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

Abstract

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

Description

[0001] References to related applications

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 726,114, filed on August 31, 2018, and U.S. Provisional Patent Application No. 62 / 731,711, filed on September 14, 2018, which are hereby incorporated by reference in their entireties. Background Art

[0003] To meet the energy and resource demands of a growing global population and reduce dependence on finite fossil energy sources, the use of renewable technologies to produce energy and consumer products must be increased.

[0004] Exemplary renewable technology includes using microbial systems to produce chemicals and fuels. Microbial systems for producing biofuels and various chemicals are well known in the art (see, for example, 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 overall benefits brought by microbial systems may be limited by the properties of the raw materials that can be used for production.

[0005] Hydrolysates are commonly used as raw materials for biochemical production of chemicals. These raw materials are cheaper than pure glucose, potentially reducing the cost of bioproduction processes. The most abundant sugars in hydrolysates are glucose and xylose. Unfortunately, however, the utilization rate of xylose is lower than that of glucose, and due to the two-stage effect, xylose is often not effectively utilized in the presence of glucose.

[0006] Therefore, in order to make the output of any renewable carbon-based product derived from the bioprocessing of hydrolysate feedstock higher, there is a need in the art for a microbial system that allows increased xylose utilization and increased co-utilization of glucose and xylose. Fortunately, as will be apparent from the disclosure below, the present invention satisfies these and other needs. Summary of the Invention

[0007] 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.

[0008] In one aspect, the present disclosure provides an 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 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.

[0009] In one aspect, the present disclosure provides a recombinant host cell comprising an 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 positions 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.

[0010] In one aspect, the present disclosure provides a method for increasing xylose utilization in a recombinant host cell. The method comprises culturing a recombinant host cell comprising a XylR protein variant in a 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 positions 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 to the recombinant host cell when the cell is cultured in the presence of xylose compared to growth of a host cell expressing SEQ ID NO: 1.

[0011] In one aspect, the present disclosure provides a method for preparing fatty acid derivatives, 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 positions 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.

[0012] In one aspect, the present disclosure provides a method for preparing 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 A method for producing fatty acid ethyl esters (FAEEs) 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 (EC 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 of SEQ ID NO: 1 selected from the group consisting of positions 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.

[0013] In some embodiments of the above aspects, 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.

[0014] In some embodiments, the XylR protein variant further comprises at least one additional mutation at positions selected from 121 and 363 corresponding to positions in 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.

[0015] In some embodiments of the above aspects, the XylR protein variant can have a combination of two or more amino acid substitutions compared to the wild-type XylR protein 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, 1313L, M333R, A336M, A336G, E337N, E337H, L351T, S364W, L365T, L365V, F372W, and E382K. 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.

[0016] In some embodiments of the above aspects, the XylR protein variant may have a combination of two or more amino acid substitutions (wherein 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, Q R121A, R121L, R121P, R121P, R121F, R121W, and P363S.

[0017] 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 to 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 to the entire sequence of SEQ ID NO: 1 for a length of and has XylR activity.

[0018] In one aspect, the present disclosure provides an 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 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.

[0019] 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 having at least one mutation at position 382 of SEQ ID NO: 1, wherein the XylR protein variant has no mutation at the position corresponding to position 121 of SEQ ID NO: 1 and no mutation at the position corresponding to position 363 of SEQ ID NO: 1, wherein expression of the XylR protein variant in the recombinant host cell confers improved growth to the recombinant host cell when the cell is cultured in the presence of xylose, compared to the growth of a host cell expressing SEQ ID NO: 1. In embodiments, the improved growth is due to increased 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.

[0020] Another aspect of the present disclosure provides a method for producing fatty acid derivatives, the method comprising: culturing a 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 having at least one mutation at position 382 of SEQ ID NO: 1 in a culture medium comprising xylose, wherein the XylR protein variant has no mutation at the position corresponding to position 121 of SEQ ID NO: 1 and no mutation at the 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 XylR protein variant having at least one mutation at position 382 of SEQ ID NO: 1 has an E382K substitution mutation. In embodiments, the culture medium comprising xylose further comprises glucose. In embodiments, the culture 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 (EC 3.1.1.67) and the fatty acid derivative is selected from fatty acid methyl esters (FAME) and fatty acid ethyl esters (FAEE), or a combination thereof.

[0021] Another aspect of the present disclosure provides an 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 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 no mutation at a position corresponding to position 363 of SEQ ID NO: 1.

[0022] Another aspect of the present disclosure provides a method for producing a C5-C24 fatty acid derivative selected from fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs), or a combination thereof, 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 (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 position 382 of SEQ ID NO: 1 and 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 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 culture medium comprising xylose further comprises glucose.

[0023] In one aspect, the present disclosure provides an 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 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.

[0024] In one aspect, the present disclosure provides a recombinant host cell comprising an 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 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.

[0025] 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 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, wherein expression of the XylR protein variant confers improved xylose utilization by the recombinant host cell when the cell is cultured in the presence of xylose compared to xylose utilization by a host cell expressing SEQ ID NO: 1.

[0026] In some embodiments, the method is for producing fatty acid derivatives, 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.

[0027] 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).

[0028] 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 invention relates to a method for producing fatty acid ethyl esters (FAEEs), 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 (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.

[0029] 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.

[0030] 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.

[0031] 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 being a member selected from the group consisting of: a XylR protein variant having the substitution mutation L89K and further comprising L112R; a XylR protein variant having the substitution mutation E150G and further comprising H88G; a XylR protein variant having the substitution mutation R280V and further comprising D305G; a XylR protein variant having the substitution mutation A286F and further comprising Q306K; a XylR protein variant having the substitution mutation Q289V and further comprising D305M; and a XylR protein variant having the substitution mutation S364W and further comprising R295C.

[0032] 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.

[0033] In some embodiments, the XylR protein variant, recombinant host cell, or method of any embodiment confers improved growth of the recombinant host cell when the cell is cultured in the presence of xylose, 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: positions 112, 141, 145, 146, 247, 286, 289, 336, 337, 364, and 365 of SEQ ID NO: 1, wherein at least one mutation is selected from the group consisting of: L112R, Y141R, Q145R, L146R, A247V, A286F, A286M, Q289V, A336G, E337H, S364W, and L365V.

[0034] In some embodiments, the recombinant host cell of any of the embodiments 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 positions 112, 145, 146, 247, 286, 336, 337, and 365 of SEQ ID NO: 1, wherein when cultured in the presence of xylose, the recombinant host cell produces an increased amount of a fatty acid species (FAS) compared to an isogenic host cell that is otherwise identical except for expressing 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 positions 112, 145, 146, 247, 286, 336, 337, and 365 of SEQ ID NO: 1, wherein at least one mutation is selected from the group consisting of L112R, Q145R, L146R, A247V, A286M, A336G, E337H, and L365V.

[0035] In some embodiments, the method of any embodiment comprises a XylR protein variant having at least one mutation at a position selected from positions 112, 145, 146, 247, 286, 336, 337, and 365 of SEQ ID NO: 1, wherein 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 of the embodiments comprises a culture medium comprising xylose, further comprising glucose. In some embodiments, the culture medium is derived from cellulosic biomass.

[0037] 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.

[0038] 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 to the recombinant host cell when the cell is cultured in the presence of xylose, compared to the growth of a host cell expressing SEQ ID NO: 1.

[0039] In some embodiments, the XylR protein variant, recombinant host cell, or method of any embodiment comprises a XylR protein variant further comprising 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.

[0040] 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 position 382 corresponding to a position of SEQ ID NO: 1, wherein expression of the XylR protein variant confers improved xylose utilization to the recombinant host cell when the cell is cultured in the presence of xylose, compared to xylose utilization by a host cell expressing SEQ ID NO: 1.

[0041] In some embodiments, the method is for producing fatty acid derivatives, 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.

[0042] 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).

[0043] 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 The invention relates to a method for producing fatty acid ethyl esters (FAEEs), 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 (EC 3.1.1.67) and a XylR protein variant having at least one mutation at position 382 of SEQ ID NO: 1.

[0044] 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, wherein the at least one mutation is an E382K substitution mutation.

[0045] 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.

[0046] In some embodiments, the method of any of the embodiments comprises a culture medium comprising xylose and further comprising glucose.In some embodiments, the culture medium is derived from cellulosic biomass.

[0047] Other features, objects and advantages of the present invention will become apparent from the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 is a graph illustrating the improved co-utilization of glucose and xylose by the E382K XylR mutant (xylR1) compared to the wild-type control.

[0049] Figure 2 is a graph illustrating improved growth in the presence of xylose, and the improved growth is due to improved xylose utilization.

[0050] Figure 3 Graph illustrating growth of xylR mutants L146R, F372W, N120C, L365V, S364W, V83C, (Q289V, D305M), Q145R, L89V, and Y141R compared to WT xylR growth in minimal medium containing xylose.

[0051] Figure 4 Graphs illustrating growth of xylR mutants (S364W, R295C), (A286F, Q306K), E337H, L112R, (R280V, D305G), A286M, and I313L compared to WT xylR in minimal medium containing xylose.

[0052] Figure 5 Graph illustrating growth of xylR mutants A247V, A336M, A336G, (L89K, L112R), E150W, S130Y, (H88G, E150G, A246A*), and A310L compared to WT xylR in minimal medium containing xylose. * indicates a silent mutation (GCG->GCA) at the nucleic acid level.

[0053] Figure 6 Graph illustrating growth of xylR mutants (A247T, S352S*) and G154C compared to WT xylR in minimal medium containing xylose. * indicates silent mutation at the nucleic acid level (TCG->TCC).

[0054] Figure 7 is a graph illustrating the growth of xylR mutants L351T, M333R and V155E compared to WT xylR in minimal medium containing xylose.

[0055] Figure 8 is a graph illustrating the growth of xylR mutants L365T, R270E, E337N and V147M compared to WT xylR in minimal medium containing xylose.

[0056] Figure 9Xylose utilization by XylR mutants L365V, Q145R, L146R and (Q289, D305M) compared to WT xylR grown in minimal medium containing xylose is illustrated.

[0057] Figure 10 The productivity of XylR mutants L365V, Q145R L146R and (Q289, D305M) in terms of fatty acid species (FAS) production compared to WT xylR grown in minimal medium containing xylose is illustrated.

[0058] Figure 11 Xylose utilization by XylR mutants F372W, N120C, V83C, S364W, L89V, and Y141R compared to WT xylR grown in minimal medium containing xylose is illustrated.

[0059] Figure 12 The productivity of XylR mutants R372W, N120C, V83C, S364W, L89V and Y141R in terms of fatty acid species (FAS) production compared to WT xylR grown in minimal medium containing xylose is illustrated.

[0060] Figure 13 Xylose utilization by XylR mutants E337H, L112R, and A286M compared to WT xylR grown in minimal medium containing xylose is illustrated.

[0061] Figure 14 The productivity of XylR mutants E337H, L112R and A286M in terms of fatty acid species (FAS) production compared to WT xylR grown in minimal medium containing xylose is illustrated.

[0062] Figure 15 Xylose utilization by XylR mutants (S364W, R295C), (A286F, Q306K), A247V, A336M and A336G compared to WT xylR grown in minimal medium containing xylose is illustrated.

[0063] Figure 16 The productivity of XylR mutants (S364W, R295C), (A286F, Q306K), A247V, A336M and A336G in terms of fatty acid species (FAS) production compared to WT xylR grown in minimal medium containing xylose is illustrated.

[0064] Figure 17 A schematic pathway for xylose utilization in metabolic pathways is shown. DETAILED DESCRIPTION

[0065] definition

[0066] As used herein and in the appended claims, singular articles such as "a," "an," and "the," and similar references in the context of describing elements are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Thus, for example, reference to "a host cell" includes two or more such host cells, reference to "a nucleic acid sequence" includes one or more nucleic acid sequences, reference to "an enzyme" includes one or more enzymes, and so forth.

[0067] As used herein, "about" is understood by one of ordinary skill in the art and may vary to some extent depending on the context in which it is used. If the use of the term is ambiguous in the context in which the term "about" is used by one of ordinary skill in the art, "about" will mean up to plus or minus 10% of the specific term (including the specific term). Thus, about 100 will mean 90 to 110, and will include 100.

[0068] As will be understood by one skilled in the art, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof for any and all purposes. Furthermore, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 atoms refers to a group having 1, 2, or 3 atoms. Similarly, a group having 1-5 atoms refers to a group having 1, 2, 3, 4, or 5 atoms, and so on.

[0069] Unless otherwise defined, the technology and scientific terms used herein generally have the same meaning as understood by those of ordinary skill in the art. In particular, the present disclosure utilizes the conventional techniques in recombinant genetics, organic chemistry, fermentation and biochemistry. The basic literature of public molecular biology and genetics general terms includes, for example, Lackie, Dictionary of Cell and Molecular Biology [cell and molecular biology dictionary], Elsevier [Elsevier Press] (2013 the 5th edition). The basic literature of public biochemistry general methods and terms includes, for example, Lehninger Principles of Biochemistry Sixth edition [Leninger biochemistry principles sixth edition], David L. Nelson and Michael M. Cox edited .WH Freeman (2012). The basic literature of public fermentation general methods and terms includes, for example, Principles of Fermentation Technology [fermentation technology principle], the 3rd edition, Peter F Stanbury edited, Allan Whitaker and Stephen J Hall. Butterworth-Heinemann (2016). Basic texts disclosing general methods and terminology in organic chemistry include, for example, Favre, Henri A. and Powell, Warren H. Nomenclature of Organic Chemistry. IUPAC Recommendations and Preferred Name 2013. Cambridge, UK: The Royal Society of Chemistry, 2013; Practical Synthetic Organic Chemistry: Reactions, Principles, and Techniques, ed. Stephane Caron, John Wiley and Sons Inc. (2011); Organic Chemistry, 9th ed. - Francis Carey and Robert Giuliano, McGraw Hill (2013).

