isolated and chemically converted back to the more toxic compounds.

[0235] As shown below, engineering cells to modify the functional groups of toxic medium-chain fatty acid derivatives, for example by esterification with short-chain acids or alcohols, eliminates the toxic response of the cells to the unesterified compounds and allows the engineered cells to survive the expression of highly productive biochemical pathways leading to the toxic compounds, enabling a novel and selective process for producing these medium-chain fatty acid derivatives at concentrations well above their inhibitory levels.

[0236] Moreover, modifying medium chain fatty acids and / or medium chain fatty alcohols by esterification to provide esterified medium chain fatty acids and / or esterified medium chain fatty alcohols further reduces the toxicity of the medium chain intermediates by acting as extractants in the biosynthetic pathway.

[0237] Esterified medium-chain fatty acid derivatives as extractants Figure 3 shows the results of an experiment designed to test whether the presence of octyl acetate could protect cells from 1-octanol toxicity. As can be seen from Figure 3, after 5 hours of exposure to 1-octanol at a concentration of 0.5 grams per liter (g / L), E. coli cell viability was completely lost. Interestingly, however, when 50 g / L of octyl acetate (a concentration nontoxic to E. coli cells) was also added, cell viability remained at 100% of control levels even when cells were exposed to 1-octanol at a concentration of 0.5 g / L and even 1 g / L. When cells were exposed to 10 g / L of 1-octanol (well above the MIC observed for 1-octanol), viability decreased by less than 20%.

[0238] Altering tolerance to medium-chain fatty alcohols by expressing fatty alcohol acetyltransferase As discussed above, microbial production of medium-chain (C6–C10) fatty alcohols is limited by their toxicity. Significant efforts have been devoted to identifying genetic and biochemical mechanisms that increase tolerance to these medium-chain fatty acid derivative compounds (see, e.g., Lennen and Pflefer, 2013; Royce et al., 2015; Tan, et al., 2016; Tan, et al., 2017). However, to date, no solutions have been found that enable production at commercial titers (e.g., concentrations between approximately 10 g / L and 200 g / L or higher).

[0239] In Example 1, we demonstrated that medium-chain fatty alcohol acetate esters are less toxic than the corresponding medium-chain fatty alcohols when added to culture media. In the experiments described below, we demonstrate that expression of a pathway that produces medium-chain fatty alcohols in cells is cytotoxic, resulting in poor cell growth and limited medium-chain alcohol production by these cells. We further demonstrate that when the same strain is further engineered to express a biochemical pathway that converts medium-chain alcohols to alcohol acetate esters, the cells grow well and produce significant amounts of fatty alcohol acetate esters. Thus, biochemical conversion of medium-chain fatty alcohols synthesized in cells to their acetate alcohols eliminates the toxicity of the intermediate medium-chain fatty alcohols and enables the production of high levels of fatty alcohol acetate esters. This further demonstrates that the gene encoding medium-chain alcohol-O-acetyltransferase confers tolerance to medium-chain fatty alcohols produced in cells (Figure 4).

[0240] Cells can be engineered to produce fatty alcohols through a variety of biochemical pathways (see, e.g., Figure 4). These biochemical pathways include, but are not limited to, pathways involving thioesterases (TEs), which hydrolyze fatty acid thioesters intracellularly to produce fatty acids (see, e.g., PCT / US1998 / 011697, U.S. Patent No. 9,765,368, PCT / US2010 / 04049), carboxylic acid reductases, which catalyze the ATP and NAD(P)H reduction of fatty acids to fatty aldehydes, and alcohol dehydrogenases, which catalyze the NAD(P)H-dependent reduction of fatty aldehydes to fatty alcohols (note that most cells have sufficient alcohol dehydrogenase activity to catalyze this reaction intracellularly, but overexpression of these or similar enzymes can ensure that fatty aldehydes do not accumulate; see, e.g., WO 2010 / 062480). Other pathways that can be engineered to produce fatty alcohols include fatty acyl reductases, which catalyze the reduction of fatty acyl thioesters (see, e.g., Kim et al., 2015) to fatty aldehydes.

[0241] Acetylation of fatty alcohols can be achieved, for example, by expression of alcohol-O-acetyltransferase (EC 2.3.1.84), which catalyzes the acetyl-coenzyme A (CoA)-dependent acetylation of alcohols (FIG. 4). Alcohol acetyltransferases (AATs) are diverse, and an appropriate AAT can be selected from families such as plant AATs (e.g., strawberry SAAT or FaAAT2, petunia PhcFATB2, etc.), yeast ATF (Saccharomyces cerevisiae ATF1) (see, e.g., PCT / US2014 / 053587), etc. As a non-limiting example, we demonstrate here the effect of expressing S. cerevisiae ATF1 in E. coli cells engineered to produce fatty alcohols.

[0242] To determine whether expressing the acetylation pathway was beneficial, the viability of a strain expressing a pathway for the biosynthesis of medium-chain fatty alcohols was compared to that of an isogenic strain expressing ATF, which would convert (toxic) medium-chain fatty alcohols to (less toxic) fatty alcohol acetate esters. In this evaluation, the sRG.674 strain produces fatty alcohol species in which 85-90% of the total fatty alcohol species (FAS) produced are of 8 or 10 carbon chain length (C8 + C10 fatty alcohols (C8 + C10 FALCs)). The sJN.209 strain is isogenic to sRG.674, except that the S. cerevisiae atf1 gene was added to a plasmid expressing the fatty alcohol (FALC) pathway.

[0243] Table 3. Strains producing medium-chain fatty alcohols (FALCs) or fatty alcohol acetates (FACEs) TIFF0007727616000009.tif25128

[0244] Strains sRG.674 and sJN.209 were grown in 5 L bioreactors using minimal salts medium containing glucose as the carbon source, fed at maximum consumption rates, as described in Examples 9 and 10 (Figure 5). The FALC-producing strain (sRG.674) was unable to grow even before the addition of IPTG, which induces FALC pathway expression and medium-chain compound production (Figure 5A). Without being bound by theory, the growth failure of the FALC-producing strain may be due to low levels of constitutive expression of enzymes involved in medium-chain FALC synthesis and the premature production of inhibitory concentrations of C8 and C10 FALC. In contrast, this growth inhibition was not observed in strain sJN.209 upon expression of AAT; instead, full growth and fatty alcohol acetate ester (FACE) production were observed throughout the entire 72-hour fermentation.

[0245] Comparison of the levels and composition of the lipid species produced (Figures 5B and 5C) further demonstrates the powerful ability of the acetyltransferase gene to enable transient, but high-level, production of medium-chain-length fatty alcohols in cells by converting them to fatty alcohol acetates, which are less toxic compounds.

[0246] Modifications for medium-chain free fatty acid tolerance Like medium-chain fatty alcohols, medium-chain free fatty acids are toxic to microbial cells (see, e.g., FIG. 1). Here, we show that the production of such compounds can be greatly improved by providing cells with the ability to convert free fatty acids to less toxic alkyl esters, such as fatty acid methyl esters (FAMEs) or fatty acid ethyl esters (FAEEs).

[0247] Esterification of medium-chain FFAs can be achieved through expression of a fatty acyl-CoA synthetase (e.g., FadD from E. coli), which catalyzes the coenzyme A (CoA)- and adenosine triphosphate (ATP)-dependent acyl-CoA synthesis, and an ester synthase, which catalyzes the alcoholysis of thioesters such as acyl-CoA (products of fatty acyl-CoA synthetase or intermediates in the β-oxidation or reverse β-oxidation pathways) (Figure 6).

[0248] Esterification of medium-chain FFAs can also be achieved through expression of a medium-chain-length selective ester synthase (e.g., Examples?) that catalyzes the direct alcoholysis of medium-chain-length acyl-ACPs (also alkylthioesters).

[0249] The benefit of expressing the ester synthesis pathway was demonstrated by comparing viability and medium-chain fatty acid derivatives produced between a strain engineered to express a thioesterase with improved activity for the production of medium-chain fatty acid derivatives and an isogenic strain that also expressed an acyl-CoA synthetase and ester synthase.

[0250] Strain sRS.786 was engineered to express a medium-chain-length thioesterase (SEQ ID NO:49), which produces FFAs, mostly C8 and C10 FFAs (Figure 7C). Strain Stpay.179 is isogenic to sRS.786 but also expresses fatty acyl-CoA synthetase and ester synthase. Stpay.179 produces medium-chain-length fatty alkyl esters when fed short-chain alcohols, such as methanol and ethanol, in the medium (Figure 7C).

[0251] Table 4. Strains producing medium-chain fatty acids (FFA) or fatty alkyl esters TIFF0007727616000010.tif29128

[0252] Strains sRS.786 and Stpay.179 were grown in 5 L fed-batch bioreactors using minimal salts medium containing glucose as the carbon source fed at a rate of 14 g / h as described below in Examples 11 and 12. In addition, either ethanol (FIG. 7) or methanol (not shown) was fed to the fermentation process to maintain the alcohol concentration around 2 g / L.

[0253] The FFA-only strain (sRS.786) stopped growing and consuming glucose approximately 10 hours after the addition of IPTG to induce expression of the medium-chain acyl-ACP thioesterase (SEQ ID NO:49) and produced approximately 5 g of C8 + C10 FFAs. Meanwhile, the Stpay.179 strain, which expressed the esterification pathway, was able to grow and produce a total fatty acid species titer of over 84 g / kg, 93% of which were C8–C10 FFAs (Figures 7B and 7C). Similar results were observed when ethanol or methanol was used as the alcohol supplement for ester synthesis.

[0254] These data demonstrate that expression of an ester synthesis pathway, which catalyzes the conversion of toxic intracellular medium-chain free fatty acids to less toxic alkyl esters (e.g., fatty acid methyl esters or fatty acid ethyl esters), enables the production of high levels of medium-chain-length fatty acid derivatives. These data further demonstrate that expression of an ester synthesis pathway enables high levels of medium-chain-length-selective thioesterase expression by eliminating its toxicity.

[0255] Example 4 The following examples illustrate engineered thioesterase variants that contain single amino acid substitutions and have improved activity and / or selectivity for the production of medium-chain length fatty acid derivatives.

[0256] The production of medium-chain (C6-C10) fatty acid derivatives using biotechnology is currently limited in part by the activity and selectivity of available thioesterases (TEs). One of the most active and selective TEs available is the Cuphea hookeriana thioesterase chFatB2, an enzyme having the amino acid sequence described by SEQ ID NO:1.

[0257] Unfortunately, this enzyme as found in nature has many limitations. It is not well expressed as a soluble protein in microorganisms, its specific activity is low, and it is more selective for hydrolysis of C10 thioesters than C8 thioesters. To create new TEs with improved activity, selectivity, and solubility, the inventors have conducted extensive engineering efforts to identify amino acid substitutions in SEQ ID NO:1 that can result in novel engineered TE variants with improved activity for the production of medium-chain fatty acid derivatives. Such TE variants with improved activity for the production of medium-chain fatty acid derivatives may achieve their improved activity for the production of medium-chain fatty acid derivatives through any one or more of improved catalytic activity, improved selectivity, and / or improved solubility.

[0258] SEQ ID NO:1 has 328 amino acids (6560 possible single amino acid variants), but no three-dimensional crystal structure has been reported that could support rational enzyme engineering efforts. The inventors first undertook efforts to identify and engineer single mutations into SEQ ID NO:1 that would result in engineered TE variants that exhibit significantly increased enzyme activity and / or medium chain length selectivity compared to that of the parent sequence SEQ ID NO:1.

[0259] To evaluate such mutations, genes encoding novel engineered TEs with selected single amino acid substitutions were expressed in E. coli and grown under conditions that support TE-dependent fatty acid derivative production. The amount and composition of medium-chain fatty acid derivatives produced by the strain were then quantified and compared to the amount and composition of medium-chain fatty acid derivatives produced by an identical control strain that expressed the enzyme having SEQ ID NO:1.

[0260] E. coli, which does not naturally produce free fatty acids, can produce free fatty acids when engineered to express heterologous TEs, and the amount and composition of these fatty acids directly correlates with the activity and selectivity of the expressed TE (see, e.g., Yuan et al., (1995), supra; International Patent Application Publication No. WO2007136762; International Patent Application Publication No. WO2008119082).

[0261] As discussed above in Examples 1-3, the production of medium-chain fatty acids and medium-chain fatty alcohols is toxic to microorganisms such as E. coli. To ensure that the host E. coli used to evaluate engineered TEs could tolerate engineered TEs that produce potentially toxic levels of medium-chain fatty acids, the E. coli used were also engineered to express genes that increase cell tolerance to medium-chain fatty acids by affecting the conversion of medium-chain fatty acids to fatty alcohol acetate esters, such that the level and composition of fatty alcohol acetate esters produced by the engineered cells directly correlates with the activity and selectivity of the expressed TE.

[0262] Generation of control evaluator strains A gene encoding the polypeptide of SEQ ID NO:1 was synthesized for optimal translation in E. coli and is set forth as SEQ ID NO:60. This gene was cloned into a pACYC-based plasmid (Genbank Accession No. X06403) conferring resistance to kanamycin so that the gene was under the transcriptional control of the Ptrc promoter (see, e.g., Camsund et al. Journal of Biological Engineering 2014, 8:4), which is induced in the presence of isopropylthiogalactoside (IPTG). The resulting plasmid, pIR.108 (FIG. 8), was transformed into E. coli derived from MG1655, which had been engineered to overexpress the gene EntD from the chromosome (see, e.g., International Patent Application Publication No. WO2010062480) and harbors a Ptrc-regulated operon expressing the genes carB, alrA, and aftA1, which together affect the biochemical conversion of free fatty acids (FFA) to fatty alcohol acetates (FACE), as described in Example 3 above.

[0263] To enable effective testing of engineered TEs with high activity and specificity, several control evaluation strains were used, each with different fatty acid derivative production capacities, i.e., engineered to support different levels of carbon flux through the fatty acid pathway (Table 5). Furthermore, in some cases, novel engineered TE variants with improved activity for medium-chain fatty acid derivative production were used as control TEs in place of SEQ ID NO: 1 to identify highly active, improved engineered TE variants. For example, a TE with a single amino acid substitution was compared to SEQ ID NO: 1 expressed in a strain engineered to have moderate fatty acid flux. Once novel, highly active TE variants were developed, SEQ ID NO: 1 expressed in a strain with moderate fatty acid flux no longer served adequately as a control. Instead, novel, highly active engineered TE variants with multiple amino acid substitutions expressed in a strain with high fatty acid capacity were used as controls. In summary, the inventors used five different TEs and strains to support the evaluation of novel engineered TE variants, allowing them to best quantify and identify the performance improvements of each TE variant. A list of control base strains with various fatty acid derivative production capabilities is shown in Table 5. Table 6 shows the performance of the control TEs compared to the wild-type sequence (SEQ ID NO: 1).

