XYLR mutants for improved xylose utilization or improved glucose and xylose co-utilization

Abstract

The present disclosure relates to one or more mutant genes that confer improved ability to utilize xylose and improved ability to co-utilize glucose and xylose to microorganisms expressing them, resulting in improved growth of the microorganisms. Further included are methods of producing fatty acids and fatty acid derivatives from cellulosic biomass, xylose and / or glucose / xylose mixtures by using host cells expressing the engineered XylR variants, and compositions of biologically produced fatty acids and fatty acid derivatives.

Description

[0001] This application is a divisional application of application no. 201980056032.4, entered into Chinese national phase on February 25, 2021.

[0002] Reference to Related Applications

[0003] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 726,114, filed August 31, 2018, and U.S. Provisional Patent Application No. 62 / 731,711, filed September 14, 2018, which are incorporated by reference herein in their entireties. BACKGROUND

[0004] To meet the growing global population’s demand for energy and resources, and to reduce dependence on finite fossil energy sources, the use of renewable technologies to produce energy products and consumer products must be increased.

[0005] Exemplary renewable technologies include the use of microbial systems to produce chemicals and fuels. Microbial systems for producing biofuels and various chemicals are well known in the art (see, e.g., 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; 8,530,221; 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; etc.). Unfortunately, however, the full benefits of microbial systems can be limited by the nature of the feedstocks available for production.

[0006] Hydrolysates are often used as feedstocks for the biological production of chemicals. These feedstocks are less expensive than pure glucose, potentially reducing the cost of the biological production process. The most abundant sugars in hydrolysates are glucose and xylose. Unfortunately, however, the utilization of xylose is lower than that of glucose, and due to two-stage action, xylose is often not effectively utilized in the presence of glucose.

[0007] Accordingly, to make the production of any renewable carbon-based products from biological processing of hydrolysate feedstocks higher, there is a need in the art for microbial systems that allow for increased utilization of xylose and increased co-utilization of glucose and xylose. Fortunately, as will be clear from the disclosure below, the present invention meets these and other needs. SUMMARY

[0008] One aspect of the present disclosure provides engineered XylR variants having improved ability to utilize xylose and improved ability to co-utilize glucose and xylose.

[0009] In one aspect, the present disclosure provides XylR protein variants, wherein the XylR protein variant has at least one mutation at a position corresponding to a position selected from the group consisting of: position 83, 88, 89, 112, 120, 141, 145, 146, 147, 150, 154, 155, 247, 270, 280, 286, 289, 295, 305, 306, 313, 333, 336, 337, 351, 364, 365, 372, and 382 of SEQ ID NO: 1.

[0010] In one aspect, the present disclosure provides recombinant host cells comprising XylR protein variants, wherein the XylR protein variant has at least one mutation at a position corresponding to a position selected from the group consisting of: position 83, 89, 112, 120, 141, 145, 146, 147, 150, 154, 155, 247, 270, 280, 286, 289, 295, 305, 306, 313, 333, 336, 337, 351, 364, 365, 372, and 382 of SEQ ID NO: 1.

[0011] In one aspect, the present disclosure provides methods for increasing xylose utilization in a recombinant host cell. The method comprises culturing a recombinant host cell comprising a XylR protein variant in a culture medium comprising xylose, wherein the XylR protein variant has at least one mutation at a position corresponding to a position selected from the group consisting of: position 83, 89, 112, 120, 141, 145, 146, 147, 150, 154, 155, 247, 270, 280, 286, 289, 295, 305, 306, 313, 333, 336, 337, 351, 364, 365, 372, and 382 of SEQ ID NO: 1. Expression of the XylR protein variant in the recombinant host cell confers improved growth of the recombinant host cell as compared to growth of a host cell expressing SEQ ID NO: 1 when the cell is cultured in the presence of xylose.

[0012] In one aspect, the present disclosure provides a method of making a fatty acid derivative, the method comprising culturing a recombinant host cell comprising at least one heterologous fatty acid derivative biosynthetic enzyme and a XylR protein variant in a culture medium comprising xylose, wherein the XylR protein variant has at least one mutation at a position corresponding to a position selected from the group consisting of: position 83, 89, 112, 120, 141, 145, 146, 147, 150, 154, 155, 247, 270, 280, 286, 289, 295, 305, 306, 313, 333, 336, 337, 351, 364, 365, 372, and 382 of SEQ ID NO: 1 of SEQ ID NO: 1.

[0013] In one aspect, the present disclosure provides a method for making a C5-C 24 fatty acid methyl ester (FAME) or C5-C 24 fatty acid ethyl ester (FAEE) or C5-C 24 fatty acid methyl ester (FAME) and C5-C 24 fatty acid ethyl ester (FAEE) in combination, the method comprising culturing in a culture medium comprising xylose: a recombinant host cell comprising at least one heterologous fatty acid derivative biosynthetic enzyme having ester synthase activity (E.C. 3.1.1.67) and a XylR protein variant having the following characteristics, wherein the XylR protein variant has at least one mutation at a position corresponding to a position selected from the group consisting of: position 83, 89, 112, 120, 141, 145, 146, 147, 150, 154, 155, 247, 270, 280, 286, 289, 295, 305, 306, 313, 333, 336, 337, 351, 364, 365, 372, and 382 of SEQ ID NO: 1 of SEQ ID NO: 1.

[0014] In some embodiments of the above aspects, the at least one mutation in the XylR protein is selected from the group consisting of: V83C, L89V, L89K, L112R, N120C, Y141R, Q145R, L146R, V147M, E150W, E150G, G154C, V155E, A247V, A247T, R270E, R280V, A286M, A286F, Q289V, R295C, E305M, Q306K, I313L, M333R, A336M, A336G, E337N, E337H, L351T, S364W, L365T, L365V, F372W, and E382K.

[0015] In some embodiments, the XylR protein variant further comprises at least one additional mutation at a position selected from the group consisting of positions 121 and 363 corresponding to positions of SEQ ID NO: 1. In some embodiments, the at least one additional mutation at positions 121 and 363 is selected from the group consisting of at least one additional mutation selected from the group consisting of R121C, R121S, R121T, R121G, R121H, R121V, R121M, T121Y, R121I, R121A, R121L, R121P, R121P, R121F, R121W, and P363S.

[0016] In some embodiments of the above aspects, the XylR protein variant can have a combination of two or more amino acid substitutions selected from the group consisting of V83C, L89V, L89K, L112R, N120C, Y141R, Q145R, L146R, V147M, E150W, E150G, G154C, V155E, A247V, A247T, R270E, R280V, A286M, A286F, Q289V, R295C, E305M, Q306K, I313L, M333R, A336M, A336G, E337N, E337H, L351T, S364W, L365T, L365V, F372W, and E382K, as compared to a wild-type XylR protein. In some embodiments, the two or more amino acid substitutions can be selected from the group consisting of L89K and L112R; E150G, H88G, and A246A; R280V and D305G; A286F and Q306K; Q289V and E305M; S364W and R295C.

[0017] In some embodiments of the above aspects, the XylR protein variant can have a combination of two or more amino acid substitutions (where at least one substitution is selected from the group consisting of V83C, H88G, L89V, L89K, L112R, N120C, Y141R, Q145R, L146R, V147M, E150W, E150G, G154C, V155E, A247V, A247T, R270E, R280V, A286M, A286F, Q289V, R295C, E305M, Q306K, I313L, M333R, A336M, A336G, E337N, E337H, L351T, S364W, L365T, L365V, F372W, and E382K) and at least one other substitution (which is selected from the group consisting of R121C, R121S, R121T, R121G, R121H, R121V, R121M, T121Y, R121I, R121A, R121L, R121P, R121P, R121F, R121W, and P363S).

[0018] In some embodiments of the above aspects, the XylR protein variant has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% sequence identity over a length of 50, 75, 100, 125, 150, 175, 200, 250, 275, 300 or more contiguous amino acids of SEQ ID NO: 1 and has XylR activity. In some embodiments of the above aspects, the XylR protein variant has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% sequence identity over the entire sequence of SEQ ID NO: 1 and has XylR activity.

[0019] In an aspect, the present disclosure provides a XylR protein variant having at least 90% sequence identity to SEQ ID NO: 1, wherein the XylR protein variant has at least one mutation at a position corresponding to position 382 of SEQ ID NO: 1, and wherein the XylR protein variant has no mutation at a position corresponding to position 121 of SEQ ID NO: 1 and also no mutation at a position corresponding to position 363 of SEQ ID NO: 1. In embodiments, the XylR protein variant has 95% sequence identity to SEQ ID NO: 1. In embodiments, the XylR protein variant has 98% sequence identity to SEQ ID NO: 1. In some embodiments, the at least one mutation at position 382 of SEQ ID NO: 1 is an E382K substitution mutation.

[0020] Another aspect of the present disclosure provides a recombinant host cell comprising a XylR protein variant having at least 90% sequence identity to SEQ ID NO: 1 and at least one mutation at position 382 of SEQ ID NO: 1, wherein the XylR protein variant has no mutation at a position corresponding to position 121 of SEQ ID NO: 1 and also has no mutation at a position corresponding to 363 of SEQ ID NO: 1, wherein expression of the XylR protein variant in the recombinant host cell confers improved growth of the recombinant host cell when the cell is cultured in the presence of xylose as compared to growth of a host cell expressing SEQ ID NO: 1. In embodiments, the improved growth is due to an increase in xylose utilization. In embodiments, the improved growth occurs in the presence of glucose. In embodiments, the XylR protein variant has 95% sequence identity to SEQ ID NO: 1. In embodiments, the XylR protein variant has 98% sequence identity to SEQ ID NO: 1. In embodiments, the at least one mutation at position 382 of SEQ ID NO: 1 is an E382K substitution mutation.

[0021] Another aspect of the present disclosure provides a method of making a fatty acid derivative, the method comprising: culturing a recombinant host cell in a medium comprising xylose, the recombinant host cell comprising at least one heterologous fatty acid derivative biosynthetic enzyme and a XylR protein variant having at least 90% sequence identity to SEQ ID NO: 1 and at least one mutation at position 382 of SEQ ID NO: 1, wherein the XylR protein variant has no mutation at a position corresponding to position 121 of SEQ ID NO: 1 and also has no mutation at a position corresponding to 363 of SEQ ID NO: 1. In embodiments, the XylR protein variant has 95% sequence identity to SEQ ID NO: 1. In embodiments, the XylR protein variant has 98% sequence identity to SEQ ID NO: 1. In embodiments, the XylR protein variant having at least one mutation at position 382 of SEQ ID NO: 1 has an E382K substitution mutation. In embodiments, the medium comprising xylose further comprises glucose. In embodiments, the medium is derived from cellulosic biomass. In embodiments, the fatty acid derivative is a C5-C24 fatty acid derivative. In embodiments, the at least one heterologous fatty acid derivative biosynthetic enzyme has ester synthase activity (E.C. 3.1.1.67) and the fatty acid derivative is selected from the group consisting of fatty acid methyl ester (FAME) and fatty acid ethyl ester (FAEE) or a combination thereof.

[0022] Another aspect of the present disclosure provides a XylR protein variant having at least 90% sequence identity to SEQ ID NO: 1, wherein the XylR protein variant has at least one mutation at a position corresponding to position 382 of SEQ ID NO: 1 and the at least one mutation at position 382 of SEQ ID NO: 1 is an E382K substitution mutation, and wherein the XylR protein variant has no mutation at a position corresponding to position 121 of SEQ ID NO: 1 and also has no mutation at a position corresponding to position 363 of SEQ ID NO: 1.

[0023] Another aspect of the present disclosure provides a method of making a C5-C24 fatty acid derivative selected from the group consisting of fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs), or combinations thereof, the method comprising: culturing in a xylose- containing medium: a recombinant host cell comprising at least one heterologous fatty acid derivative biosynthetic enzyme having ester synthase activity (E.C. 3.1.1.67) and a XylR protein variant having at least 90% sequence identity to SEQ ID NO: 1, wherein the XylR protein variant has at least one mutation at position 382 of SEQ ID NO: 1 and the at least one mutation at position 382 of SEQ ID NO: 1 is an E382K substitution mutation, and the XylR protein variant has no mutation at a position corresponding to position 121 of SEQ ID NO: 1 and also has no mutation at a position corresponding to position 363 of SEQ ID NO: 1. In embodiments, the XylR protein variant has 95% sequence identity to SEQ ID NO: 1. In embodiments, the XylR protein variant has 98% sequence identity to SEQ ID NO: 1. In embodiments, the xylose-containing medium further comprises glucose.

[0024] In one aspect, the present disclosure provides a XylR protein variant, wherein the XylR protein variant has at least one mutation at a position corresponding to a position of SEQ ID NO: 1 selected from the group consisting of: position 83, 88, 89, 112, 120, 141, 145, 146, 147, 150, 154, 155, 247, 270, 280, 286, 289, 295, 305, 306, 313, 333, 336, 337, 351, 364, 365, 372, and 382 of SEQ ID NO: 1.

[0025] In one aspect, the present disclosure provides a recombinant host cell comprising a XylR protein variant, wherein the XylR protein variant has at least one mutation at a position corresponding to a position selected from the group consisting of: position 83, 88, 89, 112, 120, 141, 145, 146, 147, 150, 154, 155, 247, 270, 280, 286, 289, 295, 305, 306, 313, 333, 336, 337, 351, 364, 365, 372, and 382 of SEQ ID NO: 1 of SEQ ID NO: 1.

[0026] In one aspect, the present disclosure provides a method for increasing xylose utilization in a recombinant host cell, the method comprising culturing a recombinant host cell comprising a XylR protein variant in a culture medium comprising xylose, wherein the XylR protein variant has at least one mutation at a position corresponding to a position selected from the group consisting of: position 83, 88, 89, 112, 120, 141, 145, 146, 147, 150, 154, 155, 247, 270, 280, 286, 289, 295, 305, 306, 313, 333, 336, 337, 351, 364, 365, 372, and 382 of SEQ ID NO: 1, wherein expression of the XylR protein variant confers improved xylose utilization to the recombinant host cell as compared to xylose utilization of a host cell expressing SEQ ID NO: 1 when the cell is cultured in the presence of xylose.

[0027] In some embodiments, the method is for making a fatty acid derivative, the method comprising culturing a recombinant host cell in a culture medium comprising xylose, the recombinant host cell further comprising at least one heterologous fatty acid derivative biosynthetic enzyme.

[0028] In some embodiments, the fatty acid derivative is: a fatty acid ester and wherein at least one heterologous fatty acid derivative biosynthetic enzyme has ester synthase activity, and optionally wherein at least one heterologous fatty acid derivative biosynthetic enzyme is a thioesterase; an ω-hydroxy fatty acid and wherein at least one heterologous fatty acid derivative biosynthetic enzyme has ω-hydroxylase activity (EC 1.14.15.3); a fatty aldehyde and wherein at least one heterologous fatty acid derivative biosynthetic enzyme has carboxylic acid reductase (CAR) activity; a fatty amine and wherein at least one heterologous fatty acid derivative biosynthetic enzyme has carboxylic acid reductase (CAR) activity and another heterologous fatty acid derivative biosynthetic enzyme has carboxylic acid reductase (CAR) activity, an aminotransferase, or an amine dehydrogenase activity; or a fatty alcohol acetate and wherein at least one heterologous fatty acid derivative biosynthetic enzyme has carboxylic acid reductase (CAR) activity and another heterologous fatty acid derivative biosynthetic enzyme has fatty alcohol O-acetyltransferase activity (which converts the fatty alcohol to fatty alcohol acetate).

[0029] In some embodiments, the method is used to prepare C5-C 24 Fatty acid methyl ester (FAME) or C5-C 24 Fatty acid ethyl ester (FAEE) or C5-C 24 Fatty acid methyl esters (FAME) and C5-C 24 1. The method comprises culturing in a medium comprising xylose: a recombinant host cell comprising at least one heterologous fatty acid derivative biosynthetic enzyme having ester synthase activity (EC 3.1.1.67) and a XylR protein variant having at least 90% sequence identity to SEQ ID NO: 1, wherein the XylR protein variant has at least one mutation at a position corresponding to a position of SEQ ID NO: 1 selected from the group consisting of positions 83, 88, 89, 112, 120, 141, 145, 146, 147, 150, 154, 155, 247, 270, 280, 286, 289, 295, 305, 306, 313, 333, 336, 337, 351, 364, 365, 372, and 382 of SEQ ID NO: 1.

[0030] In some embodiments, the XylR protein variant, recombinant host cell, or method of any embodiment comprises a XylR protein variant having 90% sequence identity to SEQ ID NO:1.

[0031] In some embodiments, the XylR protein variant, recombinant host cell, or method of any embodiment comprises a XylR protein variant having 95% sequence identity to SEQ ID NO:1.

[0032] In some embodiments, the XylR protein variant, recombinant host cell, or method of any embodiment comprises at least one mutation selected from the group consisting of V83C, H88G, L89V, L89K, L112R, N120C, Y141R, Q145R, L146R, V147M, E150W, E150G, G154C, V155E, A247V, A247T, R270E, R280V, A286M, A286F, Q289V, R295C, D305M, Q306K, I313L, M333R, A336M, A336G, E337N, E337H, L351T, S364W, L365T, L365V, F372W, and E382K. In some embodiments, the XylR protein variant, recombinant host cell, or method comprises a XylR protein variant having more than one substitution mutation and is a member selected from the group consisting of a XylR protein variant having substitution mutation L89K and further comprising L112R; a XylR protein variant having substitution mutation E150G and further comprising H88G; a XylR protein variant having substitution mutation R280V and further comprising D305G; a XylR protein variant having substitution mutation A286F and further comprising Q306K; a XylR protein variant having substitution mutation Q289V and further comprising D305M; and a XylR protein variant having substitution mutation S364W and further comprising R295C.