[0070] The sequence accession numbers throughout this specification are obtained from the following: the database provided by NCBI (National Center for Biotechnology Information) maintained by the National Institutes of Health of the United States (referred to herein as "NCBI accession numbers" or alternatively "GenBank accession numbers" or alternatively simply as "accession numbers"), and the UniProt Knowledge Base (UniProtKB) and Swiss-Prot database provided by the Swiss Institute of Bioinformatics (referred to herein as "UniProtKB accession numbers").

[0071] Enzyme Classification (EC) numbers are assigned by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB), and their descriptions can be found on the IUBMB Enzyme Nomenclature website on the World Wide Web. EC numbers classify enzymes based on the reaction they catalyze. For example, the enzymatic activity of thioesterases is classified as EC 3.1.2.1-3.1.2.27 and 3.1.2.-. The specific classifications are based on the activity of different thioesterases on different substrates.

[0072] For example, in some exemplary embodiments, thioesterases that catalyze the hydrolysis of thioester bonds in C6-C18 alkyl thioesters, such as acyl-acyl carrier protein thioesters (acyl-ACP) and acyl-coenzyme A thioesters (acyl-CoA), are classified under EC 3.1.2.-, e.g., 3.1.2.14. Thioesterases are found in most prokaryotes and in the chloroplasts of most plants and algae. Thioesterase function is conserved from species to species in most prokaryotes. Therefore, different microbial species can perform the enzymatic activity of the same thioesterase classified under EC 3.1.2.-.

[0073] As used herein, the term "fatty acid" refers to an aliphatic carboxylic acid having the formula RCOOH, wherein 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 monounsaturated or polyunsaturated.

[0074] As used herein, "fatty acids" are produced in cells by the process of fatty acid biosynthesis, by reversal of fatty acid β-oxidation, or they can be supplied to cells. As is well known in the art, fatty acid biosynthesis is typically malonyl-CoA-dependent acyl-ACP synthesis, and reversal of β-oxidation produces acyl-CoA. Fatty acids supplied to cells are converted to acyl-CoA and acyl-ACP.

[0075] The biosynthesis and degradation of fatty acids occurs in all life forms, including prokaryotes, unicellular eukaryotes, higher eukaryotes, and archaea. The tools and methods disclosed herein can be used to produce fatty acid derivatives derived from one or more of fatty acid synthesis, degradation, or production in any organism that naturally produces alkyl thioesters.

[0076] As used herein, term " fatty acid derivative " refers to the product derived from fatty acid.Therefore, " fatty acid derivative " comprises " fatty acid " as defined above.Usually, " fatty acid derivative " comprises the compound derived from malonyl-CoA, comprises acyl-ACP or acyl-ACP derivative. " fatty acid derivative " also comprises the compound derived from malonyl-CoA, for example acyl-CoA or acyl-CoA derivative.Exemplary fatty acid derivative comprises fatty acid, fatty acid ester (wax, fatty acid ester, fatty acid methyl ester (FAME), fatty acid ethyl ester (FAEE)), fatty alcohol acetate (FACE), fatty amine, fatty aldehyde, fatty alcohol, hydrocarbon (for example alkane, alkene etc.), ketone, terminal olefin, internal olefin, 3-hydroxy fatty acid derivative, difunctional fatty acid derivative (for example ω-hydroxy fatty acid, 1,3 fatty glycol, α, ω-glycol, α, ω-3-hydroxy triol, ω-hydroxy FAME, ω-OHFAEE etc.) and unsaturated fatty acid derivative, comprises every kind of unsaturated compound in the above-mentioned fatty acid derivative.

[0077] As used herein, the expression "fatty acid derivative composition" refers to a composition of fatty acid derivatives, such as a fatty acid composition produced by an organism. A "fatty acid derivative composition" may comprise a single fatty acid derivative species or may comprise a mixture of fatty acid derivative species. In some exemplary embodiments, the mixture of fatty acid derivatives comprises more than one type of fatty acid derivative product (e.g., fatty acid, fatty acid ester, fatty alcohol, fatty alcohol acetate, fatty aldehyde, fatty amine, difunctional fatty acid derivative, etc.). In other exemplary embodiments, the mixture of fatty acid derivatives comprises a mixture of fatty acid esters (or another fatty acid derivative) with different chain lengths, saturation and / or branching characteristics. In other exemplary embodiments, the mixture of fatty acid derivatives mainly comprises 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 with different chain lengths, saturation and / or branching characteristics). In still other exemplary embodiments, the mixture of fatty acid derivatives comprises a mixture of fatty esters and β-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 FAME and / or FAEE. In still other exemplary embodiments, the fatty acid derivative composition comprises a mixture of fatty alcohol acetates (FACE).

[0078] As used herein, the term "nucleotide" has its customary meaning as known in the art. In addition to referring to naturally occurring ribonucleotide or deoxyribonucleotide monomers, the term "nucleotide" also encompasses nucleotide analogs and modified nucleotides, such as amino-modified nucleotides. In addition, "nucleotide" includes non-naturally occurring analog structures. Thus, for example, each individual unit of a peptide nucleic acid containing a base may be referred to herein as a nucleotide.

[0079] The term "polynucleotide" refers to a polymer of ribonucleotides (RNA) or deoxyribonucleotides (DNA) typically in phosphodiester linkages, which can be single-stranded or double-stranded and 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 of RNA or DNA. These terms refer to the primary structure of the molecule and therefore include polynucleotides that are single-stranded, double-stranded, triple-stranded, quadruple-stranded, partially double-stranded, branched, hairpin, circular, in a padlocked conformation, and the like. The term equivalently includes analogs of RNA or DNA made from nucleotide analogs and modified polynucleotides (such as, but not limited to, methylated and / or capped polynucleotides). Polynucleotides 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 a combination thereof, as well as using any purification method known in the art.

[0080] As used herein, the terms "polypeptide" and "protein" are used interchangeably and refer to polymers of amino acid residues that are typically 12 or more amino acids in length. Polypeptides of less than 12 amino acids in length are referred to herein as "peptides". The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, and to amino acid polymers comprising naturally occurring amino acids. The term "recombinant polypeptide" refers to a polypeptide produced by recombinant technology, for example, wherein a DNA or RNA encoding an expressed protein is inserted into a suitable expression vector, which in turn is used to transform a host cell to produce the polypeptide. In some embodiments, a DNA or RNA encoding an expressed peptide, polypeptide, or protein is inserted into a host chromosome by homologous recombination or other means well known in the art, and is therefore used to transform a host cell to produce the peptide or polypeptide. Similarly, the term "recombinant polynucleotide" or "recombinant nucleic acid" or "recombinant DNA" is produced by recombinant techniques well known to those skilled in the art (see, for example, the methods described in Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Press, 4th ed. (Cold Spring Harbor, NY 2012) or Current Protocols in Molecular Biology, Vols. 1-3, John Wiley & Sons, Inc. (1994-1998) and Supplements 1-115 (1987-2016)).

[0081] 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, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure (i.e., an alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group) as a naturally occurring amino acid, such as homoserine, norleucine, methionine sulfoxide, and methylsulfoniummethionine. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. The amino acid of natural coding is 20 kinds of 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) and pyrrolysine and selenocysteine.In some exemplary embodiments, the single letter code listed in table 1 below is used to refer to 20 kinds of common naturally occurring amino acid whose specific member.Single letter amino acid code is well known in the art (see, for example, Leninger (Lehninger), the same).

[0082] Table 1

[0083] amino acids Single-letter code amino acids 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 glutamate E Aspartic acid D Serine S Threonine T

[0084] When referring to two nucleotide or polypeptide sequences, the "percent sequence identity" between the two sequences is determined by comparing the two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) for the optimal alignment of the two sequences. The "percent sequence identity" can be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in the two sequences to produce 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 percent sequence identity.

[0085] Thus, 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 that are identical or have a specified percentage of identical nucleotides or amino acids (e.g., about 50% identity over a specified region, preferably 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 200%, 201%. 5%, 96%, 97%, 98%, 99% or more identity) to sequences or subsequences thereof as measured, for example, using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters (see, e.g., Altschul et al. (1990) J. Mol. Biol. 215(3): 403-410) and / or the NCBI website at ncbi.nlm.nih.gov / BLAST / ) or by manual alignment and visual inspection. The percent sequence identity between two nucleic acid or amino acid sequences can also be determined using, for example, the Needleman and Wunsch algorithm in the GAP program, which has been integrated into the GCG software package, using a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6 (Needleman and Wunsch (1970) J. Mol. Biol. 48:444-453). The percent sequence identity between two nucleotide sequences can also be determined using the GAP program in the GCG software package, using the NWSgapdna.CMP matrix, and a gap weight of 40, 50, 60, 70, or 80, and a length weight of 1, 2, 3, 4, 5, or 6. One of ordinary skill in the art can perform an initial sequence identity calculation and adjust the algorithm parameters accordingly. If the practitioner is unsure which parameters should be used to determine whether a molecule is within the homology limits of a claim, one set of parameters that can be used is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. Additional methods of sequence alignment are known in the field of biotechnology (see, e.g., Rosenberg (2005) BMC Bioinformatics 6: 278; Altschul et al. (2005) FEBS J. 272(20): 5101-5109).

[0086] When two or more nucleic acid or amino acid sequences are aligned and analyzed as described above and found to 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, the two or more nucleic acid or amino acid sequences are said to be "substantially identical". Two nucleic acid sequences or polypeptide sequences are said to be "identical" if the sequences of the nucleotides or amino acid residues of the two sequences are respectively the same when the maximum correspondence alignment is performed as described above. This definition may also refer to or may be used to supplement a test sequence. Identity is typically calculated over a region of at least about 25 amino acids or nucleotides in length, or more preferably over a region of 50-100 amino acids or nucleotides in length, or over the entire length of a given sequence.

[0087] The expression "hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions" describes hybridization and washing conditions. Guidance for conducting hybridization reactions can be found, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, New York (1989), 6.3.1-6.3.6. Aqueous and nonaqueous methods are described in the cited references, and either method can be used. Specific hybridization conditions referred to herein are as follows: (1) low stringency hybridization conditions—6X sodium chloride / sodium citrate (SSC) at 45°C, followed by two washes in 0.2X SSC, 0.1% SDS at at least 50°C (for low stringency conditions, the temperature of the washes can be increased to 55°C); (2) moderate stringency hybridization conditions—6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60°C; (3) high stringency hybridization conditions—6X SSC at about 45°C, followed by one or more washes in 0.2.X 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 one or more washes in 0.2X SSC, 1% SDS at 65°C. Unless otherwise indicated, very high stringency conditions (4) are preferred.

[0088] As used herein, the term "endogenous" refers to a substance produced from within a cell, for example, a nucleic acid, a protein, etc. Therefore, an "endogenous" polynucleotide or polypeptide refers to a polynucleotide or polypeptide produced by a cell. In some exemplary embodiments, the "endogenous" polypeptide or polynucleotide is encoded by the genome of the parent cell (or host cell). In other exemplary embodiments, the "endogenous" polypeptide or polynucleotide is encoded by an autonomously replicating plasmid carried by the parent cell (or host cell). In some exemplary embodiments, an "endogenous" gene is a gene that is present in the cell when the cell is initially isolated from nature, that is, the gene is "natural to the cell". In other exemplary embodiments, the "endogenous" gene has been altered by recombinant technology, for example, by changing the relationship between a control sequence and a coding sequence. Therefore, in some exemplary embodiments, a "heterologous" gene can be "endogenous" to a host cell.

[0089] In contrast, an "exogenous" polynucleotide or polypeptide or other substance (e.g., fatty acid derivatives, small molecule compounds, etc.) is one that is not produced by the parent cell and, therefore, is added to the cell, cell culture, or assay from outside the cell.

[0090] As used herein, the term "native" refers to a form of nucleic acid, protein, polypeptide, or fragment thereof that is isolated from nature or a form of nucleic acid, protein, polypeptide, or fragment thereof into which no mutations are introduced.

[0091] 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 present disclosure, a fragment refers to the entire amino acid sequence of a domain (e.g., a substrate binding domain or a catalytic domain) of a polypeptide or protein.

[0092] 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-coding nucleic acid sequences are mutagenized to produce mutant nucleic acid sequences, which produce mutant proteins. 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.

[0093] Therefore, as used herein, "mutation" refers to a change in the nucleic acid position of a gene or the amino acid position (residue) of a polypeptide or protein relative to a control nucleic acid or amino acid sequence. In the context of polynucleotides, the term "mutation" refers to a modification of a polynucleotide sequence that causes the polynucleotide sequence to change relative to a control or reference polynucleotide sequence. In some exemplary embodiments, a mutated polynucleotide sequence refers to a change that does not change the encoded amino acid sequence, such as optimizing codons for expression purposes. In other exemplary embodiments, the mutation in the polynucleotide sequence modifies codons in a manner that causes the encoded amino acid sequence to be modified.

[0094] In the context of protein, the term "mutation" or "mutated" refers to the modification of the amino acid sequence, resulting in a change in the sequence of the protein relative to a control or reference protein sequence. Mutation can refer to the replacement of an amino acid by another amino acid, or the insertion or deletion of one or more amino acid residues. In some exemplary embodiments, "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, "mutation" is a sequence or subsequence relative to a precursor sequence (e.g., a deletion or interruption) or a sequence shortening caused by a deletion from one end or the other. In other exemplary embodiments, mutation is the addition of an amino acid or subsequence (e.g., two or more amino acids in a segment, which are inserted between two consecutive amino acids in a precursor protein sequence) within a protein or at either end of the protein, thereby increasing the length (or extension) of the protein. Mutations can be introduced into polynucleotides by methods known to those 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, etc.

[0095] As used herein, the term "mutant" or equivalently "variant" refers to a polynucleotide sequence or polypeptide sequence comprising at least one mutation. Thus, an engineered XylR variant or XylR (having an improved ability to utilize xylose and an improved ability to co-utilize glucose and xylose for the 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.

[0096] As used herein, 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" refers to an engineered polypeptide / protein variant of the E. coli xylose repressor (XylR) with improved ability to utilize xylose and / or with improved ability to co-utilize glucose and xylose, as measured, for example, 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 via measurement of the total amount of sugar 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" may exhibit improved xylose utilization, improved co-utilization of glucose and xylose, or may have both properties simultaneously.

