Methods of producing lipid-derived compounds and host cells thereof

Abstract

The present disclosure relates to genetically engineered host cells and methods of producing a lipid-derived compound by employing such host cells. In particular embodiments, the host cell includes a first mutant gene encoding a cytoplasmic tRNA thiolation protein. Optionally, the host cell can include other mutant genes for decreasing fatty alcohol catabolism, decreasing re-importation of secreted fatty alcohol, or displaying other useful characteristics, as described herein.

Claims

1. An isolated, genetically engineered host cell comprising: a first mutant gene that is a deletion of a gene encoding a cytoplasmic tRNA thiolation protein; and one or more expressed nucleic acids encoding 1) a fatty acyl-CoA reductase, or 2) a thioesterase, a carboxylicacid reductase and an aldehyde reductase.

2. The host cell of claim 1, wherein the cytoplasmic tRNA thiolation protein is cytoplasmic tRNA 2-thiolation protein 2.

3. The host cell of claim 1 wherein the cytoplasmic tRNA thiolation protein comprises a polypeptide sequence having at least 90% sequence identity to any one of the following SEQ ID NOs: 1, 3-8, 10-16 and 18-20.

4. The host cell of claim 1, wherein the engineered host cell further comprises a second mutant gene comprising insertion of a nucleic acid encoding an acetyl-CoA carboxylase, thereby providing overexpression of the acetyl-CoA carboxylase.

5. The host cell of claim 4, wherein the acetyl-CoA carboxylase comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:30.

6. The host cell of claim 1, wherein the engineered host cell further comprises a second mutant gene comprising deletion of a nucleic acid encoding a lysophospholipid acyltransferase, a fatty alcohol oxidase, an aldehyde dehydrogenase, an isocitrate dehydrogenase, or a pyruvate decarboxylase.

7. The host cell of claim 6, wherein the lysophospholipid acyltransferase comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 31; the fatty alcohol oxidase comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 32; the aldehyde dehydrogenase comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 33 or SEQ ID NO: 36; the isocitrate dehydrogenase comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 34; or the pyruvate decarboxylase comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 35.

8. A method of producing a fatty alcohol, the method comprising: incubating an isolated, genetically engineered host cell in a culture; and isolating one or more fatty alcohols from the culture, wherein the genetically engineered host cell comprises a first mutant gene that is a deletion of a gene encoding a cytoplasmic tRNA thiolation protein, and further one or more expressed nucleic acids encoding 1) a fatty acyl-CoA reductase, or 2) a thioesterase, a carboxylic acid reductase and an aldehyde reductase.

9. The method of claim 8, said incubating comprises 0 to 2 μM of zinc, 0 to 20 μM of cobalt, 0 to 20 μM of copper and/or 0.5 to 5 g/L ammonium in the culture.

10. The method of claim 8, wherein the host cell provides an increased amount of the one or more fatty alcohols, as compared to a corresponding control cell lacking deletion of the first mutant gene.

11. The method of claim 8, wherein the cytoplasmic tRNA thiolation protein is cytoplasmic tRNA 2-thiolation protein 2.

12. The method of claim 8, wherein the cytoplasmic tRNA thiolation protein comprises a polypeptide sequence having at least 90% sequence identity to any one of the following SEQ ID NOs: 1, 3-8, 10-16 and 18-20.

13. The method of claim 8, wherein the host cell further comprises a second mutant gene comprising insertion of a nucleic acid encoding an acetyl-CoA carboxylase or a fatty alcohol reductase, thereby providing overexpression of the acetyl-CoA carboxylase or overexpression of the fatty alcohol reductase.

14. The method of claim 8, wherein the host cell further comprises a second mutant gene comprising deletion of a nucleic acid encoding a lysophospholipid acyltransferase, a fatty alcohol oxidase, an aldehyde dehydrogenase, an isocitrate dehydrogenase, or a pyruvate decarboxylase.

15. The method of claim 8, wherein the fatty alcohol comprises a structure of R′OH, in which R′ is a C4-32 aliphatic that is optionally substituted.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A-1B show non-limiting examples of cytoplasmic tRNA 2-thiolation protein 2 (NCS2). Provided is (A) an unrooted phylogenetic tree of NSC2 homologs in model eukaryotic species. Protein identifiers are provided as UniProt short IDs for CTU2, which is the human ortholog of S. cerevisiae ncs2 and is used as the base of the UniProt name for the family. Protein identifiers include those for Homo sapiens (identified as “CTU2 Human,” UniProtKB Entry No. Q2VPK5); Caenorhabditis elegans (identified as “CTU2 CAEEL,” UniProtKB Entry No. Q19906); Drosophila melanogaster (identified as “CTU2 DROME,” UniProtKB Entry No. Q9VIV3); Rhizopus delemar (identified as “I1CPB8 RHIO9,” UniProtKB Entry No. I1CPB8); Arabidopsis thaliana (identified as “CTU2 ARATH,” UniProtKB Entry No. 065628); Chlamydomonas reinhardtii (identified as “A0A2K3E7L8 CHLRE,” UniProtKB Entry No. A0A2K3E7L8); Dictyostelium discoideum (identified as “CTU2 DICDI,” UniProtKB Entry No. Q55EX7); Tetrahymena thermophila (identified as “I7M9N8 TETTS,” UniProtKB Entry No. I7M9N8); Ustilago maydis (identified as “AOAOD1EOS3 USTMA,” UniProtKB Entry No. AOAOD1EOS3); Cryptococcus neoromans (identified as “Q5KJL1 CRYNJ,” UniProtKB Entry No. Q5KJL1); Rhodosporidium toruloides (identified as “A0A2S9ZXY4 RHOTO,” UniProtKB Entry No. A0A2S9ZXY4); Schizosaccharomyces pombe (identified as “CTU2 SCHPO,” UniProtKB Entry No. Q9UUC7); Neurospora crassa (identified as “U9W2Q7 NEUCR,” UniProtKB Entry No. U9W2Q7); Emericella (Aspergillus) nidulans (identified as “CTU2 EMENI,” UniProtKB Entry No. Q5BHB8); Yarrowia lipolytica (identified as “CTU2 YARLI,” UniProtKB Entry No. Q6CF50); Saccharomyces cerevisiae (identified as “CTU2 YEAST,” UniProtKB Entry No. P53923); and Candida albicans (identified as “CTU2 CANAL,” UniProtKB Entry No. Q59ZY9). Also provided is (B) the sequence for cytoplasmic tRNA 2-thiolation protein 2 (NCS2) for Rhodosporidium toruloides (SEQ ID NO: 1).

(2) FIGS. 2A-2F show non-limiting amino acid sequences for various cytoplasmic tRNA 2-thiolation protein 2. Provided are sequences for R. toruloides (A0A2S9ZXY4 (RHOTO), SEQ ID NO: 1); H. sapiens (Q2VPK5.1 (HUMAN), SEQ ID NO: 2); C. neoromans (Q5KJL1 (CRYNJ), SEQ ID NO: 3); U. maydis (A0A0D1EOS3 (USTMA), SEQ ID NO: 4); S. pombe (Q9UUC7.1 (SCHPO), SEQ ID NO: 5); R. delemar (I1CPB8 (RHIO9), SEQ ID NO: 6); C. albicans (Q59ZY9.2 (CANAL), SEQ ID NO: 7); D. discoideum (Q55EX7.1 (DICDI), SEQ ID NO: 8); D. melanogaster (Q9VIV3.1 (DROME), SEQ ID NO: 9); E. nidulans (Q5BHB8.2 (EMENI), SEQ ID NO: 10); C. reinhardtii (A0A2K3E7L8 (CHLRE), SEQ ID NO: 11); Y. lipolytica (Q6CF50.1 (YARLI), SEQ ID NO: 12); A. thaliana (065628.3 (ARATH), SEQ ID NO: 13); N. crassa (U9W2Q7 (NEUCR), SEQ ID NO: 14); Ashbya gossypii (Q75BK0.1 (ASHGO), SEQ ID NO: 15); S. cerevisiae (strain YJM789) (Baker's yeast) (A6ZRW4.1 (YEAST Y), SEQ ID NO: 16); C. elegans (Q19906.2 (CAEEL), SEQ ID NO: 17); S. cerevisiae (strain AWRI1631) (Baker's yeast) (B5VQS7.1 (YEAST A), SEQ ID NO: 18); S. cerevisiae (strain ATCC 204508/S288c) (Baker's yeast) (P53923.1 (YEAST S), SEQ ID NO: 19); and S. cerevisiae (strain RM11-1a) (Baker's yeast) (B3LNX6.1 (YEAST R), SEQ ID NO: 20).