[0033] In some embodiments, the XylR protein variant, recombinant host cell, or method of any embodiment further comprises at least one additional mutation at a position corresponding to a position selected from positions 121 and 363 of SEQ ID NO: 1. In some embodiments, the at least one additional mutation is selected from the group consisting of R121C, R121S, R121T, R121G, R121H, R121V, R121M, T121Y, R121I, R121A, R121L, R121P, R121P, R121F, R121W, and P363S.

[0034] In some embodiments, the XylR protein variant, recombinant host cell, or method of any embodiment, when the cells are cultured in the presence of xylose, expression of the XylR protein variant in the recombinant host cell confers improved growth of the recombinant host cell compared to the growth of a host cell expressing SEQ ID NO: 1. In some embodiments, the XylR protein variant has at least one mutation at a position of SEQ ID NO: 1 selected from the group consisting of position 112, 141, 145, 146, 247, 286, 289, 336, 337, 364, and 365 of SEQ ID NO: 1, wherein the at least one mutation is selected from the group consisting of L112R, Y141R, Q145R, L146R, A247V, A286F, A286M, Q289V, A336G, E337H, S364W, and L365V.

[0035] In some embodiments, the recombinant host cell of any embodiment expresses at least one heterologous fatty acid derivative biosynthetic enzyme, wherein the XylR protein variant has at least one mutation at a position selected from the group consisting of position 112, 145, 146, 247, 286, 336, 337, and 365 of SEQ ID NO: 1, wherein the recombinant host cell produces an increased amount of a fatty acid species (FAS) when cultured in the presence of xylose compared to an otherwise identical isogenic host cell that does not express SEQ ID NO: 1. In some embodiments, the XylR protein variant has at least one mutation at a position selected from the group consisting of position 112, 145, 146, 247, 286, 336, 337, and 365 of SEQ ID NO: 1, wherein the at least one mutation is selected from the group consisting of L112R, Q145R, L146R, A247V, A286M, A336G, E337H, and L365V.

[0036] In some embodiments, the method of any embodiment includes a XylR protein variant having at least one mutation at a position selected from the group consisting of position 112, 145, 146, 247, 286, 336, 337, and 365 of SEQ ID NO: 1, wherein the at least one mutation is selected from the group consisting of L112R, Q145R, L146R, A247V, A286M, A336G, E337H, and L365V.

[0037] In some embodiments, the method of any embodiment includes a culture medium comprising xylose, further comprising glucose. In some embodiments, the culture medium is derived from cellulosic biomass.

[0038] In one aspect, the present disclosure provides a XylR protein variant, wherein the XylR protein variant has at least one mutation at a position corresponding to position 382 of SEQ ID NO: 1.

[0039] In one aspect, the present disclosure provides a recombinant host cell comprising a XylR protein variant having at least one mutation at position 382 of SEQ ID NO: 1, wherein expression of the XylR protein variant in the recombinant host cell confers improved growth of the recombinant host cell when the cell is cultured in the presence of xylose as compared to growth of a host cell expressing SEQ ID NO: 1.

[0040] In some embodiments, the XylR protein variant, recombinant host cell, or method of any embodiment comprises a XylR protein variant that further comprises at least one additional mutation at position 121 or 363. In some embodiments, the at least one additional mutation is selected from the group consisting of R121C, R121S, R121T, R121G, R121H, R121V, R121M, T121Y, R121I, R121A, R121L, R121P, R121P, R121F, R121W, and P363S.

[0041] In one aspect, the present disclosure provides a method for increasing xylose utilization in a recombinant host cell, the method comprising culturing a recombinant host cell comprising a XylR protein variant in a culture medium comprising xylose, wherein the XylR protein variant has at least one mutation at a position corresponding to position 382 of SEQ ID NO: 1, wherein expression of the XylR protein variant confers improved xylose utilization of the recombinant host cell when the cell is cultured in the presence of xylose as compared to xylose utilization of a host cell expressing SEQ ID NO: 1.

[0042] In some embodiments, the method is for making a fatty acid derivative, the method comprising culturing a recombinant host cell in a culture medium comprising xylose, the recombinant host cell further comprising at least one heterologous fatty acid derivative biosynthetic enzyme.

[0043] In some embodiments, the fatty acid derivative is: a fatty acid ester and wherein the at least one heterologous fatty acid derivative biosynthetic enzyme has ester synthase activity, and optionally wherein the at least one heterologous fatty acid derivative biosynthetic enzyme is a thioesterase; an omega-hydroxy fatty acid and wherein the at least one heterologous fatty acid derivative biosynthetic enzyme has omega-hydroxylase activity (EC 1.14.15.3); a fatty aldehyde and wherein the at least one heterologous fatty acid derivative biosynthetic enzyme has carboxylic acid reductase (CAR) activity; a fatty amine and wherein the at least one heterologous fatty acid derivative biosynthetic enzyme has carboxylic acid reductase (CAR) activity, and another heterologous fatty acid derivative biosynthetic enzyme has carboxylic acid reductase (CAR) activity, amino transferase or amine dehydrogenase activity; or a fatty alcohol acetate and wherein the at least one heterologous fatty acid derivative biosynthetic enzyme has carboxylic acid reductase (CAR) activity, and another heterologous fatty acid derivative biosynthetic enzyme has fatty alcohol O-acetyltransferase activity that converts a fatty alcohol to a fatty alcohol acetate.

[0044] In some embodiments, the method is for making C5-C 24 fatty acid methyl ester (FAME) or C5-C 24 fatty acid ethyl ester (FAEE) or C5-C 24 fatty acid methyl ester (FAME) and C5-C 24 fatty acid ethyl ester (FAEE), the method comprising culturing in a medium comprising xylose: a recombinant host cell comprising at least one heterologous fatty acid derivative biosynthetic enzyme having ester synthase activity (E.C. 3.1.1.67) and a XylR protein variant having at least one mutation at position 382 of SEQ ID NO: 1.

[0045] In some embodiments, the XylR protein variant, recombinant host cell, or method of any embodiment comprises at least one mutation at position 382 of SEQ ID NO: 1, which is an E382K substitution mutation.

[0046] In some embodiments, the recombinant host cell of any embodiment comprises improved growth results due to increased xylose utilization. In some embodiments, the improved growth occurs in the presence of glucose.

[0047] In some embodiments, the method of any embodiment comprises a medium comprising xylose and further comprising glucose. In some embodiments, the medium is derived from cellulosic biomass.

[0048] Other features, objects, and advantages of the application will be apparent from the detailed description that follows. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 Figure 1 1 is a graph illustrating improved co-utilization of glucose and xylose by the E382K XylR mutant (xylRl) compared to wild type control.

[0050] Figure 2 Figure 12 is a graph illustrating improved growth in the presence of xylose and that the improved growth is due to improved xylose utilization.

[0051] Figure 3 Figure 13 is a graph illustrating growth of xylR mutants L146R, F372W, N120C, L365V, S364W, V83C, (Q289V, D305M), Q145R, L89V, and Y141R compared to WT xylR growth in a basal medium containing xylose.

[0052] Figure 4 Figure 14 is a graph illustrating growth of xylR mutants (S364W, R295C), (A286F, Q306K), E337H, L112R, (R280V, D305G), A286M, and I313L compared to WT xylR in a basal medium containing xylose.

[0053] Figure 5 Figure 15 is a graph illustrating growth of xylR mutants A247V, A336M, A336G, (L89K, L112R), E150W, S130Y, (H88G, E150G, A246A*), and A310L compared to WT xylR in a basal medium containing xylose. * indicates a silent mutation at the nucleic acid level (GCG->GCA).

[0054] Figure 6 Figure 16 is a graph illustrating growth of xylR mutants (A247T, S352S*) and G154C compared to WT xylR in a basal medium containing xylose. * indicates a silent mutation at the nucleic acid level (TCG->TCC).

[0055] Figure 7 Figure 17 is a graph illustrating growth of xylR mutants L351T, M333R, and V155E compared to WT xylR in a basal medium containing xylose.

[0056] Figure 8 Figure 18 is a graph illustrating growth of xylR mutants L365T, R270E, E337N, and V147M compared to WT xylR in a basal medium containing xylose.

[0057] Figure 9XylR mutants L365V, Q145R, L146R, and (Q289, D305M) xylose utilization compared to WT xylR grown in a basal medium containing xylose.

[0058] Figure 10 XylR mutants L365V, Q145R, L146R, and (Q289, D305M) productivity in fatty acid species (FAS) production compared to WT xylR grown in a basal medium containing xylose.

[0059] Figure 11 XylR mutants F372W, N120C, V83C, S364W, L89V, and Y141R xylose utilization compared to WT xylR grown in a basal medium containing xylose.

[0060] Figure 12 XylR mutants R372W, N120C, V83C, S364W, L89V, and Y141R productivity in fatty acid species (FAS) production compared to WT xylR grown in a basal medium containing xylose.

[0061] Figure 13 XylR mutants E337H, L112R, and A286M xylose utilization compared to WT xylR grown in a basal medium containing xylose.

[0062] Figure 14 XylR mutants E337H, L112R, and A286M productivity in fatty acid species (FAS) production compared to WT xylR grown in a basal medium containing xylose.

[0063] Figure 15 XylR mutants (S364W, R295C), (A286F, Q306K), A247V, A336M, and A336G xylose utilization compared to WT xylR grown in a basal medium containing xylose.

[0064] Figure 16 XylR mutants (S364W, R295C), (A286F, Q306K), A247V, A336M, and A336G productivity in fatty acid species (FAS) production compared to WT xylR grown in a basal medium containing xylose.

[0065] Figure 17 A schematic pathway of xylose utilization in metabolic pathways is shown. DETAILED DESCRIPTION

[0066] Definitions

[0067] As used herein and in the appended claims, singular articles, such as "a," "an," and "the," and classes of articles such as "said" and "the said" shall encompass both the singular and plural, unless otherwise indicated herein or clearly contradicted by the context. Thus, for example, a reference to "a host cell" includes two or more such host cells, a reference to "a nucleic acid sequence" includes one or more nucleic acid sequences, a reference to "an enzyme" includes one or more enzymes, and so on.

[0068] As used herein, "about" is understood by those of ordinary skill in the art and can vary to some extent depending upon the context in which the term is used. If the use of the term "about" in a context is unclear, "about" shall mean up to plus or minus 10% of a particular term (including the particular term). Thus, for example, about 100 would mean 90 to 110, and would include 100.

[0069] As will be understood by those of ordinary skill in the art, for any and all purposes, all ranges disclosed herein are also meant to include any and all possible sub-ranges comprising at least two endpoints of the stated range. Further, ranges include each and every possible sub-range. For example, a range of 1 -3 includes any and all possible sub-ranges, such as 1-2, 2-3, 3, etc. Likewise, a range of 1-5 includes any and all possible sub-ranges, such as 1-2, 2-3, 3-4, 4-5, 5, etc. Further, a range includes each and every individual member. For example, a range of 1-3 includes 1, 2, and 3. Likewise, a range of 1-5 includes 1, 2, 3, 4, and 5. Further, a range includes each and every individual member of the range. For example, a range of 1-5 includes 1, 2, 3, 4, and 5.

[0070] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In particular, the present disclosure utilizes conventional techniques in the fields of recombinant genetics, organic chemistry, fermentation, and biochemistry. Basic texts disclosing molecular biology and genetics general terms include, e.g., Lackie, Dictionary of Cell and Molecular Biology, Elsevier (5th edition 2013). Basic texts disclosing general methods and terms of biochemistry include, e.g., Lehninger Principles of Biochemistry Sixth edition, edited by David L. Nelson and Michael M. Cox. W. H. Freeman (2012). Basic texts disclosing general methods and terms of fermentation include, e.g., Principles of Fermentation Technology, 3rd edition, edited by Peter F Stanbury, Allan Whitaker and Stephen J Hall. Butterworth-Heinemann (2016). Basic texts disclosing general methods and terms of organic chemistry include, e.g., Favre, Henri A. and Powell, Warren H. Nomenclature of Organic Chemistry. IUPAC Recommendations and Preferred Name 2013. The Royal Society of Chemistry, Cambridge, UK, 2013; Practical Synthetic Organic Chemistry: Reactions, Principles, and Techniques, edited by Stephane Caron, John Wiley and Sons Inc. (2011); Organic Chemistry, 9th edition - Francis Carey and Robert Giuliano, McGraw Hill (2013).

[0071] The sequence accession numbers throughout this specification are obtained from the databases provided by the National Center for Biotechnology Information (NCBI) maintained by the National Institutes of Health (referred to herein as “NCBI accession numbers” or alternatively “GenBank accession numbers” or alternatively can also be referred to simply as “accession numbers”), and the UniProt Knowledgebase (UniProtKB) and Swiss-Prot databases provided by the Swiss Institute of Bioinformatics (referred to herein as “UniProtKB accession numbers”).

[0072] Enzyme Classification (EC) numbers are determined by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB), the explanation of which can be found on the IUBMB Enzyme Nomenclature website on the World Wide Web. EC numbers classify enzymes according to the reactions they catalyze. For example, the enzyme activity of thioesterases is classified as E.C. 3.1.2.1-3.1.2.27 and 3.1.2.-. The specific classification is based on the activity of different thioesterases on different substrates.

[0073] 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 under E.C. 3.1.2.- such as 3.1.2.14. Thioesterases are found in most prokaryotes and in the chloroplasts of most plants and algae. The function of thioesterases is conserved from one species to another in most prokaryotes. Thus, different microbial species can perform the same enzymatic activity of a thioesterase classified under E.C. 3.1.2.-.

[0074] 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 4 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 mono-unsaturated or poly-unsaturated.

[0075] “Fatty acid” or “fatty acids” as used herein are produced within the cell by the process of fatty acid biosynthesis, by reversal of fatty acid beta-oxidation, or they can be fed into the cell. Fatty acid biosynthesis is typically malonyl-CoA dependent acyl-ACP synthesis, while reversal of beta-oxidation results in acyl-CoA, as is well known in the art. Fatty acids fed into the cell are converted to acyl-CoA and acyl-ACP.

[0076] The biosynthesis and degradation of fatty acids occurs in all forms of life, including prokaryotes, single-celled eukaryotes, higher eukaryotes, and archaea. The tools and methods disclosed herein can be used to produce fatty acid derivatives that are derived by one or more of fatty acid synthesis, degradation, or provision in any organism that naturally produces alkyl thioesters.

[0077] The term "fatty acid derivative" as used herein refers to a product derived from a fatty acid. Thus, "fatty acid derivative" includes "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. Exemplary fatty acid derivatives include fatty acids, fatty acid esters (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 (e.g., alkanes, alkenes, etc.), ketones, terminal alkenes, internal alkenes, 3-hydroxy fatty acid derivatives, bifunctional fatty acid derivatives (e.g., omega-hydroxy fatty acids, 1,3 fatty diols, alpha, omega-diol, alpha, omega-3-hydroxy triols, omega-hydroxy FAMEs, omega-OH FAEEs, etc.), and unsaturated fatty acid derivatives, including unsaturated compounds of each of the above fatty acid derivatives.

[0078] As used herein, the expression "fatty acid derivative composition" refers to a composition of fatty acid derivatives, such as a composition of fatty acids produced by an organism. The "fatty acid derivative composition" can comprise a single fatty acid derivative species or can comprise a mixture of fatty acid derivative species. In some exemplary embodiments, the mixture of fatty acid derivatives includes more than one type of fatty acid derivative product (e.g., fatty acids, fatty acid esters, fatty alcohols, fatty alcohol acetates, fatty aldehydes, fatty amines, bifunctional fatty acid derivatives, etc.). In other exemplary embodiments, the mixture of fatty acid derivatives includes a mixture of fatty acid esters (or another fatty acid derivative) having different chain length, saturation, and / or branching characteristics. In other exemplary embodiments, the mixture of fatty acid derivatives comprises primarily one type of fatty acid derivative. In still other exemplary embodiments, the mixture of fatty acid derivatives comprises a mixture of more than one type of fatty acid derivative product (e.g., fatty acid derivatives having different chain length, saturation, and / or branching characteristics). In still other exemplary embodiments, the mixture of fatty acid derivatives comprises a mixture of fatty esters and beta-hydroxy esters. In still other exemplary embodiments, the fatty acid derivative composition comprises a mixture of fatty alcohols and fatty aldehydes. In still other exemplary embodiments, the fatty acid derivative composition comprises a mixture of FAMEs and / or FAEEs. In still other exemplary embodiments, the fatty acid derivative composition comprises a mixture of fatty alcohol acetates (FACEs).

[0079] As used herein, the term "nucleotide" has its customary meaning known in the art. Except when referring to naturally occurring ribonucleotide or deoxyribonucleotide monomers, the term "nucleotide" also encompasses nucleotide analogs and modified nucleotides, such as, for example, amino-modified nucleotides. In addition, "nucleotide" includes non-naturally occurring analog structures. Thus, for example, the individual units of a peptide nucleic acid each containing a base can be referred to herein as nucleotides.