[0097] A "XylR mutant with improved xylose utilization and / or improved co-utilization of glucose and xylose" or equivalently, a "XylR protein variant with 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 the position corresponding to position 382 of SEQ ID NO: 1, has no mutation at the position corresponding to position 121 of SEQ ID NO: 1, and has no mutation at the 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 with improved xylose utilization and / or improved co-utilization of glucose and xylose" has an amino acid sequence according to SEQ ID NO: 3. In an embodiment, a "XylR protein variant with 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 the position corresponding to position 382 of SEQ ID NO: 1 and which has no mutation at the position corresponding to position 121 of SEQ ID NO: 1 and no mutation at the position corresponding to position 363 of SEQ ID NO: 1. In other embodiments, a "XylR protein variant with 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 the position corresponding to position 382 of SEQ ID NO: 1 and which has no mutation at the position corresponding to position 121 of SEQ ID NO: 1 and no mutation at the position corresponding to position 363 of SEQ ID NO: 1. In embodiments, the XylR protein variant with 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 a feedstock.

[0098] As used herein, the term "gene" refers to a nucleic acid sequence encoding an RNA product or a protein product, e.g., a DNA sequence, and an operably linked nucleic acid sequence that affects expression of the RNA or protein product (e.g., an expression control sequence, e.g., a promoter, an enhancer, a ribosome binding site, a translation control sequence, etc.). The term "gene product" refers to an RNA, e.g., tRNA, mRNA, and / or protein expressed from a particular gene.

[0099] As used herein, the term "expression" or "expressed" with respect to a gene refers to the production of one or more transcription products and / or translation products of a gene. In an exemplary embodiment, the expression level of a DNA molecule in a cell is determined based on the amount of corresponding mRNA present in the cell or the amount of protein encoded by the DNA produced by the cell. The term "expressed gene" refers to a gene that is transcribed into messenger RNA (mRNA) and then translated into protein, as well as genes that are transcribed into other types of RNA, such as transfer RNA (tRNA), ribosomal RNA (rRNA), and regulatory RNA, which are not translated into protein.

[0100] The expression level of a nucleic acid molecule in a cell or cell-free system is affected by an "expression control sequence" or equivalently, a "regulatory sequence". "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 RNA stability, internal ribosome entry sites (IRES), etc., which provide for the expression of a polynucleotide sequence in a host cell. In exemplary embodiments, an "expression control sequence" specifically interacts with a cellular protein involved in transcription (see, e.g., Maniatis et al., Science, 236: 1237-1245 (1987); Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, CA (1990)). In exemplary methods, an expression control sequence is operably linked to a polynucleotide sequence. "Operably linked" refers to a functional connection between a polynucleotide sequence and one or more expression control sequences so as to allow expression of the polynucleotide sequence when an appropriate molecule (e.g., a transcriptional activator) contacts the one or more expression control sequences. In an exemplary embodiment, the operably linked promoter is located upstream of the selected polynucleotide sequence in terms of the direction of transcription and translation. In some exemplary embodiments, the operably linked enhancer can be located upstream, within, or downstream of the selected polynucleotide.

[0101] As used herein, a "modified activity" or "altered activity level" of a protein / polypeptide (e.g., an engineered XylR variant) refers to a difference in one or more characteristics of the protein / polypeptide activity 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, the difference in activity of a protein with a "modified activity" compared to a corresponding control protein is determined by measuring the activity of the modified protein in a recombinant host cell and comparing it to 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 the binding affinity of the protein to nucleic acids; a change in the protein structure (e.g., a change in the primary structure, such as, for example, a change in the nucleotide coding sequence of the protein, which 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, the polypeptide with a "modified activity" is a mutant or variant XylR enzyme disclosed herein.

[0102] In exemplary embodiments, the polypeptides disclosed herein have a "modified activity," i.e., for example, an "improved level of activity." As used herein, the expression "improved level of activity" refers to a polypeptide having a higher level of a biochemical or biological function (e.g., DNA binding or enzymatic activity) than the level of the 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.

[0103] " improved activity " can refer to the catalytic activity of the improvement of polypeptide or the catalytic efficiency of improvement, wherein catalytic efficiency refers to the increase of the reaction rate of the enzyme-catalyzed reaction of such polypeptide.Catalytic activity / catalytic efficiency can be improved for example by improving one or more kinetic parameters (measured or calculated) of reaction, such as Vmax (maximum rate that reaction can be carried out), Km (Michaels constant), kcat (the number of substrate molecules that each enzyme molecule turns over per second), etc., or any ratio between such parameters, such as kcat / Km (measurement of enzyme efficiency).Therefore, " improved catalytic activity " or " improved catalytic efficiency " of polypeptide can be measured in many ways.For example, " improved activity " can be measured by following: titer (concentration under specific conditions: g / L, or mg / L, or g / Kg) increase, the improvement of growth rate; Specific substrates such as the utilization of the improvement of wood sugar; The variation of composition (under certain conditions, such as the amount of specific fatty acid species / total fatty acid derivatives (FAS) produced in the presence of wood sugar).

[0104] A "control" sample, such as a "control" nucleotide sequence, a "control" polypeptide sequence, a "control" cell, or the like, or a 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, the test sample comprises a "XylR mutant for improved xylose utilization or improved co-utilization of glucose and xylose," while the control sample comprises the corresponding or designated unmodified / non-variant XylR protein / enzyme (e.g., SEQ ID NO: 1). The skilled artisan will recognize that controls can be designed for evaluating any number of parameters. Furthermore, one skilled in the art will understand which controls are valuable in a given situation and will be able to analyze data based on comparisons to control values.

[0105] As used herein, the term "recombinant" refers to a genetically modified polynucleotide, polypeptide, cell, tissue, or organism. The term "recombinant" applies equally to the first generation of genetically modified polynucleotides, polypeptides, cells, tissues, or organisms, as well as subsequent generations of genetically modified polynucleotides, polypeptides, cells, tissues, or organisms that carry the genetic modifications.

[0106] When used with respect to cells, the term "recombinant" means that the cell has been modified by introducing heterologous nucleic acids or proteins, or modified by changing native nucleic acids or proteins, or the cell is derived from the cell so modified and the derived cell comprises modification. Thus, for example, "recombinant cells" or equivalently "recombinant host cells" can be modified to express genes that are not found in the natural (non-recombinant) form of the cell, or can be modified to express abnormal natural genes, such as natural genes that can be overexpressed, underexpressed, or not expressed at all. In exemplary embodiments, "recombinant cells" or "recombinant host cells" are engineered to express "XylR mutants for improved xylose utilization or improved glucose and xylose co-utilization." Recombinant cells can be derived from microorganisms, such as bacteria, viruses, or fungi. In addition, recombinant cells can be derived from plant or animal cells. In exemplary embodiments, "recombinant host cells" or "recombinant cells" are 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 (FAME), fatty acid ethyl esters (FAEE)), fatty alcohol acetates (FACE), fatty alcohols, fatty aldehydes, hydrocarbons, fatty amines, terminal olefins, internal olefins, ketones, difunctional fatty acid derivatives (e.g., ω-hydroxy fatty acids, ω-hydroxy diols, ω-hydroxy FAME, ω-hydroxy FAEE), etc. Thus, in some exemplary embodiments, the "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 encoding a polypeptide having fatty acid biosynthetic enzyme activity, wherein when cultured in the presence of a carbon source under conditions effective to express the polynucleotides, the recombinant cell produces a fatty acid derivative composition.

[0107] When used in relation to polynucleotides, the term "recombinant" or equivalently "heterologous" means that the polynucleotide has been modified by comparison with the native form or naturally occurring form of the polynucleotide, or has been modified by comparison with a naturally occurring variant of the nucleotide. In an exemplary embodiment, a recombinant polynucleotide (or a copy or complementary sequence of a recombinant polynucleotide) is a recombinant polynucleotide that has been artificially manipulated to be different from its naturally occurring form. Therefore, in an exemplary embodiment, a recombinant polynucleotide is a mutant form of a natural gene or a mutant form of a naturally occurring variant of a natural gene, wherein the mutation is carried out by intentional human manipulation, such as by saturation mutagenesis using a mutagenic oligonucleotide, by using UV radiation or mutagenic chemicals, etc. Relative to the native form or naturally occurring variant form of a gene, such a recombinant polynucleotide can include one or more point mutations, substitutions, deletions and / or insertions. Similarly, a polynucleotide comprising a promoter operably linked to a second polynucleotide (e.g., a coding sequence) is a "recombinant" polynucleotide. Therefore, a recombinant polynucleotide comprises a polynucleotide combination not found in nature. Recombinant proteins (discussed above) are typically proteins expressed from recombinant polynucleotides, and recombinant cells, tissues, and organisms are those that contain recombinant sequences (polynucleotides and / or polypeptides).

[0108] As used herein, the term "microorganism" generally refers to a microscopic organism. A microorganism can be prokaryotic or eukaryotic. Exemplary prokaryotic microorganisms include, for example, bacteria, archaea, cyanobacteria, and the like. Exemplary bacteria are 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 thereby expresses or encompasses heterologous nucleic acid sequences and / or heterologous proteins.

[0109] A "production host" or equivalently a "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 to have attenuated expression of a selected gene. Thus, a "production host" or "production host cell" is a "recombinant host" or equivalently a "recombinant host cell." Non-limiting examples of production hosts include plants, animals, humans, bacteria, yeast, cyanobacteria, algae, and / or filamentous fungal cells. An exemplary "production host" is a recombinant E. coli cell.

[0110] As used herein, "acyl-ACP" refers to an acyl thioester formed between the carbonyl carbon of the acyl chain and the sulfhydryl group of the phosphopantetheine portion of an acyl carrier protein (ACP). In some embodiments, acyl-ACP is an intermediate in the synthesis of fully saturated acyl-ACP. In other exemplary embodiments, acyl-ACP is an intermediate in the synthesis of unsaturated acyl-ACP. 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 is 8 carbons in length. In other exemplary embodiments, the carbon chain length of the acyl group of the acyl-ACP is 10 carbons. Each of these acyl-ACPs is a substrate for an enzyme, such as an ester synthase, a thioesterase, or the like, that converts the acyl-ACP into a fatty acid derivative.

[0111] As used herein, the expression "fatty acid derivative biosynthetic pathway" refers to a biochemical pathway that produces fatty acid derivatives. Thus, an enzyme comprising a "fatty acid derivative biosynthetic pathway" is referred to herein as a "fatty acid derivative biosynthetic polypeptide" or equivalently a "fatty acid derivative enzyme." 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 an enzyme that can be expressed or overexpressed to produce fatty acid derivatives. Non-limiting examples of "fatty acid derivative enzymes" or equivalently "fatty acid derivative biosynthetic polypeptides" include, for example, fatty acid synthases, thioesterases, acyl-CoA synthetases, acyl-CoA reductases, acyl ACP reductases, alcohol dehydrogenases, alcohol O-acyltransferases, acyl-CoA reductases to form fatty alcohols, fatty acid decarboxylases, fatty aldehyde decarbonylases and / or oxidative deformylases, carboxylic acid reductases, fatty alcohol O-acetyltransferases, ester synthases, and the like. "Fatty acid derivative enzymes" or equivalently "fatty acid derivative biosynthetic polypeptides" convert a substrate into a fatty acid derivative. In an exemplary embodiment, a suitable substrate for the fatty acid derivative enzyme can be a first fatty acid derivative, which is converted into a different second fatty acid derivative by the fatty acid derivative enzyme.

[0112] As used herein, the term "culture" refers to a liquid culture medium containing living cells. In one embodiment, a culture comprises cells grown under controlled conditions in a predetermined culture medium, for example, a culture of recombinant host cells grown in a liquid culture medium containing a selected carbon source and nitrogen. "Cultivating" or "culturing" refers to growing a population of host cells (e.g., recombinant host cells) in a liquid or solid culture medium under suitable conditions. In certain embodiments, culturing refers to the bioconversion of a substrate into an end product. Culture media are well known, and the individual components of such culture media can be obtained from commercial sources, such as Difco. TM Culture medium and BBL TM Culture medium. In one non-limiting example, the aqueous nutrient medium is a "rich medium" including complex sources of nitrogen, salts, and carbon, such as YP medium, which contains 10 g / L of peptone and 10 g / L of yeast extract.

[0113] As used herein, the term "titer" refers to the amount of the fatty acid derivative produced per unit volume of host cell culture. Titer can refer to the amount of a specific fatty acid derivative or a combination of fatty acid derivatives of different chain lengths or different functionalities (such as, for example, a mixture of saturated and unsaturated fatty acid derivatives produced by a given recombinant host cell culture) or the amount of a fatty acid derivative composition.

[0114] As used herein, statement "commercial titer (commercial titers or commercial titer)" refers to the amount of the derivative of fatty acid that per unit volume of host cell culture produces, which makes commercial production economically viable. Typically, commercial titer is in the range of about 10 g / L (or equivalently 10 g / Kg) to about 200 g / L or higher. 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.

[0115] As used herein, " the productive rate of fatty acid derivative " refers to the efficiency that input carbon source is converted into product in host cell.Therefore, statement " the productive rate of fatty acid derivative " refers to the amount of the product produced from the carbon substrate of a given amount.Percent yield is the percentage of theoretical yield (product synthesized under ideal conditions, without carbon or energy loss).Therefore, percent yield=(product mass / theoretical yield mass) X100.Productive rate can refer to a specific fatty acid derivative or a combination of fatty acid derivatives.