(3) FIGS. 3A-3F show non-limiting amino acid sequences for various cytoplasmic tRNA 2-thiolation protein 2. Provided are sequences for C. neoromans (Q5KJL1 (CRYNJ), SEQ ID NO: 3); S. pombe (Q9UUC7.1 (SCHPO), SEQ ID NO: 5); C. albicans (Q59ZY9.2 (CANAL), SEQ ID NO: 7); E. nidulans (Q5BHB8.2 (EMENI), SEQ ID NO: 10); Y. lipolytica (Q6CF50.1 (YARLI), SEQ ID NO: 12); A. thaliana (065628.3 (ARATH), SEQ ID NO: 13); A. gossypii (Q75BK0.1 (ASHGO), SEQ ID NO: 15); S. cerevisiae (strain YJM789) (Baker's yeast) (A6ZRW4.1 (YEAST Y), SEQ ID NO: 16); S. cerevisiae (strain AWRI1631) (Baker's yeast) (B5VQS7.1 (YEAST A), SEQ ID NO: 18); S. cerevisiae (strain ATCC 204508/S288c) (Baker's yeast) (P53923.1 (YEAST S), SEQ ID NO: 19); S. cerevisiae (strain RM11-1a) (Baker's yeast) (B3LNX6.1 (YEAST R), SEQ ID NO: 20); R. toruloides (A0A2S9ZXY4 (RHOTO), SEQ ID NO: 1); U. maydis (AOAOD1EOS3 (USTMA), SEQ ID NO: 4); and N. crassa (U9W2Q7 (NEUCR), SEQ ID NO: 14). Also provided are consensus sequences, including CONS1 (SEQ ID NO: 21), CONS2 (SEQ ID NO: 22), CONS3 (SEQ ID NO: 23), CONS4 (SEQ ID NO: 24), CONS5 (SEQ ID NO: 25), CONS6 (SEQ ID NO: 26), CONS7 (SEQ ID NO: 27), and CONS8 (SEQ ID NO: 28). In another embodiment, for each consensus sequence (SEQ ID NOs: 21-28), each X at each position is an amino acid (or a modified form thereof) that is provided in an aligned reference sequence. For instance, this X can be any amino acid provided in an aligned reference sequence (e.g., aligned reference sequences SEQ ID NOs: 1-20 or SEQ ID NOs: 1, 3-5, 7, 10, 12-16, and 18-20 for the consensus sequence in one of SEQ ID NOs: 21-28). A black background indicates a conserved amino acid, a gray background indicates a similar amino acid, and a dash indicates an absent amino acid.

(4) FIGS. 4A-4G show non-limiting amino acid sequences for (A) acetyl-CoA carboxylase 1 (ACC1) for R. toruloides (SEQ ID NO: 30), (B) lysophospholipid acyltransferase (ALE1) for R. toruloides (SEQ ID NO: 31), (C) fatty alcohol oxidase (FAO1) for R. toruloides (SEQ ID NO: 32), (D) aldehyde dehydrogenase (HFD1) for R. toruloides (SEQ ID NO: 33), (E) isocitrate dehydrogenase (IDH) for R. toruloides (SEQ ID NO: 34), (F) pyruvate decarboxylase (PDC) for R. toruloides (SEQ ID NO: 35), and (G) aldehyde dehydrogenase (ALD) for R. toruloides (SEQ ID NO: 36).

(5) FIG. 5 shows a non-limiting pathway to provide a fatty alcohol (FOH).

(6) FIG. 6 shows fatty alcohol production in the Ancs2 strain versus a parent strain. The total fatty alcohol content was measured by GC-FID (gas chromatography-flame ionization detection).

(7) FIGS. 7A-7B show (A) hierarchical clustering of 883 proteins with significantly altered abundance (P value <0.5, fold change > two-fold between mutant and parent strains in at least one condition). Provided are data for a strain having deletion of the ncs2 gene (identified as “Ancs2”), a strain having over expression of a lipase (identified as “Lipase OE”), and a strain having over expression of NNT transhydrogenase (identified as “NNT OE”). Conditions include a high nitrogen (about 5 g/L) or a low nitrogen (about 1 g/L) environment. Also provided are (B) principal component analysis (PCA) of global proteomics.

(8) FIG. 8 shows hierarchical clustering of 165 lipids with significantly altered abundance (P value <0.5, at least 25% difference between mutant and parent strains in at least one condition). Provided are data for a strain having deletion of the ncs2 gene (identified as “Ancs2”), a strain having over expression of a lipase (identified as “Lipase OE”), and a strain having over expression of NNT transhydrogenase (identified as “NNT OE”). Conditions include a high nitrogen (about 5 g/L) or a low nitrogen (about 1 g/L) environment.

(9) FIG. 9 shows a non-limiting pathway showing synergistic effects of the Ancs2 mutation on competing pathways for fatty-acyl-CoA.

(10) FIGS. 10A-10D show relative abundance of proteins from pathways shown in FIG. 9 in the Ancs2 mutant versus the parental strain by global proteomics. Provided are graphs showing log base 2 ratios of total protein intensities for (A) proteins related to fatty-acyl-CoA synthesis; (B) proteins related to lipid synthesis; (C) proteins related to beta oxidation; and (D) proteins related to lipases and long chain fatty acids (LCFA).

(11) FIG. 11 shows relative abundance of diacylglycerol species in a Ancs2 mutant versus the parent strain as determined by global proteomics.

(12) FIG. 12 shows fatty alcohol (FOH) production in various strains that overexpress particular target genes.

(13) FIG. 13 shows fatty alcohol (FOH) production in various knock-out strains that lack particular target genes.

(14) FIG. 14 shows fatty alcohol (FOH) production in single knock-out strains (ΔALE1 or ΔIDH2) and a multi-knock-out strain (ΔALE1 and ΔIDH2), as compared to the control parental strain.

(15) FIG. 15 shows the effect of culture conditions of fatty alcohol (FOH) production for the NCS2 mutant strain (indicated as “STC0179” and “STC0180”) and its parental strain (indicated as “STC0113”).

(16) FIGS. 16A-16B show C16 and C18 fatty alcohol (FOH) production in single knock-out strains (ΔFAO1 or ΔHFD1) or a multi-knock-out strain (ΔFAO1 and ΔHFD1), as compared to the control parental strain. Provided are data for corn stover hydrolysate media (indicated as “DMR”) and for a defined media (indicated as “Mock”) under high nitrogen (HN) conditions (5 g/L ammonium sulfate).

(17) FIGS. 17A-17B show C16 and C18 fatty alcohol (FOH) production in single knock-out strains (ΔFAO1 or ΔHFD1) or a multi-knock-out strain (ΔFAO1 and ΔHFD1), as compared to the control parental strain. Provided are data for corn stover hydrolysate media (indicated as “DMR”) and for a defined media (indicated as “Mock”) under low nitrogen (LN) conditions (1 g/L ammonium sulfate).

DETAILED DESCRIPTION

(18) The present disclosure relates to host cells having one or more mutant genes. In one embodiment, the mutant gene includes a target gene related to a tRNA thiolation protein (e.g., the ncs2 gene). Such host cells can be used to produce lipid-derived compounds, such as fatty alcohols.

(19) In some non-limiting embodiments, the tRNA thiolation protein is the ncs2 gene, which plays a role in 2-thiolation of tRNA. Without wishing to be limited by mechanism, the modification of tRNA wobble positions (e.g., by way of thiolation) has been implicated in regulation of gene expression in response to heat shock, but the overall effect of this metabolic modification is unclear. In culture, the ncs2 deletion (Ancs2) mutant provides overall reduced lipid content. However, surprising, the same mutant also provide overall increased fatty alcohol (FOH) content. In some embodiments, the deletion of ncs2 resulted in at least a two- to three-fold increase in FOH production over the parent strain.

(20) This observation is supported by combined metabolomic, proteomic, and lipidomic analysis, as described herein. This analysis shows a global shift in lipid and proteomic profiles in the Ancs2 mutant with decreased flux from fatty-acyl-CoA to storage lipids (e.g., thereby providing reduced fatty-acyl-CoA incorporation into diacylglycerides), reduced fatty-acyl-CoA consumption by beta-oxidation, and increased fatty-acyl-CoA production through higher expression of malic enzyme (NADPH generating). In some embodiments, deletion of ncs2 coordinately reduces the expression of several enzymes essential for triacylglycerides biosynthesis, while maintaining fatty-acyl-CoA production.

(21) To investigate how various mutants with altered lipid accumulation might enhance or inhibit production of fatty-acyl-CoA derived chemicals, deletion mutants for several genes identified in a functional genomic screen of R. toruloides were created in a fatty alcohol producing strain expressing fatty acyl-CoA reductase for Marinobacter aquaeolei. In particular embodiments, the Ancs2 mutant include one or more further mutant genes. In particular embodiments, the further mutant gene includes deletion or overexpression of proteins that provides low FOH catabolism and/or low re-importation of secreted FOH. Overexpression can include random or targeted integration of the gene to be expressed.

(22) Accordingly, the host cell can include any useful mutant having a mutant gene encoding a cytoplasmic tRNA thiolation protein. The mutant gene can include deletion of the gene that encodes the cytoplasmic tRNA protein or modification of that gene that results in lower expression of the NCS2 protein. In one embodiment, the host cell include a ncs2 gene deletion or a ncs2 gene modification, which results in lowered expression of the NCS2 protein. The ncs2 gene or NCS2 protein can include any provided herein, such as homologs. For instance, FIG. 1A provides an unrooted phylogenetic tree of NCS2 homologs in model eukaryotic species. The NCS2 protein for R. toruloides is provided in FIG. 1B (SEQ ID NO: 1).

(23) Further non-limiting amino acid sequences for various NCS2 proteins are provided in FIGS. 2A-2F (SEQ ID NOs: 1-20). In some embodiments, the host cell includes a mutant gene encoding a cytoplasmic tRNA thiolation protein, in which the protein includes a polypeptide sequence having at least 90% sequence identity to one of SEQ ID NOs: 1-20. In particular embodiments, the mutant gene includes deletion of the gene encoding the cytoplasmic tRNA thiolation protein. In other embodiments, the mutant gene includes lower expression or under expression of the gene encoding the cytoplasmic tRNA thiolation protein.