[0080] The term "polynucleotide" refers to a polymer typically of ribonucleotides (RNA) or deoxyribonucleotides (DNA) in phosphodiester linkage, which can be single-stranded or double-stranded and which can contain natural and / or non-natural and / or altered nucleotides. The terms "polynucleotide," "nucleic acid sequence," and "nucleotide sequence" are used interchangeably herein to refer to a polymeric form of nucleotides of any length, either RNA or DNA. These terms refer to the primary structure of the molecule, and thus include single-stranded, double-stranded, triple-stranded, quadruple-stranded, partially double-stranded, branched, hairpinned, looped, circular, in a padlock configuration, and the like. The terms equally apply to RNA or DNA analogs made from nucleotide analogs and to modified polynucleotides such as, but not limited to, methylated and / or capped polynucleotides. A polynucleotide can be in any form, including but not limited to, plasmids, viruses, chromosomes, ESTs, cDNAs, mRNAs, and rRNAs, and can be prepared by any known method, including synthesis, recombination, in vitro production, or combinations thereof, as well as any purification method known in the art

[0081] As used herein, the terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues typically 12 or more in length. Polypeptides less than 12 amino acids in length are referred to herein as "peptides." The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to

[0082] 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 the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, such as the twenty amino acids found in proteins, as well as those encoded by modifications including hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds which have the same basic chemical structure as a naturally occurring amino acid (i.e., an alpha carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group) but having modified R groups (e.g., norleucine) or modified peptide backbones. Such analogs are inositol, norleucine, methionine sulfoxide, methylsulfonium methionine. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but otherwise retain the same basic chemical structure as naturally occurring amino acids. Naturally encoded amino acids are the twenty 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 one-letter codes listed in Table 1 below are used to refer to specific members of the twenty common naturally occurring amino acids. The one-letter amino acid codes are well known in the art (see, e.g., Lehninger, supra).

[0083] Table 1

[0084] Amino acid Single letter code Amino acid Single letter code Glycine G Proline P Alanine A Valine V Leucine L Isoleucine I Methionine M Cysteine C Phenylalanine F Tyrosine Y Tryptophan W％ Histidine H Lysine K Arginine R Glutamine Q Asparagine N Glutamic acid E Aspartic acid D Serine S Threonine T

[0085] When referring to two nucleotide or polypeptide sequences, the "percentage of sequence identity" between two sequences is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window can comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage of sequence identity is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs 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.

[0086] Accordingly, the expression "percent identity" or equivalently "percent sequence identity" in the context of two or more nucleic acid sequences or peptides or polypeptides refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids in common, for example, 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 and aligned for maximum correspondence, for example, using the BLAST or BLAST 2.0 sequence comparison algorithms 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. The percent sequence identity between two nucleotide or amino acid sequences can also be determined using the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:444-453, as implemented in the GAP program in the GCG software package (available at accelryds.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and the length weight of 1, 2, 3, 4, 5, or 6. The percent sequence identity between two nucleotide sequences can also be determined using the GAP program in the GCG software package (available at accelryds.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and the length weight of 1, 2, 3, 4, 5, or 6. A person of ordinary skill in the art can perform initial sequence identity calculations and adjust the algorithm parameters as appropriate. If the practitioner is unsure which parameters should be used to determine whether a molecule is within the homology limitations of the claims, a set of parameters can be used that are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. Additional methods of sequence alignment are known in the biotechnology art (see, e.g., Rosenberg (2005) BMC Bioinformatics 6:278; Altschul et al. (2005) FEBS J. 272(20):5101-5109).

[0087] Two or more nucleic acid or amino acid sequences are said to be "substantially identical" if they have about 50% identity, preferably 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity within a specified region when aligned and analyzed as described above. Two nucleotide or amino acid sequences are said to be "identical" if the sequence of nucleotides or amino acid residues in the two sequences is the same when aligned for maximum correspondence, as described above. This definition also can refer or can be used to refer to the complement of a test sequence. Identity is typically calculated over a region of at least about 25 amino acids or nucleotides, or more preferably over a region of 50-100 amino acids or nucleotides, or over the entire length of a given sequence.

[0088] The expressions "hybridize specifically" and "specific hybridization" describe conditions under which nucleic acid sequences hybridize to each other and wash conditions. Guidance in determining conditions for hybridization reactions can be found in, for example, Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY (1989), 6.3.1-6.3.6. Both aqueous and nonaqueous methods are described in the cited reference and either can be used. Specific hybridization conditions referred to herein are as follows: (1) low stringency hybridization conditions - 6X sodium chloride / sodium citrate (SSC), at 45°C, followed by a wash in 0.2X SSC, 0.1% SDS at 50°C (for low stringency conditions, the temperature can be increased to 55°C); (2) medium stringency hybridization conditions - 6X SSC, at about 45°C, followed by a wash in 0.2X SSC, 0.1% SDS at 60°C; (3) high stringency hybridization conditions - 6X SSC, at about 45°C, followed by a wash in 0.2X 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 a wash in 0.2X SSC, 1% SDS at 65°C. Unless otherwise specified, very high stringency conditions (4) are preferred.

[0089] 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 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 carried by a parent cell (or host cell). In some exemplary embodiments, an "endogenous" gene is a gene that is present in a cell when the cell is initially isolated from nature, i.e., the gene is "naturally-occurring" to the cell. In other exemplary embodiments, an "endogenous" gene has been altered by recombinant techniques, e.g., by changing the relationship of control sequences and coding sequences. Thus, in some exemplary embodiments, a "heterologous" gene can be "endogenous" to a host cell.

[0090] 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 a parent cell and thus is added to a cell, cell culture, or assay from outside the cell.

[0091] As used herein, the term "native" refers to the form of a nucleic acid, protein, polypeptide, or fragment thereof that is isolated from nature, or that is not intended to have mutations introduced.

[0092] 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 embodiments of the disclosure, a fragment refers to the entire amino acid sequence of a domain of a polypeptide or protein (e.g., a substrate binding domain or a catalytic domain).

[0093] The term "mutagenesis" refers to the process of altering the genetic information of an organism in a stable manner to produce a "mutant" or "variant." Protein-encoding nucleic acid sequence mutagenesis to produce a mutant nucleic acid sequence that produces a mutant protein. Mutagenesis also refers to changes in non-coding nucleic acid sequences. In some exemplary embodiments, mutations in non-coding nucleic acid sequences result in modified protein activity.

[0094] Accordingly, as used herein, a "mutation" refers to a change in a nucleic acid position or an amino acid position (residue) of a polypeptide or protein relative to a control nucleic acid or amino acid sequence. In the context of a polynucleotide, the term "mutation" refers to a modification to a polynucleotide sequence resulting in an alteration of the polynucleotide sequence relative to a control or reference polynucleotide sequence. In some exemplary embodiments, a mutated polynucleotide sequence refers to an alteration that does not change the encoded amino acid sequence, such as optimization of codons for expression purposes. In other exemplary embodiments, a mutation in a polynucleotide sequence modifies codons in a manner that results in a modification to the encoded amino acid sequence.

[0095] In the context of a protein, the term "mutation" or "mutated" refers to a modification to an amino acid sequence resulting in an alteration of the sequence of the protein relative to a control or reference protein sequence. A mutation can refer to the substitution of one amino acid for another, or the insertion or deletion of one or more amino acid residues. In some exemplary embodiments, a "mutation" is the replacement of an amino acid with a non-natural amino acid or a chemically modified amino acid residue. In other exemplary embodiments, a "mutation" is a truncation (e.g., deletion or interruption) in a sequence or subsequence relative to a precursor sequence or a shortening of the sequence by deletion from one or the other end. In other exemplary embodiments, a mutation is the addition of an amino acid or subsequence (e.g., two or more amino acids in a segment, which is inserted between two consecutive amino acids in a precursor protein sequence) within a protein or at either end of a protein, thereby increasing the length (or lengthening) of the protein. Mutations can be introduced into a polynucleotide by a recognized number of methods known to one of ordinary 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.

[0096] As used herein, the term "mutant" or equivalently "variant" refers to a polynucleotide sequence or a polypeptide sequence comprising at least one mutation. Accordingly, an engineered XylR variant or XylR (with improved ability to utilize xylose and improved ability to co-utilize glucose and xylose for production of, for example, fatty acids and fatty acid derivatives) will have at least one mutation in its polypeptide sequence compared to a control XylR enzyme.

[0097] The expression "XylR mutant with improved xylose utilization or improved co-utilization of glucose and xylose" or "XylR mutant with improved xylose utilization and / or improved co-utilization of glucose and xylose" or equivalently "XylR protein variant with improved xylose utilization and / or improved co-utilization of glucose and xylose" as used herein refers to an engineered polypeptide / protein variant of the E. coli xylose repressor (XylR) which has an improved ability to utilize xylose and / or has an improved ability to co-utilize glucose and xylose as for example measured by an improved rate of growth on xylose as measured by optical density (OD600) readings and improved co-consumption of glucose and xylose in a bioreactor as measured by the total amount of sugars utilized as disclosed in Examples 1 and 2 below. Thus, a "XylR mutant with improved xylose utilization and / or improved co-utilization of glucose and xylose" can show improved xylose utilization, improved co-utilization of glucose and xylose or can have both properties simultaneously.

[0098] A "XylR mutant having improved xylose utilization and / or improved co-utilization of glucose and xylose" or equivalently "XylR protein variant having improved xylose utilization and / or improved co-utilization of glucose and xylose" is a protein having at least 90% sequence identity to SEQ ID NO: 1 which has at least one mutation at a position corresponding to position 382 of SEQ ID NO: 1 and which has no mutation at a position corresponding to position 121 of SEQ ID NO: 1 and no mutation at a position corresponding to position 363 of SEQ ID NO: 1. In embodiments, the mutation at position 382 of SEQ ID NO: 1 is an E382K substitution mutation. In embodiments, the "XylR protein variant having improved xylose utilization and / or improved co-utilization of glucose and xylose" has an amino acid sequence according to SEQ ID NO: 3. In embodiments, the "XylR protein variant having improved xylose utilization and / or improved co-utilization of glucose and xylose" is a protein having at least 91% sequence identity to SEQ ID NO: 1 which has at least one mutation at a position corresponding to position 382 of SEQ ID NO: 1 and which has no mutation at a position corresponding to position 121 of SEQ ID NO: 1 and no mutation at a position corresponding to position 363 of SEQ ID NO: 1. In other embodiments, the "XylR protein variant having improved xylose utilization and / or improved co-utilization of glucose and xylose" is a protein having at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity to SEQ ID NO: 1 which has at least one mutation at a position corresponding to position 382 of SEQ ID NO: 1 and which has no mutation at a position corresponding to position 121 of SEQ ID NO: 1 and no mutation at a position corresponding to position 363 of SEQ ID NO: 1. In embodiments, the XylR protein variant having improved xylose utilization and / or improved co-utilization of glucose and xylose can be introduced into a recombinant host cell to efficiently produce fatty acids and fatty acid derivatives using, for example, lignocellulosic biomass as feedstock.

[0099] The term "gene" as used herein refers to a nucleic acid sequence, e.g., a DNA sequence, that encodes an RNA product or a protein product, as well as operably linked nucleic acid sequences that affect expression of the RNA or protein product (e.g., expression control sequences, e.g., promoters, enhancers, ribosome binding sites, translation control sequences, etc.). The term "gene product" refers to an RNA, e.g., tRNA, mRNA, and / or a protein, expressed from a particular gene.

[0100] The term "expression" or "expressed" with respect to a gene, as used herein, refers to the production of one or more transcriptional and / or translational products of a gene. In exemplary embodiments, the level of expression of a DNA molecule in a cell is determined based on the amount of the corresponding mRNA present within the cell or the amount of DNA-encoded protein produced by the cell. The term "expressed gene" refers to a gene that is transcribed into messenger RNA (mRNA) which is then translated into a 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.

[0101] The level of expression of a nucleic acid molecule in a cell or cell-free system is influenced by "expression control sequences" or equivalently "regulatory sequences." "Expression control sequences" or "regulatory sequences" are known in the art and include, for example, promoters, enhancers, polyadenylation signals, transcription terminators, nucleotide sequences that affect the stability of RNA, internal ribosome entry sites (IRES), and the like, which provide for the expression of a polynucleotide sequence in a host cell. In exemplary embodiments, "expression control sequences" specifically interact with cellular proteins 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, expression control sequences are operably linked to a polynucleotide sequence. "Operably linked" means that the polynucleotide sequence and the expression control sequence(s) are functionally connected such that the polynucleotide sequence is expressed when the appropriate molecules (e.g., transcriptional activator proteins) are contacted with the expression control sequence(s). In exemplary embodiments, a promoter that is operably linked is located upstream, in terms of the direction of transcription and translation, of the selected polynucleotide sequence. In some exemplary embodiments, an enhancer that is operably linked can be located upstream, within, or downstream of the selected polynucleotide.

[0102] As used herein, "modified activity" or "altered level of activity" of a protein / polypeptide (e.g., an engineered XylR variant) refers to a difference in one or more characteristics of the activity of the protein / polypeptide as compared to the characteristics of a suitable control protein (e.g., a corresponding parent protein or a corresponding wild-type protein). Thus, in exemplary embodiments, a difference in activity of a protein having "modified activity" as 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 measurement of the same activity of the corresponding control protein in an otherwise isogenic host cell. The modified activity can be the result of, for example, a change in binding affinity of the protein to a nucleic acid; a change in protein structure (e.g., a change in primary structure, such as, for example, a change in the nucleotide coding sequence of the protein that results in a change in substrate specificity, DNA binding, a change in observed kinetic parameters, a change in solubility, etc.); a change in protein stability (e.g., an increase or decrease in protein degradation), etc. In some exemplary embodiments, a polypeptide having "modified activity" is a mutant or variant XylR enzyme disclosed herein.

[0103] In exemplary embodiments, the polypeptides disclosed herein have "modified activity," i.e., for example, an "improved level of activity." The expression "improved level of activity" as used herein refers to a polypeptide having a higher level of biochemical or biological function (e.g., DNA binding or enzymatic activity) as compared to the level of biochemical and / or biological function of a corresponding control polypeptide under the same conditions. The degree of improvement in 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.

[0104] Accordingly, "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 rate of a reaction catalyzed by such a polypeptide. Catalytic activity / catalytic efficiency can be improved, for example, by improving one or more kinetic parameters (measured or calculated) of a reaction, such as Vmax (the maximum rate at which a reaction can proceed), Km (Michaelis constant), kcat (the number of substrate molecules turned over per enzyme molecule per second), and the like, or any ratio between such parameters, such as kcat / Km (a measure of enzyme efficiency). Accordingly, "improved catalytic activity" or "improved catalytic efficiency" of a polypeptide can be measured in a variety of ways. For example, "improved activity" can be measured by an increase in titer (concentration under certain conditions: g / L, or mg / L, or g / Kg), an improvement in growth rate; improved utilization of a particular substrate, such as xylose; a change in composition (amount of a particular fatty acid species / total fatty acid derivative (FAS) produced under certain conditions, such as in the presence of xylose).

[0105] A "control" sample, such as a "control" nucleotide sequence, "control" polypeptide sequence, "control" cell, and the like, or value refers to a sample used as a reference, typically a known reference, for comparison to a test sample. For example, in one exemplary embodiment, a test sample comprises a "XylR mutant for improved xylose utilization or improved glucose and xylose co-utilization," while a control sample comprises the corresponding or specified unmodified / non-variant XylR protein / enzyme (e.g., SEQ ID NO: 1). The skilled artisan will recognize that controls can be designed for the evaluation of any number of parameters. Moreover, the skilled artisan will understand which controls are valuable in a given situation, and will be able to analyze data based on comparison to control values.

[0106] The term "recombinant" as used herein refers to a genetically modified polynucleotide, polypeptide, cell, tissue, or organism. The term "recombinant" applies equally to a first generation genetically modified polynucleotide, polypeptide, cell, tissue, or organism, as well as to the progeny of a genetically modified polynucleotide, polypeptide, cell, tissue, or organism that carries the genetic modification.

[0107] The term "recombinant" when used with reference to cells means that the cell has been modified by the introduction of a heterologous nucleic acid or protein, or has been modified by the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified and the derived cell comprises the modification. Thus, for example, a "recombinant cell" or equivalently a "recombinant host cell" can be modified to express genes that are not found within the native (non-recombinant) form of the cell, or can be modified to express native genes in an abnormal form or at an abnormal time; for example, a native gene can be overexpressed, underexpressed, or not expressed at all. In exemplary embodiments, a "recombinant cell" or "recombinant host cell" is engineered to express a "XylR mutant for improved xylose utilization or improved glucose and xylose co-utilization." The recombinant cell can be derived from a microorganism, such as a bacterium, virus, or fungus. Additionally, the recombinant cell can be derived from a plant or animal cell. 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, bifunctional 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 to express the polynucleotides.

