Bioproduction of phenethyl alcohol, aldehyde, acid, amine, and related compounds

Patent metadata

Field	Value
Publication number	US-2020232000-A1
Application number	US-201816615689-A
Country	US
Kind code	A1
Filing date	May 23, 2018
Priority date	May 23, 2017
Publication date	Jul 23, 2020
Grant date	—

Abstract

This invention relates to the bioproduction of substituted or unsubstituted phenylacetaldehyde, 2-phenylethanol, phenylacetic acid or phenylethylamine by subjecting a starting material comprising glucose, L-phenylalanine, substituted L-phenylalanine, styrene or substituted styrene to a plurality of enzyme catalyzed chemical transformations in a one-pot reaction system, using recombinant microbial cells overexpressing the enzymes. To produce phenylacetaldehyde from styrene, the cells are modified to overexpress styrene monooxygenase (SMO) and styrene oxide isomerase (SOI). To produce phenylacetic acid from styrene, SMO, SOI and aldehyde dehydrogenase are overexpressed. Alternatively, to produce 2-phenylethanol, SMO, SOI and aldehyde reductase or alcohol dehydrogenase are overexpressed, while to produce phenylethylamine, SMO, SOI and transaminase are overexpressed.

First claim

1 . A method for bioproduction of substituted or unsubstituted phenylacetaldehyde, 2-phenylethanol, phenylacetic acid or phenylethylamine by one or more recombinant microbial cells genetically engineered to overexpress, relative to a wild type cell, at least one enzyme, which method comprises subjecting a starting material to a plurality of enzyme-catalyzed chemical transformations in a one-pot reaction system, wherein the starting material is selected from a group comprising glucose, L-phenylalanine or substituted L-phenylalanine, styrene or substituted styrene.

2 . The method of claim 1 , wherein the genetically engineered cells:
i) overexpress styrene monooxygenase and styrene oxide isomerase for generating substituted or unsubstituted phenylacetaldehyde from styrene or substituted styrene; or ii) overexpress styrene monooxygenase, styrene oxide isomerase and an aldehyde dehydrogenase for generating substituted or unsubstituted phenylacetic acid from styrene or substituted styrene; or iii) overexpress styrene monooxygenase, styrene oxide isomerase, an aldehyde reductase and/or an alcohol dehydrogenase for generating substituted or unsubstituted 2-phenylethanol from styrene or substituted styrene; or iv) overexpress styrene monooxygenase, styrene oxide isomerase and a transaminase for generating substituted or unsubstituted phenylethylamine from styrene or substituted styrene.

3 . The method of claim 2 , wherein in the styrene monooxygenase comprises an amino acid sequence set forth in SEQ ID NOs: 1 and 2, variants, mutants, or fragments thereof; styrene oxide isomerase comprises an amino acid sequence set forth in SEQ ID NO: 3, variants, mutants, or fragments thereof; the aldehyde dehydrogenase comprises an amino acid sequence set forth in SEQ ID NO: 4, variants, mutants, or fragments thereof; the alcohol dehydrogenase comprises an amino acid sequence set forth in SEQ ID NO: 5, variants, mutants, or fragments thereof and the transaminase is ω-transaminase comprises an amino acid sequence set forth in SEQ ID NO: 6, variants, mutants, or fragments thereof.

4 . (canceled)

5 . The method of claim 3 , wherein in the styrene monooxygenase is encoded by a nucleic acid sequence set forth in SEQ ID NO: 7 and 8; styrene oxide isomerase is encoded by a nucleic acid sequence set forth in SEQ ID NO: 9; the aldehyde dehydrogenase is encoded by a nucleic acid sequence set forth in SEQ ID NO: 10; the alcohol dehydrogenase is encoded by a nucleic acid sequence set forth in SEQ ID NO: 11 and the transaminase is ω-transaminase encoded by a nucleic acid sequence set forth in SEQ ID NO: 12.

6 . The method of claim 1 , wherein the genetically engineered cells produce styrene or substituted styrene from L-phenylalanine or substituted L-phenylalanine by a deamination reaction catalyzed by overexpression of an ammonia lyase and a decarboxylation reaction catalyzed by overexpression of a decarboxylase.

7 . The method of claim 6 , wherein the ammonia lyase comprises an amino acid sequence set forth in SEQ ID NO: 13, variants, mutants, or fragments thereof and the decarboxylase comprises an amino acid sequence set forth in SEQ ID NO: 14, variants, mutants, or fragments thereof.

8 . The method of claim 1 , wherein the genetically engineered cells produce L-phenylalanine from glucose by a reaction catalyzed by overexpression of at least one enzyme selected from a group comprising DAHP synthase (AroG), shikimate kinase (AroK), shikimate dehydrogenase (YdiB), chorismate mutase/prephenate dehydratase (PheA) and tyrosine aminotransferase (TyrB), or mutants thereof.

