WO2023075814A1

WO2023075814A1 - Engineered biosynthetic pathways for production of p-coumaric acid by fermentation

Info

Publication number: WO2023075814A1
Application number: PCT/US2021/072020
Authority: WO
Inventors: Cara Ann Tracewell; Alexander Glennon SHEARER; Stepan TYMOSHENKO; Anupam Chowdhury; Steven M. EDGAR
Original assignee: Zymergen Inc.
Priority date: 2021-10-25
Filing date: 2021-10-25
Publication date: 2023-05-04

Abstract

The present disclosure describes the engineering of microbial cells to express a heterologous L-tyrosine ammonia lyase for fermentative production of p-coumaric acid. Additional genetic alterations were introduced to improve p-coumaric acid titer. The disclosure further describes novel engineered microbial cells and cultures, as well as related p-coumaric acid production methods.

Description

ENGINEERED BIOSYNTHETIC PATHWAYS FOR

PRODUCTION OF p-COUMARIC ACID BY FERMENTATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Not applicable.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with Government support under Agreement No. HR0011-15-9-0014, awarded by DARPA. The Government has certain rights in the invention.

FIELD OF THE DISCLOSURE

[0003] The present disclosure relates generally to the area of engineering microbes for overproduction of p-coumaric acid by fermentation.

BACKGROUND

[0004] p-Coumaric acid is a phenolic natural product known to exist in nature in plants where it is used in lignin, a biological cross-linked polymer material that provides structural support to plants. The solubility of p-coumaric acid in water is 1 mg/mL.

Production of aromatic molecules such as p-coumaric acid by biological fermentation can make a monomer economically accessible for newly identified materials applications. Aromatic molecules have attractive thermal and mechanical properties for novel material applications.

SUMMARY

[0005] Various embodiments contemplated herein may include, but need not be limited to, one or more of the following:

[0006] Embodiment 1: An engineered microbial cell, wherein the engineered microbial cell expresses at least one heterologous L-tyrosine ammonia lyase (TAL) that is from: a fungal phylum selected from the group consisting of Ascomycota and Basidiomycota; or a bacterial phylum selected from the group consisting of Actinobacteria and Proteobacteria; wherein the engineered microbial cell produces p-coumaric acid.

[0007] Embodiment 2: The engineered microbial cell of embodiment 1, wherein the at least one heterologous TAL is from the phylum Ascomycota.

[0008] Embodiment 3: The engineered microbial cell of embodiment 2, wherein the at least one heterologous TAL is from the genus Acremonium.

[0009] Embodiment 4: The engineered microbial cell of embodiment 3, wherein the at least one heterologous TAL is from Acremonium chrysogenum.

[0010] Embodiment 5: The engineered microbial cell of embodiment 1, wherein the at least one heterologous TAL is from the phylum Basidiomycota.

[0011] Embodiment 6: The engineered microbial cell of embodiment 5, wherein the at least one heterologous TAL is from the genus Rhodosporidium.

[0012] Embodiment 7 : The engineered microbial cell of embodiment 6, wherein the at least one heterologous TAL is from Rhodosporidium toruloides.

[0013] Embodiment 8: The engineered microbial cell of embodiment 1, wherein the at least one heterologous TAL is from the phylum Actinobacteria.

[0014] Embodiment 9: The engineered microbial cell of embodiment 8 wherein the at least one heterologous TAL is from the genus Amycolatopsis or Streptomyces .

[0015] Embodiment 10: The engineered microbial cell of embodiment 9, wherein the at least one heterologous TAL is from Amycolatopsis orientalis.

[0016] Embodiment 11 : The engineered microbial cell of embodiment 9, wherein the at least one heterologous TAL is from Streptomyces sp. WK-5344.

[0017] Embodiment 12: The engineered microbial cell of embodiment 1, wherein the at least one heterologous TAL is from the phylum Proteobacteria.

[0018] Embodiment 13: The engineered microbial cell of embodiment 12, wherein the at least one heterologous TAL is from a bacterium selected from the group consisting of an alphaproteobacterium, a betaproteobacterium, and a gamma proteobacterium.

[0019] Embodiment 14: The engineered microbial cell of embodiment 13, wherein the at least one heterologous TAL is from an alphaproteobacterium. [0020] Embodiment 15: The engineered microbial cell of embodiment 14, wherein the at least one heterologous TAL is from alpha proteobacterium Q-l.

[0021] Embodiment 16: The engineered microbial cell of embodiment 14, wherein the at least one heterologous TAL is from the genus Rhodobacter.

[0022] Embodiment 17: The engineered microbial cell of embodiment 14, wherein the at least one heterologous TAL is from Rhodobacter sphaeroides.

[0023] Embodiment 18: The engineered microbial cell of embodiment 13, wherein the at least one heterologous TAL is from a betaproteobacterium.

[0024] Embodiment 19: The engineered microbial cell of embodiment 18, wherein the at least one heterologous TAL is from the genus Cupriavidus.