[0108] The term "recombinant" or equivalently "heterologous" when used with reference to a polynucleotide indicates that the polynucleotide has been modified by human intervention as compared to the natural or native form of the polynucleotide or as compared to a naturally occurring variant of the nucleotide. In an exemplary embodiment, a recombinant polynucleotide (or a copy or complement of a recombinant polynucleotide) is a recombinant polynucleotide that has been manipulated by human intervention to differ from its naturally occurring form. Thus, in an exemplary embodiment, a recombinant polynucleotide is a mutated form of a natural gene or a mutated form of a naturally occurring variant of a natural gene, where the mutation is made by deliberate human intervention, for example, by saturation mutagenesis using mutagenic oligonucleotides, by using UV radiation or mutagenic chemicals, and the like. Such a recombinant polynucleotide can comprise one or more point mutations, substitutions, deletions, and / or insertions relative to the natural form of the gene or the naturally occurring variant form. Similarly, a polynucleotide comprising a promoter operably linked to a second polynucleotide (e.g., a coding sequence) is a "recombinant" polynucleotide. Thus, a recombinant polynucleotide comprises a combination of polynucleotides that is not found in nature. Recombinant proteins (discussed supra) are typically proteins expressed from recombinant polynucleotides, and recombinant cells, tissues, and organisms are those comprising recombinant sequences (polynucleotides and / or polypeptides).

[0109] 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, and the like. An exemplary bacterium is Escherichia coli. Exemplary eukaryotic microorganisms include, for example, yeast, protozoa, algae, and the like. In exemplary embodiments, a "recombinant microorganism" is a microorganism that has been genetically altered and thus expresses or encompasses heterologous nucleic acid sequences and / or heterologous proteins.

[0110] A "production host" or equivalently "production host cell" is a cell used to produce a product. As disclosed herein, a "production host" is typically modified to express or overexpress a selected gene, or has 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, algal, and / or filamentous fungal cells. An exemplary "production host" is a recombinant Escherichia coli cell.

[0111] As used herein, "acyl-ACP" refers to an acyl thioester formed between the carbonyl carbon of an acyl chain and the thiol group of the phosphopantetheine moiety of acyl carrier protein (ACP). In some embodiments, acyl-ACP is an intermediate in the synthesis of fully saturated acyl-ACPs. In other exemplary embodiments, acyl-ACP is an intermediate in the synthesis of unsaturated acyl-ACPs. In some exemplary embodiments, the carbon chain of the acyl group of 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 acyl-ACP has 12 carbons, 14 carbons, or 16 carbons. In other exemplary embodiments, the carbon chain of the acyl group of acyl-ACP has a length of 8 carbons. In other exemplary embodiments, the carbon chain of the acyl group of acyl-ACP has a length of 10 carbons. Each of these acyl-ACPs is a substrate for an enzyme, such as, for example, an ester synthase that converts acyl-ACP to a fatty acid derivative, a thioesterase, and the like.

[0112] As used herein, the expression "fatty acid derivative biosynthetic pathway" refers to a biochemical pathway that produces a fatty acid derivative. Thus, enzymes comprising "a fatty acid derivative biosynthetic pathway" are referred to herein as "fatty acid derivative biosynthetic polypeptides" or equivalently as "fatty acid derivative enzymes." Thus, for example, a thioesterase (e.g., an enzyme EC 3.1.2.14 having thioesterase activity) is a "fatty acid derivative biosynthetic peptide" or equivalently a "fatty acid derivative enzyme." Thus, the terms "fatty acid derivative enzyme" or equivalently "fatty acid derivative biosynthetic polypeptide" collectively and individually refer to enzymes that can be expressed or overexpressed to produce a fatty acid derivative. Non-limiting examples of "fatty acid derivative enzymes" or equivalently "fatty acid derivative biosynthetic polypeptides" include, for example, fatty acid synthases, thioesterases, acyl-CoA synthases, acyl-CoA reductases, acyl ACP reductases, alcohol dehydrogenases, alcohol O-acyltransferases, acyl CoA reductases that form fatty alcohols, fatty acid decarboxylases, fatty aldehyde decarbonylases and / or oxidative deformylases, carboxylate reductases, fatty alcohol O-acetyltransferases, ester synthases, and the like. A "fatty acid derivative enzyme" or equivalently a "fatty acid derivative biosynthetic polypeptide" converts a substrate to 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.

[0113] As used herein, the term "culture" refers to a liquid medium comprising living cells. In one embodiment, a culture comprises cells grown under controlled conditions in a predetermined medium, for example, a culture of recombinant host cells grown in a liquid medium comprising a selected carbon source and nitrogen. "Carrying out a culture" or "culturing" refers to culturing a population of host cells (e.g., recombinant host cells) in a liquid or solid medium under suitable conditions. In certain embodiments, culturing refers to the bioconversion of a substrate to an end product. Culture media are well known and individual components of such media are available from commercial sources, for example, Difco TM Culture media and BBL TM Culture media. In one non-limiting example, an aqueous nutrient medium is a "rich medium" comprising complex sources of nitrogen, salts, and carbon, for example, YP medium, which comprises 10 g / L of peptone and 10 g / L of yeast extract.

[0114] As used herein, the term "titer" refers to the amount of fatty acid derivative produced per unit volume of host cell culture. Titer can refer to the amount of a particular fatty acid derivative or the amount of a combination of fatty acid derivatives of different chain lengths or different functionalities (such as, for example, a mixture of saturated and unsaturated fatty acid derivatives produced by a given recombinant host cell culture) or the amount of a fatty acid derivative composition.

[0115] The expression "commercial titers" or "commercial titer" as used herein refers to the amount of fatty acid derivative produced per unit volume of host cell culture that makes commercial production economically feasible. 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 titers are 10 g / L or more, 20 g / L or more, 30 g / L or more, 40 g / L or more, 50 g / L or more, 60 g / L or more, 70 g / L or more, 80 g / L or more, 90 g / L or more, 100 g / L or more, 110 g / L or more, 120 g / L or more, 130 g / L or more, 140 g / L or more, 150 g / L or more, 160 g / L or more, 170 g / L or more, 180 g / L or more, 190 g / L or more, 200 g / L or more.

[0116] As used herein, "yield of fatty acid derivative" refers to the efficiency of conversion of an input carbon source into a product in a host cell. Thus, the expression "yield of fatty acid derivative" refers to the amount of product produced from a given amount of carbon substrate. The percent yield is the percent of the theoretical yield (product synthesized under ideal conditions, with no carbon or energy loss). Thus, percent yield = (mass of product / mass of theoretical yield) X 100. Yield can refer to a particular fatty acid derivative or a combination of fatty acid derivatives.

[0117] As used herein, the term "productivity" refers to the amount of fatty acid derivative produced per unit volume of host cell culture per unit time. Productivity can be for one particular fatty acid derivative, or a combination of fatty acid derivatives or one or more other compounds produced by a given host cell culture. Thus, in exemplary embodiments, expression of a XylR mutant having improved utilization of xylose and / or improved co-utilization of glucose and xylose in a recombinant host cell, e.g., E. coli, results in improved productivity of a fatty acid derivative and / or other compound compared to a recombinant host cell expressing the corresponding control XylR enzyme or other suitable control. As used herein, the terms "total fatty species" and "total fatty acid product" and "total fatty acid derivative" are used interchangeably herein with respect to the amount (titer) of fatty acid derivative produced by a host cell, e.g., a host cell having a XylR mutant with improved utilization of xylose and / or improved co-utilization of glucose and xylose. Total fatty species, etc., can be assessed by gas chromatography with flame ionization detection (GC-FID). When referring to total fatty acid derivative analysis, the same terms can be used to mean, for example, total fatty esters, total fatty alcohols, total fatty aldehydes, total fatty amines, and total free fatty acids. In particular, the same terms can be used to mean total fatty acid methyl esters, fatty acid ethyl esters, or fatty alcohol acetates.

[0118] As used herein, the term "carbon source" 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 CO2). 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 thereof, 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 product of photosynthesis, such as glucose. In certain embodiments, the carbon source is biomass. In other embodiments, the carbon source is glucose. In other embodiments, the carbon source is sucrose. In other embodiments, the carbon source is glycerol. In other embodiments, the carbon source is a simple carbon source. In other embodiments, the carbon source is a renewable carbon source. In other embodiments, the carbon source is a cellulosic hydrolysate. In other examples, the carbon source is natural gas or a component of natural gas, such as methane, ethane, propane, and the like.

[0119] As used herein, the term "biomass" refers to any biological material from which a carbon source is derived. In some embodiments, the biomass is processed into a carbon source suitable for bioconversion. In some embodiments, the biomass is processed into a cellulosic hydrolysate. In other embodiments, the biomass does not require further processing into a carbon source. The carbon source can be converted into a composition comprising fatty acid derivatives.

[0120] Exemplary sources of biomass are plant matter or vegetation, such as derived from corn, sugar cane, switchgrass, rice, wheat, hardwood, softwood, palm, hemp, and the like. Another exemplary source of biomass is metabolic waste, such as animal matter (e.g., cow manure). Other exemplary sources of biomass include algae and other marine plants, such as macroalgae and kelp. Biomass also includes waste from industry, agriculture, forestry, and households, including but not limited to glycerol, fermentation waste, silage, straw, wood, pulp, sewage, garbage, cellulosic municipal waste, municipal solid waste, oleochemical waste, and leftover food (e.g., soap, oil, and fatty acids). The term "biomass" can also refer to a carbon source, such as a carbohydrate (e.g., a monosaccharide, a disaccharide, or a polysaccharide).

[0121] As used herein, the term "isolated" with respect to a product (e.g., a fatty acid derivative) refers to a product that is separated from cellular components, cell culture media, or chemical or synthetic precursors. The fatty acid derivatives produced by the methods disclosed herein can be relatively immiscible in the fermentation broth as well as in the cytoplasm. Thus, in exemplary embodiments, the fatty acid derivatives are extracellularly aggregated in an organic phase and thus are "isolated."

[0122] As used herein, the terms "purifying," "purified," or "purification" refer to the removal or isolation of a molecule from its environment, e.g., by separation or partitioning. A "substantially purified" molecule is at least about 60% free from (e.g., at least about 65% free from, at least about 70% free from, at least about 75% free from, at least about 80% free from, at least about 85% free from, at least about 90% free from, at least about 95% free from, at least about 96% free from, at least about 97% free from, at least about 98% free from, 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, removal of contaminants can result in an increase in the percentage of fatty acid derivatives or other compounds in a sample. For example, when a fatty acid derivative or other compound is produced in a recombinant host cell, the fatty acid derivative or other compound can be purified by removing host cell biomass or components thereof (e.g., proteins, nucleic acids, and other cellular components). After purification, the percentage of malonyl-CoA-derived compounds comprising the fatty acid derivative or other compound in the sample is increased. The terms "purifying," "purified," or "purification" are relative terms and do not require absolute purity. Thus, for example, when a fatty acid derivative is produced in a recombinant host cell, the fatty acid derivative is "purified" when it is substantially separated from other cellular components (e.g., nucleic acids, polypeptides, lipids, carbohydrates, or other hydrocarbons).

[0123] As used herein, the term "attenuate" refers to weakening, reducing, or decreasing. For example, the activity of a polypeptide can be attenuated, e.g., by modifying the structure of the polypeptide to reduce its activity (e.g., by modifying a nucleotide sequence encoding the polypeptide).

[0124] I. INTRODUCTION

[0125] The use of hydrolysis product feedstocks can greatly reduce the cost of producing renewable chemicals by microbial fermentation. However, although hydrolysis products of lignocellulosic biomass can be converted to biofuels and chemicals by microbial fermentation, hydrolysis product feedstocks typically comprise mixed sugars, e.g., glucose, xylose, mannose, etc., and fermentation of mixed sugars presents significant challenges to the economical production of biofuels and chemicals by microbial fermentation.

[0126] In particular, the presence of glucose in growth media inhibits E. coli and other industrial microbial species from utilizing other sugars. Only after the glucose in the growth media is completely consumed do these microorganisms begin to consume other sugars, such as xylose, pentose sugars. The preferential utilization of glucose over non-glucose sugars often results in lower overall yields and productivity; a phenomenon known as catabolite repression or diauxic growth (see, e.g., Kremling, A., et al. (2015) Volume 23(2): 99-109; Bruckner R, Titgemeyer F. (2002) FEMS Microbiol. Lett. 209: 141-14).

[0127] Accordingly, to make the production of any renewable carbon-based product derived from a hydrolyzate feedstock more efficient, there is a need in the art for microbial systems that allow for increased xylose utilization and increased co-utilization of glucose and xylose.

[0128] Fortunately, the present disclosure provides these and other needs.

[0129] II. XylR Mutants with Improved Xylose Utilization or Improved Co-Utilization of Glucose and Xylose

[0130] A. General Methods

[0131] The present disclosure utilizes routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods and terminology of molecular biology and genetics include, for example, Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Press 4thedition (Cold Spring Harbor, NY 2012); Current Protocols in Molecular Biology Vols. 1-3, John Wiley & Sons (1994-1998) and Supplements 1-115 (1987-2016). The present disclosure also utilizes routine techniques in the field of biochemistry. Basic texts disclosing the general methods and terminology of biochemistry include, for example, Lehninger Principles of Biochemistry Sixth edition, David L. Nelson and Michael M. Cox, editors. W. H. Freeman (2012). The present disclosure also utilizes routine techniques in industrial fermentation. Basic texts disclosing the general methods and terminology of fermentation include, for example, Principles of Fermentation Technology, 3rdedition, Peter F. Stanbury, editor, Allan Whitaker and Stephen J. Hall. Butterworth-Heinemann (2016); Fermentation Microbiology and Biotechnology, 2ndedition, E. M. T. El-Mansi, C. F. A. Bryce, Arnold L. Demain, and A. R. Allman, editors CRC Press (2007). The present disclosure also utilizes routine techniques in the field of organic chemistry.A basic text disclosing general methods and terminology of organic chemistry includes, for example, Practical Synthetic Organic Chemistry: Reactions, Principles, and Techniques, edited by Stephane Caron, 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).

[0132] For nucleic acids, sizes are given in kilobases (kb) or base pairs (bp). Estimates are typically derived from agarose or acrylamide gel electrophoresis, sequenced nucleic acids, or published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or number of amino acid residues. Protein sizes are estimated from gel electrophoresis, sequenced proteins, derived amino acid sequences, or published protein sequences.

[0133] Noncommercially available oligonucleotides can be chemically synthesized, e.g., using 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). Purification of oligonucleotides is done, e.g., by natural polyacrylamide gel electrophoresis or by anion-exchange HPLC, as described in Pearson & Reanier, J. Chrom. 255: 137-149 (1983).

[0134] Sequences of cloned genes and synthetic oligonucleotides can be verified after cloning using, e.g., the chain termination method of Wallace et al., Gene 16: 21-26 (1981) for sequencing double-stranded templates.

[0135] B. XylR mutants with improved xylose utilization and / or improved co-utilization of glucose and xylose

[0136] 1. General

[0137] The sequence of wild-type E. coli is provided as SEQ ID NO: 1 below. The E. coli XylR protein also has Uniprot Accession Number: UniProtKB - P0ACI3

[0138]

[0139] (SEQ ID NO: 1:

[0140] The wild-type E. coli xylose repressor (XylR) SEQ ID NO: 1 is known to activate D-xylose-responsive genes (see, e.g., Song S, Park C. (1998) FEMS Microbiol. Lett. 163:255-264). In particular, XylR-xylose activates transcription of XylAB and XylFGH by binding to specific sites near the start of each operon. The xylAB and xylFGH operons are in opposite directions on the genome. XylR uses a single dimer linked to two xylose molecules to recruit RNA polymerase to both binding sites, which causes DNA circularization.

[0141] It is also believed that CRP-cAMP is required to co-activate xylAB and xylFGH transcription with XylR-xylose. cAMP can only reach high intracellular concentrations when glucose is depleted from the growth medium. It is believed that the lack of availability of cAMP causes a two-stage effect whereby the presence of glucose effectively inhibits the uptake of xylose (see, e.g., Sievert et al. (2017) PNAS 114 (28) 7349-7354, July 11, 2017).

[0142] The linear protein sequence has been analyzed and the 3-dimensional structure of the E. coli xylose repressor protein (XylR) has been determined (see, e.g., Ni et al. (2013) Nuc. Acid. Res. 41 (3): 1998-2008).

[0143] XylR is a 392 amino acid protein that forms a homodimer that interacts with two xylose molecules and DNA. The protein comprises an N-terminal domain (residues 1-274) and a C-terminal domain (residues 285-392) connected by a linker formed by residues 275-284.

[0144] The C-terminal domain is the DNA binding domain. The C-terminal domain spans amino acids 285-392, with amino acid residues 304-323 forming a helix-turn-helix binding motif. Since residues 304-323 are directly involved in DNA binding, mutations in this region can disrupt the function of the XylR protein. However, amino acid substitutions outside the helix-turn-helix region within the DNA binding domain (e.g., residues 285 to 392) can have similar properties as XylR1, e.g., see Sievert et al. (2017) supra.

[0145] Without being bound by theory, it is believed that the XylR E382K (XylR1) mutation mapped to the XylR DNA binding domain affects protein binding, increasing the affinity of the XylR1 protein for the xylAB and xylFGH upstream promoter binding sites, thus increasing the expression of XylAB and XylFGH. This stronger interaction also makes the system less sensitive to the need for the coactivator CRP-cAMP binding at the promoter sites, and is indicated by the ability of XylR1 to simultaneously co-utilize glucose and xylose at high rates, e.g., see Examples 1 and 2 herein.