9 . The method of claim 8 , wherein AroG comprises an amino acid sequence set forth in SEQ ID NO: 17, variants, mutants, or fragments thereof; AroK comprises an amino acid sequence set forth in SEQ ID NO: 18, variants, mutants, or fragments thereof; YdiB comprises an amino acid sequence set forth in SEQ ID NO: 19, variants, mutants, or fragments thereof; PheA comprises an amino acid sequence set forth in SEQ ID NO: 20, variants, mutants, or fragments thereof and TyrB comprises an amino acid sequence set forth in SEQ ID NO: 21, variants, mutants, or fragments thereof.

10 . The method of claim 8 , wherein AroG is replaced by a feedback inhibition resistant mutant AroG* encoded by a nucleic acid comprising SEQ ID NO: 27 and/or PheA is replaced by a feedback inhibition resistant mutant PheA* encoded by a nucleic acid comprising SEQ ID NO: 28.

11 . The method of claim 8 , further comprising deletion or inactivation of crr and/or prephenate dehydrogenase (tyrA) genes.

12 . (canceled)

13 . (canceled)

14 . (canceled)

15 . (canceled)

16 . The method of claim 1 , wherein the one-pot reaction system comprises the use of a tri-phasic medium comprising:
(a) an aqueous: organic solvent: solid resin medium; or (b) an aqueous: organic solvent: functionalized nanoparticles medium.

17 . An isolated strain of genetically engineered cells capable of increased bioproduction of substituted or unsubstituted phenylacetaldehyde, 2-phenylethanol, phenylacetic acid or phenylethylamine in a one-pot reaction system compared to wild type cells, wherein the cells overexpress a combination of enzymes selected from the group (i) to (iv) comprising:
i) styrene monooxygenase and styrene oxide isomerase for generating substituted or unsubstituted phenylacetaldehyde from styrene or substituted styrene; ii) styrene monooxygenase, styrene oxide isomerase and an aldehyde dehydrogenase for generating substituted or unsubstituted phenylacetic acid from styrene or substituted styrene; iii) styrene monooxygenase, styrene oxide isomerase, an aldehyde reductase and/or an alcohol dehydrogenase for generating substituted or unsubstituted 2-phenylethanol from styrene or substituted styrene; and iv) styrene monooxygenase, styrene oxide isomerase and a transaminase for generating substituted or unsubstituted phenylethylamine from styrene or substituted styrene.

18 . (canceled)

19 . (canceled)

20 . The isolated strain of genetically engineered cells of claim 17 , wherein the cells produce styrene or substituted styrene from L-phenylalanine or substituted L-phenylalanine by a deamination reaction catalyzed by overexpression of an ammonia lyase and a decarboxylation reaction catalyzed by overexpression of a decarboxylase.

21 . The isolated strain of genetically engineered cells of claim 20 , wherein the ammonia lyase comprises an amino acid sequence set forth in SEQ ID NO: 13, variants, mutants, or fragments thereof and the decarboxylase comprises an amino acid sequence set forth in SEQ ID NO: 14, variants, mutants, or fragments thereof.

22 . The isolated strain of genetically engineered cells of claim 17 , wherein the genetically engineered cells produce L-phenylalanine from glucose by a reaction catalyzed by overexpression of at least one enzyme selected from a group comprising DAHP synthase (AroG), shikimate kinase (AroK), shikimate dehydrogenase (YdiB), chorismate mutase/prephenate dehydratase (PheA) and tyrosine aminotransferase (TyrB), or mutants thereof.

23 . The isolated strain of genetically engineered cells of claim 22 , wherein AroG comprises an amino acid sequence set forth in SEQ ID NO: 17, variants, mutants, or fragments thereof; AroK comprises an amino acid sequence set forth in SEQ ID NO: 18, variants, mutants, or fragments thereof; YdiB comprises an amino acid sequence set forth in SEQ ID NO: 19, variants, mutants, or fragments thereof; PheA c

Assignees

• Nat Univ Singapore

Inventors

• Li Zhi
• Wu Shuke
• Zhou Yi
• Lukito Benedict Ryan

Classifications

• C12N9/0008

  acting on the aldehyde or oxo group of donors (1.2) · CPC title

• C12Y503/99007

  Styrene-oxide isomerase (5.3.99.7) · CPC title

• C12Y402/01051

  Prephenate dehydratase (4.2.1.51) · CPC title

• C12Y101/01282

  Quinate/shikimate dehydrogenase (1.1.1.282) · CPC title

• C12P7/54

  Acetic acid (vinegar C12J) · CPC title