[0116] As used herein, term " productivity " refers to the amount of the derivative of fatty acid that per unit volume host cell culture produces per unit time.Productivity can be for the combination of a kind of specific derivative of fatty acid or multiple derivative of fatty acid or one or more other compounds produced by given host cell culture.Therefore, in exemplary embodiments, in recombinant host cell for example in intestinal bacteria, have the xylose that improves and / or the glucose that improves and xylose utilize together expression of XylR mutant and cause with the recombinant host cell of expressing corresponding contrast XylR enzyme or other suitable contrasts, compared, the productivity of derivative of fatty acid and / or other compounds improves.As used herein, term " total fat kind " and " total fatty acid product " and " total fatty acid derivative " can be used interchangeably in this article about the amount (titre) of the derivative of fatty acid that host cell (for example, XylR mutant has the xylose that improves and / or the glucose that improves and xylose utilizes together) produces.Total fat kind etc. can be assessed by gas chromatography (GC-FID) with flame ionization detector. When referring to total fatty acid derivative analysis, the same term can be used to represent, for example, total fatty esters, total fatty alcohols, total fatty aldehydes, total fatty amines, and total free fatty acids. In particular, the same term can be used to represent total fatty acid methyl esters, fatty acid ethyl esters, or fatty alcohol acetates.

[0117] As used herein, the term "carbon source" refers to a substrate or compound suitable for use as a carbon source for prokaryotic or simple eukaryotic cell growth. A carbon source can be in various forms, including but not limited to polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides, and gases (e.g., CO and CO 2 ). Exemplary carbon sources include but are not limited to monosaccharides such as glucose, fructose, mannose, galactose, xylose, and arabinose; oligosaccharides such as oligofructose and oligogalactose; 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. A 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 cellulose hydrolysate. In other examples, the carbon source is natural gas or a component of natural gas, such as methane, ethane, propane, etc.

[0118] As used herein, the term "biomass" refers to any biological material from which a carbon source is derived. In some embodiments, biomass is processed into a carbon source suitable for bioconversion. In some embodiments, biomass is processed into cellulose hydrolysate. In other embodiments, biomass does not need to be further processed into a carbon source. The carbon source can be converted into a composition comprising fatty acid derivatives.

[0119] Biomass can be derived from plant matter or vegetation, for example, derived from corn, sugarcane, switchgrass, rice, wheat, hardwood, softwood, palm, hemp, etc. Another exemplary source of biomass is metabolic waste, for example, animal matter (for example, cow dung). Other exemplary sources of biomass include algae and other marine plants, for example, macroalgae and kelp. Biomass also includes waste from industry, agriculture, forestry and family, including but not limited to glycerol, fermentation waste, silage, straw, timber, paper pulp, sewage, rubbish, cellulose municipal waste, municipal solid waste, grease chemical waste and remaining food (for example, soap, oil and fatty acid). Term " biomass " can also refer to carbon source, for example, carbohydrate (for example, monosaccharide, disaccharide or polysaccharide).

[0120] As used herein, the term "separated" about a product (e.g., fatty acid derivative) refers to a product separated from a cellular component, a cell culture medium, or a chemical or synthetic precursor. The fatty acid derivative produced by the method disclosed herein can be relatively immiscible in fermentation liquid and cytoplasm. Therefore, in an exemplary embodiment, the fatty acid derivative is gathered in an organic phase outside the cell, and is therefore "separated."

[0121] As used herein, the terms "purify", "purified" or "purification" refer to removing or separating molecules from their environment by, for example, separation or separation. "Substantially purified" molecules are at least about 60% free of (for example, at least about 65% free of, at least about 70% free of, at least about 75% free of, at least about 80% free of, at least about 85% free of, at least about 90% free of, at least about 95% free of, at least about 96% free of, at least about 97% free of, at least about 98% free of, at least about 99% free of) other components associated with them. As used herein, these terms also refer to removing contaminants from a sample. For example, removing contaminants can result in an increase in the percentage of fatty acid derivatives or other compounds in the sample. For example, when producing fatty acid derivatives or other compounds in a recombinant host cell, fatty acid derivatives or other compounds can be purified by removing host cell biomass or its components (for example, proteins, nucleic acids and other cellular components). After purification, the percentage of the malonyl-CoA derivative compound comprising the fatty acid derivatives or other compounds in the sample increases. The terms "purify", "purify" or "purification" are relative terms and do not require absolute purity. Thus, for example, when produced in recombinant host cells, 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).

[0122] As used herein, the term "attenuate" means to weaken, reduce or decrease. For example, the activity of a polypeptide can be attenuated, for example, by modifying the polypeptide structure to reduce its activity (eg, by modifying the nucleotide sequence encoding the polypeptide).

[0123] I. Introduction

[0124] The use of hydrolysate feedstocks can significantly reduce the cost of producing renewable chemicals through microbial fermentation. However, although the hydrolysate of lignocellulosic biomass can be converted into biofuels and chemicals through microbial fermentation, the hydrolysate feedstock typically contains mixed sugars, such as glucose, xylose, mannose, etc., and mixed sugar fermentation poses a significant challenge to the cost-effective production of biofuels and chemicals through microbial fermentation.

[0125] In particular, the presence of glucose in the growth medium inhibits E. coli and other industrial microbial species from utilizing other sugars. Only after the glucose in the growth medium has been completely consumed do these microorganisms begin to consume other sugars, such as xylose and pentose sugars. The preferential utilization of glucose relative to non-glucose sugars generally results in lower overall yields and productivity; a phenomenon known as catabolite repression or two-stage growth (see, e.g., Kremling, A., et al. (2015) Vol. 23(2): 99-109; Bruckner R, Titgemeyer F. (2002) FEMS Microbiol. Lett. [FEMS Microbiology Letters] 209: 141-14).

[0126] Therefore, to achieve higher yields of any renewable carbon-based products derived from bioprocessing of hydrolysate feedstocks, there is a need in the art for microbial systems that allow for increased xylose utilization and increased co-utilization of glucose and xylose.

[0127] Fortunately, the present disclosure provides for these and other needs.

[0128] II. XylR mutants with improved xylose utilization or improved co-utilization of glucose and xylose

[0129] A. General Methods

[0130] The present disclosure utilizes conventional techniques in the field of recombinant genetics. The basic literature of public molecular biology and genetics general methods and terminology includes, for example, Sambrook et al., Molecular Cloning, a Laboratory Manual [Molecular Cloning-Laboratory Manual], Cold Spring Harbor Press 4th edition (Cold Spring Harbor, New York 2012); Current Protocols in Molecular Biology [Molecular Biology Current Program] Vol. 1-3, John Wiley & Sons (1994-1998) and Supplement 1-115 (1987-2016). The present disclosure also utilizes conventional techniques in the field of biochemistry. The basic literature of public biochemistry general methods and terminology includes, for example, Lehninger Principles of Biochemistry Sixth edition [Lenninger Biochemistry Principles Sixth Edition], David L. Nelson and Michael M. Cox, ed. WH Freeman (2012). The present disclosure also utilizes conventional techniques in industrial fermentation. Basic literature disclosing general methods and terminology of fermentation includes, for example, Principles of Fermentation Technology, 3rd edition, edited by Peter F. Stanbury, Allan Whitaker and Stephen J. Hall. Butterworth-Heinemann (2016); Fermentation Microbiology and Biotechnology, 2nd edition, edited by EMTEl-Mansi, CFA Bryce, Arnold L. Demain and AR Allman (CRC Press, 2007). The present disclosure also utilizes conventional techniques in the field of organic chemistry.Basic texts disclosing general methods and terminology of organic chemistry include, 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. Pirmng, John Wiley and Sons Inc. (2007); Organic Chemistry, 9th edition - Francis Carey and Robert Giuliano, McGraw Hill (2013).

[0131] 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 based on gel electrophoresis, sequenced proteins, derived amino acid sequences, or published protein sequences.

[0132] Oligonucleotides that are not commercially available can be chemically synthesized, for example, according to the solid-phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Letts. [Tetrahedron Letters] 22: 1859-1862 (1981) using an automated synthesizer such as Van Devanter et al., Nucleic Acids Res. [Nucleic Acids Research] 12: 6159-6168 (1984). Purification of the oligonucleotides is, for example, by native acrylamide gel electrophoresis or by anion exchange HPLC as described in Pearson & Reanier, J. Chrom. [Chromatography Journal] 255: 137-149 (1983).

[0133] The sequence of cloned genes and synthetic oligonucleotides can be verified after cloning using, for example, the chain termination method of Wallace et al., Gene 16:21-26 (1981) of sequencing double-stranded templates.

[0134] B. XylR mutants with improved xylose utilization and / or improved glucose and xylose co-utilization

[0135] 1. General

[0136] The sequence of wild-type E. coli is provided below as SEQ ID NO: 1. The E. coli XylR protein also has the Uniprot accession number: UniProtKB-P0ACI3

[0137]

[0138] (SEQ ID NO: 1:

[0139] 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). Specifically, 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 orientations of the genome. XylR uses a single dimer attached to two xylose molecules to recruit RNA polymerase to both binding sites, which circularizes the DNA.

[0140] It is also believed that CRP-cAMP is required together with XylR-xylose to activate xylAB and xylFGH transcription. Only when glucose is depleted from the growth medium can cAMP reach high intracellular concentrations. It is believed that the lack of cAMP availability causes a two-stage effect, whereby the presence of glucose effectively inhibits the uptake of xylose (see, for example, Sievert et al. (2017) PNAS [Proceedings of the National Academy of Sciences of the United States of America], July 11, 2017. 114(28): 7349-7354).

[0141] The linear protein sequence has been analyzed and the 3-dimensional structure of the Escherichia coli xylose repressor (XylR) has been determined (see, e.g., Ni et al. (2013) Nuc. Acid. Res. 41(3): 1998-2008).

[0142] 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.

[0143] 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. Because residues 304-323 are directly involved in DNA binding, mutations in this region may 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 properties similar to those of XylR1, as described, for example, in Sievert et al. (2017) supra.

[0144] Without being bound by theory, it is believed that the XylRE382K (XylR1) mutation, which maps to the XylR DNA binding domain, affects protein binding, increasing the affinity of the XylR1 protein for the promoter binding sites upstream of xylAB and xylFGH, thereby increasing the expression of XylAB and XylFGH. This stronger interaction also makes the system less sensitive to the requirement for coactivator CRP-cAMP binding at the promoter site, and is demonstrated by the ability of XylR1 to simultaneously co-utilize glucose and xylose at high rates, as shown in Examples 1 and 2 herein below.

[0145] The xylose-binding domain of the XylR protein comprises residues 221-229. This region dimerizes in an antiparallel fashion and ultimately modulates the structure of the DNA-binding domain to allow DNA binding following interaction with xylose. Therefore, mutations in this region are expected to affect the protein's response to the presence of xylose.

[0146] Other functional regions include those with helical domains, such as the region containing the E382K (XylR1) mutation. Mutations in helical domains can affect protein function. Protein regions with β-strand structures are also functional domains, so mutations in these regions can lead to altered function.

[0147] Certain regions of the XylR protein are located outside of the helical and / or B-strand structural regions and may 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.

[0148] In addition, generally speaking, with respect to amino acid sequences, those skilled in the art 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 number of amino acids in the encoded sequence) are "conservatively modified variants," wherein the alteration results in the replacement of an amino acid with a chemically similar amino acid. Such "conservatively modified variants" may have little to no effect on protein function, particularly if they occur in regions outside of the helical and / or beta-stranded structural regions.

[0149] Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, the polymorphic variants, interspecies homologs, and alleles of the present invention. The following eight groups each contain amino acids that are conservative substitutions for each other: 1) Alanine (A), Glycine (G); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Thomas E. (1992) Proteins: Structures and Molecular Properties).

[0150] Thus, in exemplary embodiments, the present disclosure provides engineered XylR mutant polypeptides with 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 (FAME) and fatty acid ethyl esters (FAEE), fatty alcohol acetates (FACE), fatty amines, fatty aldehydes, fatty alcohols, hydrocarbons, fatty ketones, alkanes, terminal olefins, internal olefins, hydroxy fatty acid derivatives, difunctional fatty acid derivatives, such as fatty diacids, fatty diols, unsaturated fatty acid derivatives, compared to the enzyme having SEQ ID NO: 1.

[0151] 2. Determination of XylR mutants with improved xylose utilization and / or improved glucose and xylose co-utilization

[0152] In an exemplary embodiment, XylR mutants with improved xylose utilization and / or improved co-utilization of glucose and xylose are identified by measuring glucose and xylose utilization as disclosed in Examples 1 and 2 below.

[0153] In some embodiments, XylR mutants with 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 (FFA), fatty acid ethyl esters (FAEE), fatty acid methyl esters (FAME), etc.) produced by a bacterial strain comprising a XylR mutant with improved xylose utilization and / or improved co-utilization of glucose and xylose (i.e., a test strain), and comparing these fatty acid derivatives with the titer of fatty acid derivatives (e.g., FFA, FAEE, FAME, etc.) produced by an appropriate control strain (which is isogenic to the test strain except for the XylR protein it comprises). When the strain is cultured in the presence of xylose, the XylR mutant with improved xylose utilization or improved co-utilization of glucose and xylose will produce more fatty acid derivatives (FFA, FAEE, FAME) than the control strain.

[0154] 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 comprising a specific fatty acid derivative (e.g., a C14 fatty acid derivative) produced by the test strain is measured and compared to the percentage of the total titer of fatty acid derivatives comprising a specific fatty acid derivative (e.g., a C14 fatty acid derivative) produced by a suitable control strain (which is isogenic to the test strain except for the control XylR (e.g., SEQ ID NO: 1) included).

[0155] In an exemplary embodiment, gas chromatography with flame ionization detection (GC-FID) is used to measure fatty acid derivatives. GC-FID is known in the art (see, for example, Adlard, ER; Handley, Alan J. (2001). Gas chromatographic techniques and applications. London: Sheffield Academic. However, any suitable 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.

[0156] C. Methods for Making XylR Mutants with Improved Xylose Utilization and / or Improved Glucose and Xylose Co-Utilization

[0157] Engineered XylR mutants with improved xylose utilization or improved co-utilization of glucose and xylose can be prepared by any method known in the art (see, e.g., Current Protocols in Molecular Biology, supra). Thus, in an exemplary embodiment, mutagenesis is used to prepare polynucleotide sequences encoding XylR mutants / variants with improved xylose utilization or improved co-utilization of glucose and xylose, which can then be screened for improved xylose utilization or improved co-utilization of glucose and xylose. In other exemplary embodiments, polynucleotide sequences encoding XylR mutants / variants with improved xylose utilization or improved co-utilization of glucose and xylose are prepared by chemically synthesizing the polynucleotide sequence, which can then be screened for improved xylose utilization or improved co-utilization of glucose and xylose (see, e.g., MH Caruthers et al. (1987) Methods in Enzymology, Vol. 154, pp. 287-313; Beaucage, SL and Iyer, RP (1992) Tetrahedron 48(12): 2223-2311).