[0146] The xylose binding domain of the XylR protein comprises residues 221-229. This region dimerizes in an anti-parallel fashion and ultimately modulates the DNA binding domain structure to allow DNA binding after interaction with xylose. Thus, mutations in this region are expected to affect the protein’s response to the presence of xylose.

[0147] Other functional regions include regions with helix domains, such as the region comprising the E382K (XylR1) mutation. Mutations in the helix region can affect protein function. Protein regions with beta-strand structures are also functional domains, so mutations in these regions can result in functional changes.

[0148] Certain regions of the XylR protein are outside of the helix and / or beta strand structure regions and can have little to no effect on protein function. Some of these regions include, for example, the first five N-terminal amino acid residues, residues 40-57, residues 74-79, residues 126-133, residues 158-166, and / or residues 181-184.

[0149] Furthermore, in general, for amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alter, add or delete a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Such "conservatively modified variants" can have little to no effect on the function of the protein, particularly if they are in regions of the protein which are outside of the helical and / or beta sheet structural regions.

[0150] Conservative substitutions of functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the present application and are not meant to exclude those variations that result from mutations or changes in the nucleic acid sequences. The following eight groups each contain amino acids that are conservative substitutions of one another:

[0151] Thus, in exemplary embodiments, the present disclosure provides engineered XylR mutant polypeptides having improved xylose utilization and / or improved glucose and xylose co-utilization. Such mutants can be used to produce, for example, fatty esters, such as fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs), fatty alcohol acetates (FACEs), fatty amines, fatty aldehydes, fatty alcohols, hydrocarbons, fatty ketones, alkanes, terminal olefins, internal olefins, hydroxyl fatty acid derivatives, bifunctional fatty acid derivatives, such as fatty diacids, fatty diols, unsaturated fatty acid derivatives, compared to the enzyme having SEQ ID NO: 1.

[0152] 2. Assay for XylR mutants having improved xylose utilization and / or improved glucose and xylose co-utilization

[0153] In exemplary embodiments, XylR mutants having improved xylose utilization and / or improved glucose and xylose co-utilization are identified by measuring glucose and xylose utilization as disclosed in Examples 1 and 2 below.

[0154] In some embodiments, XylR mutants having improved xylose utilization or improved co-utilization of glucose and xylose are identified by measuring the titer of fatty acid derivatives (e.g., free fatty acids (FFAs), fatty acid ethyl esters (FAEEs), fatty acid methyl esters (FAMEs), etc.) produced by a bacterial strain comprising a XylR mutant having improved xylose utilization and / or improved co-utilization of glucose and xylose (i.e., a test strain) and comparing these fatty acid derivatives to the titer of fatty acid derivatives (e.g., FFAs, FAEEs, FAMEs, etc.) produced by an appropriate control strain that is isogenic to the test strain except for the XylR protein it comprises. When the strain is cultured in the presence of xylose, a XylR mutant having improved xylose utilization or improved co-utilization of glucose and xylose will produce more fatty acid derivatives (FFAs, FAEEs, FAMEs) than the control strain.

[0155] In some embodiments, the total titer of fatty acid derivatives is measured and compared between the test strain and the control strain. In other embodiments, the percentage of the total titer of fatty acid derivatives produced by the test strain that comprises a particular fatty acid derivative (e.g., a C14 fatty acid derivative) is measured and compared to the percentage of the total titer of fatty acid derivatives produced by the appropriate control strain that comprises a particular fatty acid derivative (e.g., a C14 fatty acid derivative), which is isogenic to the test strain except for the control XylR (e.g., SEQ ID NO: 1) it comprises.

[0156] In exemplary embodiments, gas chromatography with flame ionization detection (GC-FID) is used to determine the fatty acid derivatives. GC-FID is known in the art (see, e.g., Adlard, E. R.; Handley, Alan J. (2001). Gas chromatographic techniques and applications. London: Sheffield Academic). However, any appropriate quantitative and analytical method can be used, such as mass spectrometry (MS), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), thin layer chromatography (TLC), etc.

[0157] C. Methods of making XylR mutants having improved xylose utilization and / or improved co-utilization of glucose and xylose

[0158] Engineered XylR mutants with improved xylose utilization or improved glucose and xylose co-utilization can be made by any method known in the art (see, e.g., Current Protocols in Molecular Biology, supra). Thus, in exemplary embodiments, mutagenesis is used to make a polynucleotide sequence encoding a XylR mutant / variant with improved xylose utilization or improved glucose and xylose co-utilization, which can then be screened for improved xylose utilization or improved glucose and xylose co-utilization. In other exemplary embodiments, a polynucleotide sequence encoding a XylR mutant / variant with improved xylose utilization or improved glucose and xylose co-utilization is made by chemically synthesizing a polynucleotide sequence, which can then be screened for improved xylose utilization or improved glucose and xylose co-utilization (see, e.g., M. H. Caruthers et al. (1987) Methods in Enzymology vol. 154, pp. 287-313; Beaucage, S. L. and Iyer, R. P. (1992) Tetrahedron 48(12): 2223-2311).

[0159] Mutagenesis methods are well known in the art. Exemplary mutagenesis techniques for making engineered XylR mutants with improved xylose utilization or improved glucose and xylose co-utilization include, e.g., site-saturation mutagenesis (see, e.g., Chronopoulou EG1, Labrou NE. Curr. Protoc. Protein Sci. 2011 Feb;Chapter 26:Unit 26.6, John Wiley and Sons, Inc; Steffens, D. L. and Williams., J. G. K (2007) J Biomol Tech. 18(3): 147-149; Siloto, R. M. P and Weselake, R. J. (2012) Biocatalysis and Agricultural Biotechnology 1(3): 181-189).

[0160] Another exemplary mutagenesis technique for making XylR mutants with improved xylose utilization or improved glucose and xylose co-utilization includes Transitive PCR (tPCR) see, e.g., Erijman A., et al. (2011) J. Struct. Biol. 175(2): 171-7.

[0161] 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).

[0162] Another exemplary mutagenesis technique for preparing engineered XylR variants having improved xylose utilization or improved co-utilization of glucose and xylose includes the use of oligonucleotide-directed mutagenesis (see, e.g., Reidhaar-Olson et al. (1988) Science 241 :53-57) to create site-specific mutations in any cloned DNA of interest.

[0163] The mutagenized polynucleotides produced by any of the synthetic or mutagenesis methods described above are then cloned into a suitable vector or inserted into the genome of a host cell and the affected polypeptides encoded by the mutagenized polynucleotides are evaluated for activity as disclosed above.

[0164] One of ordinary skill in the art will recognize that the protocols and procedures disclosed herein can be modified and such modifications are variants according to the present disclosure. For example, when method steps are described in a particular order, the order of the steps can be modified and / or performed in parallel, sequentially.

[0165] III. Host Cells and Host Cell Cultures

[0166] In view of the present disclosure, one of ordinary skill in the art will appreciate that any of the embodiments contemplated herein can be practiced with any host cell or microorganism that can be genetically modified by the introduction of one or more nucleic acid sequences encoding the disclosed XylR mutants having improved xylose utilization or improved co-utilization of glucose and xylose. Accordingly, the recombinant microorganisms disclosed herein are used as host cells and comprise one or more polynucleotide sequences including an open reading frame encoding a XylR mutant having improved xylose utilization and / or improved co-utilization of glucose and xylose and operably linked regulatory sequences that facilitate expression of the engineered XylR mutant polypeptide in the host cell.

[0167] Exemplary microorganisms that provide suitable host cells include, but are not limited to, cells from Escherichia, Bacillus, Lactobacillus, Pseudomonas, Aspergillus, Marinobacter. 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 licheniformis cell, a Bacillus alkalophilus cell, a Bacillus coagulans cell, a Bacillus circulans cell, a Bacillus pumilus cell, a Bacillus thuringiensis cell, a Bacillus clausii cell, a Bacillus megaterium cell, a Bacillus subtilis cell, or a Bacillus amyloliquefaciens cell.

[0168] In other exemplary embodiments, the host cell is a cell from a cyanobacterium, a green sulfur bacterium, a green non-sulfur bacterium, a purple sulfur bacterium, a purple non-sulfur bacterium, an extremophile, an engineered organism thereof, or a synthetic organism. In some exemplary embodiments, the host cell is an Escherichia coli cell. In some exemplary embodiments, the Escherichia coli cell is an Escherichia coli strain B, strain C, strain K, or strain W cell.

[0169] In some example embodiments, depending on what other heterologous enzymes and what native enzyme pathways are present in the host cell, the host cell includes optional genetic manipulations and changes that are used interchangeably from one host cell to another. In one example embodiment, the host cell optionally contains a fadE and / or fhuA deletion. In other example embodiments, the host cell is optionally manipulated to have the ability to produce more than 200 mg / L fatty acid derivative, more than 1000 mg / L fatty acid derivative, more than 1200 mg / L fatty acid derivative, more than 1700 mg / L fatty acid derivative, more than 2000 mg / L fatty acid derivative, or more than 3000 mg / L fatty acid derivative.

[0170] As will be discussed in detail herein below, in some example embodiments, the host cell or host microorganism for expressing the XylR mutant / variant having improved xylose utilization or improved co-utilization of glucose and xylose further expresses a gene having an enzyme activity that can increase the production of one or more specific fatty acid derivatives such as, for example, fatty esters, fatty alcohols, fatty alcohol acetates, fatty acid methyl esters, fatty acid ethyl esters, fatty amines, fatty aldehydes, bifunctional fatty acid derivatives, diacids, alkanes, alkenes or alkynes, ketones, and the like.

[0171] In an exemplary embodiment, the host cell or host microorganism used to express the XylR mutant with improved xylose utilization or improved co-utilization of glucose and xylose further expresses ester synthase activity (E.C. 2.3.1.75) for production of fatty esters. In another exemplary embodiment, the host cell has acyl-ACP reductase (AAR) (E.C. 1.2.1.80) activity and / or alcohol dehydrogenase activity (E.C. 1.1.1.1.) and / or fatty alcohol acyl-CoA reductase (FAR) (E.C. 1.1.1.*) activity and / or carboxylic acid reductase (CAR) (EC 1.2.99.6) activity for production of fatty alcohols. In another exemplary embodiment, the host cell has acyl-ACP reductase (AAR) (E.C. 1.2.1.80) activity for production of fatty aldehydes. In another exemplary embodiment, the host cell has acyl-ACP reductase (AAR) (E.C. 1.2.1.80) activity and decarbonylase or fatty aldehyde oxidizing deformylase activity EC 4.1.99.5) for production of alkanes and alkenes. In another exemplary embodiment, the host cell has acyl-CoA reductase (E.C. 1.2.1.50) activity and acyl-CoA synthase (FadD) (E.C. 2.3.1.86) activity for production of fatty alcohols. In another exemplary embodiment, the host cell has ester synthase activity (E.C. 2.3.1.75) and acyl-CoA synthase (FadD) (E.C. 2.3.1.86) activity for production of fatty esters. In another exemplary embodiment, the host cell has OleA activity for production of ketones. In another exemplary embodiment, the host cell has OleABCD activity for production of internal olefins. In another exemplary embodiment, the host cell has acyl-ACP reductase (AAR) (E.C. 1.2.1.80) activity and alcohol dehydrogenase activity (E.C. 1.1.1.1) for production of fatty alcohols. In another exemplary embodiment, the host cell has decarbonylase activity for production of terminal olefins. Expression of enzyme activities in microorganisms and microbial cells is taught, for example, by 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; 8,530,221; 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.

[0172] In some example embodiments, the host cell or microorganism for expressing a XylR mutant having improved xylose utilization or improved glucose and xylose co-utilization 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 acetates, fatty amines, fatty amides, fatty aldehydes, bifunctional fatty acid derivatives, diacids, and the like.

[0173] In some example embodiments, the recombinant host cell produces a fatty ester, such as, for example, a fatty acid methyl ester (FAME) or fatty acid ethyl ester (FAEE), a fatty alcohol acetate (FACE), a fatty alcohol (FALC), a fatty amine, a fatty aldehyde, a bifunctional fatty acid derivative, a diacid, an alkane, an alkene, and the like.

[0174] The fatty acid derivatives are typically recovered from the culture medium and / or isolated from the host cell. In one example embodiment, the fatty acid derivatives are recovered from the culture medium (extracellularly). In another example embodiment, the fatty acid derivatives are isolated from the host cell (intracellularly). In another example embodiment, the fatty acid derivatives or non-fatty acid compounds are recovered from the culture medium and isolated from the host cell.

[0175] The fatty acid derivative composition produced by the host cell can be analyzed using methods known in the art (e.g., gas chromatography with flame ionization detection (GC-FID)) to determine the distribution of specific fatty acid derivatives as well as the chain length and saturation of the components of the fatty acid derivative composition. Similarly, other compounds can be analyzed by methods known in the art.

[0176] IV. Methods of making recombinant host cells and cultures

[0177] Any method known in the art can be used to engineer a host cell to express a XylR mutant having improved xylose utilization and / or improved glucose and xylose co-utilization to produce, for example, a fatty acid derivative and / or a fatty acid derivative composition or other compound. Exemplary methods include, for example, using a vector, such as an expression vector, comprising a polynucleotide sequence encoding a XylR mutant having improved xylose utilization and / or improved glucose and xylose co-utilization and / or a polynucleotide sequence disclosed herein. Those skilled in the art will appreciate that a variety of viral and non-viral vectors can be used in the methods disclosed herein.

[0178] In some exemplary embodiments, a polynucleotide (or gene) sequence encoding a XylR mutant having improved utilization of xylose and / or improved co-utilization of glucose and xylose is provided to a host cell by a recombinant vector comprising a promoter operably linked to the polynucleotide sequence encoding a XylR mutant having improved utilization of xylose and / or improved co-utilization of glucose and xylose. In some exemplary embodiments, the promoter is a developmentally regulated, organelle-specific, tissue-specific, inducible, constitutive, or cell-specific promoter. In some exemplary embodiments, the promoter is induced by the addition of lactose or isopropyl thiogalactoside (IPTG).

[0179] Once the polynucleotide sequence encoding a XylR mutant having improved utilization of xylose and / or improved co-utilization of glucose and xylose has been prepared and isolated, a variety of methods can be used to construct expression cassettes, vectors, and other DNA constructs. Expression cassettes comprising a polynucleotide sequence encoding a XylR mutant / variant having improved utilization of xylose or improved co-utilization of glucose and xylose can be constructed in a variety of ways. The skilled artisan is well aware of the genetic elements that must be present on an expression construct / vector in order to successfully transform, select for, and propagate the expression construct in a host cell. Techniques for manipulating polynucleotide sequences, such as those encoding a XylR mutant / variant having improved utilization of xylose or improved co-utilization of glucose and xylose, such as subcloning nucleic acid sequences into expression vectors, labeling probes, DNA hybridization, and the like, are generally described in, for example, Sambrook, et al., supra; Current Protocols in Molecular Biology, supra.

[0180] DNA constructs comprising a polynucleotide sequence (e.g., SEQ ID NO: 4) encoding a XylR mutant / variant having improved utilization of xylose or improved co-utilization of glucose and xylose linked to a heterologous DNA sequence, such as a promoter sequence, can be inserted into a variety of vectors. In some exemplary embodiments, the vector selected is an expression vector that can be used to transform bacteria, such as E. coli. The expression vector can be a plasmid, virus, cosmid, artificial chromosome, nucleic acid fragment, or the like. Such vectors can be readily constructed by using recombinant DNA techniques well known to those of ordinary skill in the art (see, e.g., Sambrook et al., supra). The expression vector comprising the polynucleotide sequence encoding the mutant or engineered XylR variant can then be transfected / transformed into a target host cell. Successfully transformed cells are then selected by methods well known in the art based on the presence of a suitable marker gene.

[0181] One skilled in the art can use a number of recombinant vectors for stable transformation / transfection of bacteria and other microorganisms (see, e.g., Sambrook et al., supra). The skilled artisan readily selects an appropriate vector. In an exemplary embodiment, known vectors are used to generate expression constructs comprising a polynucleotide sequence encoding a mutant or engineered XylR variant.

[0182] Typically, the transformation vector comprises one or more polynucleotide sequences encoding a XylR mutant having improved xylose utilization and / or improved glucose and xylose co-utilization operably linked to, e.g., a promoter sequence and a selectable marker. Such transformation vectors typically also comprise, as appropriate, a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and / or a polyadenylation signal.

[0183] Accordingly, in addition to the polynucleotide sequence encoding a XylR mutant having improved xylose utilization and / or improved glucose and xylose co-utilization, the expression constructs prepared as disclosed herein can also comprise other elements. In exemplary embodiments, the expression constructs comprising a polynucleotide sequence encoding a XylR mutant having improved xylose utilization and / or improved glucose and xylose co-utilization also comprise an enhancer sequence, whereby expression of a heterologous protein can be enhanced. As is known in the art, enhancers are typically found 5' of the start of transcription, and they can typically be inserted in either the forward or reverse orientation, i.e., 5' or 3' of the coding sequence.

[0184] As noted above, the transformation / expression vectors typically comprise a selectable and / or screenable marker gene to allow easy identification of transformants. Exemplary selectable marker genes include, but are not limited to, those encoding antibiotic resistance, e.g., resistance to kanamycin, ampicillin, etc. An exemplary screenable marker includes, e.g., a hexaamino acid histidine tag introduced at the C-terminus of a recombinant protein.