[0025] Embodiment 20: The engineered microbial cell of embodiment 19, wherein the at least one heterologous TAL is from the genus Cupriavidus metallidurans .

[0026] Embodiment 21: The engineered microbial cell of embodiment 13, wherein the at least one heterologous TAL is from a gamma proteobacterium.

[0027] Embodiment 22: The engineered microbial cell of embodiment 21, wherein the at least one heterologous TAL is from the genus Halomonas.

[0028] Embodiment 23 : The engineered microbial cell of embodiment 22, wherein the at least one heterologous TAL is from Halomonas chromatireducens .

[0029] Embodiment 24: The engineered microbial cell of any one of embodiments 1-23, wherein the engineered microbial cell comprises increased activity of one or more upstream enzyme(s) in the tyrosine biosynthesis pathway, said increased activity being increased relative to a control cell.

[0030] Embodiment 25 : The engineered microbial cell of embodiment 24, wherein the one or more upstream enzyme(s) comprises one or more enzyme(s) selected from the group consisting of a 3-phosphogly cerate kinase, a transketolase, a transaldolase, an enolase, a phospoenolpyruvate (PEP) synthase, a phospoenolpyruvate (PEP)carboxykinase, a 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, a dehydroquinate synthase, a dehydroquinate dehydratase, a shikimate dehydrogenase, a shikimate kinase, 3- phosphoshikimate 1 -carboxy vinyl transferase, EPSP synthase, aromatic pentafunctional enzyme, a chorismate synthase, a chorismate mutase, a phenyalananine aminotransferase, a prephenate dehydrogenase, a prephenate aminotransferase, an arogenate dehydrogenase, a phenylalanine 4-hydroxylase, and a tyrosine aminotransferase.

[0031] Embodiment 26: The engineered microbial cell of any one of embodiments 1-25, wherein the engineered microbial cell expresses one or more feedback-deregulated enzyme(s).

[0032] Embodiment 27 : The engineered microbial cell of embodiment 26, wherein the one or more feedback-deregulated enzyme(s) are selected from the group consisting of DAHP synthase, chorismate mutase, prephrenate dehydrogenase, 6-phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase.

[0033] Embodiment 28: The engineered microbial cell of any one of embodiments 1-27, wherein the engineered microbial cell comprises reduced activity of one or more enzymes(s) that reduce the concentration of one or more upstream pathway precursor(s) or of p-coumaric acid, said reduced activity being reduced relative to a control cell.

[0034] Embodiment 29: The engineered microbial cell of embodiment 28, wherein the one or more upstream enzymes(s) are selected from the group consisting of anthranilate synthase, indole-3-glycerol phosphate synthase, prephenate dehydratase, phenylpyruvate decarboxylase, SIT4 phosphatase, pyruvate decarboxylase, pyruvate dehydrogenase, citrate synthase, alcohol dehydrogenase, aldehyde oxidase, ferulic acid decarboxylase, phenylacrylic acid decarboxylase, and alcohol acetyl transferase.

[0035] Embodiment 30: The engineered microbial cell of any one of embodiments 1-29, wherein the engineered microbial cell comprises increased activity of one or more enzyme(s) that increase the supply of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), said increased activity being increased relative to a control cell.

[0036] Embodiment 31: The engineered microbial cell of embodiment 30, wherein the one or more enzyme(s) that increase the supply of NADPH are selected from the group consisting of pentose phosphate pathway enzymes, NADP⁺-dependent D-glyceraldehyde-3- phosphate dehydrogenase (NADP-GAPDH), and NADP+-dependent glutamate dehydrogenase.

[0037] Embodiment 32: The engineered microbial cell of any one of embodiments 1-31, wherein the engineered microbial cell comprises reduced activity of one or more enzyme(s) that reduce the supply of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), said reduced activity being reduced relative to a control cell.

[0038] Embodiment 33: The engineered microbial cell of embodiment 32, wherein the one or more enzyme(s) that reduce the supply of NADPH comprise NAD+-dependent D-glyceraldehyde-3 -phosphate dehydrogenase (NAD+-GAPDH).

[0039] Embodiment 34: The engineered microbial cell of any one of embodiments 1-33, wherein the engineered microbial cell is one that uses a phospoenolpyruvate: sugar phosphotransferase system (PTS) to import sugar into the cell, wherein the activity of the PTS is reduced as compared to a control cell.

[0040] Embodiment 35: The engineered microbial cell of any one of embodiments 1-33, wherein the engineered microbial cell comprises a fungal cell.

[0041] Embodiment 36: The engineered microbial cell of embodiment 35, wherein the engineered microbial cell comprises a yeast cell.

[0042] Embodiment 37: The engineered microbial cell of embodiment 36, wherein the yeast cell comprises a cell of the genus Saccharomyces or Yarrowia.

[0043] Embodiment 38: The engineered microbial cell of embodiment 37, wherein the yeast cell is a Saccharomyces cerevisiae cell or a Yarrowia lipolytica cell.