[0158] Mutagenesis methods are well known in the art. Exemplary mutagenesis techniques for preparing engineered XylR mutants with improved xylose utilization or improved glucose and xylose co-utilization include, for example, site-saturation mutagenesis (see, e.g., Chronopoulou EGI, Labrou NE. Curr. Protoc. Protein Sci. February 2011; Chapter 26: Unit 26.6, John Wiley and Sons, Inc; Steffens, DL and Williams., JGK (2007) J Biomol Tech. 18(3): 147-149; Siloto, RMP and Weselake, RJ (2012) Biocatalysis and Agricultural Biotechnology 1(3): 181-189).

[0159] Another exemplary mutagenesis technique for preparing XylR mutants with improved xylose utilization or improved glucose and xylose co-utilization includes transfer PCR (tPCR). See, e.g., Erijman A., et al. (2011) J. Struct. Biol. 175(2): 171-7.

[0160] Other exemplary mutagenesis techniques include, for example, the 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).

[0161] Another exemplary mutagenesis technique for preparing engineered XylR variants with improved xylose utilization or improved glucose and xylose co-utilization involves the use of oligonucleotide-directed mutagenesis (see, e.g., Reidhaar-Olson et al. (1988) Science 241:53-57) to generate site-specific mutations in any cloned DNA of interest.

[0162] The mutagenized polynucleotide generated by any of the synthetic or mutagenesis methods described above is then cloned into a suitable vector or inserted into the host cell genome, and the activity of the affected polypeptide encoded by the mutagenized polynucleotide is evaluated as disclosed above.

[0163] Those skilled in the art will recognize that the schemes and procedures disclosed herein can be modified, and such modifications are variations 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 or sequentially.

[0164] III. Host Cells and Host Cell Cultures

[0165] In view of this 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 introducing one or more nucleic acid sequences encoding the disclosed XylR mutants with improved xylose utilization or improved co-utilization of glucose and xylose. Thus, the recombinant microorganisms disclosed herein serve as host cells and comprise one or more polynucleotide sequences comprising an open reading frame encoding a XylR mutant with 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.

[0166] Exemplary microorganisms providing suitable host cells include, but are not limited to, cells from the genus Escherichia, Bacillus, Lactobacillus, Pseudomonas, Aspergillus, and 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 lichenoformis cell, a Bacillus alkalophilus cell, a Bacillus coagulans cell, a Bacillus circulans cell, a Bacillus pumilis cell, a Bacillus thuringiensis cell, a Bacillus clausii cell, a Bacillus megaterium cell, a Bacillus subtilis cell, or a Bacillus amyloliquefaciens cell.

[0167] In other exemplary embodiments, the host cell is a cell from a cyanobacteria, a green sulfur bacteria, a green non-sulfur bacteria, a purple sulfur bacteria, a purple non-sulfur bacteria, an extreme microorganism, 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 a strain B, strain C, strain K, or strain W Escherichia coli cell.

[0168] In some exemplary embodiments, the host cells include optional genetic manipulations and alterations that can be used interchangeably from one host cell to another, depending on what other heterologous enzymes and what native enzyme pathways are present in the host cell. In one exemplary embodiment, the host cell optionally comprises a deletion of fadE and / or fhuA. In other exemplary embodiments, the host cell is optionally manipulated to have the ability to produce greater than 200 mg / L fatty acid derivatives, greater than 1000 mg / L fatty acid derivatives, greater than 1200 mg / L fatty acid derivatives, greater than 1700 mg / L fatty acid derivatives, greater than 2000 mg / L fatty acid derivatives, or greater than 3000 mg / L fatty acid derivatives.

[0169] As will be discussed in detail herein below, in some exemplary embodiments, the host cell or host microorganism used to express an XylR mutant / variant with improved xylose utilization or improved glucose and xylose co-utilization 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, difunctional fatty acid derivatives, diacids, alkanes, alkenes or chain alkenes, ketones, etc.).

[0170] In an exemplary embodiment, the host cell or host microorganism used to express an XylR mutant with improved xylose utilization or improved glucose and xylose co-utilization further expresses ester synthase activity (EC 2.3.1.75) for producing fatty esters. In another exemplary embodiment, the host cell has acyl-ACP reductase (AAR) (EC 1.2.1.80) activity and / or alcohol dehydrogenase activity (EC 1.1.1.1.) and / or fatty alcohol acyl-CoA reductase (FAR) (EC 1.1.1.*) activity and / or carboxylic acid reductase (CAR) (EC 1.2.99.6) activity for producing fatty alcohols. In another exemplary embodiment, the host cell has acyl-ACP reductase (AAR) (EC 1.2.1.80) activity for producing fatty aldehydes. In another exemplary embodiment, the host cell has acyl-ACP reductase (AAR) (EC 1.2.1.80) activity and decarbonylase or fatty aldehyde oxidative deformylation activity (EC 4.1.99.5) for the production of alkanes and alkenes. In another exemplary embodiment, the host cell has acyl-CoA reductase (EC 1.2.1.50) activity and acyl-CoA synthetase (FadD) (EC 2.3.1.86) activity for the production of fatty alcohols. In another exemplary embodiment, the host cell has ester synthase activity (EC 2.3.1.75) and acyl-CoA synthetase (FadD) (EC 2.3.1.86) activity for the production of fatty esters. In another exemplary embodiment, the host cell has OleA activity for the production of ketones. In another exemplary embodiment, the host cell has OleABCD activity for the production of internal olefins. In another exemplary embodiment, the host cell has acyl-ACP reductase (AAR) (EC 1.2.1.80) activity and alcohol dehydrogenase activity (EC 1.1.1.1) for producing fatty alcohols. In another exemplary embodiment, the host cell has decarboxylase activity for producing terminal olefins. Microorganisms and the expression of enzyme activities in microbial cells are 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.

[0171] In some exemplary embodiments, the host cell or microorganism used to express the XylR mutant with 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, difunctional fatty acid derivatives, diacids, etc.

[0172] In some exemplary embodiments, the recombinant host cell produces fatty esters, such as fatty acid methyl esters (FAME) or fatty acid ethyl esters (FAEE), fatty alcohol acetates (FACE), fatty alcohols (FALC), fatty amines, fatty aldehydes, difunctional fatty acid derivatives, diacids, alkanes, alkenes, and the like.

[0173] Derivative of fatty acid typically reclaims and / or separates from host cell from substratum.In an exemplary embodiment, derivative of fatty acid reclaims (extracellular) from substratum.In another exemplary embodiment, derivative of fatty acid separates (intracellular) from host cell.In another exemplary embodiment, derivative of fatty acid or non-fatty acid compound reclaims and separates from host cell from substratum.

[0174] Can use methods known in the art (for example, gas chromatography (GC-FID) with flame ionization detection) to analyze the fatty acid derivative compositions that host cell produces, to determine the distribution of specific fatty acid derivative and the chain length and the saturation ratio of the component of fatty acid derivative compositions.Similarly, can analyze other compounds by methods well known in the art.

[0175] IV. Methods of Preparing Recombinant Host Cells and Cultures

[0176] Any method known in the art can be used to engineer host cells to express XylR mutants with improved xylose utilization and / or improved glucose and xylose co-utilization to produce, for example, fatty acid derivatives and / or fatty acid derivative compositions or other compounds. Exemplary methods include, for example, the use of vectors, such as expression vectors, comprising polynucleotide sequences encoding XylR mutants with improved xylose utilization and / or improved glucose and xylose co-utilization and / or polynucleotide sequences 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.

[0177] In some exemplary embodiments, a polynucleotide (or gene) sequence encoding a XylR mutant with improved xylose utilization and / or improved glucose and xylose co-utilization is provided to a host cell by a recombinant vector comprising a promoter operably linked to a polynucleotide sequence encoding a XylR mutant with improved xylose utilization and / or improved glucose and xylose co-utilization. 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 adding lactose or isopropylthiogalactoside (IPTG).

[0178] Once a polynucleotide sequence encoding an XylR mutant with improved xylose utilization and / or improved glucose and xylose co-utilization 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 an XylR mutant / variant with improved xylose utilization or improved glucose and xylose co-utilization 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, and propagate the expression construct in a host cell. Techniques for manipulating polynucleotide sequences (e.g., encoding XylR mutants / variants with improved xylose utilization or improved glucose and xylose co-utilization), 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.

[0179] A DNA construct comprising a polynucleotide sequence encoding a XylR mutant / variant with improved xylose utilization or improved glucose and xylose co-utilization (e.g., SEQ ID NO: 4) 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 of choice is an expression vector that can be used to transform bacteria, such as E. coli. The expression vector can be a plasmid, a virus, a cosmid, an artificial chromosome, a nucleic acid fragment, or the like. Such vectors can be readily constructed using recombinant DNA techniques well known to those skilled in the art (see, for example, Sambrook et al., supra). The expression vector comprising the polynucleotide sequence encoding the mutant or engineered XylR variant can then be transfected / transformed into the target host cell. Successfully transformed cells can then be selected based on the presence of an appropriate marker gene by methods well known in the art.

[0180] Many recombinant vectors are available to those skilled in the art for stable transformation / transfection of bacteria and other microorganisms (see, for example, Sambrook et al., supra). Suitable vectors can be readily selected by those skilled in the art. In an exemplary embodiment, a known vector is used to generate an expression construct comprising a polynucleotide sequence encoding a mutant or engineered XylR variant.

[0181] Typically, the conversion vector comprises one or more polynucleotide sequences operably linked to, for example, a promoter sequence and a selectable marker, the polynucleotide sequence encoding an XylR mutant with improved xylose utilization and / or improved glucose and xylose co-utilization. Such conversion vectors typically also include, where appropriate, a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and / or a polyadenylation signal.

[0182] Thus, in addition to the polynucleotide sequence encoding the XylR mutant with improved xylose utilization and / or improved co-utilization of glucose and xylose, the expression construct prepared as disclosed herein may also contain other elements. In an exemplary embodiment, the expression construct comprising the polynucleotide sequence encoding the XylR mutant with improved xylose utilization and / or improved co-utilization of glucose and xylose further comprises an enhancer sequence, thereby enhancing the expression of the heterologous protein. As is known in the art, enhancers are typically found 5' to the start of transcription and can generally be inserted in either the forward or reverse orientation, i.e., 5' or 3' to the coding sequence.

[0183] As mentioned above, conversion / expression vectors typically include selectable and / or screenable marker genes 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.). Exemplary screenable markers include, for example, a six-amino acid histidine tag introduced at the C-terminus of the recombinant protein.

[0184] In an exemplary embodiment, a selectable or screenable marker gene is used as or in conjunction with a specific target gene to provide or enhance the ability to identify transformants. Many selectable marker genes are known in the art (see, for example, Sambrook et al., supra).

[0185] In some exemplary embodiments, the expression vector further comprises a sequence connected to the coding sequence of the heterologous nucleic acid to be expressed, which is removed from the initial translation product after translation. In an exemplary embodiment, the sequence removed after translation helps protein transport into or through the cell or extracellular membrane, thereby helping protein transport into the compartment inside and / or outside the cell. In an exemplary embodiment, the sequence removed after translation protects the nascent protein from intracellular proteolytic degradation. In an exemplary embodiment, a nucleic acid segment is used in the recombinant expression of the coding sequence selected in the host cell, the nucleic acid segment encoding the upstream of the coding sequence and its leader peptide sequence in the reading frame.

[0186] In another exemplary embodiment, the expression construct comprises a bacterial origin of replication, such as the ColE1 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.

[0187] As well known in the art, expression constructs typically comprise restriction endonuclease sites to help vector construction.Exemplary restriction endonuclease recognition sites include but are not limited to the recognition sites of restriction endonucleases NotI, AatII, SacII, PmeI HindIII, PstI, EcoRI and BamHI.

[0188] DNA constructs (polynucleotide sequences encoding mutant or engineered XylR variants operably linked to heterologous DNA sequences, such as promoter sequences, marker sequences; purification moieties; secretion sequences operably coupled to the polynucleotide sequences; targeting sequences, etc.) are used to transform cells and generate recombinant host cells with improved xylose utilization or improved co-utilization of glucose and xylose. Exemplary host cells for transformation with expression constructs comprising polynucleotide sequences encoding XylR mutants with improved xylose utilization and / or improved co-utilization of glucose and xylose are discussed in detail above.

[0189] The skilled artisan can readily select an appropriate transformation technique. Exemplary transformation / transfection methods available to those skilled in the art include, for example, electroporation, calcium chloride transformation, and the like, which are well known to those skilled in the art (see, for example, Sambrook, supra). Thus, a polynucleotide sequence comprising an open reading frame encoding a protein and operably linked regulatory sequences can be integrated into the chromosome of a recombinant host cell, incorporated into one or more plasmid expression systems resident in the recombinant host cell, or both.

[0190] The expression vectors disclosed herein typically include a polynucleotide sequence encoding an XylR mutant having improved xylose utilization and / or improved glucose and xylose co-utilization in a format suitable for expressing the polynucleotide sequence in a host cell. As will be appreciated by those skilled in the art, the design of the expression vector may depend on factors such as, for example, the choice of the host cell to be transformed, the desired expression level of the polypeptide, etc.

[0191] V. Evaluation of Recombinant Host Cells

[0192] In an exemplary embodiment, the activity of XylR mutants with improved xylose utilization and / or improved co-utilization of glucose and xylose is determined by culturing recombinant host cells in the presence of xylose and measuring properties such as improved xylose and glucose utilization as disclosed in Examples 1 and 2 herein below.

[0193] Another assessment method 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 assessment method is to measure the concentration of the desired biochemical product (e.g., fatty acid derivative) synthesized from the carbon source of xylose or a mixture of glucose and xylose by an analytical instrument disclosed herein.