[0264] Table 5. Description of control base strains with different fatty acid derivative production capabilities TIFF0007727616000011.tif27131The range of FAS titers (mg / L) in the HTP screens in "Quantifying the relative performance of engineered TE variants" below depends on the level of flux to the engineered alkyl thioester in each strain.

[0265] Table 6. Engineered thioesterase variants of the control and their performance compared to the wild-type sequence (SEQ ID NO: 1). TIFF0007727616000012.tif67147

[0266] Identification of engineered TEs with improved activity compared to SEQ ID NO:1 Strains expressing the engineered TEs listed in Table 7 were each grown under conditions that resulted in expression of the genes encoding their unique engineered TEs, which affect the production of medium-chain fatty acids, as well as the genes encoding CarB, AlrA, and Aft1, which affect the conversion of medium-chain fatty acids to medium-chain fatty alcohol acetate esters. The resulting fatty acid-derived products were extracted, quantified, and then compared to the fatty acid derivative products produced by control evaluation strain 1 expressing SEQ ID NO:1 (Table 6), which was grown under the same conditions. Detailed methods for growth and analysis of the resulting fatty acid derivatives are described below.

[0267] Table 7 lists engineered TEs with improved performance in terms of (1) activity, i.e., total fatty acid-derived products produced by the culture, (2) C8 selectivity, i.e., %C8 FAS relative to total FAS produced by the culture, and (3) selectivity for C8 products relative to C10 products (%C8 FAS / %C10 FAS), where performance is reported as fold over control (FOC). The single mutants shown in Table 7 are relative to SEQ ID NO:1. Thus, for example, P3K represents a substitution mutation (proline to lysine) at amino acid position 3 of SEQ ID NO:1.

[0268] Table 7. Engineered thioesterase variants with improved ability to produce total FAS, %C8 FAS relative to total FAS, and / or %C8 FAS / %C10 FAS. FOC: fold over control TIFF0007727616000013.tif218159TIFF0007727616000014.tif232159TIFF0007727616000015.tif255157

[0269] Quantifying the relative performance of engineered TE variants To quantify the performance of each engineered TE variant, cultures of cells expressing the variants were grown under conditions that support expression of the TE, CarB, AlrA, and Atf1, as described below, and the resulting fatty acid derivatives were extracted and quantified by gas chromatography with a flame ionization detector (GC / FID).

[0270] The composition and quantity of the resulting fatty acid derivatives (fatty acids, fatty alcohols, and fatty alcohol acetate esters) were determined and then compared to those produced under identical conditions by a control evaluation strain expressing a control TE. Briefly, a single colony of each strain was inoculated into a well of a 96-well plate containing 200 μL of Luria Bertani medium containing the appropriate antibiotic. 40 μL of this culture was used to inoculate 360 ​​μL of the same medium in a 96-deep-well plate, which was shaken at 32°C for 4 hours. 40 μL of this culture was used to inoculate 360 ​​μL of production medium (Table 8) in a final 96-deep-well plate. These cultures were overlaid with 60 μL of hexadecane and shaken at 32°C for 2 hours. IPTG was added (to 1 mM) to induce expression of the TE, CarB, AlrA, and Atf1, and shaking was continued for an additional 20 hours. The cultures were then evaluated as described below.

[0271] Table 8: Production medium TIFF0007727616000016.tif123128

[0272] Sample preparation and quantification of fatty acid derivatives (FAS) 400 μL of butyl acetate (containing 500 mg / L undecanol as an internal analytical standard) was added to each well, the plate was heat-sealed, shaken at 2000 rpm for 15 minutes, centrifuged at 4500 rpm for 10 minutes at room temperature, and 100 μL of the upper organic layer was transferred to a 96-well plate containing 100 μL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) (see, e.g., Stalling DL, et al. Biochemical and Biophysical Research Communications. 1968 May 23;31(4):616-22). The plate was sealed and analyzed by gas chromatography with a flame ionization detector (GC-FID).

[0273] A control evaluator strain was included on each plate as an "internal plate control" for strains expressing engineered TE variants. To determine the relative performance of the engineered TE variants, the total amount of fatty acid derivatives (products resulting from the action of the expressed TE and downstream convertases CarB, AlrA, and Atf1: fatty acids, fatty aldehydes, fatty alcohols, and fatty alcohol acetate esters) or specific fatty acid derivatives (specific chain lengths, e.g., C8 or C10) was quantified and then compared to the same parameters for the control evaluator strain, reported as fold over control (FOC). For example, the FOC for the total FAS titer of mutant A was determined by adding the total titers of all fatty acid species identified in extracts of mutant A and dividing it by the total FAS titer of the internal control evaluator strain. Engineered TE variants with improved activity relative to the control would exhibit an FOC greater than 1.0 for the reported parameter. The FOC of total C8 FAS of mutant A was determined by adding the total concentrations of all fatty acid species with a C8 chain length identified in extracts of mutant A and dividing it by the total concentration of all fatty acid species with a C8 chain length identified for the internal control evaluation strain. For engineered thioesterase variants containing single amino acid substitutions, the primary metrics used to identify hits were: (a) improved FOC of total FAS, (b) improved FOC of %C8 FAS relative to total FAS, and / or (c) improved %C8 / %C10.

[0274] The mutations shown in Table 7 (above) were surprisingly identified as having the ability to significantly (a) improve the FOC of total FAS; and (b) improve the FOC of %C8 FAS relative to total FAS. Thus, engineered thioesterase (TE) variants containing the mutations listed in Table 7 represent novel engineered TE variants with improved activity for the production of fatty acid derivatives. In particular, the engineered TE variants shown in Table 7 represent novel TE variants with improved activity for the production of C8 and / or C10 fatty acid derivatives.

[0275] Example 5 TE variants were engineered to contain multiple amino acid substitutions that resulted in new TEs with improved activity for the production of medium-chain fatty acid derivatives. The variants had improved activity and selectivity relative to the native thioesterase enzyme (SEQ ID NO:1) and relative to engineered TE variants with single amino acid substitutions (Example 4, Table 7).

[0276] As in Example 4, genes encoding engineered TE variants with multiple amino acid substitutions were synthesized and cloned into expression vectors that affected their expression when grown in the presence of IPTG. These were transformed into an E. coli strain derived from MG1655 that had been engineered to overexpress the gene EntD from the chromosome (see, e.g., WO2010062480) and harbors a Ptrc-regulated operon expressing the genes carB, alrA, and aftA1, which together affect the biochemical conversion of free fatty acids (FFA) to fatty alcohol acetate esters (FACE), as described in Example 3 above. The engineered TEs with multiple amino acid substitutions were compared to specific control evaluation strains that were identical except for the TE expression.

[0277] Table 7 lists novel engineered TEs that have multiple amino acid substitutions and that show improved activity for the production of medium-chain fatty acid derivatives (FAS) and improved selectivity for producing medium-chain length fatty acid derivatives (SEQ ID NO: 2 through SEQ ID NO: 15) compared to the listed control evaluator strains and TEs. Thus, because the novel engineered TEs are thioesterases with improved activity for the production of medium-chain length fatty acid derivatives, the novel engineered TEs, their individual mutations, and unique combinations of these mutations are each useful tools for the production of medium-chain length fatty acid derivatives.

[0278] Example 6 The following examples illustrate engineered TE variants with increased surface charge and improved activity for the production of medium-chain fatty acid derivatives. Three-dimensional modeling was used to engineer thioesterase variants / mutants with improved activity for the production of medium-chain fatty acid derivatives.

[0279] In some embodiments, SEQ ID NO:1 appears to be toxic when overexpressed in E. coli. Without being bound by theory, it is believed that SEQ ID NO:1 may be unstable or easily aggregate at high concentrations within cells. Therefore, to reduce the potential toxicity of the protein, a three-dimensional model was constructed on a computer and used to modify the surface charge of SEQ ID NO:1.

[0280] In this example, a three-dimensional molecular model of SEQ ID NO:1 was constructed by computer using the x-ray crystal structures of other acyl-ACP thioesterases as templates. Based on this model, specific residues of the SEQ ID NO:1 enzyme that were predicted to alter the net surface charge were identified. Specifically, negatively charged residues (Asp or Glu) on the enzyme surface were mutated to positively charged residues (Arg or His), thereby modifying the net surface charge from +15 to +25. As shown in Table 7, the resulting engineered TE variants with increased surface charge produce a higher percentage of C8 fatty acid derivatives. Therefore, the engineered TE variants have improved activity for the production of medium-chain fatty acid derivatives.

[0281] 3-D Modeling of SEQ ID NO:1 Thioesterase: Because an experimental 3D structure of SEQ ID NO:1 was not available, a homology-based 3D model of this enzyme was constructed in silico as disclosed in steps 1-5 below.

[0282] (1) Identification of thioesterases with known structural homology The Protein Data Bank (PDB) is the only worldwide archive of structural data for biological macromolecules (see Berman, HM et al, Nucl. Acids Res. (2000) 28 (1): 235-242). The PDB Protein Data Bank is available on the World Wide Web at rcsb.org / pdb / home / home.do. The PDB data bank was used to identify three solved x-ray crystal structures of thioesterases. In particular, three solved structures of identified thioesterases were (1) acyl-ACP thioesterase from Bacteroides thetaiotaomicron with Protein Data Bank identification number (PDB ID: 2ESS), (2) oleoyl thioesterase from Lactobacillus plantarum (PDB ID: 2OWN), and (3) acyl-ACP thioesterase from Spirosoma linguale (PDB ID: 4GAK). These structures, which show approximately 25% overall sequence identity to SEQ ID NO: 1, were used as templates.

[0283] (2) Alignment of the query sequence against the template structure The three solved 3D structures of thioesterases identified in the PDB (2ESS, 2OWN, and 4GAK) and their sequences were aligned using the PROMALS3D multiple sequence and structure alignment server, available on the World Wide Web at prodata.swmed.edu / promals3d / promals3d.php (see, e.g., J. Pei and NV Grishin (2007) Bioinformatics. 23(7): 802-808; J. Pei et al., (2008) Nucl. Acids Res. 36(7): 2295-2300). After aligning the sequences and structures of 2ESS, 2OWN, and 4GAK, the query sequence of SEQ ID NO: 1 was aligned to the existing structure-based sequence alignment using MMFFT version 7 (see, e.g., Katoh, K., et al. (2013) Mol. Biol. Evol. Apr; 30(4): 772-780). The software is available on the World Wide Web at mafft.cbrc.jp / alignment / software. An alignment of SEQ ID NO:1 and the acyl-ACP thioesterases (2ESS, 2OWN and 4GAK) identified in the PDB is shown in FIG.

[0284] (3) Construction of a homology 3D structural model of SEQ ID NO:1 thioesterase A homology model of amino acids 37-310 was constructed using MODELLER software (see, e.g., B. Webb, A. Sali. Comparative Protein Structure Modeling Using Modeller. Current Protocols in Bioinformatics, John Wiley & Sons, Inc., 5.6.1-5.6.32, 2014) using all three templates 2ESS, 2OWN, and 4GAK and the structure-based alignment described in step 2 above. Further structural refinement was performed using the MODELLER built-in refinement mode. Refinement was performed with all default parameters using the VTFM optimization and MD refinement modules. Information about the MODELLER software and downloads is available on the World Wide Web at salilab.org / modeller.

[0285] (4) Construction of ab initio models of the N- and C-terminal domains As shown in Figure 9, the SEQ ID NO:1 thioesterase used in these experiments has N- and C-terminal residues (36 amino acids at the N-terminus and 18 amino acids at the C-terminus) that are not included in the template x-ray crystal structure. Therefore, there are no suitable templates for constructing homology-based models for these portions. Therefore, ab initio models for both the N- and C-termini (see, e.g., J. Lee et al., (2009) Ab Initio Protein Structure Prediction pgs. 3-25 In: From Protein Structure to Function with Bioinformatics, DJ Rigden (ed.) Springer) were constructed using the ROBETTA server (see, e.g., Kim, DE, et al. (2004) Nucleic Acids Res. Jul 1; 32 (Web Server issue): W526-W531; available on the World Wide Web at robetta.org).

[0286] (5) Building a Full-Length Model for SEQ ID NO:1 Thioesterase and Engineered TE Variants A full-length model was generated using MODELLER software using three templates: a major segment homology model, an N-terminal ab initio model, and a C-terminal ab initio model.

[0287] The improved engineered TE variant with amino acid substitutions P3K, L176V, D196V, K203R, and V282S (SEQ ID NO:4) compared to the wild-type control demonstrated improved medium-chain fatty acid derivative production capacity (Example 5, Table 7). Therefore, the model of SEQ ID NO:1 was remodeled to SEQ ID NO:4 by essentially replacing the five variant residues and re-performing structural refinement in the MODELLER built-in refinement mode. Surface residues were then defined based on the final model (Figure 10).

[0288] Creation of engineered TE variants with increased modeled surface charge Based on the 3D structural model for SEQ ID NO:4 described above, the 12 aspartic acid (D) and glutamic acid (E) residues were modeled to confer a negative charge on the surface of SEQ ID NO:4. Genes encoding engineered TE variants with various positive to negative substitutions of these 12 residues were then synthesized as described in Examples 4 and 5 and evaluated for improved production of medium-chain length fatty acid derivatives compared to the control TE (SEQ ID NO:4).

[0289] Table 7 lists a series of engineered TE variants (SEQ ID NO:16-SEQ ID NO:46) that have amino acid substitutions that result in increased modeled surface charge compared to SEQ ID NO:4 and have improved activity for the production of medium-chain length fatty acid derivatives. Thus, the TEs listed in Table 7 as SEQ ID NO:16-SEQ ID NO:46 are novel engineered TE variants useful for the production of medium-chain length fatty acid derivatives. Furthermore, engineered variant TEs with amino acid substitutions that increase the modeled surface charge are useful for the improved production of medium-chain length fatty acid derivatives compared to TEs that do not have the altered increased modeled surface charge.

