Modified host cells for high efficiency production of vanillin
Provided herein are genetically modified host cells, compositions, and methods for improved production of vanillin and/or glucovanillin. The host cells, compositions, and methods described herein provide an efficient route for the heterologous production of vanillin and/or glucovanillin and any compound that can be synthesized or biosynthesized from either or both.
1 . A genetically modified yeast host cell capable of producing vanillin or glucovanillin comprising:
(a) one or more nucleic acids comprising nucleic acids capable of overexpressing SHM2, SAH1, MET6, and MET13; or SHM2, SAH1, MET6, and a chimeric MET13; and
(b) deletion of ADH6.
2 . The genetically modified host cell of claim 1 , wherein the one or more nucleic acids further comprise nucleic acids capable of overexpressing SAM1.
3 . The genetically modified host cell of claim 2 , wherein SAM1 encodes an amino acid sequence at least 80, 85, 90, 95, 99, or 100% identical to the Sam1 amino acid sequence encoded by nucleotides 7394-6051 of SEQ ID NO:8.
4 . The genetically modified host cell of claim 1 , wherein the one or more nucleic acids further comprise nucleic acids capable of overexpressing SAM2.
5 . The genetically modified host cell of claim 4 , wherein SAM2 encodes an amino acid sequence at least 80, 85, 90, 95, 99, or 100% identical to the Sam2 amino acid sequence encoded by nucleotides 8087-9440 of SEQ ID NO:8.
6 . The genetically modified host cell of claim 1 , wherein the one or more nucleic acids further comprise nucleic acids capable of overexpressing SAM1 and SAM2.
7 . The genetically modified host cell of claim 1 that is capable of overexpressing SHM2, SAH1, MET6 under one or more inducible promoters.
8 . The genetically modified host cell of claim 1 , wherein the one or more nucleic acids further comprise nucleic acids capable of overexpressing MET12.
9 . The genetically modified host cell of claim 1 , wherein the chimeric MET13 comprises a S. cerevisiae MET13 N-terminal domain and an Arabadopsis MTHFR C-terminal domain.
10 . The genetically modified host cell of claim 1 , wherein the nucleic acids capable of overexpressing MET6 comprise two copies of MET6.
11 . The genetically modified host cell of claim 1 , wherein the one or more nucleic acids further comprise nucleic acids capable of overexpressing MET12; and
wherein the one or more nucleic acids capable of overexpressing MET6 comprise two or more copies of MET6.
12 . The genetically modified host cell of claim 11 wherein MET12 and the two or more copies of MET6 are overexpressed under one inducible promoter.
13 . The genetically modified host cell of claim 12 wherein, SAM1 and SAM2 are overexpressed under one inducible promoter.
14 . The genetically modified host cell of claim 1 wherein, SHM2, SAH1, MET6, and MET13 or chimeric MET13 are overexpressed under one or more inducible promoters.
15 . The genetically modified host cell of claim 1 wherein, SHM2, SAH1, MET6, and MET13 or chimeric MET13 are overexpressed under one inducible promoter.
16 . The genetically modified host cell of claim 1 further comprising deletion of GRE2.
17 . The genetically modified host cell of claim 1 further comprising deletion of YGL039W.
18 . The genetically modified host cell of claim 1 further comprising one or more nucleic acids expressing AroB, AroD, and AroZ.
19 . The genetically modified host cell of claim 18 , wherein the AroB gene product comprises an amino acid sequence at least 80, 85, 90, 95, 99, or 100% identical to the AroB amino acid sequence encoded by nucleotides 5596-6684 of SEQ ID NO:5, nucleotides 2930-4143 of SEQ ID NO:11, or nucleotides 2906-4119 of SEQ ID NO:17.
20 . The genetically modified host cell of claim 18 , wherein the AroD gene product comprises an amino acid sequence at least 80, 85, 90, 95, 99, or 100% identical to the AroD amino acid sequence encoded by nucleotides 7951-7193 of SEQ ID NO:5, nucleotides 5775-4892 of SEQ ID NO:11, or nucleotides 5751-4868 of SEQ ID NO:17.
21 . The genetically modified host cell of claim 18 , wherein the AroZ gene product comprises an amino acid sequence at least 80, 85, 90, 95, 99, or 100% identical to the AroZ amino acid sequence encoded by nucleotides 1134-2237 of SEQ ID NO:5, nucleotides 1095-2323 of SEQ ID NO:11, or nucleotides 1780-55218.