(24) The NCS2 protein can also be characterized by one or more consensus sequences. In one embodiment, NCS2 protein has one or more consensus sequences provided as SEQ ID NOs: 21-28 (FIGS. 3A-3F). In some embodiments, the host cell includes a mutant gene encoding a cytoplasmic tRNA thiolation protein, in which the protein includes a polypeptide sequence having at least 90% sequence identity to one or more of the following SEQ ID NOs: 21-28.

(25) In one embodiment, protein includes a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 21:
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6SX.sub.8X.sub.9X.sub.10X.sub.11X.sub.12SX.sub.14X.sub.15X.sub.16LX.sub.18X.sub.19X.sub.20, wherein:

(26) X.sub.1 is A, V, I, L, R, H, K, P, N, Q, or absent;

(27) each of X.sub.2, X.sub.6, and X.sub.20 is, independently, A, V, I, L, F, Y, or W;

(28) each of X.sub.3, X.sub.4, and X.sub.16 is, independently, A, V, I, L, or M;

(29) X.sub.5 is G, A, V, I, L, or P;

(30) X.sub.8 is G, A, V, I, L, R, H, K, S, T, F, Y, or W;

(31) each of X.sub.9 and X.sub.11 is, independently, G, C, S, T, or absent;

(32) X.sub.10 is A, V, I, L, D, E, C, S, T, N, or Q;

(33) X.sub.12 is G or absent;

(34) X.sub.14 is A, V, I, L, M, C, S, T, R, H, or K;

(35) X.sub.15 is A, V, I, L, S, or T;

(36) X.sub.18 is R, H, K, D, or E; and

(37) X.sub.19 is A, V, I, L, M, S, T, F, Y, or W.

(38) In one embodiment, protein includes a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 22:
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7, wherein:

(39) X.sub.1 is G, A, V, I, L, R, H, K, or absent;

(40) X.sub.2 is R, H, K, D, E, S, T, N, or Q;

(41) X.sub.3 is G, A, V, I, L, D, or E;

(42) X.sub.4 is A, V, I, L, R, H, K, D, E, N, Q, P, F, Y, or W;

(43) X.sub.5 is A, V, I, or L;

(44) X.sub.6 is A, V, I, L, C, S, T, F, Y, or W; and

(45) X.sub.7 is G, A, V, I, L, R, H, K, S, T, N, Q, F, Y, or W.

(46) In one embodiment, protein includes a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 23:
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.sub.11X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17X.sub.18X.sub.19X.sub.20X.sub.21, wherein:

(47) X.sub.1 is any amino acid or absent;

(48) each of X.sub.2, X.sub.7, X.sub.15, and X.sub.16 is, independently, A, V, I, L, M, S, or T;

(49) X.sub.3 is G, A, V, I, L, R, H, K, S, or T;

(50) X.sub.4 is A, V, I, L, R, H, or K;

(51) X.sub.5 is A, V, I, L, D, E, N, Q, S, T, or P;

(52) X.sub.6 is D, E, N, Q, S, or T;

(53) X.sub.8 is A, V, I, L, D, E, R, H, or K;

(54) each of X.sub.9 and X.sub.21 is, independently, A, V, I, L, R, H, K, S, T, F, Y, or W;

(55) X.sub.10 is A, V, I, L, R, H, K, F, Y, or W;

(56) X.sub.11 is A, V, I, L, F, Y, or W;

(57) X.sub.12 is A, V, I, L, R, H, K, N, Q, F, Y, or W;

(58) X.sub.13 is D, E, N, Q, S, T, R, H, K, or M;

(59) each of X.sub.14 and X.sub.18 is, independently, A, V, I, L, D, E, R, H, K, N, Q, S, or T;

(60) X.sub.17 is A, V, I, L, R, H, K, N, or Q;

(61) X.sub.19 is A, V, I, L, D, E, R, H, K, S, T, F, Y, or W; and

(62) X.sub.20 is A, V, I, or L.

(63) In one embodiment, protein includes a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 24:
X.sub.1X.sub.2X.sub.3GX.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.sub.11X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17X.sub.18X.sub.19X.sub.20GX.sub.22GX.sub.24X.sub.25X.sub.26, wherein:

(64) each of X.sub.1 and X.sub.26 is, independently, A, V, I, or L;

(65) each of X.sub.2, X.sub.11, X.sub.14, X.sub.15, X.sub.18, and X.sub.19 is, independently, A, V, I, L, M, S, or T;

(66) X.sub.3 is A, V, I, L, M, S, T, F, Y, or W;

(67) X.sub.5 is R, H, K, D, E, S, or T;

(68) X.sub.6 is C, S, T, N, or Q;

(69) each of X.sub.7 and X.sub.16 is, independently, G, A, V, I, L, M, D, E, S, or T;

(70) X.sub.8 is D, E, S, or T;

(71) each of X.sub.9 and X.sub.22 is, independently, A, V, I, L, R, H, K, S, or T;

(72) X.sub.10 is A, V, I, L, N, Q, S, or T;

(73) each of X.sub.12 and X.sub.17 is, independently, G, A, V, I, L, D, E, N, Q, S, or T;

(74) each of X.sub.13 and X.sub.20 is, independently, A, V, I, L, R, H, K, D, E, S, or T; and each of X.sub.24 and X.sub.25 is, independently, A, V, I, L, R, H, K, S, T, N, Q, F, Y, or W.

(75) In one embodiment, protein includes a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 25:
PX.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.sub.11X.sub.12X.sub.13, wherein:

(76) X.sub.2 is A, V, I, L, M, S, or T;

(77) each of X.sub.3 and X.sub.7 is, independently, A, V, I, L, R, H, K, N, Q, S, or T;

(78) X.sub.4 is R, H, K, D, E, S, or T;

(79) X.sub.5 is A, V, I, L, R, H, K, C, S, or T;

(80) each of X.sub.6 and X.sub.13 is, independently, A, V, I, L, S, T, F, Y, or W;

(81) X.sub.8 is any amino acid;

(82) X.sub.9 is D or E;

(83) X.sub.10 is A, V, I, or L;

(84) X.sub.11 is A, V, I, L, R, H, K, D, E, N, Q, S, T, or P; and

(85) X.sub.12 is A, V, I, L, R, H, K, S, T, F, Y, or W.

(86) In one embodiment, protein includes a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 26:
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.sub.11X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17X.sub.18X.sub.19X.sub.20X.sub.21X.sub.22X.sub.23X.sub.24X.sub.25X.sub.26X.sub.27X.sub.28
X.sub.29X.sub.30KL, wherein:

(87) each of X.sub.1 and X.sub.28 is, independently, M, S, T, or absent;

(88) each of X.sub.2, X.sub.5, and X.sub.25 is, independently, A, V, I, or L;

(89) X.sub.3 is A, V, I, L, R, H, K, D, E, N, or Q;

(90) X.sub.4 is G, M, R, H, K, D, E, N, or Q;

(91) each of X.sub.6, X.sub.23, and X.sub.29 is, independently, G, A, V, I, L, C, M, S, or T;

(92) each of X.sub.7, X.sub.11, X.sub.16, and X.sub.30 is, independently, G, A, V, I, L, M, R, H, K, D, E, N, Q, S, or T;

(93) each of X.sub.8 and X.sub.17 is, independently, G, R, H, K, D, E, N, Q, S, or T;

(94) X.sub.9 is F, Y, or W;

(95) X.sub.10 is A, V, I, L, F, Y, or W;

(96) X.sub.12 is G, A, V, I, L, D, E, N, Q, S, or T;

(97) X.sub.13 is A, V, I, L, N, or Q;

(98) X.sub.14 is G, D, E, N, or Q;

(99) X.sub.15 is G, A, V, I, L, or absent;

(100) X.sub.18 is A, V, I, L, R, H, K, N, Q, F, Y, or W;

(101) X.sub.19 is A, V, I, L, P, S, or T;

(102) each of X.sub.20 and X.sub.26 is, independently, G, A, V, I, L, N, Q, S, or T;

(103) X.sub.21 is A, V, I, L, R, H, K, S, or T;

(104) X.sub.22 is A, V, I, L, M, D, or E;

(105) X.sub.24 is N, Q, S, or T; and

(106) X.sub.27 is R, H, or K.

(107) In one embodiment, protein includes a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 27:
CX.sub.2X.sub.3CX.sub.5X.sub.6X.sub.7X.sub.8, wherein:

(108) X.sub.2 is G, A, V, I, L, P, N, Q, S, or T;

(109) X.sub.3 is A, V, I, or L;

(110) X.sub.5 is G, A, V, I, L, D, E, N, Q, S, or T;

(111) X.sub.6 is G, A, V, I, L, M, N, Q, S, or T;

(112) X.sub.7 is R, H, K, D, E, P, F, Y, or W; and

(113) X.sub.8 is A, V, I, L, M, S, or T.

(114) In one embodiment, protein includes a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 28:
X.sub.1CX.sub.3X.sub.4CX.sub.6X.sub.7X.sub.8X.sub.9, wherein:

(115) X.sub.1 is A, V, I, L, F, Y, W, or absent;

(116) X.sub.3 is S, T, F, Y, or W;

(117) X.sub.4 is G, A, V, I, L, S, or T;

(118) X.sub.6 is A, V, I, L, R, H, K, D, E, S, or T;

(119) X.sub.7 is A, V, I, L, R, H, K, S, T, F, Y, or W;

(120) X.sub.8 is A, V, I, L, N, Q, S, or T; and

(121) X.sub.9 is A, V, I, L, R, H, or K.