[0194] IV. Products Derived from Recombinant Host Cells

[0195] Strategies to increase xylose utilization or improve the co-utilization of glucose and xylose can be used to utilize different carbon sources to produce fatty acid derivatives by recombinant host cells. Xylose can be used as a carbon source. Figure 17 A schematic diagram of an exemplary metabolic pathway utilizing xylose is shown.

[0196] XylR is a regulatory protein that induces the expression of the XylFGH and XylAB genes, which control the transport and subsequent utilization pathways of xylose, respectively. The gene product of xylB, such as Figure 17 As shown, xylulose-5-phosphate enters the central metabolism of Escherichia coli via the well-described pentose phosphate pathway. Ultimately, acetyl CoA produced by the pentose phosphate pathway enters the fatty acid biosynthesis pathway to produce fatty acid derivatives.

[0197] Thus, in exemplary embodiments, the recombinant host cell is engineered to contain, in addition to a XylR mutant with improved xylose utilization and / or improved glucose and xylose co-utilization, one or more polynucleotide sequences encoding one or more "fatty acid derivative biosynthetic polypeptides" or equivalently, "fatty acid derivative enzymes." Metabolic engineering of fatty acid derivative biosynthetic pathways to produce fatty acid derivative compounds (e.g., fatty acid esters, alkanes, alkenes, fatty ketones, fatty alcohols, fatty alcohol acetates, etc.) using microorganisms to convert biomass-derived sugars into desired products is known in the art, see, for example, U.S. Patent Nos. 9,133,406; 9,340,801; 9,200,299; 9,068,201; 8,999,686; 8,658,404; 8,597,922; 8,535,916; 8,530,221; 8,372,610; 8,323,924; 8,313,934; 8,283,143; 8,268,599; 8,183,028; 8,110,670; 8,110,093; and 8,097,439. Metabolically engineered strains can be cultured in industrial-scale bioreactors, and the resulting products purified using traditional chemical and biochemical engineering techniques.

[0198] Thus, 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 in the presence of a carbon source comprising xylose under conditions effective to express the XylR mutant with improved xylose utilization and / or improved co-utilization of glucose and xylose.

[0199] In some embodiments, all fatty acid derivatives that produce by cultivating recombinant host cell (it comprises the XylR mutant that the glucose and wood sugar that have improvement utilize and / or improve utilize altogether) under the condition that comprises wood sugar are extracellular production.Therefore, in some exemplary embodiments, from substratum, reclaim the fatty acid derivative that produces.In some exemplary embodiments, use any suitable method known in the art (for example GC FID) that the fatty acid derivative composition that reclaims is analyzed, so that determine and quantify the distribution of specific fatty acid derivative and the chain length and the saturation ratio of the component of fatty acid derivative composition.

[0200] Production of fatty acid derivatives

[0201] As described above, recombinant host cells comprising an engineered XylR variant with improved xylose utilization or improved co-utilization of glucose and xylose produce increased amounts of fatty acids compared to a suitable control host cell that does not comprise the engineered XylR variant (e.g., an isogenic control host cell having a control XylR enzyme (e.g., SEQ ID NO: 1)).

[0202] In other exemplary embodiments, discussed in detail below, in addition to engineered XylR variants with improved xylose utilization or improved glucose and xylose co-utilization, the recombinant host cell further comprises additional fatty acid derivative biosynthetic polypeptides that facilitate the production of specific types of fatty acid derivatives.

[0203] Production of fatty aldehydes

[0204] In some exemplary embodiments, the recombinant host cell further comprises carboxylic acid reductase ("CAR") activity in addition to an engineered XylR variant with improved xylose utilization or improved glucose and xylose co-utilization, and thus, the recombinant host cell synthesizes fatty aldehydes and fatty alcohols, see, e.g., U.S. Patent 9,340,801.

[0205] Therefore, in some exemplary embodiments, fatty aldehydes are produced by expressing or overexpressing a polynucleotide encoding a polypeptide having fatty aldehyde biosynthetic activity, such as carboxylic acid reductase (CAR) activity, in a recombinant host cell. Exemplary carboxylic acid reductase (CAR) polypeptides and polynucleotides encoding them include, for example, FadD9 (EC 6.2.1.-, UniProtKBQ50631, GenBank NP_217106), CarA (GenBank ABK75684), CarB (GenBank YP889972), and related polypeptides disclosed in, for example, U.S. Patent No. 8,097,439 and U.S. Patent No. 9,340,801.

[0206] In some exemplary embodiments, the fatty aldehydes produced by the recombinant host cell are then converted to fatty alcohols or hydrocarbons. Thus, in some exemplary embodiments, in addition to the XylR variants having improved xylose utilization or improved glucose and xylose co-utilization, the recombinant host cell further comprises acyl-CoA reductase ("FAR" or "ACR") activity, and thus the recombinant host cell synthesizes 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 No. 2015 / 0361454).

[0207] In some embodiments, fatty aldehydes produced by the recombinant host cell are converted to fatty alcohols through the activity of a native or heterologous fatty alcohol biosynthetic polypeptide, such as an aldehyde reductase or alcohol dehydrogenase (see, e.g., U.S. Patent Application Publication No. 2011 / 0250663). Thus, in some exemplary embodiments, in addition to an XylR variant with improved xylose utilization or improved glucose and xylose co-utilization, 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 fatty alcohols. Exemplary fatty alcohol biosynthesis genes include, but are not limited to, alcohol dehydrogenases, such as AlrA or an AlrA homolog of Acenitobacter sp. M-1; and endogenous E. coli alcohol dehydrogenases, such as DkgA (Np.sub.--417485), DkgB (NP.sub.--414743), YjgB (AAC77226), YdjL (AAC74846), YdjJ (NP.sub.--416288), AdhP (NP.sub.--415995), YhdH (NP.sub.--417719), YahK (NP.sub.--414859), YphC (AAC75598), and YqhD (Q46856).

[0208] Production of fatty amines

[0209] In some exemplary embodiments, a recombinant host cell comprising an engineered XylR variant with improved xylose utilization or improved glucose and xylose co-utilization and which produces fatty aldehydes (e.g., as disclosed herein above) is further modified to comprise a heterologous biosynthetic enzyme with aminotransferase or amine dehydrogenase activity that converts fatty aldehydes to fatty amines (see, e.g., PCT Publication No. WO 2015 / 085271).

[0210] Production of fatty alcohols

[0211] In some exemplary embodiments, in addition to the engineered XylR variant with 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 fatty alcohols are produced by the recombinant host cell. Thus, in exemplary embodiments, a composition comprising medium-chain fatty alcohols (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 (which has improved xylose utilization or improved co-utilization of glucose and xylose) and the fatty alcohol biosynthetic enzyme.

[0212] Thus, in some exemplary embodiments, in addition to an engineered XylR variant with improved xylose utilization or improved glucose and xylose co-utilization, the recombinant host cell further comprises carboxylic acid reductase (CAR) activity and alcohol dehydrogenase activity, and thus, the recombinant host cell synthesizes fatty alcohols, such as octanol (see, e.g., U.S. Patent 9,340,801).

[0213] In some exemplary embodiments, a native fatty aldehyde biosynthetic polypeptide, such as an aldehyde reductase / alcohol dehydrogenase present in the host cell, converts the fatty aldehyde to a fatty alcohol. In other exemplary embodiments, a native fatty aldehyde reductase / alcohol dehydrogenase is overexpressed to convert the fatty aldehyde to a fatty alcohol. In other exemplary embodiments, a heterologous aldehyde reductase / alcohol dehydrogenase is introduced into a recombinant host cell and expressed or overexpressed to convert the fatty aldehyde to a fatty alcohol. Exemplary aldehyde reductase / alcohol dehydrogenase polypeptides that can be used to convert fatty aldehydes to fatty alcohols are disclosed above and in International Patent Application Publication Nos. WO 2007 / 136762; WO 2010 / 062480; U.S. Patent No. 8,110,670; and U.S. Patent No. 9,068,201.

[0214] In some exemplary embodiments, in addition to the engineered XylR variant with 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 1,3 fatty diols when grown in a fermentation broth with a simple carbon source. In other exemplary embodiments, in addition to the engineered XylR variant with 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 1,3 fatty diols when grown in a fermentation broth with a simple carbon source (see, e.g., WO 2016 / 011430).

[0215] Production of fatty alcohol acetate

[0216] In some embodiments, the fatty alcohol produced in the cell, or in some embodiments, supplied to the cell, is further processed by the recombinant cell to provide fatty alcohol acetate (FACE). In exemplary embodiments, an alcohol O-acetyltransferase (EC 2.8.1.14) processes the fatty alcohol into 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. 2014; 9(8): PMCID: PMC4122449.

[0217] An exemplary alcohol O-acetyltransferase is yeast Aftl, e.g., GenBank Accession No. AY242062; GenBank Accession No. AY242063, see e.g., Kevin J. Verstrepen KJ, et al. (2003) Appl Environ Microbiol. 2003 Sep; 69(9): 5228-5237.

[0218] In an exemplary embodiment, the 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 fatty aldehydes and fatty alcohols, and further comprises fatty alcohol O-acetyltransferase activity that converts fatty alcohols to fatty alcohol acetates.

[0219] In another exemplary embodiment, the 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 fatty alcohol O-acetyltransferase activity that converts the first fatty acid derivative into a second fatty acid derivative, wherein the second fatty acid derivative has a higher MIC than the first fatty acid derivative.

[0220] In another exemplary embodiment, the 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 fatty alcohol O-acetyltransferase activity that converts the first fatty acid derivative into a second fatty acid derivative, wherein the second fatty acid derivative has a higher LogP than the first fatty acid derivative.

[0221] In another exemplary embodiment, the 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 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.

[0222] In another exemplary embodiment, the 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 fatty alcohol O-acetyltransferase activity that converts the first fatty acid derivative into a second fatty acid derivative, wherein the second fatty acid derivative is less toxic than the first fatty acid derivative.

[0223] Production of fatty esters

[0224] In some embodiments, in addition to the engineered XylR variant with improved xylose utilization or improved glucose and xylose co-utilization, the recombinant host cell further comprises a polynucleotide encoding a polypeptide having fatty ester biosynthetic activity, and thus fatty esters are produced by the recombinant host cell.

[0225] As used herein, the term "fatty ester" or equivalently "fatty acid ester" refers to any ester made from a fatty acid. In an exemplary embodiment, the 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 where 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 an alkyl thioester.

[0226] Any alcohol can be used to form the A side of the fatty ester. In an exemplary embodiment, 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, for example, the alcohol is provided exogenously, for example, the alcohol is supplied in the fermentation broth.

[0227] The carbon chain comprising the A side or the B side can be of any length. In an exemplary embodiment, the fatty ester is a fatty acid methyl ester, wherein the B side is provided by the fatty acid biosynthetic pathway and the A side of the ester is 1 carbon in length. In an exemplary embodiment, the A side is provided by 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).

[0228] In another exemplary embodiment, the fatty ester is a fatty acid ethyl ester, wherein the B side is provided by a fatty acid biosynthetic pathway and the A side of the ester is 2 carbons in length.

[0229] In one exemplary embodiment, side A is linear. In another exemplary embodiment, side A is branched. In one exemplary embodiment, side B is linear. In another exemplary embodiment, side B is branched. The branches may have one or more branch points. In one exemplary embodiment, side A is saturated. In another exemplary embodiment, side A is unsaturated. In one exemplary embodiment, side B is saturated. In another exemplary embodiment, side B is unsaturated.

[0230] In an exemplary embodiment, 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, for example, International Patent Application Publication No. WO 2011 / 038134.

[0231] In some exemplary embodiments, fatty acid esters are produced by recombinant host cells comprising an engineered XylR variant with 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., Intl. Pat. Appl. Publ. No. WO / 2011 / 038134; Intl. Pat. Appl. Publ. No. WO 2007 / 136762; U.S. Pat. No. 8,110,670).

[0232] In an exemplary embodiment, the recombinant host cell comprising an engineered XylR variant with improved xylose utilization or improved glucose and xylose co-utilization further comprises ester synthase activity (EC 3.1.1.67) sufficient to produce fatty esters (e.g., FAME or FAEE, see, e.g., U.S. Patent 9,879,239).

[0233] Hydrocarbon production

[0234] In some embodiments, in addition to the engineered XylR variant with improved xylose utilization or improved glucose and xylose co-utilization, the recombinant host cell further comprises a polynucleotide encoding a polypeptide having fatty aldehyde biosynthesis activity (e.g., an acyl-ACP reductase polypeptide (EC 6.4.1.2)) and a polynucleotide encoding a polypeptide having hydrocarbon biosynthesis activity (e.g., a decarbonylase (EC 4.1.99.5), an oxidative deformylase, 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, the recombinant host cell comprising the engineered XylR variant with improved xylose utilization or improved glucose and xylose co-utilization produces hydrocarbons, e.g., alkanes or alkenes (e.g., terminal alkenes or internal alkenes), or ketones.

[0235] In some exemplary embodiments, fatty aldehydes produced by recombinant host cells comprising an engineered XylR variant with improved xylose utilization or improved glucose and xylose co-utilization are converted by decarbonylation, removing a carbon atom, to form hydrocarbons (see, e.g., U.S. Patent 8,110,670 and WO 2009 / 140695).

[0236] In other exemplary embodiments, fatty acids produced by the recombinant host cells are converted by decarboxylation to remove carbon atoms to form terminal olefins. Thus, in some exemplary embodiments, in addition to expressing an engineered XylR variant with improved xylose utilization or improved glucose and xylose co-utilization, the recombinant cell further expresses or overexpresses a polynucleotide encoding a hydrocarbon biosynthesis polypeptide (e.g., a polypeptide with decarboxylase activity, as disclosed, for example, in U.S. Patent No. 8,597,922).