[0044] Embodiment 39: The engineered microbial cell of any one of embodiments 35-38 wherein the engineered microbial cell is an S. cerevisiae cell and expresses a TAL having at least 70% amino acid sequence identity with a TAL from Acremoni um chrysogenum strain ATCC 11550 comprising SEQ ID NO:1.

[0045] Embodiment 40: The engineered microbial cell of embodiment 39, wherein the engineered microbial cell additionally expresses: (a) a shikimate kinase having at least 70% amino acid sequence identity with a shikimate kinase from Corynebacterium glutamicum ATCC 13032 comprising SEQ ID NO:2; (b) a 3-dehydroquinate synthase having at least 70% amino acid sequence identity with a 3-dehydroquinate synthase from Corynebacterium glutamicum ATCC 13032 comprising SEQ ID NO:3 or a chorismate synthase having at least 70% amino acid sequence identity with a chorismate synthase from Corynebacterium glutamicum ATCC 13032 comprising SEQ ID NO:4; and (c) a feedback-deregulated DAHP synthase having at least 70% amino acid sequence identity with an Saccharomyces cerevisiae DAHP synthase comprising a K229L substitution and SEQ ID NO:5. [0046] Embodiment 41 : The engineered microbial cell of embodiment 40, wherein the engineered microbial cell additionally expresses: (a) a second copy of the shikimate kinase having at least 70% amino acid sequence identity with a shikimate kinase from Corynebacterium glutamicum ATCC 13032 comprising SEQ ID NO: 2; (b) a chorismate synthase having at least 70% amino acid sequence identity with a chorismate synthase from Corynebacterium glutamicum ATCC 13032 comprising SEQ ID NO:4; and (c) a transaldolase having at least 70% amino acid sequence identity with a transaldolase from Corynebacterium glutamicum ATCC 13032 comprising SEQ ID NO:6.

[0047] Embodiment 42: The engineered microbial cell of embodiment 41, wherein the engineered microbial cell additionally expresses a histidine ammonia-lyase having at least 70% amino acid sequence identity with a histidine ammonia-lyase from Rivularia sp. comprising SEQ ID NO:20.

[0048] Embodiment 43 : The engineered microbial cell of embodiment 40, wherein the engineered microbial cell additionally expresses two copies of a feedback-deregulated DAHP synthase having at least 70% amino acid sequence identity with an Escherichia coli DAHP synthase comprising a D146N substitution and SEQ ID NO:7.

[0049] Embodiment 44: The engineered microbial cell of embodiment 40, wherein the engineered microbial cell additionally expresses: (a) a second copy of the shikimate kinase having at least 70% amino acid sequence identity with a shikimate kinase from Corynebacterium glutamicum ATCC 13032 comprising SEQ ID NO: 2; (b) a glucose-6-phosphate dehydrogenase having at least 70% amino acid sequence identity with a glucose-6-phosphate dehydrogenase from Corynebacterium glutamicum R comprising an A243T substitution and SEQ ID NO: 8; and (c) a prephenate dehydrogenase having at least 70% amino acid sequence identity with a prephenate dehydrogenase from Corynebacterium glutamicum ATCC 13032 comprising SEQ ID NO:9.

[0050] Embodiment 45 : The engineered microbial cell of embodiment 40, wherein the engineered microbial cell additionally expresses: (a) a second copy of the shikimate kinase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a shikimate kinase from Corynebacterium glutamicum ATCC 13032 comprising SEQ ID NO:2; (b) a 6-phosphogluconate dehydrogenase having at least 70% amino acid sequence identity with a 6- phosphogluconate dehydrogenase from Corynebacterium glutamicum ATCC 13032 comprising an S361F substitution and SEQ ID NO: 10; and (c) a prephenate dehydrogenase having at least 70% amino acid sequence identity with a prephenate dehydrogenase from Corynebacterium glutamicum ATCC 13032 comprising SEQ ID NO:9.

[0051] Embodiment 46: The engineered microbial cell of embodiment 40, wherein the engineered microbial cell additionally expresses: (a) a 3-dehydroquinate dehydratase having at least 70% amino acid sequence identity with a shikimate kinase from Corynebacterium glutamicum ATCC 13032 comprising SEQ ID NO: 11; and (b) two copies of a transaldolase having at least 70% amino acid sequence identity with transaldolase from Corynebacterium glutamicum ATCC 13032 comprising SEQ ID NO:6.

[0052] Embodiment 47 : The engineered microbial cell of any one of embodiments 35-38 wherein the engineered microbial cell is a Y. lipolytica cell and expresses: (a) a TAL having at least 70% amino acid sequence identity with a TAL from Acremonium chrysogenum ATCC 11550 comprising SEQ ID NO: 1 ; and (b) a phospho-2-dehydro-3- deoxy heptonate aldolase having at least 70% amino acid sequence identity with a phospho- 2-dehydro-3-deoxyheptonate aldolase from Saccharomyces cerevisiae S288c comprising SEQ ID NO:26.

[0053] Embodiment 48: The engineered microbial cell of embodiment 47, wherein the the engineered microbial cell additionally expresses a feedback-deregulated DAHP synthase having at least 70% amino acid sequence identity with an Escherichia coli DAHP synthase comprising a P150L substitution and SEQ ID NO: 13.