[0290] Creation of novel thioesterases containing multiple engineered mutations with modeled increased surface charge and multiple engineered mutations that increase activity and / or selectivity for the production of medium-chain length fatty acid derivatives Amino acid substitutions predicted to increase thioesterase surface charge, identified by 3-D modeling as described above, and resulting in improved production of medium-chain fatty acid derivatives (Table 7) were combined with an engineered TE variant having SEQ ID NO:15, which had improved activity for medium-chain fatty acid derivative production relative to its corresponding control (Example 6, Table 7).

[0291] As in Example 5, genes encoding engineered TE variants with multiple amino acid substitutions were synthesized and cloned into expression vectors that affected their expression when grown in the presence of IPTG. These were transformed into an MG1655-derived E. coli strain engineered to overexpress the gene EntD from the chromosome (see, e.g., WO2010062480) and carrying a Ptrc-regulated operon expressing the genes carB, alrA, and aftA1, which together affect the biochemical conversion of free fatty acids (FFA) to fatty alcohol acetate esters (FACE), as described in Example 3 above. The engineered TEs with multiple amino acid substitutions were compared to a control evaluation strain, the only difference being that the expressed TE was the control TE SEQ ID NO:15. Table 7 lists a series of engineered TE variants (SEQ ID NO:47 to SEQ ID NO:51) from this example with improved fatty acid derivative production activity relative to the control (SEQ ID NO:15). The TEs listed in Table 7 are thioesterases with improved activity for the production of medium-chain fatty acid derivatives and are therefore novel engineered TE variants useful for the production of medium-chain length fatty acid derivatives.

[0292] Example 7 The following examples illustrate engineered TE variants with N-terminal truncations, increased solubility, and improved activity for the production of medium-chain length fatty acid derivatives.

[0293] Plant FatB-like thioesterases have signal peptides that mediate their translocation from the endoplasmic reticulum to plastids. These enzymes are known to contain an N-terminal hydrophobic region that remains after signal peptide processing. This region is thought to be involved in the association of the thioesterase to thylakoid membranes. When expressed in microorganisms such as E. coli, the wild-type (SEQ ID NO:1) and the novel engineered TE variants described above are insoluble and associate with the membrane pellet upon cell lysis and centrifugation. The low enzyme solubility suggested that much of the enzyme may be membrane-associated or misfolded and inactive.

[0294] To generate novel engineered TE variants with improved solubility and activity for the production of medium-chain fatty acid derivatives, a polypeptide having SEQ ID NO:49 was engineered to have a truncation between amino acids 2 and 40, a region modeled to harbor key hydrophobic residues suspected to be responsible for the poor solubility of this enzyme. The solubility and activity of these engineered TE variants was then evaluated in comparison to a control TE of the same amino acid sequence that does not have the truncation between amino acids 2 and 40.

[0295] Evaluation of the solubility of engineered TE variants with truncations between amino acids 2 and 40 Genes encoding engineered TE variants with deletions between amino acids 2 and 40 of SEQ ID NO:49 were synthesized and cloned under the control of the Ptrc promoter in a medium-copy-number pACYC-based expression plasmid (GenBank Accession No. X06403). These plasmids were then transformed into E. coli and evaluated for their ability to direct expression of TE variants with increased solubility compared to the same strain carrying a plasmid directing expression of a control TE (SEQ ID NO:49). The only difference between strains expressing engineered TE variants with truncations between amino acids 2 and 40 and strains expressing the control TE (SEQ ID NO:49) was the sequence of the expressed TE. The resulting strains expressing the control and truncated TEs were each grown in 96-well plates under conditions conducive to expression of the TE-encoding gene, as described in Example 4. Cells were harvested by centrifugation and resuspended in 50 μL of 50 mM Tris-HCl (pH 7.8) containing 25 mM NaCl, 5 mM EDTA, and 1 mg / mL lysozyme. The samples were incubated at 25°C and 1500 rpm. After 20 minutes, 10 μl of 1 mg / mL DNase I solution and 10 μL of 1 M MgSO4 were added to each sample. The samples were then shaken at 1500 rpm for an additional 20 minutes. The resulting whole cell lysates (WCLs) were centrifuged at 4500 rpm for 10 minutes to separate the insoluble (pellet) and soluble (supernatant) fractions.

[0296] Western blots using an antibody directed against the C-terminus of TE were used to track the presence of the control and engineered TE truncated variants in the soluble fraction of WCL. As shown in Figure 11, the control polypeptide (thioesterase having SEQ ID NO:49) is visible in the WCL (indicating that this polypeptide is expressed in host cells) but is absent from the soluble fraction, indicating low solubility. On the other hand, the engineered truncated TE variants were found in both the WCL and the soluble fraction, and all TE variants demonstrate a significant increase in the presence of TE in the soluble fraction.

[0297] This demonstrates that the solubility of plant FatB-like thioesterases, such as SEQ ID NO:1, which are insoluble when expressed in microorganisms, can be improved by expressing engineered variants in which the N-terminus of the enzyme is truncated. It further shows that the engineered truncated variants have increased solubility when expressed in E. coli compared to TEs without this truncation, such as the control TE (SEQ ID NO:49).

[0298] Growth and fatty acid derivative production Increasing the solubility of poorly soluble medium-chain length TEs should result in more active medium-chain length TEs being present in the cell, which in turn should result in increased production of medium-chain fatty acids.

[0299] To assess the relative activity of the engineered TE truncation variants, each enzyme was cloned into a vector such that the gene was under the transcriptional control of the Ptrc promoter (Camsund et al., supra), which is induced in the presence of isopropylthiogalactoside (IPTG) as described in Example 4. These were transformed into an E. coli mother strain derived from MG1655 that had been engineered to overexpress the gene EntD from the chromosome and harbors a Ptrc-regulated operon expressing the genes carB, alrA, and aftA1 (described in Example 3) for the biochemical conversion of free fatty acids (FFA) to fatty alcohol acetate esters (FACE). The engineered E. coli mother strain was designed to accommodate the expected high activity of these more soluble engineered TE variants for high productivity of fatty acid derivatives.

[0300] The performance of each of the engineered truncated thioesterases was compared to a control evaluator strain expressing a control TE (SEQ ID NO:49). The only difference between the evaluator strains expressing the engineered truncated TE variants and the control evaluator strain expressing the control TE (SEQ ID NO:49) was the sequence of the gene encoding the expressed TE. Each of the strains was grown for the production of medium-chain fatty alcohol acetate esters, and the resulting medium-chain fatty acid-derived products were extracted and quantified as described in Example 4.

[0301] The activity of each engineered truncated TE variant was assessed by comparing the resulting fatty acid derivative products to those produced by a control evaluation strain (expressing SEQ ID NO:49) as described in Example 4.

[0302] Table 7 lists engineered truncated TE variants (SEQ ID NO:52 to SEQ ID NO:59) with improved performance, reported as fold over internal control (FOC), for (1) solubility (FIG. 11) and (2) activity for the production of medium-chain fatty acid derivatives. Thus, the truncated mutants are thioesterase variants with improved activity for the production of medium-chain fatty acid derivatives.

[0303] To further demonstrate the improved activity of the engineered truncated TE variants with increased solubility, strains expressing SEQ ID NO:55 and SEQ ID NO:56 were grown in 5 L bioreactors as described in Example 9 and compared to a control evaluation strain expressing TE SEQ ID NO:49 grown under the same conditions. Table 9 describes the performance of these strains, reported as fold over control (FOC) at 72 hours. Both SEQ ID NO:55 and SEQ ID NO:56 show improved activity (FAS FOC) and selectivity (%C8 FAS, and %C8 / %C10 FOC) at this larger scale.

[0304] Table 9. Engineered truncated TE variants showing improved in vivo activity for the production of medium-chain fatty acid derivatives when grown in 5 L bioreactors. TIFF0007727616000017.tif39141

[0305] Example 8 The following examples illustrate processes that can be used to produce fatty acid derivatives using genetically modified microorganisms with improved activity for the production of medium-chain fatty acid derivatives. The fatty acid derivative compositions produced by this process include, but are not limited to, medium-chain fatty acids, medium-chain fatty alcohols, medium-chain fatty alcohol acetates (FACEs), medium-chain fatty acid methyl esters (FAMEs), medium-chain fatty acid ethyl esters (FAEEs), and other medium-chain fatty acid esters.

[0306] Seed culture expansion generation A frozen cell bank vial of the selected engineered E. coli strain was used to inoculate 20 mL of LB medium in a 125 mL baffled shake flask containing the appropriate antibiotic. The shake flasks were incubated at 32°C in an orbital shaker for approximately 6 hours, and then 1.25 mL of medium (1% v / v) was added to a 500 mL baffled Erlenmeyer shake flask containing minimal overnight seed medium (2 g / L NH4Cl, 0.5 g / L NaCl, 0.3 g / L KH2PO4, 1 mM MgSO4, 0.1 mM CaCl2, 20 g / L glucose, 1 mL / L trace mineral solution (2 g / L ZnCl2·4H2O, 2 g / L CaCl2·6H2O, 2 g / L Na2MoO4·2H2O, 1.9 g / L CuSO4·5H2O, 0.5 g / L H3BO3, and 10 mL / L concentrated HCl), 10 mg / L ferric citrate, 100 mM Bis-Tris buffer (pH 7.0). 7.0), and the appropriate antibiotic) and incubated overnight at 32°C on a shaker.

[0307] Bioreactor Culture Protocol 75 mL (5% v / v) of the above overnight seed culture was used to inoculate a 5 L Biostat Aplus bioreactor (Sartorius BBI) initially containing 1.5 L of sterilized bioreactor fermentation medium. This medium consisted of 2 g / L KH2PO4, 0.5 g / L (NH4)2SO4, 2.2 g / L MgSO4 heptahydrate, 10 g / L sterile-filtered glucose, 80 mg / L ferric citrate, 1 mL / L of the trace mineral solution described above, 0.25 mL / L of a vitamin solution (0.42 g / L riboflavin, 5.4 g / L pantothenic acid, 6 g / L niacin, 1.4 g / L pyridoxine, 0.06 g / L biotin, and 0.04 g / L folic acid), 1 g / L NaCl, 1 g / L citric acid, 140 mg / L CaCl2 dihydrate, 10 mg / L ZnCl2, and the appropriate antibiotics. The culture pH was maintained between 6.9 and 7.2 using 28% w / v aqueous ammonia, the culture temperature was 33–35°C depending on the specific product, the aeration rate was 0.75 lpm (0.5 v / v / m), and the dissolved oxygen tension was maintained at 30% saturation using a stirring loop connected to a DO controller and oxygen supply. Foam formation was controlled by the automatic addition of a silicone emulsion antifoam agent (Dow Corning 1430).

[0308] Nutrient feed consisting of approximately 50% w / w glucose (600 g / L) was initiated once the glucose in the initial medium was completely depleted (approximately 7 hours after inoculation) and was fed as needed at a rate of 10 g / L / h using a DO stat controller strategy (each feeding shot was 1 hour in duration). Genes involved in the production of medium-chain fatty acid derivatives were induced by adding isopropylthiogalactoside (IPTG) to a final concentration of 1 mM. The bioreactor run was stopped at approximately 72 hours of elapsed fermentation time. Samples of the fermentation broth were taken into the tank throughout the fermentation process.

[0309] Analysis of broth composition The fatty acid derivatives present in the fermentation broth samples were extracted and separated in a single run using conventional GC-FID. To this end, 0.5 mL of each homogenized fermentation broth sample was divided into 15 mL Falcon tubes. The sample mass was recorded, and 5.0 mL of butyl acetate containing 500 ppm of an internal standard (C11 FAME or C9 / C11 / C15 FALC) was added to the broth to achieve a 10-fold extraction. The sample was mechanically shaken at 2500 rpm for 30 minutes and centrifuged at 4500 rpm for 10 minutes at 25°C. 50 μL of the extract (top layer) was transferred to a GC vial and derivatized with 50 μL of BSTFA w / 10% TCMS, followed by vortexing for approximately 15 seconds. The sample was then run on a conventional GC-FID system using an Agilent DB1 column, 10 m x 180 μm x 0.2 μm, to separate all fatty acid derivatives present in the extracted sample. The concentration of each fatty acid derivative is reported in g / Kg.

[0310] Example 9 The following example illustrates a process that can be used to produce medium-chain fatty alcohol acetate esters using genetically modified microorganisms with improved activity for the production of medium-chain fatty acid derivatives. The composition of the fatty acid derivatives produced by this process can include medium-chain fatty acids, medium-chain fatty alcohols, and medium-chain fatty alcohol acetate esters (FACEs) with acyl chains of 6 to 12 carbons. Production of medium-chain fatty alcohol acetate esters in a 5 L bioreactor was carried out as described in Example 8.

[0311] In this example, E. coli strains derived from MG1655 were used that had been engineered to overexpress the gene EntD from the chromosome and possessed high productivity for medium-chain fatty acid derivatives. These strains contained an operon expressing an engineered thioesterase with improved activity for the production of medium-chain fatty acids, as well as the genes carB, alrA, and aftA1 (described in Example 3) for the biochemical conversion of free fatty acids (FFA) to fatty alcohol acetate esters (FACE). The engineered thioesterase having SEQ ID NO:9 was expressed in strain sRG.825, while the engineered thioesterase having SEQ ID NO:49 was expressed in strain sDH.377. The genes encoding the engineered thioesterases, carB, alrA, and aftA1, were all under the transcriptional control of an inducible (Ptrc) promoter that was activated by adding isopropylthiogalactoside (IPTG) to the bioreactor at approximately 24 hours of elapsed fermentation time. The bioreactor run was stopped at approximately 72 hours elapsed fermentation time and the fermentation broth was collected and analyzed as described above in Example 8. The results are shown in Table 10 and Figure 12.