(122) In addition to a first mutant gene encoding a cytoplasmic tRNA thiolation protein, the host cell can include one or more second mutant genes. In one embodiment, the second mutant gene encodes a target protein selected from the group consisting of an acetyl-CoA carboxylase, a lysophospholipid acyltransferase, a fatty-acyl-CoA oxidase, a fatty acid synthase, a fatty-acyl-CoA reductase, an aldehyde reductase, a fatty-acyl-CoA synthetase, a thioesterase, a carboxylic acid reductase, a fatty alcohol oxidase, a fatty alcohol reductase, an aldehyde dehydrogenase, an isocitrate dehydrogenase, or a pyruvate decarboxylase.

(123) In particular embodiments, the second mutant gene provides a host cell having low FOH catabolism, as compared to a parent or control strain lacking the second gene. In other embodiments, the second mutant gene provides a host cell having low re-importation of secreted FOH, as compared to a parent or control strain lacking the second gene. In yet other embodiments, the second mutant gene provides a host cell having high export of FOH and/or having improved or alleviated FOH toxicity, as compared to a parent or control strain lacking the second gene.

(124) In one embodiment, the host cell includes a mutant gene that encodes a target protein that is an acetyl-CoA carboxylase. In particular embodiments, the mutant gene includes deletion of the nucleic acid encoding the acetyl-CoA carboxylase. In some embodiments, the acetyl-CoA carboxylase includes a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 30 (FIG. 4A).

(125) In another embodiment, the host cell includes a mutant gene that encodes a target protein that is a lysophospholipid acyltransferase. In particular embodiments, the mutant gene includes expression or overexpression of the nucleic acid encoding the lysophospholipid acyltransferase. In some embodiments, the lysophospholipid acyltransferase includes a polypeptide having at least 90% sequence identity to SEQ ID NO: 31 (FIG. 4B). Expression or overexpression can include random integration using a plasmid, in which the target gene can be under the control of a promoter (e.g., a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter); or include targeted integration, in which the target gene is under the control of a promoter (e.g., a translational elongation factor Ef-1 (TEF1) promoter). The location of gene integration can be at any locus, e.g., in which the target locus can include ku70, NCS2 and ALE1, all in FOH producing strains.

(126) In yet another embodiment, the host cell includes a mutant gene that encodes a target protein that is a fatty alcohol oxidase. In particular embodiments, the mutant gene includes deletion of the nucleic acid encoding the fatty alcohol oxidase. In some embodiments, the fatty alcohol oxidase includes a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 32 (FIG. 4C).

(127) In another embodiment, the host cell includes a mutant gene that encodes a target protein that is an alcohol dehydrogenase. In particular embodiments, the mutant gene includes deletion of the nucleic acid encoding the alcohol dehydrogenase. In some embodiments, the alcohol dehydrogenase includes a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 33 (FIG. 4D) or SEQ ID NO: 36 (FIG. 4G).

(128) In one embodiment, the host cell includes a mutant gene that encodes a target protein that is an isocitrate dehydrogenase. In particular embodiments, the mutant gene includes deletion of the nucleic acid encoding the isocitrate dehydrogenase. In some embodiments, the isocitrate dehydrogenase includes a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 34 (FIG. 4E).

(129) In another embodiment, the host cell includes a mutant gene that encodes a target protein that is a pyruvate decarboxylase. In particular embodiments, the mutant gene includes deletion of the nucleic acid encoding the pyruvate decarboxylase. In some embodiments, the pyruvate decarboxylase includes a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 35 (FIG. 4F).

(130) In particular embodiments, modification (e.g., over-expression) of a lipase, a transhydrogenase (e.g., a NADPH transhydrogenase), and/or an acyl-CoA synthetase/ligase may be synergistic. In some embodiments, a mutant herein include two or more mutations that exhibit additive or synergistic effects.

(131) The host cell can include a first mutant gene encoding a first target protein and a second mutant gene encoding a second target protein, in which the first and second target proteins are different. The first target protein can include a cytoplasmic tRNA thiolation protein, and the second target protein can be any in a pathway that can enhance fatty alcohol (FOH) production. FIG. 5 shows a non-limiting lipid synthesis pathway, which shows various proteins and lipid-derived compounds. Non-limiting lipid-derived compounds include acetyl-CoA, malonyl Co-A, fatty-acyl-CoA, fatty alcohol (FOH), free fatty acid (FFA), and free aldehyde (FAL). Non-limiting target proteins (e.g., which can be encoded as the second mutant gene) include acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), fatty-acyl-CoA oxidase, fatty-acyl-CoA reductase (FAR), fatty-acyl-CoA synthetase (FAA), thioesterase (TES), aldehyde dehydrogenase (ALD), carboxylic acid reductase (CAR), and aldehyde reductase (AHR).

(132) Lipid-Derived Compound

(133) The host cells and methods herein can be used to provide a lipid-derived compound. In particular embodiments, the host cell provides an increased concentration of the lipid-derived compound, as compared to a control cell. The control cell can be a parental cell or parental strain that lacks any of the modifications described herein for the first mutant gene and/or second mutant gene.

(134) Non-limiting lipid-derived compounds include a fatty alcohol, a fatty acid, a fatty aldehyde, a fatty alkene, a fatty amide, a fatty ester, a fatty alkane, and a fatty diacid. Yet other lipid-derived compounds can include an oil, a lipid, a glycerolipid, a sphingolipid, a sterol lipid, or a triacylglyceride. In some embodiments, a lipid-derived compound includes a class of molecules that are soluble in nonpolar solvents (e.g., ether or chloroform), are relatively or completely insoluble in water, and include one or more hydrocarbon chains which are hydrophobic.

(135) In particular embodiments, the lipid-derived compound is a fatty alcohol. Non-limiting fatty alcohols can include at least one hydroxyl group (—OH) and at least on aliphatic group, as defined herein. In particular embodiments, the fatty alcohol includes a structure of R′OH, in which R′ is an optionally substituted C.sub.4-32 aliphatic. In other embodiments, the fatty alcohol is lauryl alcohol (1-dodecanol), tridecyl alcohol (1-tridecanol), myristyl alcohol (1-tetradecanol), pentadecyl alcohol (1-pentadecanol), cetyl alcohol (1-hexadecanol), palmitoleyl alcohol (cis-9-hexadecen-1-ol), heptadecyl alcohol (1-n-heptadecanol), stearyl alcohol (1-octadecanol), oleyl alcohol (1-octadecenol), nonadecyl alcohol (1-nonadecanol), arachidyl alcohol (1-eicosanol), or combinations thereof.

(136) In other embodiments, the lipid-derived compound is a fatty-acyl-coenzyme A (CoA) derived chemical. Non-limiting chemicals include a fatty alcohol, as well as combinations including two or more different fatty alcohols.

(137) Host Cells

(138) The host cells herein are genetically modified in that heterologous nucleic acid have been introduced into the host cells, and as such the genetically modified host cells do not occur in nature. The suitable host cell is one capable of expressing a protein (e.g., any described herein) from a nucleic acid configured to encode that protein.

(139) In one embodiment, the host cell is a genetically modified oleaginous organism. As used herein, an oleaginous organism includes an organism that can accumulate more than about 20% (w/w) of lipid-derived compounds on a cell dry weight basis. Non-limiting oleaginous organisms include microalgae, bacteria, fungi, and yeast (e.g., an oleaginous yeast cell, Rhodosporidium, and the like).

(140) In some embodiments, the oleaginous organism is an oleaginous yeast. Non-limiting examples include Apiotrichum (e.g., A. curvatum), Candida (e.g., C. ortholopsis, C. pseudolambica, or C. viswanathii), Cryptococcus (e.g., C. albidus, C. curvatus, C. phenolicus, C. podzolicus, C. terricola, or C. vishniaccii), Cutaneotrichosporon (e.g., C. oleaginosus), Cystobasidium (e.g., C. oligophagum), Cystofilobasidium (e.g., C. informiminiatum), Debaromyces (e.g., D. hansenii), Issatchenika (e.g., I. occidentalis), Leucosporidium (e.g., L. scottii), Lipomyces (e.g., L. starkeyi), Occultifur (e.g., O. externus), Pichia (e.g., P. deserticola or P. segobiensis), Rhizopus (e.g., R. arrhizus), Rhodosporidium (e.g., R. azoricum, R. bajevae, R. diobovatum, R. fluviale, R. kratochvilovae, R. paludigenum, R. sphaerocarpum, or R. toruloides), Rhodotorula (e.g., R. araucariae, R. bogoriensis, R. colostri, R. dairenensis, R. glutinis, R. graminis, R. minuta, or R. mucilaginosa), Sporidiobolus (e.g., S. johnsonii, S. pararoseus, S. ruineniae, or S. salmonicolor), Sporobolomyces (e.g., S. bannaensis, S. carnicolor, S. metaroseus, S. odoratus, S. poonsookiae, or S. singularis), Starmerella (e.g., S. bombicola), Trichosporon (e.g., T. oleaginosus or T. porosum), and Yarrowia (e.g., Y. lipolytica).