[0054] Embodiment 49: The engineered microbial cell of any one of embodiments 1-33, wherein the engineered microbial cell is a bacterial cell.

[0055] Embodiment 50: The engineered microbial cell of embodiment 49, wherein the bacterial cell is a cell of the genus Corynebacterium or Bacillus.

[0056] Embodiment 51: The engineered microbial cell of embodiment 50, wherein the bacterial cell is a Corynebacterium glutamicum cell or a Bacillus subtilis cell.

[0057] Embodiment 52: The engineered microbial cell of any one of embodiments 49-51, wherein the engineered microbial cell is a C. glutamicum cell and expresses a TAL having at least 70% amino acid sequence identity with a TAL from Amycolatopsis orientalis HCCB10007 comprising SEQ ID NO: 12.

[0058] Embodiment 53: The engineered microbial cell of any one of embodiments 49-52, wherein the engineered microbial cell has increased chorismate mutase activity, compared to a control cell. [0059] Embodiment 54: The engineered microbial cell of any one of embodiments 49- 53, wherein the engineered microbial cell additionally expresses a feedback-deregulated DAHP synthase having at least 70% amino acid sequence identity with an Escherichia coli DAHP synthase comprising a P150L substitution and SEQ ID NO: 13.

[0060] Embodiment 55: The engineered microbial cell of any one of embodiments 1-53, wherein, when cultured, the engineered microbial cell produces p-coumaric acid at a level greater than 100 μg/L of culture medium.

[0061] Embodiment 56: The engineered microbial cell of embodiment 55, wherein the engineered microbial cell produces p-coumaric acid at a level of at least 20 mg/L of culture medium.

[0062] Embodiment 57: A culture of engineered microbial cells according to any one of embodiments 1-56.

[0063] Embodiment 58: The culture of embodiment 57, wherein the p-coumaric acid is produced from fermentation of a substrate wherein at least 50% of the substrate is not derived from protein or amino acid sources.

[0064] Embodiment 59: The culture of embodiment 58, wherein the substrate comprises a carbon source and a nitrogen source selected from the group consisting of urea, an ammonium salt, ammonia, and any combination thereof.

[0065] Embodiment 60: The culture of any one of embodiments 57-59, wherein the engineered microbial cells are present in a concentration such that the culture has an optical density at 600 nm of 10-500.

[0066] Embodiment 61: The culture of any one of embodiments 57-60, wherein the culture comprises p-coumaric acid.

[0067] Embodiment 62: The culture of any one of embodiments 57-61, wherein the culture comprises p-coumaric acid at a level greater than 100 μg/L of culture medium.

[0068] Embodiment 63: The culture of any one of embodiments 57-62, wherein the culture comprises p-coumaric acid at a level of at least 20 mg/L of culture medium.

[0069] Embodiment 64: A method of culturing engineered microbial cells according to any one of embodiments 1-63, the method comprising culturing the cells in the presence of a fermentation substrate comprising a non-protein carbon and a non-protein nitrogen source, wherein the engineered microbial cells produce p-coumaric acid. [0070] Embodiment 65 : The method of embodiment 64, wherein the method comprises fed-batch culture, with an initial glucose level in the range of 1-100 g/L, followed controlled sugar feeding.

[0071] Embodiment 66: The method of embodiment 64 or embodiment 65, wherein the fermentation substrate comprises glucose and a nitrogen source selected from the group consisting of urea, an ammonium salt, ammonia, and any combination thereof.

[0072] Embodiment 67 : The method of any one of embodiments 64-66, wherein the culture is pH-controlled during culturing.

[0073] Embodiment 68: The method of any one of embodiments 64-67, wherein the culture is aerated during culturing.

[0074] Embodiment 69: The method of any one of embodiments 64-68, wherein the engineered microbial cells produce p-coumaric acid at a level greater than 100 μg/L of culture medium.

[0075] Embodiment 70: The method of any one of embodiments 64-69, wherein the engineered microbial cells produce p-coumaric acid at a level of at least 20 mg/L of culture medium.

[0076] Embodiment 71 : The method of any one of embodiments 64-70, wherein the method additionally comprises recovering p-coumaric acid from the culture.

BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 : Biosynthetic pathway for production of p-coumaric acid by fermentation.

[0078] Figure 2: p-Coumaric acid titers measured in extracellular broth following fermentation by the first-round engineered host Corynebacterium glutamicum.

[0079] Figure 3: p-Coumaric acid titers measured in extracellular broth following fermentation by the first-round engineered host Saccharomyces cerevisiae.

[0080] Figure 4: p-Coumaric acid titers measured in extracellular broth following fermentation by the second-round engineered host Corynebacterium glutamicum.

[0081] Figure 5: p-Coumaric acid titers measured in extracellular broth following fermentation by the second-round engineered host Saccharomyces cerevisiae. [0082] Figure 6: p-Coumaric acid titers measured in extracellular broth following fermentation by the third-round engineered host Corynebacterium glutamicum.