[0312] Table 10. Total fatty acid species (FAS) concentrations produced by representative strains engineered for medium-chain fatty alcohol acetate or fatty acid alkyl ester production TIFF0007727616000018.tif22132

[0313] Example 10 The following example illustrates a process that can be used to produce medium-chain fatty alcohols using genetically modified microorganisms with improved ability to produce fatty acid derivatives. The composition of the fatty acid derivatives produced by this process can include fatty acids, fatty aldehydes, and fatty alcohols with acyl chains of 6 to 12 carbons. Production of medium-chain fatty alcohols in a 5 L bioreactor was carried out using an E. coli strain engineered to overexpress the gene EntD from the chromosome and containing an operon expressing a medium-chain thioesterase and the genes carB and alrA for the biochemical conversion of free fatty acids (FFAs) to fatty alcohols (FALCs), as described in Example 8. The thioesterase-encoding genes, carB and alrA, were all under the transcriptional control of an inducible (Ptrc) promoter that was activated by adding isopropylthiogalactoside (IPTG) to the bioreactor at approximately 7 hours of elapsed fermentation time. Medium-chain fatty alcohols are highly toxic to E. coli, and therefore, accumulation of these compounds during production in a 5 L bioreactor quickly stopped growth and production after reaching inhibitory concentrations (less than 1 g / L, see Example 3).

[0314] Example 11 The following examples illustrate a process for producing medium-chain fatty acid alkyl esters using a genetically engineered microorganism containing a thioesterase variant with improved activity for the production of medium-chain fatty acid derivatives. The composition of the fatty acid derivatives produced by this process can include medium-chain fatty acids, medium-chain fatty acid methyl esters (FAMEs), and / or medium-chain fatty acid ethyl esters (FAEEs) with acyl chains of 6 to 12 carbons.

[0315] This example illustrates the production of fatty acid ethyl esters (FAEEs) using an E. coli strain (sAZ918) derived from MG1655 that was engineered to possess high productivity for medium-chain fatty acid derivatives. This strain contained an operon expressing an engineered thioesterase with improved activity for the production of medium-chain fatty acids (SEQ ID NO:49) and an acyl-CoA synthetase and ester synthase (described in Example 3) for the biochemical conversion of free fatty acids (FFAs) to fatty acid alkyl esters (FAMEs or FAEEs). The engineered thioesterase, acyl-CoA synthetase, and ester synthase were all under the transcriptional control of an inducible (Ptrc) promoter that was activated by the addition of isopropylthiogalactoside (IPTG).

[0316] Production of medium-chain fatty acid ethyl esters in a 5L bioreactor was carried out as described in Example 8, but with the addition of ethanol to the nutrient feed. After inoculation of the 5L bioreactor with a seed culture, a nutrient feed consisting of 47.5% w / w glucose and 50 mL / L ethanol was initiated once the glucose in the initial medium was completely depleted (approximately 7 hours after inoculation) and fed on demand at a rate of 10 g / L / h using a pH stat controller strategy (each feeding shot was 1 hour in duration). Once this parameter value was achieved, the minimum agitation speed was fixed at 1200 rpm to prevent biofilm from coating the dissolved oxygen probe and resulting in a falsely low signal reading. Additional ethanol was added to the culture once the residual concentration fell below 10 g / L. The ethyl octanoate production pathway of the strain was induced by adding IPTG to a final concentration of 1 mM at approximately 24 hours of elapsed fermentation time. The bioreactor run was stopped at approximately 72 hours of elapsed fermentation time. The fermentation broth was collected and analyzed as in Example 8 above.

[0317] The results are shown in Figure 13.

[0318] Example 12 The following example illustrates a process for the production of medium-chain fatty acids using a genetically modified microorganism containing a thioesterase with improved activity for the production of medium-chain fatty acid derivatives. The fatty acid composition produced by this process includes medium-chain fatty acids with acyl chains of 6 to 12 carbons. In this example, production of medium-chain fatty acids in a 5 L bioreactor was carried out using an E. coli strain engineered to overexpress medium-chain thioesterase under the transcriptional control of an inducible (Ptrc) promoter, activated by the addition of isopropylthiogalactoside (IPTG) to the bioreactor at approximately 13 hours of elapsed fermentation time, as described in Example 8. Medium-chain fatty acids are highly toxic to E. coli; therefore, accumulation of these compounds during production in a 5 L bioreactor quickly stopped growth and production after reaching an inhibitory concentration (less than 5 g / L, see Example 3).

[0319] Appendix A: Arrays TIFF0007727616000019.tif216157TIFF0007727616000020.tif216157TIFF0007727616000021.tif206157 TIFF0007727616000022.tif215157TIFF0007727616000023.tif215157TIFF0007727616000024.tif218157 TIFF0007727616000025.tif204157TIFF0007727616000026.tif215157TIFF0007727616000027.tif215157 TIFF0007727616000028.tif218157TIFF0007727616000029.tif216157TIFF0007727616000030.tif206157

[0320] As will be apparent to those skilled in the art, various modifications and variations of the above aspects and embodiments can be made without departing from the spirit and scope of the disclosure.