(141) Any prokaryotic or eukaryotic host cell may be used in the present method so long as it remains viable after being transformed with a sequence of nucleic acids configured to encode a protein described herein (e.g., NCS2, ACC1, ALE1, or others). Prokaryotic cells include bacteria or archaea cells. Suitable eukaryotic cells include, but are not limited to, fungal, insect, or mammalian cells. Suitable fungal cells are yeast cells, which may belong to the genus Rhodosporidium, Blastomyces, Candida, Citeromyces, Crebrothecium, Cryptococcus, Debaryomyces, Eremothecium, Geotrichum, Kloeckera, Lipomyces, Pichia, Rhodotorula, Saccharomyces (e.g., S. bayanus, S. carlsbergensis, S. cerevisiae, or S. pastorianus), Schizosaccharomyces, Sporobolomyces, Trichosporon, or Wickerhamia.

(142) Preferably, the host cell is not adversely affected by the transduction of the necessary nucleic acid sequences, the subsequent expression of the proteins (e.g., enzymes), or the resulting intermediates required for carrying out the steps associated with the fatty alcohol pathway. In one embodiment, it is preferred that minimal “cross-talk” (i.e., interference) occur between the host cell's own metabolic processes and those processes involved with the fatty alcohol pathway. In another embodiment, it is preferred that the host cell includes other mutant gene(s) that provide low catabolism of fatty alcohol and/or provide minimal re-importation of secreted fatty alcohol.

(143) Incubation in a Culture

(144) The host cell can be incubated in a culture having any useful medium. Such medium can include growth medium, biomass, nutrients, micronutrients, cofactors, and such, as well as combinations thereof. Non-limiting components within the medium can include a carbon source, an amino acid, a peptide, a lipid, a vitamin, a trace element, a salt, a growth factor, a buffer, or combinations thereof.

(145) The medium can include any useful carbon source, such as and without limitation, acetate, arabinose, carboxymethylcellulose, cellulose, cellulosic material (e.g., depolymerized cellulose material), corn starch, fructose, galactose, glucose, glycerol, lactose, mannose, milk whey, molasses, potato, rhamnose, ribose, rice, sorghum, starch, sucrose, sugar alcohol, sugar beet pulp (e.g., depolymerized sugar beet pulp), sugar cane, switchgrass, wheat, xylose, a feedstock (e.g., whole whey, modified whey products, dairy permeates, crop residues, and the like), and/or a biomass (e.g., lignocellulosic biomass or a hydrolysate thereof), as well as mixtures thereof. Yet other carbon sources include monosaccharides, disaccharides, oligosaccharides, polysaccharides, monoglycerides, diglycerides, triglycerides, alkanes, fatty acids, fatty acid esters, phospholipids, vegetable oils (e.g., soybean oil), or animal fats.

(146) Any useful biomass can be employed. A biomass (e.g., a lignocellulosic biomass) may include agricultural residues (e.g., corn stover or sugarcane bagasse), energy crops (e.g., grass, such as elephant grass, silver grass, Sudan grass, or switchgrass; poplar trees; willow; maize; millet; white sweet clover; rapeseed; jatropha; or sugarcane), food waste (e.g., Brewers' spent grain), wood residues (e.g., sawmill or papermill discard), or municipal paper waste.

(147) Yet other exemplary biomass includes corn stover (e.g., deacetylation and mechanical refining (DMR) processed corn stover or de-acetylated corn stover hydrolysate from the National Renewable Energy Laboratory (NREL), Golden, Colo.), corn cob hydrolysate, fishwaste hydrolysate, paper industry effluent or waste product (e.g., black liquor), rice residue hydrolysate, sugar beet molasses, sugarcane molasses, wastewater (e.g., distillery wastewater, livestock wastewater, or municipal wastewater), distillers grains or co-products (e.g., wet distillers grains (WDGs), dried distillers grains (DDGs), dried distillers grains with solubles (DDGS), fatty acids from oil hydrolysis, lipids from evaporation of thin stillage, syrup, distillers grains, distillers grains with or without solubles, solids from a mash before fermentation, solids from a whole stillage after fermentation, biodiesel, and acyl glycerides), oilseed meals (e.g., soybean meal or canola meal), feeds (e.g., alfalfa meal, cottonseed meal, DDGS, rice bran, or wheat bran), and others.

(148) The medium may be supplemented with a nitrogen source (to increase the concentration of nitrogen) or supplemented within an agent to capture nitrogen (to decrease the concentration of nitrogen, such as with a chelating agent). For instance, nitrogen may be supplied from an inorganic source (e.g., (NH.sub.4).sub.2SO.sub.4, NH.sub.4Cl, or another ammonium source) or organic source (e.g., urea, glutamate, or an amino acid). The nitrogen source can be any nitrogen-containing composition (e.g., compound, mixture of compounds, salts, etc.) that an organism may metabolize for organism viability. The concentration of nitrogen within the medium can be controlled to provide a nitrogen-rich environment, a standardized nitrogen-containing environment, or a nitrogen-poor environment. In particular embodiments, the concentration of nitrogen is from about 0.5 to 5 g/L of ammonium (e.g., NH.sub.4SO.sub.4).

(149) In embodiments, the medium can include one or more micronutrients. Non-limiting micronutrients include cobalt, copper, zinc, iron, and/or potassium. In particular embodiments, the growth medium can include from about 0 to 2 μM of zinc, 0 to 20 μM of cobalt, and/or 0 to 20 μM of copper.

(150) In one embodiment, the medium includes corn stover hydrolysate medium (mechanically refined de-acetylated corn stover hydrolysate from NREL) diluted to a concentration, such that final glucose concentration is approximately 75 g/L glucose and xylose is approximately 40 g/L, plus 100 mM potassium phosphate and 1 g/L ammonium sulfate.

(151) In another embodiment, the medium includes a mixture (e.g., a 10:1 to 5:1 mixture) of Difco™ Yeast Nitrogen Base (YNB) without amino acids (includes a long list of trace elements and some vitamins like thiamine and 5 g/L ammonium sulfate) with Complete Supplement Mix (CSM, several amino acids and some nucleotides, from Sunrise Science Products, Inc.) plus 100 mM potassium phosphate plus 75 g/L glucose plus 40 g/L xylose.

(152) The host cell can be incubated in any useful medium. The terms “culture,” “cultivate,” “ferment”, and “incubate” are used interchangeably and refer to the intentional growth, propagation, proliferation, and/or enablement of metabolism, catabolism, and/or anabolism of one or more host cells. The combination of both growth and propagation may be termed proliferation. Culture does not refer to the growth or propagation of microorganisms in nature or otherwise without human intervention. Exemplarily, host cells may be cultivated in a suspension culture or on plates such as, e.g., agar plates. The suspension medium or agar may contain nutrients suitable for the host cells. The cells may be cultivated at aerobic or anaerobic conditions.

(153) Preferably, the cultivation of cells leads to the reproduction of the cells. Reproduction may occur form cell division of the yeast cell(s), budding of the yeast cell(s), formation of spores, formation of one or more gamete(s) and/or sexual reproduction. More preferably, the reproduction of the yeast cell(s) is cell division or budding.

(154) Cultivation of the cells may include cultivation in a laboratory scale, e.g., cultivation of several culture plates or suspension cultures of several milliliters up to few liters culture broth. Cultivation of the cells may further include cultivation in a semi-technical scale, e.g., cultivation of suspension cultures of several liters culture broth and cultivation in an industrial scale, e.g., cultivation of suspension cultures of several liters or even several square meters culture broth. A culture broth can include both host cells and the medium. A suspension culture may optionally be stirred or shaken. A suspension culture may optionally be aerated, ventilated and/or degassed. The cells may be cultivated at a suitable pressure, the pressure may be atmospheric pressure, excess pressure or underpressure. Typically, the cells may be cultivated at atmospheric pressure or slight excess pressure.

(155) Conditions for cultures can be optimized to promote growth. For instance, non-limiting temperatures for cultures can be from about 28° C. to 32° C., and non-limiting culture times can be from three to ten days (e.g., from four to seven days).

(156) Isolation from a Culture

(157) The host cells or byproducts of the host cell can be isolated from the culture. Non-limiting byproducts can be a lipid-derived compound, such as a fatty alcohol or a combination of different fatty alcohols.

(158) In one embodiment, the host cell is cultured in the presence of an organic solvent (e.g., a hydrocarbon solvent, such as dodecane or pentadecane) as an overlay. Upon mixing, the aqueous media and the organic overlay can form an emulsion. As FOH is produced from the cells, it can partition into the organic layer. After mixing is stopped, the organic layer and aqueous layer can be easily separated by way of any isolating methods described herein.

(159) Isolation from culture can include separating the host cells from other components within the suspension, culture, or culture broth. Such separating can include harvesting the host cells or harvesting the lipid-derived compound from the culture. Isolating can include any useful methodology, e.g., centrifugation, chromatography (e.g., affinity, size exclusion, ion-exchange chromatography, and others), crossflow filtration, filtration, or abrasion or swabbing off a solid surface or culture plate. Alternatively, the cells may descent over time or may float due to gassing of the container including such cells. Alternatively, the cells are not isolated, but the cells and the medium are treated further together.

(160) The cells can be harvested and optionally washed. Subsequently, the cells may be optionally lysed by any means known in the art and indicated above. Optionally, the lipid-derived compound(s) may be extracted by solvent extraction, e.g., with an organic solvent. Optionally, the organic solvent may be evaporated subsequently. Alternatively or additionally, the lipid-derived compound(s) may be isolated, depending on their specific chemical nature, by chromatographic methods (e.g., phase chromatography, ion-exchange chromatography, reverse phase chromatography, size exclusion chromatography, high performance liquid chromatography (HPLC), ultrahigh pressure liquid chromatography (UPLC), fast protein chromatography (FPLC)), by electrophoresis, capillary electrophoresis (CE), or by distillation.