[0083] Figure 7: p-Coumaric acid titers measured in extracellular broth following fermentation by the third-round engineered host Saccharomyces cerevisiae.

[0084] Figure 8: p-Coumaric acid titers measured in extracellular broth following fermentation by host evaluation-round engineered host Yarrowia lipolytica.

[0085] Figure 9: p-Coumaric acid titers measured in extracellular broth following fermentation by the host evaluation-round engineered host Saccharomyces cerevisiae.

[0086] Figure 10: p-Coumaric acid titers measured in extracellular broth following fermentation by the improvement-round engineered host Saccharomyces cerevisiae.

[0087] Figure 11: A “split-marker, double-crossover” genomic integration strategy, which was developed to engineer Saccharomyces cerevisiae and Yarrowia lipolytica strains. Two plasmids with complementary 5’ and 3’ homology arms and overlapping halves of a URA3 selectable marker (direct repeats shown by the hashed bars) were digested with meganucleases and transformed as linear fragments. A triple-crossover event integrated the desired heterologous genes into the targeted locus and re-constituted the full URA3 gene. Colonies derived from this integration event were assayed using two 3-primer reactions to confirm both the 5’ and 3’ junctions (UF/IF/wt-R and DR/IF/wt-F). See Example 1.

[0088] Figure 12: Promoter replacement in Saccharomyces cerevisiae and Yarrowia lipolytica.

[0089] Figure 13: Targeted gene deletion in Saccharomyces cerevisiae and Yarrowia lipolytica.

[0090] Figure 14: A “loop-in, single-crossover” genomic integration strategy, which was developed to engineer Corynebacterium glutamicum and Bacillus subtilis strains. Loop-in only constructs (shown under the heading “Loop-in”) contained a single 2-kb homology arm (denoted as “integration locus”), a positive selection marker (denoted as “Marker”)), and gene(s) of interest (denoted as “promoter-gene-terminator”). A single crossover event integrated the plasmid into the C. glutamicum (or B. subtilis') chromosome. Integration events are stably maintained in the genome by growth in the presence of antibiotic (e.g., 25μg/ml kanamycin). Correct genomic integration in colonies derived from loop-in integration were confirmed by colony PCR with UF/IR and DR/IF PCR primers.

Loop-in, loop-out constructs (shown under the heading “Loop-in, loop-out) contained two 2-kb homology arms (5’ and 3’ arms), gene(s) of interest (arrows), a positive selection marker (denoted “Marker”), and a counter-selection marker. Similar to “loop-in” only constructs, a single crossover event integrated the plasmid into the chromosome of C. glutamicum (or B. subtilis). Note: only one of two possible integrations is shown here. Correct genomic integration was confirmed by colony PCR and counter-selection was applied so that the plasmid backbone and counter-selection marker could be excised. This results in one of two possibilities: reversion to wild-type or the desired pathway integration. Again, correct genomic loop-out is confirmed by colony PCR. (Abbreviations: Primers: UF = upstream forward, DR = downstream reverse, IR = internal reverse, IF = internal forward.) See Example 1.

DETAILED DESCRIPTION

[0091] Described herein is method for the production of the small molecule, p- coumaric acid via fermentation by a microbial host from simple carbon and nitrogen sources, such as glucose and urea, respectively. To achieve this aim, a non-native metabolic pathway is introduced into a suitable microbial host for industrial fermentation of large- scale chemical products, such as Saccharomyces cerevisiae, Corynebacterium glutamicum, Bacillus subtilis, and Yarrowia lipolytica. The engineered metabolic pathway links the central metabolism of the host to a non-native pathway to enable the production of p- coumaric acid. In a simple embodiment, the expression of an enzyme, a non-native tyrosine ammonia lyase enzyme, in a microbial host strain that can produce tyrosine allows the host to produce p-coumaric acid. Further engineering of the metabolic pathway by modifying the host central metabolism through overexpression of key upstream pathway enzymes, e.g., a 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHP synthase) harboring the amino acid substitution K229L, which reduces feedback inhibition of this enzyme; a shikimate kinase (expressed from two copies of the corresponding gene); a 3- dehydroquinate synthase; a chorismate synthase; and a transaldolase enabled titers of 37 mg/L p-coumaric acid to be achieved in S. cerevisiae.

Definitions

[0092] Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0093] The term “fermentation” is used herein to refer to a process whereby a microbial cell converts one or more substrate(s) into a desired product (such as p-coumaric acid) by means of one or more biological conversion steps, without the need for any chemical conversion step.

[0094] The term “engineered” is used herein, with reference to a cell, to indicate that the cell contains at least one targeted genetic alteration introduced by man that distinguishes the engineered cell from the naturally occurring cell.

[0095] The term “native” is used herein to refer to a cellular component, such as a polynucleotide or polypeptide, that is naturally present in a particular cell. A native polynucleotide or polypeptide is endogenous to the cell.