[0237] In other exemplary embodiments, the alkylthioester intermediate is converted by enzymatic decarboxylation condensation to form an internal olefin or ketone. Thus, in some exemplary embodiments, in addition to expressing an engineered XylR variant with improved xylose utilization or improved glucose and xylose co-utilization, the recombinant cell further expresses or overexpresses a polynucleotide encoding a hydrocarbon biosynthesis polypeptide (e.g., such as a polypeptide with OleA activity), thereby producing ketones (see, e.g., U.S. Patent No. 9,200,299). In other exemplary embodiments, in addition to expressing an engineered XylR variant with improved xylose utilization or improved glucose and xylose co-utilization, the recombinant cell further expresses or overexpresses a polynucleotide encoding a hydrocarbon biosynthesis polypeptide (e.g., such as OleCD or OleBCD) together with a polypeptide with OleA activity, thereby producing internal olefins (see, e.g., U.S. Patent No. 9,200,299).

[0238] Some exemplary hydrocarbon biosynthesis polypeptides are shown in Table 2 below.

[0239] Table 2. Exemplary hydrocarbon biosynthetic polynucleotides and polypeptides.

[0240]

[0241] Production of Omega (ω)-hydroxylated fatty acid derivatives

[0242] In some embodiments, in addition to the engineered XylR variants with improved xylose utilization or improved glucose and xylose co-utilization, the recombinant host cell further comprises a polynucleotide encoding a polypeptide having ω-hydroxylase activity (EC 1.14.15.3). In an exemplary embodiment, the modified ω-hydroxylase has the enzymatic activity of a modified cytochrome P450 monooxygenase (P450) and effectively catalyzes the hydroxylation of the ω-position of the hydrocarbon chain in vivo. 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 (ω-hydroxylated) fatty acid derivatives in vivo (see, e.g., PCT Application Publication WO 2014 / 201474).

[0243] In other exemplary embodiments, the recombinant host cell further comprises a polynucleotide encoding an alkane hydroxylase (e.g., alkA, CYP153A-reductase, or a CYP153A-reductase hybrid fusion polypeptide variant (see, e.g., WO 2015 / 195697)) in addition to the engineered XylR variant with improved xylose utilization or improved co-utilization of glucose and xylose, such that when cultured in a culture medium containing a carbon source under conditions effective to express the alkane hydroxylase (e.g., AlkA, CYP153, or a CYP153A-reductase hybrid fusion polypeptide variant) and the engineered XylR variant with improved xylose utilization or improved co-utilization of glucose and xylose, the recombinant host cell produces omega-hydroxylated (ω-hydroxylated) and bifunctional fatty acid derivatives and compositions thereof (including ω-hydroxylated fatty acids, ω-hydroxylated fatty acid esters, α,ω-diacids, α,ω-diesters, α,ω-diols, and chemicals derived therefrom, such as macrolactones and macroketones).

[0244] V. Cultivation and Fermentation of Recombinant Host Cells

[0245] As used herein, fermentation refers broadly to the conversion of organic material into a target substance by a recombinant host cell. For example, this includes converting the carbon source into a fatty acid derivative, such as, for example, a fatty acid, a fatty acid ester, a fatty alcohol, a fatty alcohol acetate, etc., by breeding a culture of the recombinant host cell in a culture medium comprising a carbon source. The conditions allowing 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 a desired product, such as a fatty acid derivative composition. Suitable conditions include, for example, typical fermentation conditions, see, for example, Principles of Fermentation Technology [principles of fermentation technology], 3rd edition (2016), ibid; Fermentation Microbiology and Biotechnology [fermentation microbiology and biotechnology], 2nd edition, (2007), ibid.

[0246] In some embodiments, the host cell of the present invention can be grown in a fermentation environment. Fermentation conditions can include many parameters well known in the art, including but not limited to temperature range, pH level, aeration rate, feed rate and culture medium composition. Each of these conditions allows host cell growth individually or in combination. Fermentation can be aerobic, anaerobic or its variation (such as microaerobic). Exemplary culture medium includes liquid culture medium (liquid) or gel (solid). Usually, culture medium includes the carbon source (such as, derived from the simple carbon source of renewable raw materials) that can be directly metabolized by host cell. In addition, enzyme can be used in culture medium to contribute to the mobilization of carbon source (such as, starch or cellulose depolymerization is fermentable sugar) and subsequent metabolism to produce fatty acid derivatives.

[0247] For small-scale production, host cells engineered to produce fatty acid derivative compositions can be grown, for example, in batches of about 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 1 mL, 5 mL, 10 mL, 15 mL, 25 mL, 50 mL, 75 mL, 100 mL, 500 mL, 1 L, 2 L, 5 L, or 10 L; fermented; and induced to express a desired polynucleotide sequence, for example, a polynucleotide encoding a polypeptide having a specific enzymatic activity, such as a thioesterase (TE), an ester synthase (ES), a carboxylic acid reductase (CAR), an alcohol dehydrogenase (ADH), a fatty acyl-CoA / ACP reductase (FAR), an acyl-CoA reductase (ACR), an acetyl-CoA carboxylase (ACC), and / or an acyl-ACP / CoA reductase (AAR) enzymatic activity. For large-scale production, the engineered host cells can be grown in culture in volumes of about 10 L, 100 L, 1000 L, 10,000 L, 100,000 L, 1,000,000 L, or greater; fermented, and induced to express any desired polynucleotide sequence.

[0248] Fatty acid derivative compositions disclosed herein can be found in the extracellular environment of recombinant host cell culture usually, and can be easily separated from substratum.Fatty acid derivatives (such as fatty acid, fatty acid ester, fatty aldehyde, fatty ketone, fatty alcohol, fatty alcohol acetate etc.) can be secreted by recombinant host cell, transported to the extracellular environment or passively transferred to the extracellular environment of recombinant host cell culture.Can use conventional methods known in the art, including but not limited to centrifugal, separate fatty acid derivative compositions from recombinant host cell culture.

[0249] In some embodiments, the present invention provides the fatty acid derivatives of the present invention.For example, the fatty acid derivatives of the present invention can be produced by the host cell of the present invention.For example, the fatty acid derivatives of the present invention can be produced by the host cell of the present invention.For example, the fatty acid derivatives of the present invention can be produced by the host cell of the present invention.For example, the fatty acid derivatives of the present invention can be produced by the host cell of the present invention.For example, the fatty acid derivatives of the present invention can be produced by the host cell of the present invention.For example, the fatty acid derivatives of the present invention can be produced by the host cell of the present invention.For example, the fatty acid derivatives of the present invention can be produced by the host cell of the present invention.For example, the fatty acid derivatives of the present invention can be produced by the host cell of the present invention.For example, the fatty acid derivatives of the present invention can be produced by the host cell of the present invention.For example, the fatty acid derivatives of the present invention can be produced by the host cell of the present invention.

[0250] In some exemplary embodiments, host cells are produced and cultured in a culture medium (e.g., fermentation medium) comprising an initial concentration of a carbon source (simple carbon source) of about 20g / L to about 900g / L. In other embodiments, the culture medium comprises an initial concentration of a carbon source of about 2g / L to about 10g / L; about 10g / L to about 20g / L; about 20g / L to about 30g / L; about 30g / L to about 40g / L; or about 40g / L to about 50g / L. In some embodiments, the level of available carbon source in the culture medium can be monitored during fermentation. In some embodiments, the method further comprises adding a supplemental carbon source to the culture medium when the level of the initial carbon source in the culture medium is less than about 0.5g / L.

[0251] In some exemplary embodiments, when the level of carbon source in culture medium is less than about 0.4g / L, less than about 0.3g / L, less than about 0.2g / L or less than about 0.1g / L, in culture medium, add supplementary carbon source.In some embodiments, add supplementary carbon source to maintain the carbon source level of about 1g / L to about 25g / L.In some embodiments, add supplementary carbon source to maintain the carbon source level of about 2g / L or higher (for example, about 2g / L or higher, about 3g / L or higher, about 4g / L or higher).In certain embodiments, add supplementary carbon source to maintain the carbon source level of about 5g / L or lower (for example, about 5g / L or lower, about 4g / L or lower, about 3g / L or lower).In some embodiments, add supplementary carbon source to maintain the carbon source level of about 2g / L to about 5g / L, about 5g / L to about 10g / L or about 10g / L to about 25g / L.

[0252] In one exemplary embodiment, the carbon source for fermentation is derived from a renewable feedstock. In some embodiments, the carbon source is glucose. In other embodiments, the carbon source is glycerol. Other possible carbon sources include, but are not limited to, fructose, mannose, galactose, xylose, arabinose, starch, cellulose, hemicellulose, pectin, xylan, sucrose, maltose, cellobiose, turanose, acetic acid, ethane, ethanol, methane, methanol, formic acid, and carbon monoxide; cellulosic materials and their variants, such as hemicellulose, methylcellulose, and sodium carboxymethylcellulose; saturated or unsaturated fatty acids, succinates, lactates, and acetates; alcohols, such as ethanol, methanol, and glycerol, or mixtures thereof. In one embodiment, the carbon source is derived from corn, sugarcane, sorghum, sugar beets, switchgrass, silage, straw, wood, pulp, sewage, garbage, cellulosic municipal waste, flue gas, synthesis gas, or carbon dioxide. Simple carbon sources can also be products of photosynthesis, such as glucose or sucrose. In one embodiment, the carbon source is derived from waste, such as glycerol, flue gas, or synthesis gas; or from the reforming of organic materials such as biomass; or from natural gas or methane, or from the reforming of these materials into synthesis gas; or from carbon dioxide fixed by photosynthesis, for example, fatty acid derivatives can be produced by recombinant cyanobacteria or algae grown by photosynthesis and using CO2 as a carbon source.

[0253] In some embodiments, the carbon source is a cellulose hydrolyzate derived from biomass. Cellulose hydrolyzates 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, sugarcane, or switchgrass. Another exemplary source of biomass is metabolic waste, such as animal matter (e.g., cow dung). 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, straw, wood, sewage, garbage, cellulosic municipal waste, municipal solid waste, and leftover food.

[0254] In some exemplary embodiments, fatty acid derivatives, for example, fatty acids, fatty acid esters, fatty alcohols, etc., are produced at a concentration of about 0.5 g / L to about 40 g / L. In some embodiments, fatty acid derivatives are produced at a concentration of about 1 g / L or higher (e.g., about 1 g / L or higher, about 10 g / L or higher, about 20 g / L or higher, about 50 g / L or higher, about 100 g / L or higher). In some embodiments, fatty acid derivatives are produced at a concentration of about 1 g / L to about 170 g / L, about 1 g / L to about 10 g / L, about 40 g / L to about 170 g / L, about 100 g / L to about 170 g / L, about 10 g / L to about 100 g / L, about 1 g / L to about 40 g / L, about 40 g / L to about 100 g / L, or about 1 g / L to about 100 g / L.

[0255] In other exemplary embodiments, the dosage of the pharmaceutical composition is 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 60 0 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 In some embodiments, the fatty acid derivatives, e.g., fatty acids, fatty acid esters, fatty alcohols, etc., are produced at a titer of about 1775 mg / L, about 1800 mg / L, about 1825 mg / L, about 1850 mg / L, about 1875 mg / L, about 1900 mg / L, about 1925 mg / L, about 1950 mg / L, about 1975 mg / L, about 2000 mg / L (2 g / L), 3 g / L, 5 g / L, 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, 100 g / L, or a range bounded by any two of the foregoing values. In other embodiments, the fatty acid derivatives or other compounds are produced at a titer of greater than 100 g / L, greater than 200 g / L, or greater than 300 g / L.In an exemplary embodiment, the titer of the fatty acid derivative or other compound produced by the recombinant host cell according to methods disclosed herein is 5g / L to 200g / L, 10g / L to 150g / L, 20g / L to 120g / L and 30g / L to 100g / L. Titre can refer to the specific fatty acid derivative or the combination of fatty acid derivative or another compound or the combination of other compounds produced by a given recombinant host cell culture. In an exemplary embodiment, the expression of the engineered XylR variant in recombinant host cells, for example, in escherichia coli, causes production of a higher titer compared with the recombinant host cells expressing the corresponding wild-type polypeptide. In one embodiment, the higher titer range is at least about 5g / L to about 200g / L.

[0256] In other exemplary embodiments, host cells engineered according to the methods of the present disclosure to produce fatty acid derivatives (e.g., fatty acids, fatty acid esters, fatty alcohols, etc.) 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, produce a kind of fatty acid derivative or multiple fatty acid derivative or one or more other compounds with 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% productive rate.Alternately or additionally, productive rate is about 30% or lower, about 27% or lower, about 25% or lower or about 22% or lower.In another embodiment, productive rate is about 50% or lower, about 45% or lower or about 35% or lower.In another embodiment, productive rate is about 95% or lower, or 90% or lower, or 85% or lower, or 80% or lower, or 75% or lower, or 70% or lower, or 65% or lower, or 60% or lower, or 55% or lower, or 50% or lower. Therefore, productive rate can be any two of the above two endpoints as boundaries.For example, the productive rate of the fatty acid derivative produced by recombinant host cell according to method disclosed herein can be about 5% to about 15%, about 10% to about 25%, about 10% to about 22%, about 15% to about 27%. Productive rate can refer to the combination of specific fatty acid derivative or fatty acid derivative. In addition, productive rate will also depend on employed raw materials.

[0257] In some exemplary embodiments, the productivity of a host cell engineered according to the methods of the present disclosure to produce fatty acid derivatives (e.g., fatty acids, fatty acid esters (e.g., FAME, FAEE), fatty alcohols, etc.) is at least 100 mg / L / hour, at least 200 mg / L / hour, at least 300 mg / L / hour, at least 400 mg / L / hour, at least 500 mg / L / hour, at least 600 mg / L / hour, at least 700 mg / L / hour, at least 800 mg / L / hour, at least 900 mg / L / hour, at least 1000 mg / L / hour, at least 1100 mg / L / hour, or at least 200 mg / L / hour. In some embodiments, the productivity of the malonyl-CoA derived compound (including one or more fatty acid derivatives or one or more other compounds) produced by the recombinant host cell according to the method of the present disclosure can be from 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 specific 14- 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, such as E. coli, results in increased productivity of 14- and / or 16-carbon fatty acid derivatives or other compounds compared to a recombinant host cell expressing the corresponding wild-type polypeptide. In an exemplary embodiment, 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.