[0185] In exemplary embodiments, a selectable or screenable marker gene is used as or along with a gene of interest to provide or enhance the ability to identify transformants. A number of selectable marker genes are known in the art (see, e.g., Sambrook et al., supra).

[0186] In some exemplary embodiments, the expression vector further comprises a sequence linked to the coding sequence of the expressed heterologous nucleic acid that is removed post-translationally from the initial translation product. In one exemplary embodiment, the post-translationally removed sequence facilitates protein trafficking into or through intracellular or extracellular membranes, thereby facilitating protein trafficking into compartments inside and / or outside of the cell. In one exemplary embodiment, the post-translationally removed sequence protects the nascent protein from intracellular proteolytic degradation. In one exemplary embodiment, a nucleic acid segment encoding a leader peptide sequence upstream of and in reading frame with the coding sequence is used in the recombinant expression of the coding sequence in the host cell of choice.

[0187] In another exemplary embodiment, the expression construct comprises a bacterial origin of replication, such as the ColEl origin. In another exemplary embodiment, the expression construct / vector comprises a bacterial selectable marker, such as an ampicillin, tetracycline, hygromycin, neomycin, or chloramphenicol resistance gene.

[0188] As is well known in the art, expression constructs typically comprise restriction endonuclease sites to facilitate vector construction. Exemplary restriction endonuclease recognition sites include, but are not limited to, recognition sites for restriction endonucleases Notl, Aatll, SacII, Pmel Hindlll, Pstl, EcoRI, and BamHI, for example.

[0189] DNA constructs (polynucleotide sequences encoding a mutant or engineered XylR variant operably linked to a heterologous DNA sequence, such as a promoter sequence, a marker sequence; a purification moiety; a secretion sequence operably coupled to the polynucleotide sequence; a targeting sequence, etc.; and / or polynucleotide sequences encoding other fatty acid derivative biosynthetic pathway polypeptides) are used to transform cells and produce recombinant host cells having improved xylose utilization or improved glucose and xylose co-utilization. Exemplary host cells for transformation with expression constructs comprising polynucleotide sequences encoding XylR mutants having improved xylose utilization and / or improved glucose and xylose co-utilization are discussed in detail above.

[0190] Exemplary transformation / transfection methods available to the skilled artisan include, for example, electroporation, calcium chloride transformation, and the like, which are well known to the skilled artisan (see, e.g., Sambrook, supra). Thus, the polynucleotide sequence comprising an open reading frame encoding a protein and operably linked regulatory sequences can be integrated into the chromosome of the recombinant host cell, incorporated into one or more plasmid expression systems resident in the recombinant host cell, or both.

[0191] The expression vectors disclosed herein typically include a polynucleotide sequence encoding a XylR mutant having improved xylose utilization and / or improved glucose and xylose co-utilization in a form suitable for expression of the polynucleotide sequence in a host cell. As will be appreciated by those skilled in the art, the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of the desired polypeptide, etc.

[0192] V. Evaluating Recombinant Host Cells

[0193] In exemplary embodiments, the activity of a XylR mutant having improved xylose utilization and / or improved glucose and xylose co-utilization is determined by culturing the recombinant host cell in the presence of xylose and measuring, for example, the properties of improved xylose and glucose utilization as disclosed herein below in Examples 1 and 2.

[0194] Another method of evaluation is to measure the growth rate of the recombinant host cell by measuring optical density (OD 600) in the presence of xylose (see, e.g., Example 2). Another method of evaluation is to measure the concentration of a desired biochemical product (e.g., a fatty acid derivative) synthesized from a carbon source of xylose or a mixture of glucose and xylose by the analytical instruments disclosed herein.

[0195] IV. Products Derived from Recombinant Host Cells

[0196] Strategies to increase xylose utilization or to improve glucose and xylose co- utilization can be used to utilize different carbon sources for the production of fatty acid derivatives by recombinant host cells. Xylose can be used as a carbon source. Figure 17 A schematic diagram showing an exemplary metabolic pathway for utilizing xylose is shown.

[0197] XylR is a regulatory protein that induces the expression of XylFGH and XylAB genes, which control the transport and subsequent utilization pathway of xylose, respectively. The gene product of xylB, like Figure 17 As shown, xylulose-5-phosphate enters the central metabolism of E. coli through the well-described pentose phosphate pathway. Ultimately, acetyl-CoA produced from the pentose phosphate pathway enters the fatty acid biosynthesis pathway to produce fatty acid derivatives.

[0198] Accordingly, in exemplary embodiments, the recombinant host cell is engineered to comprise, in addition to the XylR mutant having improved xylose utilization and / or improved glucose and xylose co-utilization, one or more polynucleotide sequences encoding one or more "fatty acid derivative biosynthesis polypeptides" or equivalently "fatty acid derivative enzymes." Metabolic engineering of the fatty acid derivative biosynthesis pathway to use microorganisms to produce fatty acid derivative compounds (e.g., fatty acid esters, alkanes, alkenes, fatty ketones, fatty alcohols, fatty alcohol acetates, etc.) to convert biomass-derived sugars into desired products is known in the art, see, e.g., 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 cultivated in industrial scale bioreactors and the resulting products purified using traditional chemical and biochemical engineering techniques.

[0199] Accordingly, in some embodiments, a fatty acid derivative composition comprising, e.g., fatty acid esters, e.g., FAMEs, is produced by culturing a recombinant host cell comprising a XylR mutant having improved xylose utilization and / or improved glucose and xylose co-utilization in the presence of a carbon source comprising xylose under conditions effective for expression of the XylR mutant.

[0200] In some embodiments, substantially all of the fatty acid derivatives produced by culturing a recombinant host cell comprising a XylR mutant having improved xylose utilization and / or improved glucose and xylose co-utilization are produced extracellularly. Accordingly, in some exemplary embodiments, the produced fatty acid derivatives 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, e.g., GC FID, in order to determine and quantify the distribution of particular fatty acid derivatives as well as the chain length and saturation of the components of the fatty acid derivative composition.

[0201] Production of fatty acid derivatives

[0202] As noted above, recombinant host cells comprising engineered XylR variants having improved xylose utilization or improved glucose and xylose co-utilization produce increased amounts of fatty acids compared to suitable control host cells that do not comprise the engineered XylR variants (e.g., isogenic control host cells having a control XylR enzyme (e.g., SEQ ID NO: 1)).

[0203] In other exemplary embodiments to be discussed in detail below, in addition to engineered XylR variants having improved xylose utilization or improved glucose and xylose co-utilization, the recombinant host cells further comprise additional fatty acid derivative biosynthetic polypeptides that contribute to the production of particular types of fatty acid derivatives.

[0204] Production of fatty aldehydes

[0205] In some exemplary embodiments, in addition to engineered XylR variants having improved xylose utilization or improved glucose and xylose co-utilization, the recombinant host cells further comprise carboxylate reductase (“CAR”) activity, and as a result, the recombinant host cells synthesize fatty aldehydes and fatty alcohols, see, e.g., U.S. Patent 9,340,801.

[0206] Accordingly, 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, e.g., carboxylate reductase (CAR) activity. Exemplary carboxylate reductase (CAR) polypeptides and polynucleotides encoding them include, e.g., FadD9 (EC 6.2.1.-, UniProtKB Q50631, GenBank NP_217106), CarA (GenBank ABK75684), CarB (GenBank YP889972), and related polypeptides disclosed in, e.g., U.S. Patent No. 8,097,439 and U.S. Patent No. 9,340,801.

[0207] In some exemplary embodiments, the fatty aldehydes produced by the recombinant host cells are then converted to fatty alcohols or hydrocarbons. Accordingly, in some exemplary embodiments, in addition to XylR variants having improved xylose utilization or improved glucose and xylose co-utilization, the recombinant host cells further comprise acyl-CoA reductase (“FAR” or “ACR”) activity, and as a result, the recombinant host cells synthesize fatty aldehydes and fatty alcohols (see, e.g., U.S. Patent No. 8,658,404, U.S. Patent No. 8,268,599, U.S. Patent Application Publication 2015 / 0361454).

[0208] In some embodiments, the aldehyde produced by the recombinant host cell is converted to a fatty alcohol by the activity of a native or heterologous fatty alcohol biosynthetic polypeptide such as an aldehyde reductase or an alcohol dehydrogenase (see, e.g., U.S. Patent Application Publication 2011 / 0250663). Thus, in some exemplary embodiments, in addition to the XylR variant having improved xylose utilization or improved co-utilization of glucose and xylose, the recombinant host cell further comprises aldehyde reductase activity or equivalently alcohol dehydrogenase activity (EC 1.1.1.1), and thus the recombinant host cell synthesizes a fatty alcohol. Exemplary fatty alcohol biosynthetic genes include, but are not limited to, for example, alcohol dehydrogenases such as AlrA of Acenitobacter sp. M-1 or an AlrA homolog; 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).

[0209] Production of fatty amines

[0210] In some exemplary embodiments, the recombinant host cell (which comprises an engineered XylR variant having improved xylose utilization or improved co-utilization of glucose and xylose and which produces a fatty aldehyde (e.g., as disclosed herein above)) is further modified to comprise a heterologous biosynthetic enzyme having aminotransferase or amine dehydrogenase activity that converts the fatty aldehyde to a fatty amine (see, e.g., PCT Publication No. WO 2015 / 085271).

[0211] Production of fatty alcohols

[0212] In some exemplary embodiments, in addition to the engineered XylR variant having improved xylose utilization or improved co-utilization of glucose and xylose, the recombinant host cell further comprises a polynucleotide encoding a polypeptide having fatty alcohol biosynthetic activity, and thus a fatty alcohol is produced by the recombinant host cell. Thus, in exemplary embodiments, a composition comprising a medium chain fatty alcohol (e.g., comprising octanol) is prepared by culturing the recombinant host cell in the presence of a carbon source under conditions effective to express the engineered XylR variant having improved xylose utilization or improved co-utilization of glucose and xylose and the fatty alcohol biosynthetic enzyme.

[0213] Accordingly, in some example embodiments, in addition to an engineered XylR variant having improved xylose utilization or improved co-utilization of glucose and xylose, the recombinant host cell further comprises carboxylic acid reductase (CAR) activity and alcohol dehydrogenase activity, and as a result, the recombinant host cell synthesizes a fatty alcohol, e.g., octanol (see, e.g., U.S. Patent 9,340,801).

[0214] In some example embodiments, a native fatty aldehyde biosynthetic polypeptide, e.g., an aldehyde reductase / alcohol dehydrogenase present in the host cell, converts a fatty aldehyde to a fatty alcohol. In other example embodiments, a native fatty aldehyde reductase / alcohol dehydrogenase is overexpressed to convert a fatty aldehyde to a fatty alcohol. In other example embodiments, a heterologous aldehyde reductase / alcohol dehydrogenase is introduced into the recombinant host cell and expressed or overexpressed to convert a fatty aldehyde to a fatty alcohol. Example aldehyde reductase / alcohol dehydrogenase polypeptides useful for converting a fatty aldehyde to a fatty alcohol are disclosed in WO 2007 / 136762; WO 2010 / 062480; U.S. Patent 8,110,670; U.S. Patent 9,068,201, supra.

[0215] In some example embodiments, in addition to an engineered XylR variant having improved xylose utilization or improved co-utilization of glucose and xylose, 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, such that the recombinant host cell produces a 1,3 fatty diol when grown in a fermentation broth having a simple carbon source. In other example embodiments, in addition to an engineered XylR variant having improved xylose utilization or improved co-utilization of glucose and xylose, 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, wherein the recombinant host cell produces a 1,3 fatty diol when grown in a fermentation broth having a simple carbon source (see, e.g., WO 2016 / 011430).

[0216] Production of fatty alcohol acetates

[0217] In some embodiments, the fatty alcohol produced in the cell or in some embodiments fed to the cell is further processed by the recombinant cell to provide a fatty alcohol acetate (FACE). In exemplary embodiments, an alcohol O-acetyltransferase (EC 2.8.1.14) processes the fatty alcohol to a fatty alcohol acetate (FACE), see, e.g., Gabriel M Rodriguez, et al. (2014) Nature Chemical Biology 10, 259-265; Jyun-Liang Lin and Ian Wheeldon (2014) PLoS One. [Public Library of Science·Comprehensive] 2014; 9(8): PMCID: PMC4122449.

[0218] An exemplary alcohol O-acetyltransferase is the yeast Aftl, e.g., GenBank Accession No. AY242062; GenBank Accession No. AY242063, see, e.g., Kevin J. Verstrepen K.J., et al. (2003) Appl Environ Microbiol. [Applied and Environmental Microbiology] September 2003; 69(9):5228-5237.

[0219] In one exemplary embodiment, a recombinant host cell comprising an engineered XylR variant with improved xylose utilization or improved glucose and xylose co-utilization further comprises carboxylic acid reductase activity (EC 1.2.99.6) sufficient to produce a fatty aldehyde and a fatty alcohol, and further comprises a fatty alcohol O-acetyltransferase activity that converts the fatty alcohol to a fatty alcohol acetate.

[0220] In another exemplary embodiment, a recombinant host cell comprising an engineered XylR variant with improved xylose utilization or improved glucose and xylose co-utilization further comprises 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.

[0221] In another exemplary embodiment, a recombinant host cell comprising an engineered XylR variant with improved xylose utilization or improved glucose and xylose co-utilization further comprises 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.

[0222] In another exemplary embodiment, the recombinant host cell comprising an engineered XylR variant with improved utilization of xylose or improved co-utilization of glucose and xylose further comprises carboxylic acid reductase activity (EC 1.2.99.6) that results in the production of a first fatty acid derivative, and further comprises fatty alcohol O-acetyltransferase activity that converts the first fatty acid derivative into a second fatty acid derivative, wherein the MIC of the first fatty acid derivative is increased in the presence of the second fatty acid derivative.

[0223] In another exemplary embodiment, the recombinant host cell comprising an engineered XylR variant with improved utilization of xylose or improved co-utilization of glucose and xylose further comprises carboxylic acid reductase activity (EC 1.2.99.6) that results in the production of a first fatty acid derivative, and further comprises fatty alcohol O-acetyltransferase activity that converts the first fatty acid derivative into a second fatty acid derivative, wherein the toxicity of the second fatty acid derivative is lower than the first fatty acid derivative.

[0224] Production of fatty esters

[0225] In some embodiments, in addition to an engineered XylR variant with improved utilization of xylose or improved co-utilization of glucose and xylose, the recombinant host cell further comprises a polynucleotide encoding a polypeptide with fatty ester biosynthetic activity, and as a result, a fatty ester is produced by the recombinant host cell.

[0226] As used herein, the term "fatty ester" or equivalently "fatty acid ester" refers to any ester made from a fatty acid. In exemplary embodiments, a fatty ester comprises an "A-side" and a "B-side". As used herein, the "A-side" of an ester refers to the carbon chain attached to the carboxylic acid oxygen of the ester. As used herein, the "B-side" of an ester refers to the carbon chain comprising the parent carboxylic acid of the ester. In embodiments in which the fatty ester is derived 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 alkyl thioester.

[0227] Any alcohol can be used to form the A-side of a fatty ester. In exemplary embodiments, the alcohol is derived from a fatty acid derivative biosynthetic pathway. In other exemplary embodiments, the alcohol is produced by a non-fatty acid derivative biosynthetic pathway, e.g., the alcohol is exogenously supplied, e.g., the alcohol is supplied in a fermentation broth.

[0228] The carbon chain comprising either the A-side or the B-side can be of any length. In one exemplary embodiment, the fatty ester is a fatty acid methyl ester, where the B-side is provided through the fatty acid biosynthetic pathway, and the A-side of the ester is 1 carbon in length. In one exemplary embodiment, the A-side is provided through the action of a fatty acid O-methyltransferase (FAMT) (EC 2.1.1.15) (see, e.g., Applied and Environmental Microbiology 77(22): 8052-8061).

[0229] In another exemplary embodiment, the fatty ester is a fatty acid ethyl ester, where the B-side is provided through the fatty acid biosynthetic pathway, and the A-side of the ester is 2 carbons in length.

[0230] 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 branch 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.

[0231] In exemplary embodiments, 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., International Patent Application Publication WO 2011 / 038134.

[0232] In some exemplary embodiments, the fatty acid ester is produced by a recombinant host cell comprising an engineered XylR variant having improved xylose utilization or improved glucose and xylose co-utilization, and a thioesterase, an acyl-CoA synthetase (fadD), and an ester synthase (see, e.g., International Patent Application Publication WO / 2011 / 038134; International Patent Application Publication WO 2007 / 136762; U.S. Patent 8,110,670).

[0233] In one exemplary embodiment, the recombinant host cell comprising an engineered XylR variant having improved xylose utilization or improved glucose and xylose co-utilization further comprises ester synthase activity (EC 3.1.1.67) sufficient to produce a fatty ester (e.g., FAME or FAEE, see, e.g., U.S. Patent 9,879,239).

[0234] Production of hydrocarbons

[0235] In some embodiments, in addition to an engineered XylR variant having improved utilization of xylose or improved co-utilization of glucose and xylose, 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 deforrmylase, or a fatty acid decarboxylase), and thus, the recombinant host cell exhibits enhanced hydrocarbon production (see, e.g., U.S. Patent Application Publication 2011 / 0124071). Thus, in exemplary embodiments, a recombinant host cell comprising an engineered XylR variant having improved utilization of xylose or improved co-utilization of glucose and xylose produces a hydrocarbon, e.g., an alkane or an alkene (e.g., a terminal alkene or an internal alkene) or a ketone.