22 . The genetically modified host cell of claim 1 further comprising one or more nucleic acids expressing AroB, AroD, AroF, and AroZ.
23 . The genetically modified host cell of claim 22 , wherein the AroF gene product comprises an amino acid sequence at least 80, 85, 90, 95, 99, or 100% identical to the AroF amino acid sequence encoded by nucleotides 3761-2961 of SEQ ID NO:5, nucleotides 7577-6382 of SEQ ID NO:11, or nucleotides 6877-8072 of SEQ ID NO:17.
24 . The genetically modified host cell of claim 1 further comprising one or more nucleic acids expressing E. coli AroB, E. coli AroD, E. coli AroF, and Podospora pauciseta AroZ.
25 . The genetically modified host cell of claim 1 further comprising one or more nucleic acids expressing PPTASE and ACAR.
26 . The genetically modified host cell of claim 25 , wherein the PPTASE gene product comprises an amino acid sequence at least 80, 85, 90, 95, 99, or 100% identical to the PPTASE amino acid sequence encoded by nucleotides 1618-825 of SEQ ID NO: 6, nucleotides 7070-5883 of SEQ ID NO: 14, or nucleotides 5883-7070 of SEQ ID NO: 19.
27 . The genetically modified host cell of claim 1 further comprising one or more nucleic acids expressing UDP-glycosyltransferase (UGT).
28 . The genetically modified host cell of claim 27 , wherein the UGT gene product comprises an amino acid sequence at least 80, 85, 90, 95, 99, or 100% identical to the UGT amino acid sequence encoded by nucleotides 2214-769 and 2907-4352 of SEQ ID NO:10.
29 . The genetically modified host cell of claim 1 further comprising one or more nucleic acids expressing Arabidopsis thaliana UGT.
30 . The genetically modified host cell of claim 1 , wherein SHM2, SAH1, MET6, and MET13 or the chimeric MET13 are each expressed from a GAL promoter, and wherein a GAL80 gene is expressed from a MAL promoter.
31 . The genetically modified host cell of claim 1 , wherein the yeast host cell is Saccharomyces cerevisiae.
32 . The genetically modified host cell of claim 1 , wherein SAH1 encodes an amino acid sequence at least 80, 85, 90, 95, 99, or 100% identical to the Sah1 amino acid sequence encoded by nucleotides 2094-554 of SEQ ID NO:8.
33 . The genetically modified host cell of claim 1 , wherein MET6 encodes an amino acid sequence at least 80, 85, 90, 95, 99, or 100% identical to the Met6 amino acid sequence encoded by nucleotides 2787-5302 of SEQ ID NO:8 or nucleotides 3249-5764 of SEQ ID NO:14.
34 . The genetically modified host cell of claim 1 , wherein SHM2 encodes an amino acid sequence at least 80, 85, 90, 95, 99, or 100% identical to the Shm2 amino acid sequence encoded by nucleotides 9381-10975 of SEQ ID NO:14.
35 . The genetically modified host cell of claim 1 , wherein MET12 encodes an amino acid sequence at least 80, 85, 90, 95, 99, or 100% identical to the Met12 amino acid sequence encoded by nucleotides 8688-6513 of SEQ ID NO:14.
36 . The genetically modified host cell of claim 1 , wherein MET13 encodes an amino acid sequence at least 80, 85, 90, 95, 99, or 100% identical to the Met13 amino acid sequence encoded by nucleotides 2556-554 of SEQ ID NO: 14, and the chimeric MET13 encodes an amino acid sequence at least 80, 85, 90, 95, 99, or 100% identical to the chimeric Met13 amino acid sequence encoded by nucleotides 554-2338 of SEQ ID NO:23.
37 . The genetically modified host cell of claim 1 that produces at least a 5, 10, 15, or 20% increase in peak cumulative yield or productivity, or both, compared to a parent strain.
38 . The genetically modified host cell of claim 37 that produces up to 7% increase in peak cumulative yield and up to 17% productivity, compared to a parent strain.
39 . A method for producing vanillin or one or more glucovanillins comprising the steps:
(a) culturing a population of the host cells of claim 1 in a medium with a carbon source under conditions suitable for making vanillin or one or more glucovanillins to yield a culture broth; and
(b) recovering said vanillin or one or more glucovanillins from the culture broth.