[0321] Sequence information SEQUENCE LISTING <110> GENOMATICA, INC. <120> THIOESTERASE VARIANTS HAVING IMPROVED ACTIVITY FOR THE PRODUCTION OF MEDIUM-CHAIN FATTY ACID DERIVATIVES <150> US 62 / 481,078 <151> 2017-04-03 <160> 64 <170> PatentIn version 3.5 <210> 1 <211> 328 <212> PRT <213> Cuphea hookeriana <400> 1 Met Leu Pro Asp Trp Ser Arg Leu Leu Thr Ala Ile Thr Thr Val Phe 1 5 10 15 Val Lys Ser Lys Arg Pro Asp Met His Asp Arg Lys Ser Lys Arg Pro 20 25 30 Asp Met Leu Val Asp Ser Phe Gly Leu Glu Ser Thr Val Gln Asp Gly 35 40 45 Leu Val Phe Arg Gln Ser Phe Ser Ile Arg Ser Tyr Glu Ile Gly Thr 50 55 60 Asp Arg Thr Ala Ser Ile Glu Thr Leu Met Asn His Leu Gln Glu Thr 65 70 75 80 Ser Leu Asn His Cys Lys Ser Thr Gly Ile Leu Leu Asp Gly Phe Gly 85 90 95 Arg Thr Leu Glu Met Cys Lys Arg Asp Leu Ile Trp Val Val Ile Lys 100 105 110 Met Gln Ile Lys Val Asn Arg Tyr Pro Ala Trp Gly Asp Thr Val Glu 115 120 125 Ile Asn Thr Arg Phe Ser Arg Leu Gly Lys Ile Gly Met Gly Arg Asp 130 135 140 Trp Leu Ile Ser Asp Cys Asn Thr Gly Glu Ile Leu Val Arg Ala Thr 145 150 155 160 Ser Ala Tyr Ala Met Met Asn Gln Lys Thr Arg Arg Leu Ser Lys Leu 165 170 175 Pro Tyr Glu Val His Gln Glu Ile Val Pro Leu Phe Val Asp Ser Pro 180 185 190 Val Ile Glu Asp Ser Asp Leu Lys Val His Lys Phe Lys Val Lys Thr 195 200 205 Gly Asp Ser Ile Gln Lys Gly Leu Thr Pro Gly Trp Asn Asp Leu Asp 210 215 220 Val Asn Gln His Val Ser Asn Val Lys Tyr Ile Gly Trp Ile Leu Glu 225 230 235 240 Ser Met Pro Thr Glu Val Leu Glu Thr Gln Glu Leu Cys Ser Leu Ala 245 250 255 Leu Glu Tyr Arg Arg Glu Cys Gly Arg Asp Ser Val Leu Glu Ser Val 260 265 270 Thr Ala Met Asp Pro Ser Lys Val Gly Val Arg Ser Gln Tyr Gln His 275 280 285 Leu Leu Arg Leu Glu Asp Gly Thr Ala Ile Val Asn Gly Ala Thr Glu 290 295 300 Trp Arg Pro Lys Asn Ala Gly Ala Asn Gly Ala Ile Ser Thr Gly Lys 305 310 315 320 Thr Ser Asn Gly Asn Ser Val Ser 325 <210> 2 <211> 328 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 2 Met Leu Pro Asp Trp Ser Arg Leu Leu Thr Ala Ile Thr Thr Val Phe 1 5 10 15 Ala Lys Ser Lys Arg Pro Asp Met His Asp Arg Lys Ser Lys Arg Pro 20 25 30 Asp Met Leu Val Asp Ser Phe Gly Leu Glu Ser Thr Val Gln Asp Gly 35 40 45 Leu Val Phe Arg Gln Ser Phe Ser Ile Arg Ser Tyr Glu Ile Gly Arg 50 55 60 Asp Arg Thr Ala Ser Ile Glu Thr Leu Met Asn His Leu Gln Glu Thr 65 70 75 80 Ser Leu Asn His Cys Lys Ser Thr Gly Ile Leu Leu Asp Gly Phe Gly 85 90 95 Arg Thr Leu Glu Met Cys Lys Arg Asp Leu Ile Trp Val Val Ile Lys 100 105 110 Met Gln Ile Lys Val Asn Arg Tyr Pro Ala Trp Gly Asp Thr Val Glu 115 120 125 Ile Asn Thr Arg Phe Ser Arg Leu Gly Lys Ile Gly Met Gly Arg Asp 130 135 140 Trp Leu Ile Ser Asp Cys Asn Thr Gly Glu Ile Leu Val Arg Ala Thr 145 150 155 160 Ser Ala Tyr Ala Met Met Asn Gln Lys Thr Arg Arg Leu Ser Lys Val 165 170 175 Pro Tyr Glu Val His Gln Glu Ile Val Pro Leu Phe Val Asp Ser Pro 180 185 190 Val Ile Glu Asp Ser Asp Leu Lys Val His Arg Phe Lys Val Lys Thr 195 200 205 Gly Asp Ser Ile Gln Lys Gly Leu Thr Pro Gly Trp Asn Asp Leu Asp 210 215 220 Val Asn Gln His Val Ser Asn Val Lys Tyr Ile Gly Trp Ile Leu Glu 225 230 235 240 Ser Met Pro Thr Glu Val Leu Glu Thr Gln Glu Leu Cys Ser Leu Cys 245 250 255 Leu Glu Tyr Arg Arg Glu Cys Gly Arg Asp Ser Val Leu Glu Ser Val 260 265 270 Thr Ala Met Asp Pro Ser Lys Val Gly Ser Arg Ser Gln Tyr Gln His 275 280 285 Leu Leu Arg Leu Glu Asp Gly Thr Ala Ile Val Asn Gly Ala Thr Glu 290 295 300 Trp Arg Pro Lys Asn Ala Gly Ala Asn Gly Ala Ile Ser Thr Gly Lys 305 310 315 320 Thr Ser Asn Gly Asn Ser Val Ser 325 <210> 3 <211> 328 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 3 Met Leu Lys Asp Trp Ser Arg Leu Leu Thr Ala Ile Thr Thr Val Phe 1 5 10 15 Val Lys Ser Lys Arg Pro Asp Met His Asp Arg Lys Ser Lys Arg Pro 20 25 30 Asp Met Leu Val Pro Ser Phe Gly Leu Glu Ser Thr Val Gln Asp Gly 35 40 45 Leu Val Phe Arg Gln Ser Phe Ser Ile Arg Ser Tyr Glu Ile Gly Thr 50 55 60 Asp Arg Thr Ala Ser Ile Glu Thr Leu Met Asn His Leu Gln Glu Thr 65 70 75 80 Ser Leu Asn His Cys Lys Ser Thr Gly Ile Leu Leu Asp Gly Phe Gly 85 90 95 Arg Thr Leu Glu Met Cys Lys Arg Asp Leu Ile Trp Val Val Ile Lys 100 105 110 Met Gln Ile Lys Val Asn Arg Tyr Pro Ala Trp Gly Asp Thr Val Glu 115 120 125 Val Asn Thr Arg Phe Ser Arg Leu Gly Lys Ile Gly Met Gly Arg Asp 130 135 140 Trp Leu Ile Ser Asp Cys Asn Thr Gly Glu Ile Leu Val Arg Ala Thr 145 150 155 160 Ser Ala Tyr Ala Met Met Asn Gln Lys Thr Arg Arg Leu Ser Lys Val 165 170 175 Pro Tyr Glu Val His Gln Glu Ile Val Pro Leu Phe Val Asp Ser Pro 180 185 190 Val Ile Glu Asp Ser Asp Leu Lys Val His Lys Phe Lys Val Lys Thr 195 200 205 Gly Asp Ser Ile Gln Lys Gly Leu Thr Pro Gly Trp Asn Asp Leu Asp 210 215 220 Val Asn Gln His Val Ser Asn Val Lys Tyr Ile Gly Trp Ile Leu Glu 225 230 235 240 Ser Met Pro Thr Glu Val Leu Glu Thr Gln Glu Leu Cys Ser Leu Ala 245 250 255 Leu Glu Tyr Arg Arg Glu Cys Gly Arg Asp Ser Val Leu Glu Ser Val 260 265 270 Thr Ala Met Asp Pro Ser Lys Val Gly Val Arg Ser Gln Tyr Gln His 275 280 285 Leu Leu Arg Leu Glu Asp Gly Thr Ala Ile Val Asn Gly Ala Thr Glu 290 295 300 Trp Arg Pro Lys Asn Ala Gly Ala Asn Gly Ala Ile Ser Thr Gly Lys 305 310 315 320 Thr Ser Asn Gly Asn Ser Val Ser 325 <210> 4 <211> 328 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 4 Met Leu Lys Asp Trp Ser Arg Leu Leu Thr Ala Ile Thr Thr Val Phe 1 5 10 15 Val Lys Ser Lys Arg Pro Asp Met His Asp Arg Lys Ser Lys Arg Pro 20 25 30 Asp Met Leu Val Asp Ser Phe Gly Leu Glu Ser Thr Val Gln Asp Gly 35 40 45 Leu Val Phe Arg Gln Ser Phe Ser Ile Arg Ser Tyr Glu Ile Gly Thr 50 55 60 Asp Arg Thr Ala Ser Ile Glu Thr Leu Met Asn His Leu Gln Glu Thr 65 70 75 80 Ser Leu Asn His Cys Lys Ser Thr Gly Ile Leu Leu Asp Gly Phe Gly 85 90 95 Arg Thr Leu Glu Met Cys Lys Arg Asp Leu Ile Trp Val Val Ile Lys 100 105 110 Met Gln Ile Lys Val Asn Arg Tyr Pro Ala Trp Gly Asp Thr Val Glu 115 120 125 Ile Asn Thr Arg Phe Ser Arg Leu Gly Lys Ile Gly Met Gly Arg Asp 130 135 140 Trp Leu Ile Ser Asp Cys Asn Thr Gly Glu Ile Leu Val Arg Ala Thr 145 150 155 160 Ser Ala Tyr Ala Met Met Asn Gln Lys Thr Arg Arg Leu Ser Lys Val 165 170 175 Pro Tyr Glu Val His Gln Glu Ile Val Pro Leu Phe Val Asp Ser Pro 180 185 190 Val Ile Glu Val Ser Asp Leu Lys Val His Arg Phe Lys Val Lys Thr 195 200 205 Gly Asp Ser Ile Gln Lys Gly Leu Thr Pro Gly Trp Asn Asp Leu Asp 210 215 220 Val Asn Gln His Val Ser Asn Val Lys Tyr Ile Gly Trp Ile Leu Glu 225 230 235 240 Ser Met Pro Thr Glu Val Leu Glu Thr Gln Glu Leu Cys Ser Leu Ala 245 250 255 Leu Glu Tyr Arg Arg Glu Cys Gly Arg Asp Ser Val Leu Glu Ser Val 260 265 270 Thr Ala Met Asp Pro Ser Lys Val Gly Ser Arg Ser Gln Tyr Gln His 275 280 285 Leu Leu Arg Leu Glu Asp Gly Thr Ala Ile Val Asn Gly Ala Thr Glu 290 295 300 Trp Arg Pro Lys Asn Ala Gly Ala Asn Gly Ala Ile Ser Thr Gly Lys 305 310 315 320 Thr Ser Asn Gly Asn Ser Val Ser 325 <210> 5 <211> 328 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 5 Met Leu Pro Asp Trp Ser Arg Leu Leu Thr Ala Ile Thr Thr Val Phe 1 5 10 15 Val Lys Ser Lys Arg Pro Asp Met His Asp Arg Lys Ser Lys Arg Pro 20 25 30 Asp Met Leu Val Asp Ser Phe Gly Leu Glu Ser Thr Val Gln Asp Gly 35 40 45 Leu Val Phe Arg Gln Ser Phe Ser Ile Arg Ser Tyr Glu Ile Gly Thr 50 55 60 Asp Arg Thr Ala Ser Ile Glu Thr Leu Met Asn His Leu Gln Glu Thr 65 70 75 80 Ser Leu Asn His Cys Lys Ser Thr Gly Ile Leu Leu Asp Gly Phe Gly 85 90 95 Arg Thr Leu Glu Met Cys Lys Arg Asp Leu Ile Trp Val Leu Ile Lys 100 105 110 Met Gln Ile Lys Val Asn Arg Tyr Pro Ala Trp Gly Asp Thr Val Glu 115 120 125 Val Asn Thr Arg Phe Ser Arg Leu Gly Lys Ile Gly Met Gly Arg Asp 130 135 140 Trp Leu Ile Ser Asp Cys Asn Thr Gly Glu Ile Leu Val Arg Ala Thr 145 150 155 160 Ser Ala Tyr Ala Met Met Asn Gln Lys Thr Arg Arg Leu Ser Lys Val 165 170 175 Pro Tyr Glu Val His Gln Glu Ile Val Pro Leu Phe Val Asp Ser Pro 180 185 190 Val Ile Glu Asp Ser Asp Leu Lys Val His Arg Phe Lys Val Lys Thr 195 200 205 Gly Asp Ser Ile Gln Lys Gly Leu Thr Pro Gly Trp Asn Asp Leu Asp 210 215 220 Val Asn Gln His Val Ser Asn Val Lys Tyr Ile Gly Trp Ile Leu Glu 225 230 235 240 Ser Met Pro Thr Glu Val Leu Glu Thr Gln Glu Leu Cys Ser Leu Ala 245 250 255 Leu Glu Tyr Arg Arg Glu Cys Gly Arg Asp Ser Val Leu Glu Ser Val 260 265 270 Thr Ala Met Asp Pro Ser Lys Val Gly Val Arg Ser Gln Tyr Gln His 275 280 285 Leu Leu Arg Leu Glu Asp Gly Thr Ala Ile Val Asn Gly Ala Thr Glu 290 295 300 Trp Arg Pro Lys Asn Ala Gly Ala Asn Gly Ala Ile Ser Thr Gly Lys 305 310 315 320 Thr Ser Asn Gly Asn Ser Val Lys 325 <210> 6 <211> 328 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 6 Met Leu Lys Asp Trp Ser Arg Leu Leu Thr Ala Ile Thr Thr Val Phe 1 5 10 15 Val Lys Ser Lys Arg Pro Asp Met His Asp Arg Lys Ser Lys Arg Pro 20 25 30 Asp Met Leu Val Asp Ser Phe Gly Leu Glu Ser Thr Val Gln Asp Gly 35 40 45 Leu Val Phe Arg Gln Ser Phe Ser Ile Arg Ser Tyr Glu Ile Gly Thr 50 55 60 Asp Arg Thr Ala Ser Ile Glu Thr Leu Met Asn His Leu Gln Glu Thr 65 70 75 80 Ser Leu Asn His Cys Lys Ser Thr Gly Ile Leu Leu Asp Gly Phe Gly 85 90 95 Arg Thr Leu Glu Met Cys Lys Arg Asp Leu Ile Trp Val Val Ile Lys 100 105 110 Met Gln Ile Lys Val Asn Arg Tyr Pro Ala Trp Gly Asp Thr Val Glu 115 120 125 Val Asn Thr Arg Phe Ser Arg Leu Gly Lys Ile Gly Met Gly Arg Asp 130 135 140 Trp Leu Ile Ser Asp Cys Asn Thr Gly Glu Ile Leu Val Arg Ala Thr 145 150 155 160 Ser Ala Tyr Ala Met Met Asn Gln Lys Thr Arg Arg Leu Ser Lys Val 165 170 175 Pro Tyr Glu Val His Gln Glu Ile Val Pro Leu Phe Val Asp Ser Pro 180 185 190 Val Ile Glu Asp Ser Asp Leu Lys Val His Lys Phe Lys Val Lys Thr 195 200 205 Gly Asp Ser Ile Gln Lys Gly Leu Thr Pro Gly Trp Asn Asp Leu Asp 210 215 220 Val Asn Gln His Val Ser Asn Val Lys Tyr Ile Gly Trp Ile Leu Glu 225 230 235 240 Ser Met Pro Thr Glu Val Leu Glu Thr Gln Glu Leu Cys Ser Leu Ala 245 250 255 Leu Glu Tyr Arg Arg Glu Cys Gly Arg Asp Ser Val Leu Glu Ser Val 260 265 270 Thr Ala Met Asp Pro Ser Lys Val Gly Val Arg Ser Gln Tyr Gln His 275 280 285 Leu Leu Arg Leu Glu Asp Gly Thr Ala Ile Val Asn Gly Ala Thr Glu 290 295 300 Trp Arg Pro Lys Asn Ala Gly Ala Asn Gly Ala Ile Ser Thr Gly Lys 305 310 315 320 Thr Ser Asn Gly Asn Ser Val Lys 325 <210> 7 <211> 328 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 7 Met Leu Pro Asp Trp Arg Arg Leu Leu Thr Ala Ile Thr Thr Leu Phe 1 5 10 15 Val Lys Ser Lys Arg Pro Asp Met His Asp Arg Lys Ser Lys Arg Pro 20 25 30 Asp Met Leu Val Asp Ser Phe Gly Leu Glu Ser Thr Val Gln Asp Gly 35 40 45 Leu Val Phe Arg Gln Ser Phe Ser Ile Arg Ser Tyr Glu Ile Gly Thr 50 55 60 Asp Arg Thr Ala Ser Ile Glu Thr Leu Met Asn His Leu Gln Glu Thr 65 70 75 80 Ser Leu Asn His Cys Lys Ser Thr Gly Ile Leu Leu Asp Gly Phe Gly 85 90 95 Arg Thr Leu Glu Met Cys Lys Arg Asp Leu Ile Trp Val Val Ile Lys 100 105 110 Met Gln Ile Lys Val Asn Arg Tyr Pro Ala Trp Gly Asp Thr Val Glu 115 120 125 Ile Asn Thr Arg Phe Ser Arg Leu Gly Lys Ile Gly Met Gly Arg Asp 130 135 140 Trp Leu Ile Ser Asp Cys Asn Thr Gly Glu Ile Leu Val Arg Ala Thr 145 150 155 160 Ser Ala Tyr Ala Met Met Asn Gln Lys Thr Arg Arg Leu Ser Lys Leu 165 170 175 Pro Tyr Glu Val His Gln Glu Ile Val Pro Leu Phe Val Asp Ser Pro 180 185 190 Val Ile Glu Val Ser Asp Leu Lys Val His Lys Phe Lys Val Lys Thr 195 200 205 Gly Asp Ser Ile Gln Lys Gly Leu Thr Pro Gly Trp Asn Asp Leu Asp 210 215 220 Val Asn Gln His Val Ser Asn Val Lys Tyr Ile Gly Trp Ile Leu Glu 225 230 235 240 Ser Met Pro Thr Glu Val Leu Glu Thr Gln Glu Leu Cys Ser Leu Ala 245 250 255 Leu Glu Tyr Arg Arg Glu Cys Gly Arg Asp Ser Val Leu Glu Ser Val 260 265 270 Thr Ala Met Asp Pro Ser Lys Val Gly Val Arg Ser Gln Tyr Gln His 275 280 285 Leu Leu Arg Leu Glu Asp Gly Thr Ala Ile Val Asn Gly Ala Thr Glu 290 295 300 Trp Arg Pro Lys Asn Ala Gly Ala Asn Gly Ala Thr Ser Thr Gly Lys 305 310 315 320 Thr Lys Asn Gly Asn Ser Val Lys 325 <210> 8 <211> 328 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 8 Met Leu Lys Asp Trp Ser Arg Leu Leu Thr Ala Ile Thr Thr Val Phe 1 5 10 15 Val Lys Ser Lys Arg Pro Asp Met His Asp Arg Lys Ser Lys Arg Pro 20 25 30 Asp Met Leu Val Asp Ser Phe Gly Leu Glu Ser Thr Val Gln Asp Gly 35 40 45 Leu Val Phe Arg Gln Ser Phe Ser Ile Arg Ser Tyr