[0236] In some exemplary embodiments, a fatty aldehyde produced by a recombinant host cell comprising an engineered XylR variant having improved utilization of xylose or improved co-utilization of glucose and xylose is converted by decarboxylation to remove a carbon atom to form a hydrocarbon (see, e.g., U.S. Patent 8,110,670 and WO 2009 / 140695).

[0237] In other exemplary embodiments, a fatty acid produced by a recombinant host cell is converted by decarboxylation to remove a carbon atom to form a terminal alkene. Thus, in some exemplary embodiments, in addition to expressing an engineered XylR variant having improved utilization of xylose or improved co-utilization of glucose and xylose, the recombinant cell further expresses or overexpresses a polynucleotide encoding a hydrocarbon biosynthetic polypeptide (e.g., a polypeptide having decarboxylase activity, such as disclosed in, e.g., U.S. Patent 8,597,922).

[0238] In other exemplary embodiments, an alkyl thioester intermediate is converted by enzymatic decarboxylation condensation to form an internal alkene or a ketone. Thus, in some exemplary embodiments, in addition to expressing an engineered XylR variant having improved utilization of xylose or improved co-utilization of glucose and xylose, the recombinant cell further expresses or overexpresses a polynucleotide encoding a hydrocarbon biosynthetic polypeptide (e.g., such as a polypeptide having OleA activity) to produce a ketone (see, e.g., U.S. Patent 9,200,299). In other exemplary embodiments, in addition to expressing an engineered XylR variant having improved utilization of xylose or improved co-utilization of glucose and xylose, the recombinant cell further expresses or overexpresses a polynucleotide encoding a hydrocarbon biosynthetic polypeptide (e.g., such as OleCD or OleBCD) along with a polypeptide having OleA activity to produce an internal alkene (see, e.g., U.S. Patent 9,200,299).

[0239] Some exemplary hydrocarbon biosynthetic polypeptides are shown in Table 2 below.

[0240] Table 2. Exemplary hydrocarbon biosynthesis polynucleotides and polypeptides.

[0241]

[0242] Production of Omega (ω)-hydroxylated fatty acid derivatives

[0243] In some embodiments, in addition to the engineered XylR variant having improved xylose utilization or improved co-utilization of glucose and xylose, the recombinant host cell further comprises a polynucleotide encoding a polypeptide having omega-hydroxylase activity (EC 1.14.15.3). In exemplary embodiments, the modified omega-hydroxylase has the enzymatic activity of a modified cytochrome P450 monooxygenase (P450) and is effective in vivo to catalyze hydroxylation of the omega-position of a hydrocarbon chain. Thus, when grown in a fermentation broth in the presence of a carbon source from a renewable feedstock, the recombinant microorganism produces medium-chain omega-hydroxylated (omega-hydroxylated) fatty acid derivatives in vivo (see, e.g., PCT Application Publication WO 2014 / 201474).

[0244] In other exemplary embodiments, in addition to the engineered XylR variant having improved xylose utilization or improved co-utilization of glucose and xylose, the recombinant host cell further comprises a polynucleotide encoding an alkane hydroxylase (e.g., alkA, CYP153A-reductase or CYP153A-reductase hybrid fusion polypeptide variant (see, e.g., WO 2015 / 195697)) such that when cultured in a medium containing a carbon source under conditions effective to express the alkane hydroxylase (e.g., AlkA, CYP153 or CYP153A-reductase hybrid fusion polypeptide variant) and the engineered XylR variant having improved xylose utilization or improved co-utilization of glucose and xylose, the recombinant host cell produces omega-hydroxylated (omega-hydroxylated) and bifunctional fatty acid derivatives and compositions thereof (including omega-hydroxylated fatty acids, omega-hydroxylated fatty acid esters, alpha, omega-diacids, alpha, omega-diesters, alpha, omega-diol and chemicals derived therefrom such as macro-lactones and macro-cyclic ketones).

[0245] V. Culturing and fermentation of recombinant host cells

[0246] As used herein, fermentation broadly refers to the conversion of organic material to a target substance by a recombinant host cell. For example, this includes the conversion of a carbon source to a fatty acid derivative, such as, for example, a fatty acid, a fatty ester, a fatty alcohol, a fatty alcohol acetate, etc., by a recombinant host cell through the propagation of a culture of the recombinant host cell in a medium comprising the carbon source. The conditions that allow for the production of a target substance, such as, for example, a fatty acid, a fatty ester, a fatty alcohol, a fatty alcohol acetate, etc., are any conditions that allow the host cell to produce the desired product, e.g., a fatty acid derivative composition. Suitable conditions include, for example, typical fermentation conditions, see, e.g., Principles of Fermentation Technology, 3rd Edition (2016), supra; Fermentation Microbiology and Biotechnology, 2nd Edition, (2007), supra.

[0247] Fermentation conditions can include a number of parameters well known in the art, including, but not limited to, temperature ranges, pH levels, aeration rates, feed rates, and medium composition. Each of these conditions, individually or in combination, allows for the growth of the host cell. Fermentation can be aerobic, anaerobic, or a variation thereof (e.g., microaerophilic). Exemplary media include liquid media (liquids) or gels (solids). Typically, the medium includes a carbon source that can be directly metabolized by the host cell (e.g., a simple carbon source derived from a renewable feedstock). Additionally, enzymes can be used in the medium to aid in the mobilization of the carbon source (e.g., depolymerization of starch or cellulose into fermentable sugars) and subsequent metabolism to produce the fatty acid derivative.

[0248] For small scale production, the host cell engineered to produce a fatty acid derivative composition can be grown, for example, in a batch of about 100 pL, 200 pL, 300 pL, 400 pL, 500 pL, 1 mL, 5 mL, 10 mL, 15 mL, 25 mL, 50 mL, 75 mL, 100 mL, 500 mL, 1 L, 2 L, 5 L, or 10 L; fermented; and induced to express any desired polynucleotide sequence, e.g., a polynucleotide encoding a polypeptide having a particular enzymatic activity (e.g., thioesterase (TE), ester synthase (ES), carboxylate 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)). For large scale production, the engineered host cell can be grown in a culture having a volume 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.

[0249] The fatty acid derivative compositions disclosed herein can generally be found in the extracellular environment of the recombinant host cell culture and can be readily isolated from the culture medium. The fatty acid derivatives (e.g., fatty acids, fatty acid esters, fatty aldehydes, fatty ketones, fatty alcohols, fatty alcohol acetates, etc.) can be secreted by the recombinant host cell, transported to the extracellular environment, or passively transferred into the extracellular environment of the recombinant host cell culture. The fatty acid derivative compositions can be isolated from the recombinant host cell culture using conventional methods known in the art, including but not limited to centrifugation.

[0250] Exemplary microorganisms suitable for use as production host cells include, for example, bacteria, cyanobacteria, yeast, algae, filamentous fungi, and the like. To produce the fatty acid derivative compositions, the production host cell (or equivalently, the host cell) is engineered to comprise a fatty acid biosynthetic pathway that is modified relative to a non-engineered or native host cell, for example, as described above and as disclosed, for example, in U.S. Patent Application Publication 2015 / 0064782. The production host engineered to comprise the modified fatty acid biosynthetic pathway is capable of efficiently converting glucose or other renewable feedstocks to fatty acid derivatives. Protocols and procedures for high-density fermentation to produce 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).

[0251] In some exemplary embodiments, the production host cell is cultured in a medium (e.g., a fermentation medium) comprising an initial concentration of about 20 g / L to about 900 g / L of a carbon source (simple carbon source). In other embodiments, the medium comprises an initial 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 of the carbon source. In some embodiments, the level of available carbon source in the medium can be monitored during the fermentation process. In some embodiments, the method further comprises adding a supplemental carbon source to the medium when the level of initial carbon source in the medium is less than about 0.5 g / L.

[0252] In some example embodiments, a supplemental carbon source is added to the culture medium when the level of carbon source in the culture 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, a supplemental carbon source is added to maintain a carbon source level of about 1 g / L to about 25 g / L. In some embodiments, a supplemental 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, a supplemental 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, a supplemental 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.

[0253] In one example 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, acetic acid, ethane, ethanol, methane, methanol, formic acid, and carbon monoxide; cellulose materials and variants thereof, 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, sugar cane, sorghum, sugar beets, switchgrass, silage, stover, wood, pulp, sewage, garbage, cellulosic municipal waste, flue gas, syngas, or carbon dioxide. The simple carbon source can also be a product of photosynthesis, such as glucose or sucrose. In one embodiment, the carbon source is derived from waste, such as glycerol, flue gas, or syngas; or from reforming of organic materials, such as biomass; or from natural gas or methane, or from reforming of these materials to syngas; or from carbon dioxide fixed by photosynthesis, such as can be produced by growing recombinant blue-green bacteria or algae by photosynthesis using CO2 as a carbon source to produce fatty acid derivatives.

[0254] In some embodiments, the carbon source is a cellulosic hydrolysate derived from biomass. Cellulosic hydrolysates are known in the art (see, e.g., Yang, B., et al. (2011) Biofuels 2(4):421). An exemplary source of biomass is plant matter or vegetation, such as corn, sugar cane, or switchgrass. Another exemplary source of biomass is metabolic waste, such as animal matter (e.g., cow manure). Other exemplary sources of biomass include algae and other marine plants. Biomass also includes waste from industry, agriculture, forestry, and households, including but not limited to fermentation waste, silage, stover, wood, sewage, garbage, cellulosic municipal waste, municipal solid waste, and leftover food.

[0255] In some exemplary embodiments, the fatty acid derivative, e.g., fatty acid, fatty acid ester, fatty alcohol, etc., is produced at a concentration of about 0.5 g / L to about 40 g / L. In some embodiments, the fatty acid derivative is produced at a concentration of about 1 g / L or more (e.g., about 1 g / L or more, about 10 g / L or more, about 20 g / L or more, about 50 g / L or more, about 100 g / L or more). In some embodiments, the fatty acid derivative is 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.

[0256] In other exemplary embodiments, a titer of about 25 mg / L, about 50 mg / L, about 75 mg / L, about 100 mg / L, about 125 mg / L, about 150 mg / L, about 175 mg / L, about 200 mg / L, about 225 mg / L, about 250 mg / L, about 275 mg / L, about 300 mg / L, about 325 mg / L, about 350 mg / L, about 375 mg / L, about 400 mg / L, about 425 mg / L, about 450 mg / L, about 475 mg / L, about 500 mg / L, about 525 mg / L, about 550 mg / L, about 575 mg / L, about 600 mg / L, about 625 mg / L, about 650 mg / L, about 675 mg / L, about 700 mg / L, about 725 mg / L, about 750 mg / L, about 775 mg / L, about 800 mg / L, about 825 mg / L, about 850 mg / L, about 875 mg / L, about 900 mg / L, about 925 mg / L, about 950 mg / L, about 975 mg / L, about 1000 mg / L, about 1050 mg / L, about 1075 mg / L, about 1100 mg / L, about 1125 mg / L, about 1150 mg / L, about 1175 mg / L, about 1200 mg / L, about 1225 mg / L, about 1250 mg / L, about 1275 mg / L, about 1300 mg / L, about 1325 mg / L, about 1350 mg / L, about 1375 mg / L, about 1400 mg / L, about 1425 mg / L, about 1450 mg / L, about 1475 mg / L, about 1500 mg / L, about 1525 mg / L, about 1550 mg / L, about 1575 mg / L, about 1600 mg / L, about 1625 mg / L, about 1650 mg / L, about 1675 mg / L, about 1700 mg / L, about 1725 mg / L, about 1750 mg / L, about 1775 mg / L, about 1800 mg / L, about 1825 mg / L, about 1850 mg / L, about 1875 mg / L, about 1900 mg / L, about 1925 mg / L, about 1950 mg / L, about 1975 mg / L, about 2000 mg / L (2 g / L), 3 g / L, 5 g / L, 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, 100 g / L, or a range bounded by any two of the foregoing values, produces a fatty acid derivative, e.g., a fatty acid, a fatty acid ester, a fatty alcohol, etc. In other embodiments, a titer of greater than 100 g / L, greater than 200 g / L, or greater than 300 g / L produces a fatty acid derivative or other compound.In exemplary embodiments, the titer of a fatty acid derivative or other compound produced by a recombinant host cell according to the methods disclosed herein is 5 g / L to 200 g / L, 10 g / L to 150 g / L, 20 g / L to 120 g / L, and 30 g / L to 100 g / L. Titer can refer to a particular fatty acid derivative or combination of fatty acid derivatives or another compound or combination of other compounds produced by a given recombinant host cell culture. In exemplary embodiments, expression of an engineered XylR 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.

[0257] In other exemplary embodiments, host cells engineered to produce fatty acid derivatives (e.g., fatty acids, fatty acid esters, fatty alcohols, etc.) 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, a fatty acid derivative or fatty acid derivatives or one or more other compounds are produced at a yield 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%, 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 bounded by any two of the two endpoints. For example, the yield of a fatty acid derivative produced from a recombinant host cell according to the methods disclosed herein can be about 5% to about 15%, about 10% to about 25%, about 10% to about 22%, about 15% to about 27%. The yield can refer to a particular fatty acid derivative or a combination of fatty acid derivatives. Additionally, the yield will also depend on the feedstock used.

[0258] In some exemplary embodiments, the methods according to the present disclosure are engineered to produce a productivity of a host cell of a fatty acid derivative (e.g., a fatty acid, a fatty acid ester (e.g., FAME, FAEE), a fatty alcohol, etc.) of at least 100 mg / L / hour, at least 200 mg / L / hour, at least 300 mg / L / hour, at least 400 mg / L / hour, at least 500 mg / L / hour, at least 600 mg / L / hour, at least 700 mg / L / hour, at least 800 mg / L / hour, at least 900 mg / L / hour, at least 1000 mg / L / hour, at least 1100 mg / L / hour, at least 1200 mg / L / hour, at least 1300 mg / L / hour, at least 1400 mg / L / hour, at least 1500 mg / L / hour, at least 1600 mg / L / hour, at least 1700 mg / L / hour, at least 1800 mg / L / hour, at least 1900 mg / L / hour, at least 2000 mg / L / hour, at least 2100 mg / L / hour, at least 2200 mg / L / hour, at least 2300 mg / L / hour, at least 2400 mg / L / hour, 2500 mg / L / hour, or up to 10 g / L / hour (depending on the cell mass). For example, the productivity of a malonyl-CoA derived compound (including one or more fatty acid derivatives or one or more other compounds) produced by a recombinant host cell according to the methods of the present disclosure can be 500 mg / L / hour to 2500 mg / L / hour, or from 700 mg / L / hour to 2000 mg / L / hour. Productivity can refer to a particular 14 carbon and / or 16 carbon fatty acid derivative or combination of fatty acid derivatives or one or more other compounds produced by a given host cell culture. For example, expression of an engineered XylR variant in a recombinant host cell, e.g., E. coli, results in increased productivity of 14 carbon and / or 16 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 is in the range of about 0.3 g / L / h to about 3 g / L / h to about 10 g / L / h to about 100 g / L / h to about 1000 g / L / h.

[0259] VI. Isolation

[0260] The biological product (e.g., a composition comprising a fatty acid derivative produced using a recombinant host cell as described above disclosed herein) is typically isolated from the fermentation broth by methods known in the art. In one exemplary embodiment, the composition (comprising a fatty acid derivative produced using a recombinant host cell as described above disclosed herein) is isolated from the fermentation broth by gravity settling, centrifugation, or decanting.

[0261] VII. Compositions and Formulations of Fatty Acid Derivatives

[0262] As described above, the biological products (e.g., compositions comprising fatty acids and fatty acid derivatives produced using recombinant host cells as described in detail above) are produced from renewable sources (e.g., simple carbon sources derived from renewable feedstocks) and, thus, are new compositions of matter. According to the dual carbon isotope fingerprinting method or 14 C dating, these new biological products can be distinguished from organic compounds derived from petrochemical carbon. Additionally, the specific source of biogenic carbon (glucose versus glycerol) can be determined by dual carbon isotope fingerprinting by methods known in the art (see, e.g., U.S. Patent No. 7,169,588, WO 2016 / 011430 Al, etc.).

[0263] The following examples are provided to illustrate, but not limit, the present invention.

[0264] EMBODIMENT

[0265] The following specific embodiments are intended to be illustrative of the present disclosure and should not be construed as limiting the scope of the claims.

[0266] EMBODIMENT 1

[0267] This example demonstrates that a single amino acid substitution mutation in E. coli XylR (XylR E382K) increases xylose utilization and increases co-utilization of xylose compared to wild-type xylR control.