(161) The lipid-derived compounds from the culture can be captured by distillation, filtration, phase separation, as well as and/or solvent co-extraction. Any useful distillation and extraction techniques can be employed, including flash extraction, ionic liquid extraction, etc., to isolate one or more lipid phases, oils, aqueous phases, aqueous co-products, nutrients, etc. Phase separation can include any that separate liquid from solid phases, as well as separate two or more phases that can be differentiated based on solubility, miscibility, etc., (e.g., as those present in non-aqueous phases, aqueous phases, lipophilic phases, etc.) in any useful solvent (e.g., an organic solvent, an aqueous solvent, water, buffer, etc.). Phase separation techniques include flash separation, acid absorption, filtration, distillation, solvent extraction, ion liquid extraction, etc. The resultant products and co-products can include one or more intermediate products that can optionally be processed to form useful end-use products.

EXAMPLES

Example 1: Manipulation of tRNA Thiolation Gene Ncs2 for Enhanced Production of Fatty-Acyl-CoA Derived Chemicals in R. toruloides

(162) Fatty alcohols are a versatile class of chemicals with many consumer and industrial applications. The Agile Biofoundry is developing strains of the oleaginous yeast Rhodosporidium toruloides (also known as Rhodotorula toruloides) to convert lignocellulosic hydrolysate into fatty alcohols (see, e.g., Liu D et al., “Exploiting nonionic surfactants to enhance fatty alcohol production in Rhodosporidium toruloides,” Biotechnology and Bioengineering 2020; 117: 1418-1425).

(163) There are several aspects of bioconversion of lignocellulose derived carbon to fatty alcohols in R. toruloides that may include optimization to achieve a commercially viable process. These include process optimization of extraction and separation of hydrophobic fatty alcohols from liquid cultures, mitigation of toxic effects of high concentrations of fatty alcohols on production the production host, fermentation and media optimization, and/or optimization of expression of heterologous enzymes in a non-model yeast. In particular, we explored global remodeling of central carbon metabolism to improve flux to fatty alcohols.

(164) The immediate precursor to long chain fatty alcohols is fatty-acyl-CoA. Fatty-acyl-CoA sits at the nexus of the fatty acid biosynthesis pathway at the cytosol/endoplasmic reticulum (ER) membrane, the network of pathways that participate in membrane lipid synthesis and recycling in the ER and endomembrane network, the carbon storage pathway for triacylglyceride synthesis and the interface of the ER and the lipid droplet, and the fatty acid catabolic pathway through beta-oxidation of fatty-acyl-CoA in the peroxisome and mitochondria. Thus, fatty-acyl-CoA is the product or substrate of numerous enzymes in all cellular compartments, as well as a participant in many reactions essential for cell survival.

(165) In order to maximize carbon flux to fatty alcohol, the flux into these alternate fates for fatty acyl-CoA can be minimized. However, in many cases, crude gene deletions and elimination of the competing pathways could be lethal to the cell, and the enzymes involved are so numerous as to make direct targeting of them all prohibitively laborious given the current state of genome engineering tools for R. toruloides. Thus, as part of our genome engineering strategy, we set out to identify single gene deletions with global effects on carbon metabolism that are synergistically beneficial to increasing available fatty-acyl-CoA. Such single gene deletions can be optionally combined with other gene modifications to further tune production of desired lipid-derived compounds, such as fatty alcohol.

(166) We have identified dozens of genes with altered lipid accumulation in R. toruloides through a global functional genomics screen of cell buoyancy and fluorescence activated cell sorting (see, e.g., Coradetti S T et al., “Functional genomics of lipid metabolism in the oleaginous yeast Rhodosporidium toruloides,” eLife 2018; 7: Article No. e32110 (55 pages)). Many of the identified genes had only very general functional predictions by sequence homology or functional predictions that did not obviously explain their lipid accumulation phenotypes. Several of these mutants were selected for further study in a fatty alcohol production context, in the hopes that altered lipid accumulation would also result in altered fatty alcohol production and shed light on function the metabolic regulatory network we aim to optimize.

(167) One of these mutations was the deletion of protein ID 10764, ortholog of Saccharomyces cerevisiae gene ncs2. This gene has annotated function in the thiolation of several tRNAs. It has been noted in S. cerevisiae that carbon metabolism are altered in the ncs2 deletion mutants, with major changes in phosphate acquisition, amino acid metabolism, and storage carbohydrates, leading to a hypothesis that gene has some role in nutrient sensing (see, e.g., Gupta R et al., “A tRNA modification balances carbon and nitrogen metabolism by regulating phosphate homeostasis,” eLife 2019; 8: Article No. e44795 (33 pages)), but the mechanism and adaptive function of that regulation remains unclear. To date, we are aware of no investigation of ncs2s effect on lipid metabolism in S. cerevisiae or any other species.

(168) Protein ID 10764 is predicted to be a 612 amino acid protein containing the interpro domain IPR019407 conserved in cytoplasmic tRNA thiolation proteins. The most closely related gene in S. cerevisiae is the tRNA thiolation protein ncs2, apparently orthologous to R. toruloides protein ID 10764. The Ncs2 protein sequence is well conserved across diverse eukaryotes. FIG. 1A shows a phylogenetic tree built from significant sequence matches (BLAST) from 17 model eukaryote proteomes. Orthologs are present in single copy across fungi, plants, animals and early diverging eukaryotes, suggesting highly conserved molecular function.

(169) Thus far, the function of ncs2 function has been characterized mainly in S. cerevisiae. This function is the thiolation of the wobble position in tRNAs for glutamine, glutamate, and lysine. Thiolation enhances translation efficiency of codons using those tRNAs, but only modestly. Currently, the adaptive function of ncs2 thus remains unclear, though multiple studies have demonstrated altered carbon and amino acid metabolism. Gupta et al., supra, argued that thiolation of these tRNAs may be an indirect way of sensing sulfur availability, with hypo-thiolation serving as a signal for sulfur scarcity, which in turn triggers a phosphate-limited metabolic response through regulation of phosphate acquisition genes.

(170) Deletions for ncs2 and other proteins in the tRNA thiolation pathway had significant reductions in lipid accumulation in a high throughput functional genomics study of R. toruloides. How this low lipid phenotype might be consistent with a role in sulfur sensing is unclear, as previous studies on nutrient limitation in R. toruloides have observed increased lipid accumulation in conditions of sulfur limitation and phosphate limitation (see, e.g., Wu S et al., “Microbial lipid production by Rhodosporidium toruloides under sulfate-limited conditions,” Bioresource Technology 2011; 102(2): 1803-1807; and Wang Y et al., “Systems analysis of phosphate-limitation-induced lipid accumulation by the oleaginous yeast Rhodosporidium toruloides,” Biotechnologyfor Biofuels 2018; 11: Article No. 148 (15 pages)).

Example 2: Deletion Mutants for Ncs2 have Increased Fatty Alcohol Production

(171) The ncs2 gene was deleted by transforming a Ku70 deficient strain of R. toruloides IFO 0880 expressing fatty acyl-CoA reductase from Marinobacter aquaeolei (ABF archived strain ABF_006072) with a nourseothricin resistance cassette, replacing the ncs2 coding sequence by homologous recombination. The resulting strain is stored in the ABF strain archive as strain ABF_006749.

(172) The ncs2 deletion strain was grown on media prepared from deacetylated mechanically refined enzymatic hydrolysate (DMR-EH) from corn stover (see, e.g., Chen X et al., “DMR (deacetylation and mechanical refining) processing of corn stover achieves high monomeric sugar concentrations (230 g/L) during enzymatic hydrolysis and high ethanol concentration (>10% v/v) during fermentation without hydrolyzate purification or concentration,” Energy & Environmental Science 2016; 9(4): 1237-1245) provided by NREL.

(173) In the final media composition, concentrated DMR-EH was diluted to approximately 75 g/L glucose, 40 g/L xylose, with addition of 1 g/L or 5 g/L ammonium sulfate, 100 mM potassium phosphate, and 0.1% (v/v) Tergitol™ (an ethoxylated alcohol that serves as a linear non-ionic surfactant). Cultures were incubated 3 to 6 days at 30° C., 1000 rpm in an M2P labs 48-well flower plate with 800 μl culture volume and 200 μl dodecane overlay. Total fatty alcohol was measured from the dodecane overlay by the additional of 100 μl dodecane with 100 mg of 1-tridecanol, mixing, and then separating the organic overlay for analysis by GC-FID. Fatty alcohols of 16 and 18 carbon length were then quantified against the 1-tridecanol internal standard. Total fatty alcohol concentrations in Ancs2 cultures were 2-3 times that of the parent strain (FIG. 6).

Example 3: Deletion Mutants for Ncs2 have Globally Altered Lipid and Proteomic Profiles

(174) To explore the mechanism of increased fatty alcohol production in Ancs2 mutants, we subjected three day old DMR-EH grown cultures to metabolomic, proteomic, and lipidomic analysis and compared them to the parent strain in the same conditions. A similar analysis was carried out on several other mutant strains as part of a larger study. In FIGS. 7-8, comparable data from strain STC105 (ABF_006090, fatty alcohol producing strain over-expressing a native lipase) and strain STC153 (ABF_006597, fatty alcohol producing strain expressing a nicotinamide nucleotide transhydrogenase, NNT) are included as an informative control as STC105 and STC153 also exhibit increased fatty alcohol production through independent mechanisms.