Glu Ile Gly Thr 50 55 60 Asp Arg Thr Ala Ser Ile Glu Thr Leu Met Asn His Leu Gln Glu Thr 65 70 75 80 Ser Leu Asn His Cys Lys Ser Thr Gly Ile Leu Leu Asp Gly Phe Gly 85 90 95 Arg Thr Leu Glu Met Cys Lys Arg Asp Leu Ile Trp Val Val Ile Lys 100 105 110 Met Gln Ile Lys Val Asn Arg Tyr Pro Ala Trp Gly Asp Thr Val Glu 115 120 125 Ile Asn Thr Arg Phe Ser Arg Leu Gly Lys Ile Gly Met Gly Arg Asp 130 135 140 Trp Leu Ile Ser Asp Cys Asn Thr Gly Glu Ile Leu Val Arg Ala Thr 145 150 155 160 Ser Ala Tyr Ala Met Met Asn Gln Lys Thr Arg Arg Leu Ser Lys Val 165 170 175 Pro Tyr Glu Val His Gln Glu Ile Val Pro Leu Phe Val Asp Ser Pro 180 185 190 Val Ile Glu Val Ser Asp Leu Lys Val His Lys Phe Lys Val Lys Thr 195 200 205 Gly Asp Ser Ile Gln Lys Gly Leu Thr Pro Gly Trp Asn Asp Leu Asp 210 215 220 Val Asn Gly His Val Ser Asn Val Lys Tyr Ile Gly Trp Ile Leu Glu 225 230 235 240 Ser Met Pro Thr Glu Val Leu Glu Thr Gln Glu Leu Cys Ser Leu Ala 245 250 255 Leu Glu Tyr Arg Arg Glu Cys Gly Arg Asp Ser Val Leu Glu Ser Val 260 265 270 Thr Ala Met Asp Pro Ser Lys Val Gly Val Arg Ser Gln Tyr Gln His 275 280 285 Leu Leu Arg Leu Glu Asp Gly Thr Ala Ile Val Asn Gly Ala Thr Glu 290 295 300 Trp Arg Pro Lys Asn Ala Gly Ala Asn Gly Ala Ile Ser Thr Gly Lys 305 310 315 320 Thr Ser Asn Gly Asn Ser Val Ser 325 <210> 9 <211> 328 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 9 Met Leu Pro Asp Trp Ser Arg Leu Leu Thr Ala Ile Thr Thr Val Phe 1 5 10 15 Val Lys Ser Lys Arg Pro Asp Met His Asp Arg Lys Ser Lys Arg Pro 20 25 30 Asp Met Leu Val Asp Ser Phe Gly Leu Glu Ser Gly Val Gln Asp Gly 35 40 45 Leu Val Phe Arg Gln Ser Phe Ser Ile Arg Ser Tyr Glu Ile Gly Pro 50 55 60 Asp Arg Thr Ala Ser Ile Glu Thr Leu Met Asn His Leu Gln Glu Thr 65 70 75 80 Ser Leu Asn His Cys Lys Ser Thr Gly Ile Leu Leu Asp Gly Phe Gly 85 90 95 Arg Thr Lys Glu Met Cys Lys Arg Asp Leu Ile Trp Val Val Ile Lys 100 105 110 Met Gln Ile Lys Val Asn Arg Tyr Pro Ala Trp Gly Asp Thr Val Glu 115 120 125 Ile Asn Thr Arg Phe Ser Arg Leu Gly Lys Ile Gly Met Gly Arg Asp 130 135 140 Trp Leu Ile Ser Asp Cys Asn Thr Gly Glu Ile Leu Val Arg Ala Thr 145 150 155 160 Ser Ala Tyr Ala Met Met Asn Gln Lys Thr Arg Arg Leu Ser Lys Leu 165 170 175 Pro Tyr Glu Val His Gln Glu Ile Val Pro Leu Phe Val Asp Ser Pro 180 185 190 Val Ile Glu Val Ser Asp Leu Lys Val His Arg Phe Lys Val Lys Thr 195 200 205 Gly Asp Ser Ile Gln Lys Gly Leu Thr Pro Gly Trp Asn Asp Leu Asp 210 215 220 Val Asn Gln His Val Ser Asn Val Lys Tyr Ile Gly Trp Ile Leu Glu 225 230 235 240 Ser Met Pro Thr Glu Val Leu Glu Thr Gln Glu Leu Cys Ser Leu Ala 245 250 255 Leu Glu Tyr Arg Arg Glu Cys Gly Arg Asp Ser Val Leu Glu Ser Val 260 265 270 Thr Ala Met Asp Pro Ser Lys Val Gly Ser Arg Ser Gln Tyr Gln His 275 280 285 Leu Leu Arg Leu Glu Asp Gly Thr Ala Ile Val Asn Gly Ala Thr Glu 290 295 300 Trp Arg Pro Lys Asn Ala Gly Ala Asn Gly Ala Arg Ser Thr Gly Lys 305 310 315 320 Thr Ser Asn Gly Asn Ser Val Ser 325 <210> 10 <211> 328 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 10 Met Leu Lys Asp Trp Ser Arg Leu Leu Thr Ala Ile Thr Thr Val Phe 1 5 10 15 Val Lys Ser Lys Arg Pro Asp Met His Asp Arg Lys Ser Lys Arg Pro 20 25 30 Asp Met Leu Val Asp Ser Phe Gly Leu Glu Ser Gly Val Gln Asp Gly 35 40 45 Leu Val Phe Arg Gln Ser Phe Ser Ile Arg Ser Tyr Glu Ile Gly Pro 50 55 60 Asp Arg Thr Ala Ser Ile Glu Thr Leu Met Asn His Leu Gln Glu Thr 65 70 75 80 Ser Leu Asn His Cys Lys Ser Thr Gly Ile Leu Leu Asp Gly Phe Gly 85 90 95 Arg Thr Lys Glu Met Cys Lys Arg Asp Leu Ile Trp Val Val Ile Lys 100 105 110 Met Gln Ile Lys Val Asn Arg Tyr Pro Ala Trp Gly Asp Thr Val Glu 115 120 125 Ile Asn Thr Arg Phe Ser Arg Leu Gly Lys Ile Gly Met Gly Arg Asp 130 135 140 Trp Leu Ile Ser Asp Cys Asn Thr Gly Glu Ile Leu Val Arg Ala Thr 145 150 155 160 Ser Ala Tyr Ala Met Met Asn Gln Lys Thr Arg Arg Leu Ser Lys Leu 165 170 175 Pro Tyr Glu Val His Gln Glu Ile Val Pro Leu Phe Val Asp Ser Pro 180 185 190 Val Ile Glu Val Ser Asp Leu Lys Val His Arg Phe Lys Val Lys Thr 195 200 205 Gly Asp Ser Ile Gln Lys Gly Leu Thr Pro Gly Trp Asn Asp Leu Asp 210 215 220 Val Asn Gln His Val Ser Asn Val Lys Tyr Ile Gly Trp Ile Leu Glu 225 230 235 240 Ser Met Pro Thr Glu Val Leu Glu Thr Gln Glu Leu Cys Ser Leu Ala 245 250 255 Leu Glu Tyr Arg Arg Glu Cys Gly Arg Asp Ser Val Leu Glu Ser Val 260 265 270 Thr Ala Met Asp Pro Ser Lys Val Gly Ser Arg Ser Gln Tyr Gln His 275 280 285 Leu Leu Arg Leu Glu Asp Gly Thr Ala Ile Val Asn Gly Ala Thr Glu 290 295 300 Trp Arg Pro Lys Asn Ala Gly Ala Asn Gly Ala Arg Ser Thr Gly Lys 305 310 315 320 Thr Ser Asn Gly Asn Ser Val Ser 325 <210> 11 <211> 328 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 11 Met Leu Lys Asp Trp Ser Arg Leu Leu Thr Ala Ile Thr Thr Val Phe 1 5 10 15 Val Lys Ser Lys Arg Pro Asp Met His Asp Arg Lys Ser Lys Arg Pro 20 25 30 Asp Met Leu Val Asp Ser Phe Gly Leu Glu Ser Thr Val Gln Asp Gly 35 40 45 Leu Val Phe Arg Gln Ser Phe Cys Ile Arg Ser Tyr Glu Ile Gly Thr 50 55 60 Asp Arg Thr Ala Ser Ile Glu Thr Leu Met Asn His Leu Gln Glu Thr 65 70 75 80 Ser Leu Asn His Cys Lys Ser Thr Gly Ile Leu Leu Asp Gly Phe Gly 85 90 95 Arg Thr Leu Glu Met Cys Lys Arg Asp Leu Ile Trp Val Leu Ile Lys 100 105 110 Met Gln Ile Lys Val Asn Arg Tyr Pro Ala Trp Gly Asp Thr Val Glu 115 120 125 Ile Asn Thr Arg Phe Ser Arg Leu Gly Lys Ile Gly Met Gly Arg Asp 130 135 140 Trp Leu Ile Ser Asp Cys Asn Thr Gly Glu Ile Leu Val Arg Ala Thr 145 150 155 160 Ser Ala Tyr Ala Met Met Asn Gln Lys Thr Arg Arg Leu Ser Lys Val 165 170 175 Pro Tyr Glu Val His Gln Glu Ile Val Pro Leu Phe Val Asp Ser Pro 180 185 190 Val Ile Glu Val Ser Asp Leu Lys Val His Arg Phe Lys Val Lys Thr 195 200 205 Gly Asp Ser Ile Gln Lys Gly Leu Thr Pro Gly Trp Asn Asp Leu Asp 210 215 220 Val Asn Gln His Val Ser Asn Val Lys Tyr Ile Gly Trp Ile Leu Glu 225 230 235 240 Ser Met Pro Thr Glu Val Leu Glu Thr Gln Glu Leu Cys Ser Leu Ala 245 250 255 Leu Glu Tyr Arg Arg Glu Cys Gly Arg Asp Ser Val Leu Glu Ser Val 260 265 270 Thr Ala Met Asp Pro Ser Lys Val Gly Val Arg Ser Gln Tyr Gln His 275 280 285 Leu Leu Arg Leu Glu Asp Gly Thr Ala Ile Val Asn Gly Ala Thr Glu 290 295 300 Trp Arg Pro Lys Asn Ala Gly Ala Asn Gly Ala Ile Ser Thr Gly Lys 305 310 315 320 Thr Ser Asn Gly Asn Ser Val Ser 325 <210> 12 <211> 328 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 12 Met Leu Pro Asp Trp Ser Arg Leu Leu Thr Ala Ile Thr Thr Val Phe 1 5 10 15 Cys Lys Ser Lys Arg Pro Asp Met His Asp Arg Lys Ser Lys Arg Pro 20 25 30 Asp Met Leu Val Asp Ser Phe Gly Leu Glu Ser Thr Val Gln Asp Gly 35 40 45 Leu Val Phe Arg Gln Ser Phe Ser Ile Arg Ser Tyr Glu Ile Gly Pro 50 55 60 Asp Arg Thr Ala Ser Ile Glu Thr Leu Met Asn His Leu Gln Glu Thr 65 70 75 80 Ser Leu Asn His Cys Lys Ser Thr Gly Ile Leu Leu Asp Gly Phe Gly 85 90 95 Arg Thr Lys Glu Met Cys Lys Arg Asp Leu Ile Trp Val Leu Ile Lys 100 105 110 Met Gln Ile Lys Val Asn Arg Tyr Pro Ala Trp Gly Asp Thr Val Glu 115 120 125 Ile Asn Thr Arg Phe Ser Arg Leu Gly Lys Ile Gly Met Gly Arg Asp 130 135 140 Trp Leu Ile Ser Asp Cys Asn Thr Gly Glu Ile Leu Val Arg Ala Thr 145 150 155 160 Ser Ala Tyr Ala Met Met Asn Gln Lys Thr Arg Arg Leu Ser Lys Val 165 170 175 Pro Tyr Glu Val His Gln Glu Ile Val Gly Leu Phe Val Asp Ser Pro 180 185 190 Val Ile Glu Val Ser Asp Leu Lys Val His Arg Phe Lys Val Lys Thr 195 200 205 Gly Asp Ser Ile Gln Lys Gly Leu Thr Pro Gly Trp Asn Asp Leu Asp 210 215 220 Val Asn Gln His Val Ser Asn Val Lys Tyr Ile Gly Trp Ile Leu Glu 225 230 235 240 Ser Met Pro Thr Glu Val Leu Glu Thr Gln Glu Leu Cys Ser Leu Ala 245 250 255 Leu Glu Tyr Arg Arg Glu Cys Gly Arg Asp Ser Val Leu Glu Ser Val 260 265 270 Thr Ala Met Asp Pro Ser Lys Val Gly Ser Arg Ser Gln Tyr Gln His 275 280 285 Leu Leu Arg Leu Glu Asp Gly Thr Ala Ile Val Asn Gly Ala Thr Glu 290 295 300 Trp Arg Pro Lys Asn Ala Gly Ala Asn Gly Ala Ile Ser Thr Gly Lys 305 310 315 320 Thr Ser Asn Gly Asn Ser Val Ser 325 <210> 13 <211> 328 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 13 Met Leu Pro Asp Trp Ser Arg Leu Leu Thr Ala Ile Thr Arg Val Phe 1 5 10 15 Val Lys Ser Lys Arg Pro Asp Met His Asp Arg Lys Ser Lys Arg Pro 20 25 30 Asp Met Leu Val Asp Ser Phe Gly Leu Glu Ser Gly Val Gln Asp Gly 35 40 45 Leu Val Phe Arg Gln Ser Phe Ser Ile Arg Ser Tyr Glu Ile Gly Pro 50 55 60 Asp Arg Thr Ala Ser Ile Glu Thr Leu Met Asn Leu Leu Gln Glu Thr 65 70 75 80 Ser Leu Asn His Cys Lys Ser Thr Gly Ile Leu Leu Asp Gly Phe Gly 85 90 95 Arg Thr Lys Glu Met Cys Lys Arg Asp Leu Ile Trp Val Val Ile Lys 100 105 110 Met Gln Ile Lys Val Asn Arg Tyr Pro Ala Trp Gly Asp Thr Val Glu 115 120 125 Ile Asn Thr Arg Phe Ser Arg Leu Gly Lys Ile Gly Met Gly Arg Asp 130 135 140 Trp Leu Ile Ser Asp Cys Asn Thr Gly Glu Ile Leu Val Arg Ala Thr 145 150 155 160 Ser Ala Tyr Ala Met Met Asn Gln Lys Thr Arg Arg Leu Ser Lys Leu 165 170 175 Pro Tyr Glu Val His Gln Glu Ile Ala Pro Leu Phe Val Asp Ser Pro 180 185 190 Val Ile Glu Val Ser Asp Leu Lys Val His Arg Phe Lys Val Lys Thr 195 200 205 Gly Asp Ser Ile His Lys Gly Leu Thr Pro Gly Trp Asn Asp Leu Asp 210 215 220 Val Asn Gln His Val Ser Asn Val Lys Tyr Ile Gly Trp Ile Leu Glu 225 230 235 240 Ser Met Pro Thr Glu Val Leu Glu Thr Gln Glu Leu Cys Ser Leu Ala 245 250 255 Leu Glu Tyr Arg Arg Glu Cys Gly Arg Asp Ser Val Leu Glu Ser Val 260 265 270 Thr Ala Met Asp Pro Ser Lys Val Gly Ser Arg Ser Gln Tyr Gln His 275 280 285 Leu Leu Arg Leu Glu Asp Gly Thr Ala Ile Val Trp Gly Ala Thr Glu 290 295 300 Trp Arg Pro Lys Asn Ala Gly Ala Asn Gly Ala Arg Ser Thr Gly Lys 305 310 315 320 Thr Ser Asn Gly Lys Ser Val Ser 325 <210> 14 <211> 328 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 14 Met Leu Pro Met Trp Ser Arg Leu Leu Thr Ala Ile Thr Thr Val Phe 1 5 10 15 Cys Lys Ser Lys Arg Pro Asp Met His Asp Arg Lys Ser Lys Arg Pro 20 25 30 Asp Met Leu Val Asp Ser Phe Gly Leu Glu Ser Gly Val Gln Asp Gly 35 40 45 Leu Val Phe Arg Gln Ser Phe Ser Ile Arg Ser Tyr Glu Ile Gly Pro 50 55 60 Asp Arg Thr Ala Ser Ile Glu Thr Leu Met Asn His Leu Gln Glu Thr 65 70 75 80 Ser Leu Asn His Cys Lys Ser Thr Gly Ile Leu Leu Asp Gly Phe Gly 85 90 95 Arg Thr Lys Glu Met Cys Lys Arg Asp Leu Ile Trp Val Val Ile Lys 100 105 110 Met Gln Ile Lys Val Asn Arg Tyr Pro Ala Trp Gly Asp Thr Val Glu 115 120 125 Ile Asn Thr Arg Phe Ser Arg Leu Gly Lys Ile Gly Met Gly Arg Asp 130 135 140 Trp Leu Ile Ser Asp Cys Asn Thr Gly Glu Ile Leu Val Arg Ala Thr 145 150 155 160 Ser Ala Tyr Ala Met Met Asn Gln Lys Thr Arg Arg Leu Ser Lys Leu 165 170 175 Pro Tyr Glu Val His Gln Glu Ile Ala Pro Leu Phe Val Asp Ser Pro 180 185 190 Val Ile Glu Asp Ser Asp Leu Lys Val His Arg Phe Lys Val Lys Thr 195 200 205 Gly Asp Ser Ile Gln Lys Gly Leu Thr Pro Gly Trp Asn Asp Leu Asp 210 215 220 Val Asn Gln His Val Ser Asn Val Lys Tyr Ile Gly Trp Ile Leu Glu 225 230 235 240 Ser Met Pro Thr Glu Val Leu Glu Thr Gln Glu Leu Cys Ser Leu Ala 245 250 255 Leu Glu Tyr Arg Arg Glu Cys Gly Arg Asp Ser Val Leu Glu Ser Val 260 265 270 Thr Ala Met Asp Pro Ser Lys Val Gly Ser Arg Ser Gln Tyr Gln His 275 280 285 Leu Leu Arg Leu Glu Asp Gly Thr Ala Ile Val Trp Gly Ala Thr Glu 290 295 300 Trp Arg Pro Lys Asn Ala Gly Ala Asn Gly Ala Arg Ser Thr Gly Lys 305 310 315 320 Thr Ser Asn Gly Lys Ser Val Ser 325 <210> 15 <211> 328 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 15 Met Leu Pro Asp Trp Ser Arg Leu Leu Thr Ala Ile Thr Thr Val Phe 1 5 10 15 Ala Lys Ser Lys Arg Pro Asp Met His Asp Arg Lys Ser Lys Arg Pro 20 25 30 Asp Met Leu Val Asp Ser Phe Gly Leu Glu Ser Gly Val Gln Asp Gly 35 40 45 Leu Val Phe Arg Gln Ser Phe Ser Ile Arg Ser Tyr Glu Ile Gly Pro 50 55 60 Asp Arg Leu Ala Ser Ile Glu Thr Leu Met Asn His Leu Gln Glu Thr 65 70 75 80 Ser Leu Asn His Cys Lys Ser Thr Gly Ile Leu Leu Asp Gly Phe Gly 85 90 95 Arg Thr Lys Glu Met Cys Lys Arg Asp Leu Ile Trp Val Val Ile Lys 100 105 110 Met Gln Ile Lys Val Asn Arg Tyr Pro Ala Trp Gly Asp Thr Val Glu 115 120 125 Ile Asn Thr Arg Phe Ser Arg Leu Gly Lys Ile Gly Met Gly Arg Asp 130 135 140 Trp Leu Ile Ser Asp Cys Asn Thr Gly Glu Ile Leu Val Arg Ala Thr 145 150 155 160 Ser Ala Tyr Ala Met Met Asn Gln Lys Thr Arg Arg Leu Ser Lys Leu 165 170 175 Pro Tyr