[0268] Strains IC.200 (IC.187 XylR wild type pSven.037; control) and sven.938 (IC.187 XylR1 pSven.037; XylR1) were initially expanded in LB medium, then grown overnight at 32°C in shake flasks containing defined minimal salts medium (2 g / L NH4CI, 0.5 g / L NaCI, 0.3 g / L KH2PO4, 1 mM MgSO4, 0.1 mM CaCI2, 20 g / L glucose, 1 mL / L trace metals solution, 10 g / L ferric citrate, 100 mM Bis Tris phosphate buffer, 20 mL / L methanol and 100 mg / L spectinomycin) and inoculated at 5% v / v into a 5 L bioreactor containing defined minimal salts medium (0.5 g / L (NH4)2SO4, 2 g / L KH2PO4, 80 mg / L ferric citrate, 1 mL / L trace metals solution, 1 g / L NaCI, 140 mg / L CaCI2-H2O, 10 mg / L ZnCI2 2.2 g / L MgSO4-7H2O, 0.25 mL / L trace vitamins solution, 5 g / L each of glucose and xylose, 1 mL / L spectinomycin and 25 mL / L methanol). The trace metals solution consisted of 0.5 g / L H3BO3, 1.9 g / L CuSO4-5H2O, 1 g / L ZnCI, Na2MoO4-2H2O, CaCI2-2H2O and 2 mL / L concentrated hydrochloric acid. The trace vitamins solution consisted of 0.06 g / L riboflavin, 6 g / L nicotinic acid, 5.4 g / L pantothenic acid, 1.4 g / L pyridoxine, 0.06 g / L biotin and 0.01 g / L folic acid.

[0269] The bioreactor was run with the following operating parameters: pH = 7.2, temperature = 30.5°C, gas flow = 0.5 v / v / m, dissolved oxygen at 30% saturation.

[0270] Sugar feeds of a 50:50 mixture of glucose and xylose (total sugar concentration in feed was 610 g / L) to a total initial volume of 50 g / L were added to the bioreactor as required by a dissolved oxygen triggered controller. Each of these feeds was added automatically in response to a slowing of the metabolic activity of the culture, which was reflected in a corresponding rise in residual dissolved oxygen concentration as the sugar concentration in the bioreactor decreased. Once the residual dissolved oxygen concentration value rose above a certain predetermined offset from the dissolved oxygen set point, the feed controller triggered the next addition of sugar to the bioreactor.

[0271] The two feeds in the bioreactor were followed with additional bioreactor samples taken (indicated by black dots) at approximately 26 and 50 hours after inoculation of the bioreactor.

[0272] The cultures were typically induced around 10 hours into the fermentation, so the 26 hour time point was chosen because this is the time point at which the culture is fully induced and producing FAMEs at the maximum rate in young, healthy cells. The cells are using sugar at close to their highest capacity. The 50 hour time point was chosen because this is the time point at which the culture has been induced and producing for approximately 40 hours, so the cells are older and in a different state of health than at 26 hours. The cells are somewhat tired and the utilization is starting to drop normally (in the case of WT XylR, but not in the case of XylRl).

[0273] The duration of the feed is shown in the rectangular box Figure 1 ) on the x-axis. The glucose (solid line) and xylose (dashed line) concentrations remaining in the sample supernatant were measured at the start and end of each of these feeds using high performance liquid chromatography (HPLC), which was also used to measure the residual glucose and xylose over the subsequent period of time until the start of the next feed to quantitatively determine the glucose and xylose consumption levels of the culture.

[0274] The calculated glucose and xylose utilization rates for the two strains at the two test time points (26 and 50 hours EFT) are listed in the table below. The residual glucose at the start of all feeds was zero.

[0275]

[0276] As Figure 1 and shown in the table above, the control strain efficiently utilizes glucose, but not xylose. The residual xylose concentration in the control strain at the start of the feed at 26 hours is high (13.7 g / L), but the residual glucose is zero. This indicates that while the glucose is fully utilized with each feed, the xylose is not, so it accumulates in the bioreactor over time. Further sampling approximately 1.5 hours after the feed shows the xylose level to be reduced, but the xylose level remains elevated until the residual glucose level returns to zero. At 50 hours, the residual glucose level is again zero, but the xylose level has further increased (54.6 g / L), indicating that the control strain is not efficiently utilizing the xylose. Further sampling approximately 1.5 hours after the feed shows that the xylose level is not reduced, while the residual glucose concentration returns to zero.

[0277] In contrast, in the strain expressing the XylRl mutant (E382K), the residual xylose concentration in the control strain at the start of the feed at 26 hours was low (0.5 g / L), and the residual glucose was zero. Further sampling approximately 1.5 hours after the feed showed that both the xylose and glucose were almost down to zero. At 50 hours, the residual glucose concentration was again zero, and the residual xylose concentration was essentially so as well. Further sampling approximately 1.5 hours after the feed showed that the xylose level dropped from a high of about 6.5 g / L to a level of about 4 g / L. Thus, even in the presence of glucose, the XylRl mutant exhibited increased xylose utilization.

[0278] Example 2

[0279] This example demonstrates that cells expressing the XylRl mutant show improved growth in the presence of xylose, and that the improved growth is due to improved xylose utilization.

[0280] IC.187 and sven.903 (IC.187 XylR1)

[0281] IC.187 is an E. coli cell with an unmodified xylR locus (XylR WT). sven.903 is isogenic to IC.187, but has a point mutation E382K in the xylR locus. Both strains do not have a plasmid, so FAME is not produced in this case.

[0282] IC.187 and sven.903 were grown in high throughput. Cells were grown as seed cultures in seed minimal media containing 10 g / L glycerol or 10 g / L glucose at 32°C with 250 RPM (revolutions per minute) shaking overnight. Twenty percent inoculum was then added to minimal media containing 10 g / L xylose and grown at 32°C with 250 RPM shaking. Growth was then measured at 8 hours by OD600 reading. Seed minimal media = IX trace vitamins, 0.001 mg / mL thiamine, 0.1 mM CaCl2, 0.01 g / L ferric citrate, 1 mM MgSO4, IX trace minerals, 0.5% MeOH, 100 mM Bis-Tris (pH=7), 0.424 g / L KH2PO4, 0.376 Na2HPO4, 10 g / L (NH4)2SO4, 2 g / L NaCl. Minimal media = same as seed, but with the following differences: 0.0125% Triton, 2% MeOH, 200 mM Bis-Tris (pH=7), 0.318 g / L KH2PO4, 0.282 g / L Na2HPO4, 7.5 g / L (NH4)2SO4, 1.5 g / L NaCl.

[0283] From Figure 2As can be seen, the strain expressing wild type xylR (IC.187) grew more slowly than the strain expressing the xylRl (E382K) mutant, whether starting from a glucose or a glycerol seed. Thus, cells expressing the xylRl mutant showed improved growth in the presence of xylose, whether or not the culture was shifted from a glucose or glycerol seed.

[0284] From previous experiments, it was known that glucose or glycerol was completely consumed after the seed culture had completed growth. Glucose inhibits the xylose operon through carbon catabolite repression, while glycerol is a neutral sugar that does not inhibit the xylose operon. Thus, the faster growth of cells with the xylRl mutation, whether shifted from a sugar that shows carbon catabolite repression or from a neutral sugar, indicates a higher rate of xylose utilization in both cases.

[0285] Example 3

[0286] This example illustrates the construction and testing of new XylR mutants with improved xylose utilization and / or improved co-utilization of glucose and xylose.

[0287] Wild type XylR nucleic acid (SEQ ID NO: 2) was cloned into a template plasmid, pSven.178. Mutants were generated using Transference PCR (tPCR), followed by testing their ability to allow growth on xylose. Transference PCR was performed using methods known in the art.

[0288] Briefly, a template plasmid (pSven.178 = p15A-lacI-PxylR-xylR(WT)-KanR) was constructed. The plasmid contains homology to the upstream and downstream of the xylR genomic sequence, which is necessary for genomic integration of the resulting XylR mutants into E. coli. Transference PCR was performed using the template plasmid in combination with a forward (5'-3') primer and a reverse primer containing the desired mutation. PCR was then used to amplify from the tPCR template using external primers that amplify sequences containing the diversity and homology regions.

[0289] The amplified mutants were cut with the restriction enzyme (Dpn1) to release the mutagenized XylR gene and homology regions for integration. The linear DNA product containing the XylR mutation and homology regions was integrated into the xylR locus of E. coli strain sven.999. (See, e.g., Datsenko and Wanner, 2000, PNAS, 97(12), 6640-6645).

[0290] The sven.999 strain contains a deletion of the native XylR gene and is unable to grow on minimal media containing xylose as the sole carbon source. Therefore, only bacteria that have integrated the XylR mutant with the ability to utilize xylose will be able to grow on the minimal plates.

[0291] Once colonies containing the xylR mutant in the ΔxylR locus are obtained, a serial passaging method can be used to identify xylR variants that can support growth on minimal media containing xylose.

[0292] Colonies from the serial passaging method are isolated and screened for growth comparison to control strains containing the WT xylR protein and xylR2 (R121C).

[0293] Table 3 and Figures 3-8 illustrates how the growth of xylR mutants compared to WT xylR in minimal media containing xylose.

[0294] To measure growth on xylose, control strains KTT.560 (WT XylR), sCR.002 (XylR2 R121C), and sven.996 (ΔxylR) and various xylR mutant strains were screened for growth in minimal media with xylose. Colonies were initially grown on LB media for 4-6 hours, then transferred to minimal media containing limited phosphate and other nutrients and glycerol as the carbon source. Cultures were grown overnight and used to subculture into fresh phosphate-limited minimal media containing xylose as the carbon source. The optical density (OD) of the cultures was measured periodically at a wavelength of 600 nm (OD600) to measure the growth of the strains relative to the controls. All data shown is the average of 3 replicates for each strain.

[0295] Table 3: Ranking of point mutations in the XylR protein, nucleotide codon changes, and mutants for growth on xylose compared to controls

[0296]

[0297] Table 3 Notes: μ and FOIC ranking criteria. + up to 50% improvement; ++ 50% to 100% improvement; +++ over 100% improvement; - up to 25% decrease; -- 25% to 50% decrease; --- over 50% decrease. μ is the cell growth rate defined as the reciprocal of time units. It is a measure of the change in cell number per unit time.

[0298] All comparisons were made relative to the control strain KTT.560 with WT XylR. Controls were KTT.560 = WT xylR; sCR.002 = xylR2; sven.996 = ΔxylR; sDH.687 = KTT.560 pDH.138; sven.954 = KTT.560 xylR2 pDH.138; sven.1053 = KTT.560 ΔxylR pDH.138.

[0299] FOIC = Fold over internal control. Internal control = WT XylR (KTT.560). OD600 of mutant strains during mid-log phase is expressed as fold higher than control strain KTT.560. All final production strains contained production plasmid pDH.138, which contains Marinobacter hydrocarbonoclasticus ester synthase. Plasmid pDH.138 is a production plasmid containing SC101 origin of replication, spectinomycin resistance, IPTG inducible PTrc promoter, and ester synthase variant. Genotype of plasmid is described as follows:

[0300] ori_SC101, repA, par, aadA1-terminator (B1004), lacIq-terminator (T22), Ptrc_ linker D IGR19-ES50_term (rrnB_T1T2)

[0301] Selected XylR mutants were further tested for growth on xylose minimal medium and their ability to produce various fatty acid species (FAS). Growth was monitored by periodic measurement of OD 600 as described above. FAS production was measured using standard methods known in the art.

[0302] Table 4 and Figures 9-16 Xylose utilization and productivity of various XylR mutants in FAS production compared to WT xylR grown in minimal medium containing xylose is illustrated.

[0303] Table 4: Performance metrics of xylR mutants compared to WT xylR, and ranking compared to controls

[0304] Strains Mutations Ranking of xylose consumption Ranking of FAS production sDH.687 WT XylR sven.1042 F372W -- -- sven.1052 N120C --- --- sven.1051 V83C --- --- sven.1043 S364W + - sven.1048 Y141R + - sven.1047 L89V --- --- sven.1045 L365V ++ ++ sven.1049 Q145R + + sven.1050 L146R +++ +++ sven.1044 Q289V D305M +++ - Sven.1066 S364W,R295C - - Sven.1067 A286F,Q306K + --- Sven.1072 A247V + + Sven.1073 A336M -- -- Sven.1074 A336G + + sven.1068 E337H + + sven.1069 L112R ++ ++ sven.1070 A286M ++ ++

[0305] Table 4 Notes: Xylose consumption and FAS production ranking. + up to 10% improvement; ++ 10% to 20% improvement; +++ more than 20% improvement; - up to 10% decrease; -- 10% to 20% decrease; --- more than 20% decrease. All comparisons were made relative to the control strain sDH.687 with WT XylR.

[0306] Example 4

[0307] This example illustrates a mutation at position 121 of SEQ ID NO: 1 that, when expressed in a recombinant host cell, confers on the host cell the ability to consume xylose faster than an otherwise identical isogenic host cell that does not express SEQ ID NO: 1.

[0308] Table 5 lists specific substitutions at position 121 relative to the wild-type sequence and their effect on xylose consumption. International Application No. PCT / US2014 / 027337 is incorporated herein by reference.

[0309] Table 5: Amino acid substitutions that relieve catabolite repression

[0310] Amino acid Faster xylose consumption than WT (Arg)

[0311] Cysteine Yes

[0312] Serine Yes (original mutation)

[0313] Threonine Yes

[0314] Glycine Yes

[0315] Histidine Yes

[0316] Valine Yes

[0317] Methionine Yes

[0318] Tyrosine Yes

[0319] Isoleucine Yes

[0320] Alanine Yes

[0321] Leucine Yes

[0322] Proline Yes

[0323] Phenylalanine Yes

[0324] Tryptophan Possibly (depends on time course)

[0325] Appendix A

[0326] SEQ ID NO: 1 XylR wild-type protein sequence (Genbank # NC_000913; Escherichia coli K-12 MG1655; Blattner and Plunkett, 1997; NCBI protein ID: NP_418026)

[0327]

[0328] SEQ ID NO: 2 XylR WT DNA sequence (Genbank # NC_000913; Escherichia coli K-12 MG1655; Blattner and Plunkett, 1997; NCBI Protein ID: NP_418026)

[0329]

[0330] SEQ ID NO: 3 (E382K) protein sequence:

[0331]

[0332] SEQ ID NO: 4 XylRl (E382K) DNA sequence:

[0333]

[0334] It will be apparent to those skilled in the art that various modifications and variations can be made in the aspects and embodiments described above without departing from the spirit and scope of the disclosure.

Claims

1. An engineered XylR protein variant, wherein the mutation of the XylR protein variant relative to SEQ ID NO: 1 is E382K.

2. The engineered XylR protein variant of claim 1 , wherein expression of the engineered XylR protein variant in a recombinant host cell confers improved growth to the recombinant host cell compared to the growth of a host cell expressing SEQ ID NO: 1 when the cell is cultured in the presence of xylose.

3. The engineered XylR protein variant of claim 1 , wherein expression of the engineered XylR protein variant in a recombinant host cell confers improved xylose utilization to the recombinant host cell compared to a host cell expressing SEQ ID NO: 1 when the cell is cultured in the presence of xylose.

4. A recombinant host cell comprising the engineered XylR protein variant according to any one of claims 1 to 3.

5. The recombinant host cell of claim 4, wherein the recombinant host cell expresses at least one heterologous fatty acid derivative biosynthetic enzyme and, when cultured in the presence of a carbon source, produces a fatty acid derivative composition.

6. A method for improving xylose utilization in a recombinant host cell, the method comprising culturing a recombinant host cell comprising the engineered XylR protein variant according to any one of claims 1 to 3 in a culture medium comprising xylose, wherein expression of the engineered XylR protein variant confers improved xylose utilization to the recombinant host cell compared to xylose utilization by a host cell expressing SEQ ID NO: 1 when the cell is cultured in the presence of xylose.

7. The method of claim 6, wherein: The method is used to prepare fatty acid derivatives, and The method comprises culturing the recombinant host cell in a culture medium comprising xylose, the recombinant host cell further comprising at least one heterologous fatty acid derivative biosynthetic enzyme.

8. The method of claim 7, wherein the fatty acid derivative is: fatty acid esters and wherein at least one heterologous fatty acid derivative biosynthetic enzyme has ester synthase activity; ω-hydroxy fatty acids and wherein at least one heterologous fatty acid derivative biosynthetic enzyme has ω-hydroxylase activity; a fatty aldehyde and wherein at least one heterologous fatty acid derivative biosynthetic enzyme has carboxylic acid reductase activity; a fatty alcohol and wherein at least one heterologous fatty acid derivative biosynthetic enzyme has acyl-ACP reductase activity or carboxylic acid reductase activity, or wherein at least one heterologous fatty acid derivative biosynthetic enzyme has acyl-CoA reductase activity and at least one heterologous fatty acid derivative biosynthetic enzyme has acyl-CoA synthetase activity; a fatty amine and wherein at least one heterologous fatty acid derivative biosynthetic enzyme has carboxylic acid reductase activity and another heterologous fatty acid derivative biosynthetic enzyme has aminotransferase or amine dehydrogenase activity; or Fatty alcohol acetate and wherein at least one heterologous fatty acid derivative biosynthetic enzyme has carboxylic acid reductase activity and another heterologous fatty acid derivative biosynthetic enzyme has fatty alcohol O-acetyltransferase activity that converts the fatty alcohol to fatty alcohol acetate.

9. The method of claim 8, wherein the fatty acid ester comprises C5-C 24 Fatty acid methyl ester (FAME) or C5-C 24 Fatty acid ethyl ester (FAEE) or C5-C 24 Fatty acid methyl esters (FAME) and C5-C 24 Combinations of fatty acid ethyl esters (FAEEs).

10. The method of claim 6, wherein the culture medium is derived from cellulosic biomass.

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