[0096] When used with reference to a polynucleotide or polypeptide, the term “nonnative” refers to a polynucleotide or polypeptide that is not naturally present in a particular cell.

[0097] When used with reference to the context in which a gene is expressed, the term “non-native” refers to a gene expressed in any context other than the genomic and cellular context in which it is naturally expressed. A gene expressed in a non-native manner may have the same nucleotide sequence as the corresponding gene in a host cell, but may be expressed from a vector or from an integration point in the genome that differs from the locus of the native gene.

[0098] The term “heterologous” is used herein to describe a polynucleotide or polypeptide introduced into a host cell. This term encompasses a polynucleotide or polypeptide, respectively, derived from a different organism, species, or strain than that of the host cell. In this case, the heterologous polynucleotide or polypeptide has a sequence that is different from any sequence(s) found in the same host cell. However, the term also encompasses a polynucleotide or polypeptide that has a sequence that is the same as a sequence found in the host cell, wherein the polynucleotide or polypeptide is present in a different context than the native sequence (e.g., a heterologous polynucleotide can be linked to a different promotor and inserted into a different genomic location than that of the native sequence). “Heterologous expression” thus encompasses expression of a sequence that is non-native to the host cell, as well as expression of a sequence that is native to the host cell in a non-native context.

[0099] As used with reference to polynucleotides or polypeptides, the term “wild- type” refers to any polynucleotide having a nucleotide sequence, or polypeptide having an amino acid, sequence present in a polynucleotide or polypeptide from a naturally occurring organism, regardless of the source of the molecule; i.e., the term “wild-type” refers to sequence characteristics, regardless of whether the molecule is purified from a natural source; expressed recombinantly, followed by purification; or synthesized. The term “wildtype” is also used to denote naturally occurring cells.

[0100] A “control cell” is a cell that is otherwise identical to an engineered cell being tested, including being of the same genus and species as the engineered cell, but lacks the specific genetic modification(s) being tested in the engineered cell.

[0101] Enzymes are identified herein by the reactions they catalyze and, unless otherwise indicated, refer to any polypeptide capable of catalyzing the identified reaction. Unless otherwise indicated, enzymes may be derived from any organism and may have a native or mutated amino acid sequence. As is well known, enzymes may have multiple functions and/or multiple names, sometimes depending on the source organism from which they derive. The enzyme names used herein encompass orthologs, including enzymes that may have one or more additional functions or a different name.

[0102] The term “feedback-deregulated” is used herein with reference to an enzyme that is normally negatively regulated by a downstream product of the enzymatic pathway (i.e., feedback-inhibition) in a particular cell. In this context, a “feedback-deregulated” enzyme is a form of the enzyme that is less sensitive to feedback-inhibition than the enzyme native to the cell or a form of the enzyme that is native to the cell, but is naturally less sensitive to feedback inhibition than one or more other natural forms of the enzyme. A feedback-deregulated enzyme may be produced by introducing one or more mutations into a native enzyme. Alternatively, a feedback-deregulated enzyme may simply be a heterologous, native enzyme that, when introduced into a particular microbial cell, is not as sensitive to feedback-inhibition as the native enzyme. In some embodiments, the feedback- deregulated enzyme shows no feedback-inhibition in the microbial cell.

[0103] The term “p-coumaric acid” refers to (2E)-3-(4-hydroxyphenyl)prop-2-enoic acid, identified by CAS Number 501-98-4.

[0104] The term “sequence identity,” in the context of two or more amino acid or nucleotide sequences, refers to two or more sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. [0105] For sequence comparison to determine percent nucleotide or amino acid sequence identity, typically one sequence acts as a “reference sequence,” to which a “test” sequence is compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence relative to the reference sequence, based on the designated program parameters. Alignment of sequences for comparison can be conducted using BLAST set to default parameters.

[0106] The term “titer,” as used herein, refers to the mass of a product (e.g., p- coumaric acid) in the extracellular medium of a culture of microbial cells divided by the culture volume.

[0107] As used herein with respect to recovering p-coumaric acid from a cell culture, “recovering” refers to separating the p-coumaric acid from at least one other component of the cell culture medium.

[0108] The phrase “a second copy” is used herein with respect to a named enzyme to refer to a second copy of a gene encoding the enzyme.

Engineering Microbes for p-Coumaric Acid Production p-Coumaric Acid Biosynthesis Pathway

[0109] p-Coumaric acid is derived from the aromatic branch of amino acid biosynthesis, based on the precursors phosphoenolpyruvate (PEP) and erythrose-4- phosphate (E4P). Specifically, p-Coumaric acid is derived from the aromatic amino acid tyrosine. This pathway is illustrated in Fig. 1. The first step of the amino acid biosynthesis pathway, catalyzed by 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, is subject to feedback inhibition by the aromatic amino acids tyrosine, tryptophan and phenylalanine. Many microbes lack the enzyme that catalyzes the final step in this pathway, namely tyrosine ammonia lyase (TAL). Production of p-coumaric acid in such microbial hosts requires the addition of at least one heterologous TAL enzyme. For example, p-coumaric acid production is enabled by the addition of a single heterologous enzymatic step in Saccharomyces cerevisiae (Sc), Corynebacterium glutamicum (Cg), Bacillus subtilis (Bs), and Yarrowia lipolytica (Yl) hosts; this step is catalyzed by TAL. Engineering for Microbial p-Coumaric Acid Production

[0110] Any TAL that is active in the microbial cell being engineered may be introduced into the cell, typically by introducing and expressing the gene encoding the enzyme using standard genetic engineering techniques. Suitable TALs may be derived from any source, including plant, archaeal, fungal, gram-positive bacterial, and gram-negative bacterial sources. Exemplary sources include, but are not limited to those shown in Example 1, Table 1, below.