Glu Val His Gln Glu Ile Ala Pro Leu Phe Val Asp Ser Pro 180 185 190 Val Ile Glu Val Ser Asp Leu Lys Val His Arg Phe Lys Val Lys Thr 195 200 205 Gly Asp Ser Ile His Lys Gly Leu Thr Pro Gly Trp Asn Asp Leu Asp 210 215 220 Val Asn Gln His Val Ser Asn Val Lys Tyr Ile Gly Trp Ile Leu Glu 225 230 235 240 Ser Met Pro Thr Glu Val Leu Glu Thr Gln Glu Leu Cys Ser Leu Ala 245 250 255 Leu Glu Tyr Arg Arg Glu Cys Gly Arg Asp Ser Val Leu Glu Ser Val 260 265 270 Thr Ala Met Asp Pro Ser Lys Val Gly Ser Arg Ser Gln Tyr Gln His 275 280 285 Leu Leu Arg Leu Glu Asp Gly Thr Ala Ile Val Asn Gly Ala Thr Glu 290 295 300 Trp Arg Pro Lys Asn Ala Gly Ala Asn Gly Ala Arg Ser Thr Gly Lys 305 310 315 320 Thr Ser Asn Gly Lys Ser Val Ser 325 <210> 16 <211> 328 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 16 Met Leu Lys Asp Trp Ser Arg Leu Leu Thr Ala Ile Thr Thr Val Phe 1 5 10 15 Val Lys Ser Lys Arg Pro His Met His Asp Arg Lys Ser Lys Arg Pro 20 25 30 Asp Met Leu Val His Ser Phe Gly Leu Glu Ser Thr Val Gln Asp Gly 35 40 45 Leu Val Phe Arg Gln Ser Phe Ser Ile Arg Ser Tyr Glu Ile Gly Thr 50 55 60 Asp Arg Thr Ala Ser Ile Glu Thr Leu Met Asn His Leu Gln Glu Thr 65 70 75 80 Ser Leu Asn His Cys Lys Ser Thr Gly Ile Leu Leu His Gly Phe Gly 85 90 95 Arg Thr Leu Glu Met Cys Lys Arg Asp Leu Ile Trp Val Val Ile Lys 100 105 110 Met Gln Ile Lys Val Asn Arg Tyr Pro Ala Trp Gly Asp Thr Val Glu 115 120 125 Ile Asn Thr Arg Phe Ser Arg Leu Gly Lys Ile Gly Met Gly Arg Asp 130 135 140 Trp Leu Ile Ser Asp Cys Asn Thr Gly Glu Ile Leu Val Arg Ala Thr 145 150 155 160 Ser Ala Tyr Ala Met Met Asn Gln Lys Thr Arg Arg Leu Ser Lys Val 165 170 175 Pro Tyr Glu Val His Gln Glu Ile Val Pro Leu Phe Val Asp Ser Pro 180 185 190 Val Ile Glu Val Ser Asp Leu Lys Val His Arg Phe Lys Val Lys Thr 195 200 205 Gly Asp Ser Ile Gln Lys Gly Leu Thr Pro Gly Trp Asn Asp Leu Asp 210 215 220 Val Asn Gln His Val Ser Asn Val Lys Tyr Ile Gly Trp Ile Leu Arg 225 230 235 240 Ser Met Pro Thr Glu Val Leu Glu Thr Gln Glu Leu Cys Ser Leu Ala 245 250 255 Leu Glu Tyr Arg Arg Glu Cys Gly Arg Asp Ser Val Leu Glu Ser Val 260 265 270 Thr Ala Met Asp Pro Ser Lys Val Gly Ser Arg Ser Gln Tyr Gln His 275 280 285 Leu Leu Arg Leu Glu Asp Gly Thr Ala Ile Val Asn Gly Ala Thr Glu 290 295 300 Trp Arg Pro Lys Asn Ala Gly Ala Asn Gly Ala Ile Ser Thr Gly Lys 305 310 315 320 Thr Ser Asn Gly Asn Ser Val Ser 325 <210> 17 <211> 328 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 17 Met Leu Lys Asp Trp Ser Arg Leu Leu Thr Ala Ile Thr Thr Val Phe 1 5 10 15 Val Lys Ser Lys Arg Pro Asp Met His Asp Arg Lys Ser Lys Arg Pro 20 25 30 Asp Met Leu Val Arg Ser Phe Gly Leu Glu Ser Thr Val Gln Asp Gly 35 40 45 Leu Val Phe Arg Gln Ser Phe Ser Ile Arg Ser Tyr Glu Ile Gly Thr 50 55 60 Asp Arg Thr Ala Ser Ile Glu Thr Leu Met Asn His Leu Gln Glu Thr 65 70 75 80 Ser Leu Asn His Cys Lys Ser Thr Gly Ile Leu Leu His Gly Phe Gly 85 90 95 Arg Thr Leu Glu Met Cys Lys Arg Asp Leu Ile Trp Val Val Ile Lys 100 105 110 Met Gln Ile Lys Val Asn Arg Tyr Pro Ala Trp Gly Asp Thr Val Glu 115 120 125 Ile Asn Thr Arg Phe Ser Arg Leu Gly Lys Ile Gly Met Gly Arg Asp 130 135 140 Trp Leu Ile Ser Asp Cys Asn Thr Gly Glu Ile Leu Val Arg Ala Thr 145 150 155 160 Ser Ala Tyr Ala Met Met Asn Gln Lys Thr Arg Arg Leu Ser Lys Val 165 170 175 Pro Tyr Glu Val His Gln Glu Ile Val Pro Leu Phe Val Asp Ser Pro 180 185 190 Val Ile Glu Val Ser Asp Leu Lys Val His Arg Phe Lys Val Lys Thr 195 200 205 Gly Asp Ser Ile Gln Lys Gly Leu Thr Pro Gly Trp Asn Asp Leu Asp 210 215 220 Val Asn Gln His Val Ser Asn Val Lys Tyr Ile Gly Trp Ile Leu Glu 225 230 235 240 Ser Met Pro Thr Glu Val Leu Glu Thr Gln Glu Leu Cys Ser Leu Ala 245 250 255 Leu Glu Tyr Arg Arg Glu Cys Gly Arg Asp Ser Val Leu Glu Ser Val 260 265 270 Thr Ala Met Asp Pro Ser Lys Val Gly Ser Arg Ser Gln Tyr Gln His 275 280 285 Leu Leu Arg Leu Glu Asp Gly Thr Ala Ile Val Asn Gly Ala Thr Glu 290 295 300 Trp Arg Pro Lys Asn Ala Gly Ala Asn Gly Ala Ile Ser Thr Gly Lys 305 310 315 320 Thr Ser Asn Gly Asn Ser Val Ser 325 <210> 18 <211> 328 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 18 Met Leu Lys Asp Trp Ser Arg Leu Leu Thr Ala Ile Thr Thr Val Phe 1 5 10 15 Val Lys Ser Lys Arg Pro Asp Met His Asp Arg Lys Ser Lys Arg Pro 20 25 30 Asp Met Leu Val Asp Ser Phe Gly Leu Glu Ser Thr Val Gln Asp Gly 35 40 45 Leu Val Phe Arg Gln Ser Phe Ser Ile Arg Ser Tyr Glu Ile Gly Thr 50 55 60 Asp Arg Thr Ala Ser Ile Glu Thr Leu Met Asn His Leu Gln Glu Thr 65 70 75 80 Ser Leu Asn His Cys Lys Ser Thr Gly Ile Leu Leu His Gly Phe Gly 85 90 95 Arg Thr Leu Glu Met Cys Lys Arg Asp Leu Ile Trp Val Val Ile Lys 100 105 110 Met Gln Ile Lys Val Asn Arg Tyr Pro Ala Trp Gly Asp Thr Val Glu 115 120 125 Ile Asn Thr Arg Phe Ser Arg Leu Gly Lys Ile Gly Met Gly Arg Asp 130 135 140 Trp Leu Ile Ser Asp Cys Asn Thr Gly Glu Ile Leu Val Arg Ala Thr 145 150 155 160 Ser Ala Tyr Ala Met Met Asn Gln Lys Thr Arg Arg Leu Ser Lys Val 165 170 175 Pro Tyr Arg Val His Gln Glu Ile Val Pro Leu Phe Val Asp Ser Pro 180 185 190 Val Ile Glu Val Ser Asp Leu Lys Val His Arg Phe Lys Val Lys Thr 195 200 205 Gly Asp Ser Ile Gln Lys Gly Leu Thr Pro Gly Trp Asn Asp Leu Asp 210 215 220 Val Asn Gln His Val Ser Asn Val Lys Tyr Ile Gly Trp Ile Leu Val 225 230 235 240 Ser Met Pro Thr Glu Val Leu Glu Thr Gln Glu Leu Cys Ser Leu Ala 245 250 255 Leu Glu Tyr Arg Arg Glu Cys Gly Arg Asp Ser Val Leu Glu Ser Val 260 265 270 Thr Ala Met Asp Pro Ser Lys Val Gly Ser Arg Ser Gln Tyr Gln His 275 280 285 Leu Leu Arg Leu His Asp Gly Thr Ala Ile Val Asn Gly Ala Thr Glu 290 295 300 Trp Arg Pro Lys Asn Ala Gly Ala Asn Gly Ala Ile Ser Thr Gly Lys 305 310 315 320 Thr Ser Asn Gly Asn Ser Val Ser 325 <210> 19 <211> 328 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 19 Met Leu Lys His Trp Ser Arg Leu Leu Thr Ala Ile Thr Thr Val Phe 1 5 10 15 Val Lys Ser Lys Arg Pro Asp Met His Asp Arg Lys Ser Lys Arg Pro 20 25 30 Asp Met Leu Val Arg Ser Phe Gly Leu Glu Ser Thr Val Gln Asp Gly 35 40 45 Leu Val Phe Arg Gln Ser Phe Ser Ile Arg Ser Tyr Glu Ile Gly Thr 50 55 60 Asp Arg Thr Ala Ser Ile Glu Thr Leu Met Asn His Leu Gln Glu Thr 65 70 75 80 Ser Leu Asn His Cys Lys Ser Thr Gly Ile Leu Leu His Gly Phe Gly 85 90 95 Arg Thr Leu Glu Met Cys Lys Arg Asp Leu Ile Trp Val Val Ile Lys 100 105 110 Met Gln Ile Lys Val Asn Arg Tyr Pro Ala Trp Gly Asp Thr Val Glu 115 120 125 Ile Asn Thr Arg Phe Ser Arg Leu Gly Lys Ile Gly Met Gly Arg Asp 130 135 140 Trp Leu Ile Ser Asp Cys Asn Thr Gly Glu Ile Leu Val Arg Ala Thr 145 150 155 160 Ser Ala Tyr Ala Met Met Asn Gln Lys Thr Arg Arg Leu Ser Lys Val 165 170 175 Pro Tyr His Val His Gln Glu Ile Val Pro Leu Phe Val Asp Ser Pro 180 185 190 Val Ile Glu Val Ser Asp Leu Lys Val His Arg Phe Lys Val Lys Thr 195 200 205 Gly Asp Ser Ile Gln Lys Gly Leu Thr Pro Gly Trp Asn Asp Leu Asp 210 215 220 Val Asn Gln His Val Ser Asn Val Lys Tyr Ile Gly Trp Ile Leu Arg 225 230 235 240 Ser Met Pro Thr Glu Val Leu Glu Thr Gln Glu Leu Cys Ser Leu Ala 245 250 255 Leu Glu Tyr Arg Arg Glu Cys Gly Arg Asp Ser Val Leu Glu Ser Val 260 265 270 Thr Ala Met Asp Pro Ser Lys Val Gly Ser Arg Ser Gln Tyr Gln His 275 280 285 Leu Leu Arg Leu Glu Asp Gly Thr Ala Ile Val Asn Gly Ala Thr Glu 290 295 300 Trp Arg Pro Lys Asn Ala Gly Ala Asn Gly Ala Ile Ser Thr Gly Lys 305 310 315 320 Thr Ser Asn Gly Asn Ser Val Ser 325 <210> 20 <211> 328 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 20 Met Leu Lys Asp Trp Ser Arg Leu Leu Thr Ala Ile Thr Thr Val Phe 1 5 10 15 Val Lys Ser Lys Arg Pro Asp Met His Asp Arg Lys Ser Lys Arg Pro 20 25 30 Asp Met Leu Val His Ser Phe Gly Leu Glu Ser Thr Val Gln Asp Gly 35 40 45 Leu Val Phe Arg Gln Ser Phe Ser Ile Arg Ser Tyr Glu Ile Gly Thr 50 55 60 Asp Arg Thr Ala Ser Ile Glu Thr Leu Met Asn His Leu Gln Glu Thr 65 70 75 80 Ser Leu Asn His Cys Lys Ser Thr Gly Ile Leu Leu His Gly Phe Gly 85 90 95 Arg Thr Leu Glu Met Cys Lys Arg Asp Leu Ile Trp Val Val Ile Lys 100 105 110 Met Gln Ile Lys Val Asn Arg Tyr Pro Ala Trp Gly Asp Thr Val Glu 115 120 125 Ile Asn Thr Arg Phe Ser Arg Leu Gly Lys Ile Gly Met Gly Arg Asp 130 135 140 Trp Leu Ile Ser Asp Cys Asn Thr Gly Glu Ile Leu Val Arg Ala Thr 145 150 155 160 Ser Ala Tyr Ala Met Met Asn Gln Lys Thr Arg Arg Leu Ser Lys Val 165 170 175 Pro Tyr Glu Val His Gln Glu Ile Val Pro Leu Phe Val Asp Ser Pro 180 185 190 Val Ile Glu Val Ser Asp Leu Lys Val His Arg Phe Lys Val Lys Thr 195 200 205 Gly Asp Ser Ile Gln Lys Gly Leu Thr Pro Gly Trp Asn Asp Leu Asp 210 215 220 Val Asn Gln His Val Ser Asn Val Lys Tyr Ile Gly Trp Ile Leu Glu 225 230 235 240 Ser Met Pro Thr Glu Val Leu Glu Thr Gln Glu Leu Cys Ser Leu Ala 245 250 255 Leu Glu Tyr Arg Arg Glu Cys Gly Arg Asp Ser Val Leu Glu Ser Val 260 265 270 Thr Ala Met Asp Pro Ser Lys Val Gly Ser Arg Ser Gln Tyr Gln His 275 280 285 Leu Leu Arg Leu Glu Asp Gly Thr Ala Ile Val Asn Gly Ala Thr Glu 290 295 300 Trp Arg Pro Lys Asn Ala Gly Ala Asn Gly Ala Ile Ser Thr Gly Lys 305 310 315 320 Thr Ser Asn Gly Asn Ser Val Ser 325 <210> 21 <211> 328 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 21 Met Leu Lys Asp Trp Ser Arg Leu Leu Thr Ala Ile Thr Thr Val Phe 1 5 10 15 Val Lys Ser Lys Arg Pro Asp Met His Asp Arg Lys Ser Lys Arg Pro 20 25 30 Asp Met Leu Val His Ser Phe Gly Leu Glu Ser Thr Val Gln Asp Gly 35 40 45 Leu Val Phe Arg Gln Ser Phe Ser Ile Arg Ser Tyr Glu Ile Gly Thr 50 55 60 Asp Arg Thr Ala Ser Ile Glu Thr Leu Met Asn His Leu Gln Glu Thr 65 70 75 80 Ser Leu Asn His Cys Lys Ser Thr Gly Ile Leu Leu His Gly Phe Gly 85 90 95 Arg Thr Leu Glu Met Cys Lys Arg Asp Leu Ile Trp Val Val Ile Lys 100 105 110 Met Gln Ile Lys Val Asn Arg Tyr Pro Ala Trp Gly Asp Thr Val Glu 115 120 125 Ile Asn Thr Arg Phe Ser Arg Leu Gly Lys Ile Gly Met Gly Arg Asp 130 135 140 Trp Leu Ile Ser Asp Cys Asn Thr Gly Glu Ile Leu Val Arg Ala Thr 145 150 155 160 Ser Ala Tyr Ala Met Met Asn Gln Lys Thr Arg Arg Leu Ser Lys Val 165 170 175 Pro Tyr Glu Val His Gln Glu Ile Val Pro Leu Phe Val Asp Ser Pro 180 185 190 Val Ile Glu Val Ser Asp Leu Lys Val His Arg Phe Lys Val Lys Thr 195 200 205 Gly Asp Ser Ile Gln Lys Gly Leu Thr Pro Gly Trp Asn Asp Leu Asp 210 215 220 Val Asn Gln His Val Ser Asn Val Lys Tyr Ile Gly Trp Ile Leu Glu 225 230 235 240 Ser Met Pro Thr Glu Val Leu Glu Thr Gln Glu Leu Cys Ser Leu Ala 245 250 255 Leu Glu Tyr Arg Arg Glu Cys Gly Arg Arg Ser Val Leu Glu Ser Val 260 265 270 Thr Ala Met Asp Pro Ser Lys Val Gly Ser Arg Ser Gln Tyr Gln His 275 280 285 Leu Leu Arg Leu Glu Asp Gly Thr Ala Ile Val Asn Gly Ala Thr Glu 290 295 300 Trp Arg Pro Lys Asn Ala Gly Ala Asn Gly Ala Ile Ser Thr Gly Lys 305 310 315 320 Thr Ser Asn Gly Asn Ser Val Ser 325 <210> 22 <211> 328 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 22 Met Leu Lys Arg Trp Ser Arg Leu Leu Thr Ala Ile Thr Thr Val Phe 1 5 10 15 Val Lys Ser Lys Arg Pro Asp Met His Asp Arg Lys Ser Lys Arg Pro 20 25 30 Asp Met Leu Val Asp Ser Phe Gly Leu Glu Ser Thr Val Gln Asp Gly 35 40 45 Leu Val Phe Arg Gln Ser Phe Ser Ile Arg Ser Tyr Glu Ile Gly Thr 50 55 60 Asp Arg Thr Ala Ser Ile Glu Thr Leu Met Asn His Leu Gln Glu Thr 65 70 75 80 Ser Leu Asn His Cys Lys Ser Thr Gly Ile Leu Leu Asp Gly Phe Gly 85 90 95 Arg Thr Leu Glu Met Cys Lys Arg Asp Leu Ile Trp Val Val Ile Lys 100 105 110 Met Gln Ile Lys Val Asn Arg Tyr Pro Ala Trp Gly Asp Thr Val Glu 115 120 125 Ile Asn Thr Arg Phe Ser Arg Leu Gly Lys Ile Gly Met Gly Arg Asp 130 135 140 Trp Leu Ile Ser Asp Cys Asn Thr Gly Glu Ile Leu Val Arg Ala Thr 145 150 155 160 Ser Ala Tyr Ala Met Met Asn Gln Lys Thr Arg Arg Leu Ser Lys Val 165 170 175 Pro Tyr Glu Val His Gln Glu Ile Val Pro Leu Phe Val Asp Ser Pro 180 185 190 Val Ile Glu Val Ser Asp Leu Lys Val His Arg Phe Lys Val Lys Thr 195 200 205 Gly Asp Ser Ile Gln Lys Gly Leu Thr Pro Gly Trp Asn Asp Leu Asp 210 215 220 Val Asn Gln His Val Ser Asn Val Lys Tyr Ile Gly Trp Ile Leu Glu 225 230 235 240 Ser Met Pro Thr Glu Val Leu Glu Thr Gln Glu Leu Cys Ser Leu Ala 245 250 255 Leu Glu Tyr Arg Arg Glu Cys Gly Arg Asp Ser Val Leu Glu Ser Val 260 265 270 Thr Ala Met Asp Pro Ser Lys Val Gly Val Arg Ser Gln Tyr Gln His 275 280 285 Leu Leu Arg Leu Glu Asp Gly Thr Al...