(175) Of 100 water soluble metabolites quantified, relatively few had different abundance in the Ancs2 mutant sufficient for a P-value <0.05 with an independent T-test, without multiple hypothesis correction. In the low nitrogen condition, which had the greatest fatty alcohol production, only 3-phosphoglycerate, citrate, glycerol-3-phosphate, D-ribose-5-phosphate, and sucrose were less abundant in the Ancs2 mutant than the parent strain, and only 1-octadecanol was more abundant.

(176) Of 3375 proteins with measurable peptide abundances in global proteomics analysis, 562 had significantly different abundances in the ncs2 deletion mutant than the parent strain in the same condition and this differential abundance was similar between high and low nitrogen cultures. Notably, there was a high degree of overlap between the proteomic changes in the ncs2 mutant and the lipase over-expression mutant (FIGS. 7A-7B), but essentially no overlap with the NNT mutant. These results demonstrate that the proteomic changes are not a result of fatty alcohol toxicity or another indirect effect of increased flux to fatty alcohols.

(177) For FIGS. 7A-7B, the lipase overexpression (OE) strain was ABF_006090 (or STC0105), which included a putative triacylglycerol (TAG) lipase (R. toruloides IF00880 v4.0, Protein Id: 8386; UniProtKB No. A0A2T0AH33) driven by the Tef1 promoter. The NNT overexpression strain was ABF_006596 (aka STC0152), which included an NNT (NAD(P) transhydrogenase or nicotinamide nucleotide transhydrogenase) from Haliaeetus leucocephalus driven by the Tef1 promoter.

(178) Generally, proteomic analysis of the ncs2 mutant were more comparable to the global expression profile for the lipase overexpression (OE) mutant than the NNT OE mutant. Without wishing to be limited by mechanism, lipid metabolism is likely perturbed in the ncs2 mutant, which may provide the higher FOH production.

(179) Of 301 lipid species measured with global proteomics, 60 had significantly altered abundance in the ncs2 deletion strain versus its parent, particularly in low nitrogen conditions (FIG. 8). This pattern of altered lipid abundance was markedly different in the lipase over expression strain. These results suggest that while altered lipid abundance is sufficient to trigger many elements of the Ancs2 mutants proteomic shift, it is unlikely that the Ancs2 mutant achieves this through a shared mechanism with the lipase over expression strain, as their lipid profiles are globally divergent and in particular that Ancs2 mutant has only modest lipid changes in the high nitrogen condition, yet a very consistent proteomic profile between high and low nitrogen conditions. Without wishing to be limited by mechanism, the Ancs2 mutant is altering proteomic abundance through an unknown, lipid independent mechanism.

(180) Regardless of mechanism, the Ancs2 mutant exhibit a synergistic combination of altered protein abundance that shifted carbon flux from lipid synthesis towards fatty alcohol synthesis by inhibiting several early steps in the diacylglycerol and phospholipid synthesis, thus removing a major sink for fatty-acyl-CoA, while increasing abundance of NADPH though activity of malic enzyme Meal, thus promoting fatty-acyl-CoA synthesis. Liberation of fatty-acyl-CoA from storage lipids acids is also reduced by down regulation of several lipases and long chain fatty acyl-CoA synthetases, but that is balanced by concomitant down regulation of fatty-acyl-CoA degradation through beta-oxidation. These changes are summarized in FIG. 9.

(181) Quantitative changes in relative protein intensity for several proteins (provided in Table 1) in these pathways are shown in FIGS. 10A-10D. While many of these changes in abundance are modest (less than 2-fold, or smaller than 1 in log 2 space), the combined effect across multiple pathways is significant. This effect can be evidenced by the consistent depletion of several lipid classes, particularly diacylglycerides, the final shared precursor of triacylglycerides and several membrane lipids (FIG. 11).

(182) In Table 1, the Protein Id correspond to protein IDs provided for the JGI's genome assembly for Rhodosporidium toruloides, which can be accessed at mycocosm.jgi.doe.gov/Rhoto_IFO0880_4/Rhoto_IF00880_4.home.html.

(183) TABLE-US-00001 TABLE 1 List of genes and Protein Id Abbreviation Annotation Group Protein Id Mea1 malate dehydrogenase (oxaloacetate- Fatty Acid (FA) 12761 decarboxylating) (NADP+); malic Synthesis enzyme (EC: 1.1.1.40); NAD-dependent malic enzyme (EC: 1.1.1.38) Gpd1-1 glycerol-3-phosphate dehydrogenase Lipid Synthesis 12154 (NAD+) (EC:1.1.1.8) Gpd1-2 glycerol-3-phosphate dehydrogenase Lipid Synthesis 14576 (NAD+)(EC:1.1.1.8) Ayr1 1-acyl dihydroxyacetone phosphate Lipid Synthesis 15575 reductase and related dehydrogenases; acylglycerone-phosphate reductase (EC:1.1.1.101) Sct1 phospholipid/glycerol acyltransferase; Lipid Synthesis 15435 glycerol-3-phosphate O-acyltransferase/ dihydroxyacetone phosphate acyltransferase Slc1 lysophosphatidate acyltransferase; 1- Lipid Synthesis 10427 acylglycerol-3-phosphate O- acyltransferase (EC:2.3.1.51) Pah1 LPIN phosphatidate phosphatase; Lipid Synthesis 12485 phosphatidate phosphatase (EC:3.1.3.4) Ale1 lysophospholipid acyltransferase; Lipid Synthesis 16030 membrane-bound O-acyltransferase (MBOAT) family; acyltransferase Lcb1 serine C-palmitoyltransferase Lipid Synthesis 10303 (EC:2.3.1.50) Are1 sterol O-acyltransferase/diacyl O- Lipid Synthesis 11799 acyltransferase (EC:2.3.1.26); MBOAT family Dga1 MGAT2 2-acylglycerol O- Lipid Synthesis 16460 acyltransferase 2; 2-acylglycerol O- acyltransferase (EC:2.3.1.22) Lro1 phospholipid:diacylglycerol Lipid Synthesis 16477 acyltransferase (EC:2.3.1.158); lechitin: cholesterol acyltransferase Acc1 acetyl-CoA carboxylase; acetyl-CoA FA Synthesis 8639 carboxylase, biotin carboxylase subunit (EC:6.4.1.2, 6.3.4.14); acetyl-CoA carboxyl transferase domain of homomeric ACCase (EC 6.4.1.2) Fas1 fatty-acyl-CoA synthase system FA Synthesis 8670 (EC:2.3.1.86); fatty acid synthase subunit beta, fungi type Fas2 fatty-acyl-CoA synthase system FA Synthesis 8777 (EC:2.3.1.86); fatty acid synthase subunit alpha, fungi type ACAD10 acyl-CoA dehydrogenase family Beta-Oxidation 10408 member 10; medium-chain acyl-CoA dehydrogenase (EC:1.3.8.7); acyl-CoA dehydrogenase (EC: 1.3.99.3) FOX2 multifunctional beta-oxidation protein Beta-Oxidation 11362 (EC: 1.1.1.-4.2.1.-]); short-chain dehydrogenase/reductase (SDR) POT1-1 acetyl-CoA C-acyltransferase 1 Beta-Oxidation 13813 (EC:2.3.1.16) POT1-2 acetyl-CoA C-acyltransferase Beta-Oxidation 9065 (EC:2.3.1.16); 3-oxoacyl CoA thiolase; 3-ketoacyl-CoA thiolase (EC:2.3.1.16) ACADM acd acyl-CoA dehydrogenase; acyl-CoA Beta-Oxidation 12570 dehydrogenase (EC: 1.3.99.3); medium- chain acyl-CoA dehydrogenase (EC:1.3.8.7) EHD3 enoyl-CoA hydratase (EC:4.2.1.17) Beta-Oxidation 14805 HADH 3-hydroxyacyl-CoA dehydrogenase Beta-Oxidation 11203 (EC: 1.1.1.35) ACAA2 acetyl-CoA acyltransferase 2; acetyl- Beta-Oxidation 8885 CoA C-acyltransferase (EC: 2.3.1.16) TGL2 triacylglycerol lipase (EC:3.1.1.3) Lipases and 10393 LCFA TGL2 triacylglycerol lipase (EC:3.1.1.3) Lipases and 14317 LCFA YEH2 lysosomal acid lipase/cholesteryl ester Lipases and 14617 hydrolase; sterol esterase (EC:3.1.1.13) LCFA TGL5 Predicted esterase of the alpha-beta Lipases and 9746 hydrolase superfamily; patatin-like LCFA phospholipase RTO4_8386 arylacetamide deacetylase; Lipases and 8386 triacylglycerol lipase (EC:3.1.1.3) LCFA RTO4_8726 arylacetamide deacetylase; alpha/beta Lipases and 8726 hydrolase fold LCFA RTO4_8745 arylacetamide deacetylase; alpha/beta Lipases and 8745 hydrolase fold LCFA RTO4_8919 arylacetamide deacetylase; alpha/beta Lipases and 8919 hydrolase fold LCFA RTO4_9174 arylacetamide deacetylase; alpha/beta Lipases and 9174 hydrolase fold LCFA RTO4_9181 arylacetamide deacetylase; alpha/beta Lipases and 9181 hydrolase fold; 2-oxoglutarate LCFA dehydrogenase E1 component (EC: 1.2.4.2) RTO4_10002 arylacetamide deacetylase; alpha/beta Lipases and 10002 hydrolase fold LCFA RTO4_11473 arylacetamide deacetylase; alpha/beta Lipases and 11473 hydrolase fold LCFA RTO4_11568 arylacetamide deacetylase; alpha/beta Lipases and 11568 hydrolase fold LCFA RTO4_14459 arylacetamide deacetylase; Lipases and 14459 triacylglycerol lipase (EC:3.1.1.3) LCFA RTO4 14706 arylacetamide deacetylase; alpha/beta Lipases and 14706 hydrolase fold LCFA RTO4_16608 arylacetamide deacetylase; alpha/beta Lipases and 16608 hydrolase fold LCFA RTO4_11739 predicted lipase/calmodulin-binding Lipases and 11739 heat-shock protein; DAGL sn1-specific LCFA diacylglycerol lipase RTO4_15065 hormone-sensitive lipase (HSL) Lipases and 15065 LCFA MGLL lysophospholipase; acylglycerol lipase Lipases and 14158 (EC:3.1.1.23) LCFA YJU3 lysophospholipase; acylglycerol lipase Lipases and 9728 (EC:3.1.1.23) LCFA Faa2-1 long-chain acyl-CoA synthetase (AMP- Lipases and 12538 forming) LCFA Faa2-2 long-chain acyl-CoA synthetase (AMP- Lipases and 12555 forming); long-chain-fatty-acid - CoA LCFA ligase (EC:6.2.1.3) Faa1-1 acyl-CoA synthetase Lipases and 15746 LCFA Faa1-2 acyl-CoA synthetase; long-chain-fatty- Lipases and 11167 acid---CoA ligase (EC:6.2.1.3) LCFA Faa1-3 acyl-CoA synthetase; long-chain-fatty- Lipases and 15748 acid---CoA ligase (EC:6.2.1.3) LCFA