[0111] One or more copies of a TAL can be introduced into a selected microbial host cell. If more than one copy of a TAL gene is introduced, the copies can be copies of the same or different TAL genes. In some embodiments, the TAL gene(s) is/ are expressed from a strong, constitutive promoter. In some embodiments, the TAL gene(s) is/are expressed from inducible promoters. The TAL gene(s) can optionally be codon-optimized to enhance expression in the selected microbial host cell. Codon-optimization tables are available for common microbial host cells. The codon-optimization tables used in the Examples are as follows: Bacillus subtilis Kazusa codon table:

Yarrowia lipolytica Kazusa codon table:

Corynebacterium glutamicum Kazusa codon table:

Saccharomyces cerevisiae Kazusa codon table:

. Also used, was a modified, combined codon usage scheme for S. cereviae and C. glutamicum, which is reproduced below.

Engineering for Increased p-Coumaric Acid Production

Increasing the Activity of Upstream Pathway Enzymes

[0112] One approach to increasing p-coumaric acid production in a microbial cell which expresses a TAL is to increase the activity of one or more upstream enzymes in the p- coumaric acid biosynthesis pathway. In this context, “increasing the activity” refers increasing the enzymatic function attributable to an enzyme in an engineered cell, as compared to a control cell. Various ways of increasing a given enzymatic function in a cell are known to those of skill in the art. For example, increased enzymatic function can conveniently be achieved by expressing one or more additional copies of the gene encoding the corresponding enzyme. The expression of native genes can be upregulated by replacing a native promoter with a stronger one. Both modifications tend to increase the amount of the enzyme in the cell. Alternatively or in addition, the activity of the enzyme itself can be modulated. For example, as discussed further below, mutations or enzymes bearing mutations that reduce feedback inhibition can be introduced into an engineered cell to increase an enzyme’s function.

[0113] Upstream pathway enzymes include all enzymes involved in the conversions from a feedstock all the way to tyrosine. In certain embodiments, the upstream pathway enzymes refer specifically to the enzymes involved in the conversion of key precursors (i. e. , E4P and PEP) into the last native metabolite (i.e. tyrosine) in the pathway leading to p- coumaric acid. In some embodiments, the activity of one or more upstream pathway enzymes is increased by modulating the expression or activity of the endogenous enzyme(s). In some embodiments, the activity of one or more upstream pathway enzymes is supplemented by introducing one or more of the corresponding genes into the TAL- expressing microbial host cell. Such genes include those encoding a 3-phosphogly cerate kinase, a transketolase, a transaldolase, an enolase, a phospoenolpyruvate (PEP) synthase, a phospoenolpyruvate (PEP)carboxykinase, a 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, a dehydroquinate synthase, a dehydroquinate dehydratase, a shikimate dehydrogenase, a shikimate kinase, 3-phosphoshikimate 1 -carboxy vinyl transferase, EPSP synthase, aromatic pentafunctional enzyme, a chorismate synthase, a chorismate mutase, a phenyalanine aminotransferase, a prephenate dehydrogenase, a prephenate aminotransferase, an arogenate dehydrogenase, a phenylalanine 4-hydroxylase, and a tyrosine aminotransferase. Suitable upstream pathway genes may be derived from any source, including, for example, those discussed above as sources for a heterologous TAL gene and those described in Example 1, Tables 1-3. If the upstream pathway enzyme is normally subject to feedback inhibition, flux through the pathway can be increased by introducing either a feedback-sensitive or a feedback-deregulated form to of the enzyme (see below).

[0114] An introduced upstream pathway gene may be heterologous or may simply be an additional copy of a native gene. In some embodiments, one or more such genes are introduced into the TAL-expressing microbial host cell and expressed from a strong constitutive promoter and/or can optionally be codon-optimized to enhance expression in the selected microbial host cell. A TAL-expressing microbial cell can, for example, be engineered to express one or more copies of one or more upstream pathway genes.