Claims

1. an engineered thioesterase variant, the engineered thioesterase variant having an amino acid sequence that has at least 90% sequence identity with SEQ ID NO:1 when the sequences are optimally aligned for maximum correspondence and compared over a comparison window; The engineered thioesterase variants include P3K, D4M, S6R, T14G, T14R, V15L, V15W, V17A, V17C, P22R, D37P, T44G, V45S, V50W, S54R, S56C, S56K, T64P, T64R, T67L, H76L, L91M, C102I, V110L, I129V, G137C, R158Q, L176V, Y178P, P186G, D196V, D198W, K203R, Q213R, T217R, V225L, Q227G, G236T, T244M, T244R, S254G, A256C, E258T, containing a substitution mutation of E258V, S278K, S278V, V282S, L292F, A297T, A297V, I298C, I298V, V299L, N300L, N300W, A302T, I316R, T321R, or S322K; and An engineered thioesterase variant, wherein the engineered thioesterase variant has improved catalytic activity, selectivity and / or solubility for the production of medium-chain fatty acid derivatives compared to the enzyme having SEQ ID NO:

1.

2. 2. The engineered thioesterase variant of claim 1, further comprising a substitution mutation of D4H, D4R, D23H, D23R, D37H, D37R, L73V, H76F, H76Y, D93H, D93R, L99K, L99P, E179H, E179R, D210H, D210R, E240H, E240R, E240V, E245H, E245R, E248H, D266H, D266R, E293H, E293R, D294H, I316T, N325K, or S328K.

3. 3. The engineered thioesterase variant of claim 1 or 2, wherein the engineered thioesterase variant has improved activity for the production of C8 fatty acid derivatives, C10 fatty acid derivatives, or both.

4. The engineered thioesterase variants may be selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO: NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, or SEQ ID 4. The engineered thioesterase variant of any one of claims 1 to 3, having the amino acid sequence of NO:

59.

5. (a) the engineered thioesterase variant has an increased overall net positive charge compared to the thioesterase of SEQ ID NO:1 or SEQ ID NO:4; or (b) the engineered thioesterase variant has an increased surface positive charge compared to SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:15; The engineered thioesterase variant of any one of claims 1 to 4.

6. (a) the engineered thioesterase variant has improved solubility; (b) the engineered thioesterase variant has improved solubility compared to SEQ ID NO:49; (c) the engineered thioesterase variant comprises a truncation mutation at amino acids 2 to 40 of SEQ ID NO:49; or (d) the engineered thioesterase variant has the amino acid sequence of SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, or SEQ ID NO:59; The engineered thioesterase variant of any one of claims 1 to 5.

7. A recombinant microorganism comprising an engineered thioesterase variant according to any one of claims 1 to 6.

8. 8. The recombinant microorganism of claim 7, the recombinant microorganism further expresses one or more of an ester synthase, an acyl-ACP reductase, an alcohol dehydrogenase, an aldehyde reductase, an acyl-CoA reductase, an acyl-CoA synthetase, a fatty alcohol forming acyl-CoA reductase, a carboxylic acid reductase, an alcohol O-acetyltransferase, an O-methyltransferase, a decarbonylase, an oxidative deformylase, a decarboxylase, OleA, OleCD, or OleBCD; and the recombinant microorganism produces a fatty acid derivative that is a fatty acid, a fatty ester, a fatty alcohol, a fatty alcohol acetate ester (FACE), a fatty acid methyl ester (FAME), a fatty acid ethyl ester (FAEE), a fatty amine, a fatty aldehyde, a difunctional fatty acid derivative, a diacid, a diol, a hydrocarbon, an alkane, an alkene, an olefin, or a ketone; Recombinant microorganisms.

9. 8. The recombinant microorganism of claim 7, further comprising a biochemical pathway for converting a first fatty acid derivative into a second fatty acid derivative, wherein the first fatty acid derivative and the second fatty acid derivative are medium-chain fatty acid derivatives; and the second fatty acid derivative has a higher minimum inhibitory concentration (MIC) than the first fatty acid derivative, and the presence of the second fatty acid derivative increases the MIC of the first fatty acid derivative; or the second fatty acid derivative has a higher LogP than the first fatty acid derivative; or the second fatty acid derivative has lower toxicity than the first fatty acid derivative; Recombinant microorganisms.

10. the biochemical pathway a) carboxylic acid reductase, b) carboxylic acid reductase and alcohol dehydrogenase, c) carboxylic acid reductase and alcohol-O-acetyltransferase, d) carboxylic acid reductase, and alcohol dehydrogenase, and alcohol O-acetyltransferase; e) ester synthase, f) ester synthases and acyl-CoA synthetases, g) acyl-CoA reductase, h) acyl-CoA reductase and acyl-CoA synthetase, i) acyl-CoA reductase and alcohol O-acetyltransferase, j) acyl-CoA reductase, alcohol O-acetyltransferase, and acyl-CoA synthetase; k) O-methyltransferase, l) acyl-ACP reductase, m) acyl-ACP reductase and aldehyde decarbonylase, n) acyl-ACP reductase and aldehyde oxidative deformylase, o) acyl-ACP reductase and alcohol O-acetyltransferase, p) acyl-ACP reductase, alcohol-O-acetyltransferase, and alcohol dehydrogenase, q) OleA r) OleA, OleC, and OleD s) OleA and acyl-CoA synthetase, or t) OleA, OleC, OleD, and acyl-CoA synthetase 10. The recombinant microorganism of claim 9, comprising:

11. (i) the first fatty acid derivative is a fatty acid, the second fatty acid derivative is a fatty acid alkyl ester, a fatty acid methyl ester, or a fatty acid ethyl ester, and the biochemical pathway comprises an ester synthase or comprises an ester synthase and an acyl-CoA synthetase; or (ii) the first fatty acid derivative is a fatty alcohol, the second fatty acid derivative is a fatty alcohol acetate, and the biochemical pathway comprises a carboxylic acid reductase and an alcohol-O-acetyltransferase, or comprises a carboxylic acid reductase, an alcohol dehydrogenase, and an alcohol-O-acetyltransferase; A recombinant microorganism according to claim 9 or 10.

12. 12. A method for producing a medium-chain fatty acid derivative, the method comprising culturing the recombinant microorganism of any one of claims 7 to 11 in the presence of a carbon source under conditions suitable for the production of a medium-chain fatty acid derivative.

13. The method of claim 12, further comprising recovering the medium-chain fatty acid derivatives from the culture or isolating the medium-chain fatty acid derivatives from the recombinant microorganism.

14. 14. The method of claim 12 or 13, wherein the medium-chain fatty acid derivative is a medium-chain fatty acid, medium-chain fatty ester, medium-chain fatty acid methyl ester (FAME), medium-chain fatty acid ethyl ester (FAEE), fatty alcohol acetate ester (FACE), medium-chain fatty alcohol, medium-chain fatty aldehyde, medium-chain fatty amine, medium-chain hydrocarbon, medium-chain ketone, medium-chain alkane, medium-chain terminal olefin, medium-chain internal olefin, medium-chain hydroxy fatty acid derivative, medium-chain difunctional fatty acid derivative, medium-chain fatty diacid, medium-chain fatty diol, or unsaturated medium-chain fatty acid derivative.

Images