Example 4: Further Mutants for Increasing Fatty Alcohol Production

(184) The host cell can include one, two, or more gene modifications to promote fatty alcohol (FOH) production. Such gene modification can result in overexpression, under expression, or no expression of the target gene. Expression, including overexpression, of the target gene can include insertion of the gene using a plasmid, in which expression can include use of a promoter (e.g., an inducible promoter). To reduce or remove expression, the target gene can be removed or modified.

(185) FIG. 12 shows FOH production in various strains that overexpress particular target genes, and Table 2 provides a list of these target genes. Such targets can include proteins that are implicated in increasing acetyl-CoA, NADPH (nicotinamide adenine dinucleotide phosphate), and/or fatty-acyl-CoA production. In Table 2, sources include the JGI's genome assembly for Rhodosporidium toruloides (accessed at mycocosm.jgi.doe.gov/Rhoto_IFO0880_4/Rhoto_IF00880_4.home.html), in which proteins are provided as RTO4_XXX and XXX indicates the protein ID. Also provided are RefSeq numbers (accessed at ncbi.nlm.nih.gov/protein) and UniProtKB numbers (accessed at uniprot.org).

(186) TABLE-US-00002 TABLE 2 List of genes in FIG. 12 Abbreviation Annotation Source PDC pyruvate decarboxylase RTO4_15791 ALD aldehyde dehydrogenase (NAD+) RTO4_12042 ACS1 acetyl-CoA synthetase RTO4_14597 Cat2 carnitine O-acetyltransferase RT04_14245 ScCat2 carnitine O-acetyltransferase, UniProtKB No. P32796 mitochondrial ACL ATP citrate (pro-S)-lyase RTO4_9726 ME malate dehydrogenase RTO4_12761 (oxaloacetate-decarboxylating) (NADP+) IDH isocitrate dehydrogenase RTO4_11129 NNT_human NAD(P) transhydrogenase, UniProtKB No. Q13423 mitochondrial NNT_A_aegypti proton-translocating NAD(P)(+) RefSeqNo. XP_001662741.2; transhydrogenase UniProtKB No. Q16LL0 NNT_H_leucocephalus proton-translocating NAD(P)(+) RefSeqNo. XP_009919164.1; transhydrogenase UniProtKB No. A0A091Q7M4 NNT_G_trabeum Proton-translocating NAD(P)(+) RefSeqNo. XP_007863552.1; transhydrogenase UniProtKB No. S7QGM1 EcfadD long-chain-fatty-acid-CoA ligase UniProtKB No. P69451 FAA3 long-chain acyl-CoA synthetase RTO4_12555 FAA2 long-chain acyl-CoA synthetase RTO4_11167 FAA2-2 long-chain acyl-CoA synthetase RTO4_15748 FAA3_Nc long-chain acyl-CoA synthetases RTO4 _12538 (AMP-forming) Lip_Rt_8386 arylacetamide deacetylase RTO4_8386 TGL2_S288C triacylglycerol lipase 2 UniProtKB No. P54857 Lip_Tlanuginosus lipase UniProtKB No. O59952 Lip_Mouse patatin-like phospholipase UniProtKB No. Q8BJ56 domain-containing protein 2 Lip_Creinhardtii Lipase-3 domain-containing JGI Phytosome protein Cre09.g390615; UniProtKB No. A0A2K3DE56 Lip_human hormone-sensitive lipase UniProtKB No. Q05469-2 (without tag) Lip_human_tag3 hormone-sensitive lipase UniProtKB No. Q05469-2 (with tag at the C-terminus) Lip_human_tag5 hormone-sensitive lipase UniProtKB No. Q05469-2 (with tag at the N-terminus) Rt_Lip_15065 hormone-sensitive lipase RTO4_15065 Rt_Lip_8919 arylacetamide deacetylase RTO4_8919 Rt_Lip_9374 predicted lipase RTO4_9374 Rt_Lip_10393 Superfamily SSF53474 protein RTO4_10393 (alpha/beta-hydrolases) Rt_Lip_8386 arylacetamide deacetylase RTO4_8386 Rt_Lip_12712 Superfamily SSF53474 protein RTO4_12712 (secretory lipase) SCD stearoyl-CoA desaturase RTO4_9730 ACC acetyl-CoA carboxylase/biotin RTO4_8639 carboxylase l Stacked FAR NAD-dependent epimerase/ Multiple copies of FAR dehydratase (RefSeq No. YP_959486.1) FAR NAD-dependent epimerase/ Parent strain expressing FAR dehydratase (RefSeq No. YP_959486.1)

(187) Knock-out strains were also characterized. FIG. 13 shows FOH production in various knock-out strains that lack particular target genes. Such targets can include proteins that are implicated in triacylglyceride (TAG) biosynthesis, β-oxidation of fatty acid molecules, and other processes. FIG. 14 shows single knock-out of a target gene, as well as multiple knock-out of two target genes. Multi-knock-out strains can be developed to further enhance FOH production, and the present disclosure encompasses strains having a plurality of mutant genes, in which each gene encodes a different target protein. Non-limiting target proteins can be any combination of proteins described herein.

Example 5: Effect of Culture Conditions on Fatty Alcohol Production

(188) FIG. 15 shows the effect of culture conditions of fatty alcohol (FOH) production for the NCS2 mutant strain (indicated as “STC0179”), its parental strain (indicated as “STC0113”), and STC0180. The culture can include various media, including DMR hydrolysate media or “mock DMR” media having entirely synthetic components to simulate the composition of a corn stover hydrolysate. As can be seen in FIG. 15, the type of culture media and concentration of nitrogen within the culture can affect FOH production. Such differences can be controlled by using host cells having low catabolism of FOH, in which such catabolism may be influenced by minor components of the media (e.g., trace metals and byproducts of the biomass hydrolysis process).

(189) The effect of culture conditions was also assessed for different single knock-out and multi-knock-out strains. These strains included deletion of a fatty alcohol oxidase (RTO4_10253, FAO1) and/or an aldehyde dehydrogenase (RTO4_16323, HFD1) in a strain expressing fatty alcohol reductase (Maq_2220). The strain displayed dramatically reduced growth on fatty alcohols as a sole carbon source (˜90% reduction in growth rate on 1-hexadecanol, demonstrating that we did indeed disrupt fatty alcohol catabolism) and significantly increased fatty alcohol production (2-4 fold depending on condition tested) (FIGS. 16A-16B and FIGS. 17A-17B).

(190) The defined media (indicated as “Mock” in FIG. 16B and FIG. 17B) included Difco™ Yeast Nitrogen Base (YNB) without amino acids (includes a long list of trace elements and some vitamins like thiamine and 5 g/L ammonium sulfate) plus Complete Supplement Mix (CSM, several amino acids and some nucleotides, from Sunrise Science Products, Inc., Knoxville, Tenn.) plus 100 mM potassium phosphate plus 75 g/L glucose plus 40 g/L xylose.

(191) Notably, fatty alcohol production was more consistent between media conditions than for the parental strain, consistent with our hypothesis that variation in fatty alcohol production between those conditions can be strongly influenced by variation in fatty alcohol catabolism.

Other Embodiments

(192) All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

(193) While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

(194) Other embodiments are within the claims.

CONCLUSION

(195) Although the foregoing embodiments have been described in some detail for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims. It should be noted that there are many alternative ways of implementing the processes, systems, and apparatus of the present embodiments. Accordingly, the present embodiments are to be considered as illustrative and not restrictive, and the embodiments are not to be limited to the details given herein.