[0258] VI. Separation

[0259] Bioproducts (e.g., compositions comprising the fatty acid derivatives of the recombinant host cell disclosed herein) are typically separated from fermentation broth by methods known in the art. In an exemplary embodiment, compositions (comprising the fatty acid derivatives of the recombinant host cell disclosed herein) are separated from fermentation broth by gravity sedimentation, centrifugation, or decantation.

[0260] VII. Compositions and Formulations of Fatty Acid Derivatives

[0261] As described above, bioproducts (e.g., compositions comprising fatty acids and fatty acid derivatives produced using recombinant host cells as described above) are produced from renewable sources (e.g., simple carbon sources derived from renewable raw materials) and are therefore novel compositions of matter. 14 C dating can distinguish these novel bioproducts from organic compounds derived from petrochemical carbon. Additionally, the specific origin of biogenic carbon (glucose versus glycerol) can be determined by dual carbon isotope fingerprinting using methods known in the art (see, e.g., U.S. Patent No. 7,169,588, WO 2016 / 011430 A1, etc.).

[0262] The following examples are offered to illustrate, but not to limit, the present invention.

[0263] Example

[0264] The following specific examples are intended to illustrate the present disclosure and should not be construed as limiting the scope of the claims.

[0265] Example 1

[0266] This example demonstrates that a single amino acid substitution mutation in E. coli XylR (XylR E382K) increases xylose utilization and increases xylose co-utilization compared to a wild-type xylR control.

[0267] Strains IC.200 (IC.187XylR wild-type pSven.037; control) and sven.938 (IC.187XylR1pSven.037; XylR1) were initially expanded in LB medium and then grown overnight at 32°C in shake flasks containing basal salts medium (2 g / L NH4Cl, 0.5 g / L NaCl, 0.3 g / L KH2PO4, 1 mM MgSO4, 0.1 mM CaCl2, 20 g / L glucose, 1 mL / L trace metal 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 basal salts medium (0.5 g / L (NH4)2SO4, 2 g / L The sample consisted of KH2PO4, 80 mg / L ferric citrate, 1 mL / L trace metal solution, 1 g / L NaCl, 140 mg / L CaCl2-H2O, 10 mg / L ZnCl2, 2.2 g / L MgSO4-7H2O, 0.25 mL / L trace vitamin solution, 5 g / L each of glucose and xylose, 1 mL / L spectinomycin, and 25 mL / L methanol. The trace metal solution consisted of 0.5 g / L H3BO3, 1.9 g / L CuSO4-5H2O, 1 g / L ZnCl, Na2MoO4-2H2O, CaCl2-2H2O, and 2 mL / L concentrated hydrochloric acid. The trace vitamin solution consisted of 0.06 g / L riboflavin, 6 g / L niacin, 5.4 g / L pantothenic acid, 1.4 g / L pyridoxine, 0.06 g / L biotin, and 0.01 g / L folic acid.

[0268] The bioreactor was operated with the following operating parameters: pH = 7.2, temperature = 30.5°C, air flow = 0.5 v / v / m, and dissolved oxygen at 30% saturation.

[0269] A sugar feed (to a total initial volume of 50 g / L) consisting of a 50:50 mixture of glucose and xylose (total sugar concentration in the feed of 610 g / L) was added to the bioreactor on demand via a dissolved oxygen-triggered controller. Each of these feeds was added automatically in response to a slowdown in the metabolic activity of the culture, which was reflected in a corresponding increase in the residual dissolved oxygen concentration due to a decrease in sugar concentration in the bioreactor. Once the residual dissolved oxygen concentration value rose to a predetermined offset above the dissolved oxygen set point, the feed controller would trigger the next sugar addition to the bioreactor.

[0270] Two of these feeds were followed with additional bioreactor samples taken approximately 26 and 50 hours after inoculation of the bioreactor (indicated by black dots).

[0271] Cultures are typically induced around 10 hours into fermentation, so the 26-hour time point was chosen because this is when the culture is fully induced and producing FAMEs at maximum rate in young, healthy cells. The cells are using sugars at near peak capacity. The 50-hour time point was chosen because this is when 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 fatigued, and utilization begins to decline normally (in the case of WT XylR, but not in the case of XylR1).

[0272] The duration of the feed is shown in the rectangular box ( Figure 1 ), the end of feed addition is zero on the x-axis. High performance liquid chromatography (HPLC) was used to measure the residual glucose (solid line) and xylose (dashed line) concentrations in the sample supernatant at the beginning and end of each of these feeds. HPLC was also used to measure the residual glucose and xylose over a period of time thereafter until the next feed was started to quantitatively determine the glucose and xylose consumption levels of the culture.

[0273] The calculated glucose and xylose utilization for the two strains at the two time points tested (26 and 50 hours EFT) are listed in the table below. The residual glucose at the start of all feeds was zero.

[0274]

[0275] like Figure 1 As shown in the table above, the control strain effectively utilizes glucose, but does not utilize wood sugar. At 26 hours when the feeding started, the residual wood sugar concentration in the control strain was very high (13.7g / L), but residual glucose was zero. This shows that although each feeding glucose is fully utilized, wood sugar is not like this, so wood sugar accumulates in bioreactor over time. After the feeding, about 1.5 hours further sampling showed that the wood sugar level reduced, but when the residual glucose level returned to zero, the wood sugar level still kept raising, increased to about 17.5g / L. At 50 hours, the residual glucose level was zero again, but the wood sugar level further increased (54.6g / L), showing that the control strain did not effectively utilize wood sugar. After the feeding, about 1.5 hours further sampling showed that the wood sugar level did not reduce, and the residual glucose concentration returned to zero.

[0276] In contrast, in the strain expressing the XylR1 mutant (E382K), at the start of the feed at 26 hours, the residual xylose concentration in the control strain was very low (0.5 g / L), and the residual glucose was zero. Further sampling approximately 1.5 hours after the feed showed that both xylose and glucose had dropped to almost zero. At 50 hours, the residual glucose concentration was again zero, and the residual xylose concentration was essentially the same. Further sampling approximately 1.5 hours after the feed showed that the xylose level had dropped from a high of approximately 6.5 g / L to a level of approximately 4 g / L. Thus, even in the presence of glucose, the XylR1 mutant exhibited increased xylose utilization.

[0277] Example 2

[0278] This example demonstrates that cells expressing the XylR1 mutant show improved growth in the presence of xylose and that the improved growth is due to improved xylose utilization.

[0279] IC.187 and sven.903 (IC.187 XylR1)

[0280] 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 lack the plasmid, so no FAMEs are produced in this case.

[0281] IC.187 and sven.903 are grown at high throughput. Cells are grown as seed cultures in a seed basal medium containing 10 g / L glycerol or 10 g / L glucose at 32°C with 250 RPM (revolutions per minute) shaking overnight. Twenty percent of the inoculum is then added to the basal medium containing 10 g / L xylose and cultured at 32°C with 250 RPM shaking. Growth is then measured at 8 hours by OD600 readings. Seed basal medium = 1X trace vitamins, 0.001 mg / mL thiamine, 0.1 mM CaCl2, 0.01 g / L ferric citrate, 1 mM MgSO4, 1X 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. Basal medium = same as seed 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.

[0282] from Figure 2As can be seen in the Figure 3, the strain expressing wild-type xylR (IC.187) grew slower (whether started from glucose or glycerol seeds) than the strain expressing the xylR1(E382K) mutant. Thus, cells expressing the xylR1 mutant showed improved growth in the presence of xylose regardless of whether they were switched from glucose or glycerol seed cultures.

[0283] From previous experiments, it was known that after seed cultures have completed growth, glucose or glycerol is completely consumed. Glucose represses the xylose operon through carbon catabolite repression, while glycerol is a neutral sugar that does not repress the xylose operon. Therefore, whether switching to a sugar that exhibits carbon catabolite repression or a neutral sugar, cells grew faster on xylose when the xylR1 mutant was present, indicating that xylose utilization was higher in both cases.

[0284] Example 3

[0285] This example illustrates the construction and testing of new XylR mutants with improved xylose utilization and / or improved co-utilization of glucose and xylose.

[0286] The wild-type XylR nucleic acid (SEQ ID NO: 2) was cloned into the template plasmid pSven.178. Mutants were generated using transfer PCR (tPCR) and subsequently tested for their ability to allow growth on xylose. Transfer PCR was performed using methods known in the art.

[0287] Briefly, a template plasmid (pSven.178 = p15A-lacI-PxylR-xylR(WT)-KanR) was constructed. The plasmid contains upstream and downstream homology to the xylR genomic sequence, which is necessary for genomic integration of the resulting XylR mutant into E. coli. Transfer PCR was performed using the template plasmid and a combination of forward (5'-3') and reverse primers containing the desired mutation. PCR was then used to amplify the tPCR template using external primers (which amplify sequences containing regions of diversity and homology).

[0288] The amplified mutant was cleaved with a restriction enzyme (Dpn1) to release the mutagenized XylR gene and the homology region for integration. The linear DNA product containing the XylR mutation and the homology region was integrated into the xylR locus of Escherichia coli strain sven.999 (see, e.g., Datsenko and Wanner, 2000, PNAS [Proceedings of the National Academy of Sciences of the United States of America], 97(12), 6640-6645).

[0289] The sven.999 strain contains a deletion of the native XylR gene and is unable to grow on minimal medium containing xylose as the sole carbon source. Therefore, only bacteria that have integrated a XylR mutant that confers the ability to utilize xylose can grow on minimal plates.

[0290] Once colonies containing the xylR mutant in the ΔxylR locus are obtained, serial passaging methods can be used to identify xylR variants that can support growth on minimal medium containing xylose.

[0291] Colonies from the serial passage method were isolated and screened for growth comparison with control strains containing WT xylR protein and xylR2(R121C).

[0292] Table 3 and Figure 3-8 It illustrates how the growth of the xylR mutants compared to WT xylR in minimal medium containing xylose.

[0293] In order to measure the growth with wood sugar, for the growth screening control strain KTT.560 (WT XylR), sCR.002 (XylR2 R121C) and sven.996 (Δ xylR) and various xylR mutant strains in the basal medium with wood sugar. Bacterial colony was initially grown 4-6 hours on LB substratum, then transferred to the basal medium containing limited phosphate and other nutrients and glycerol as a carbon source. Culture was grown overnight, and used for subculture to contain the fresh phosphate restricted basal medium of wood sugar as a carbon source. Regularly at 600nm wavelength (OD600), the optical density (OD) of culture was measured, to measure the growth of bacterial strain relative to control. All data shown are the mean values of 3 repetitions of each bacterial strain.

[0294] Table 3: Point mutations in the XylR protein, nucleotide codon changes, and ranking of mutants grown on xylose compared to controls

[0295]

[0296] Table 3 Notes: μ and FOIC ranking criteria. + Up to 50% increase; ++ 50% to 100% increase; +++ More than 100% increase; - Up to 25% decrease; -- 25% to 50% decrease; -- More than 50% decrease. μ is the cell growth rate, defined in reciprocal units of time. It is a measure of the change in cell number per unit time.

[0297] All comparisons were made relative to the control strain KTT.560 with WT XylR. Controls are KTT.560 = WTxylR; sCR.002 = xylR2; sven.996 = ΔxylR; sDH.687 = KTT.560pDH.138; sven.954 = KTT.560xylR2 pDH.138; sVen.1053 = KTT.560 ΔxylR pDH.138.

[0298] FOIC = multiple compared to the internal control. Internal control = WTXylR (KTT.560). The OD600 of the mutant strains during mid-log phase is expressed as a multiple higher than the control strain KTT.560. All final production strains contained the production plasmid pDH.138, which contains the Marinobacter hydrocarbonoclasticus ester synthase. Plasmid pDH.138 is a production plasmid containing the SC101 origin of replication, spectinomycin resistance, IPTG-inducible PTrc promoter, and ester synthase variants. The genotypes of the plasmids are described below:

[0299] ori_SC101, repA, par, aadA1-terminator (B1004), lacIq-terminator (T22), Ptrc_linker D_IGR19-ES50_term (rrnB_T1T2)

[0300] The selected XylR mutants were further tested for growth on xylose basal medium and their ability to produce various fatty acid species (FAS) was determined. Growth was monitored by regularly measuring OD 600 as described above. FAS production was measured using standard methods known in the art.

[0301] Table 4 and Figure 9-16 Xylose utilization and productivity of various XylR mutants in terms of FAS production compared to WT xylR grown in minimal medium containing xylose are illustrated.

[0302] Table 4: Performance indicators of xylR mutants compared with WT xylR, and ranking compared with the control

[0303] strain mutation Xylose consumption ranking FAS generates rankings 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 ++ ++

[0304] Table 4 Notes: Ranking of xylose consumption and FAS production. + Up to 10% increase; ++ 10% to 20% increase; +++ More than 20% increase; - 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.

[0305] Example 4

[0306] This example illustrates that a mutation at position 121 of SEQ ID NO: 1, when expressed in a recombinant host cell, confers upon the host cell the ability to consume xylose faster than an isogenic host cell that is otherwise identical except for the expression of SEQ ID NO: 1.

[0307] Specific substitutions at position 121 relative to the wild-type sequence and their effect on xylose consumption are listed in Table 5. International Application No. PCT / US2014 / 027337 is incorporated herein by reference.

[0308] Table 5: Amino acid substitutions that relieve catabolite repression

[0309]

[0310] Appendix A

[0311] 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)

[0312]

[0313] SEQ ID NO: 2XylR WT DNA sequence (Genbank #NC_000913; Escherichia coli K-12 MG1655; Blattner and Plunkett, 1997; NCBI Protein ID: NP_418026)

[0314]

[0315] SEQ ID NO: 3 (E382K) protein sequence:

[0316]

[0317] SEQ ID NO: 4XylR1(E382K) DNA sequence:

[0318]

[0319] It will be apparent to those skilled in the art that various modifications and variations can be made to the above-described aspects and embodiments 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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