[0115] In various embodiments, the engineering of a TAL-expressing microbial cell to increase the activity of one or more upstream pathway enzymes increases the p-coumaric acid titer by at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 percent or by at least 2-fold, 2.5- fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8- fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 30-fold, 35- fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, or 100-fold. In various embodiments, the increase in p-coumaric acid titer is in the range of 10 percent to 100-fold, 2-fold to 50-fold, 5-fold to 40-fold, 10-fold to 30- fold, or any range bounded by any of the values listed above. (Ranges herein include their endpoints.) These increases are determined relative to the p-coumaric acid titer observed in a p-coumaric acid-producing microbial cell that lacks any increase in activity of upstream pathway enzymes. This reference cell may have one or more other genetic alterations aimed at increasing p-coumaric acid production, e.g., the cell may express a feedback- deregulated enzyme.

[0116] In various embodiments, the p-coumaric acid titers achieved by increasing the activity of one or more upstream pathway genes are at least 10, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, or 900 μg/L or at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg/L. In various embodiments, the titer is in the range of 10 μg/L to 100 mg/L, 100 μg/L to 75 mg/L, 200 μg/L to 50 mg/L, 300 μg/L to 40 mg/L, or any range bounded by any of the values listed above.

Introduction of Feedback-Deregulated Enzymes

[0117] Since aromatic amino acid biosynthesis is subject to feedback inhibition, another approach to increasing p-coumaric acid production in a microbial cell engineered to express a heterologous TAL is to introduce feedback-deregulated forms of one or more enzymes that are normally subject to feedback inhibition in the TAL-expressing microbial cell. Examples of such enzymes include DAHP synthase, chorismate mutase, 6- phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase.

[0118] A feedback-deregulated form can be a heterologous, wild-type enzyme that is less sensitive to feedback inhibition (“feedback-insensitive”) than the endogenous enzyme in the particular microbial host cell. Alternatively, a feedback-deregulated form can be a variant of an endogenous or heterologous enzyme that has one or more mutations rendering it less sensitive to feedback inhibition than the corresponding wild-type enzyme.

[0119] Specific examples of enzymes useful in this regard include variant DAHP synthases that have known point mutations rendering them resistant to feedback inhibition, e.g., Saccharomyces cerevisiae ARO4K226L, S. cerevisiae ARO4Q166K, Escherichia coli AroGD146N, and E. coli AroP150L (the last 5 characters of these designations indicate amino acid substitutions, using the standard one-letter code for amino acids, with the first letter referring to the wild-type residue and the last letter referring to the replacement reside; the numbers indicate the position of the amino acid substitution in the translated protein); variant chorismate mutases S. cerevisiae ARO7T226I or I225T, a variant chorismate mutase from E. coli containing the amino acid substitution G141S; a variant chorismate mutase/prephenate dehydrogenase (UniProt ID P0A9J8) from A’, coli containing the amino acid substitutions Q306L and G309C; a prephrenate dehydrogenase from Zymomonas mobilis that is known to be feedback-insensitive to tyrosine; a 6-phosphogluconate dehydrogenase (UniProt ID Q8NQI2) from Corynebacterium glutamicum containing the amino acid substitution S361F; a glucose-6-phosphate dehydrogenase (UniProt ID A4QEF2) from C. glutamicum with the amino acid substitution A243T. [0120] In various embodiments, the engineering of a TAL-expressing microbial cell to express a feedback-deregulated enzymes increases the p-coumaric acid titer by at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 percent or by at least 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19- fold, 20-fold, 21 -fold, 22-fold, 23-fold, 24-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, or 100-fold. In various embodiments, the increase in p-coumaric acid titer is in the range of 10 percent to 100-fold, 2-fold to 50-fold, 5-fold to 40-fold, 10-fold to 30-fold, or any range bounded by any of the values listed above. These increases are determined relative to the p- coumaric acid titer observed in a p-coumaric acid-producing microbial cell that does not express a feedback-deregulated enzyme. This reference cell may (but need not) have other genetic alterations aimed at increasing p-coumaric acid production, i.e., the cell may have increased activity of an upstream pathway enzyme resulting from some means other than feedback-insensitivity.

[0121] In various embodiments, the p-coumaric acid titers achieved by using a feedback-deregulated enzyme to increase flux though the p-coumaric acid biosynthetic pathway are at least 10, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, or 900 μg/L or at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg/L. In various embodiments, the titer is in the range of 10 μg/L to 100 mg/L, 100 μg/L to 75 mg/L, 200 μg/L to 50 mg/L, 300 μg/L to 40 mg/L, or any range bounded by any of the values listed above.

[0122] The approaches of supplementing the activity of one or more upstream pathway enzymes and/or introducing one or more feedback-deregulated enzymes can be combined in TAL-expressing microbial cells to achieve even higher p-coumaric acid production levels.

Reducing the Consumption of Precursors and/or p-Coumaric Acid

[0123] Another approach to increasing p-coumaric acid production in a microbial cell that is capable of such production is to decrease the activity of one or more enzymes that consume one or more p-coumaric acid pathway or that consume p-coumaric acid itself (see those discussed above in the Summary). In some embodiments, the activity of one or more such enzymes is reduced by modulating the expression or activity of the native enzyme(s). The activity of such enzymes can be decreased, for example, by substituting the native promoter of the corresponding gene(s) with a less active or inactive promoter or by deleting the corresponding gene(s). Promoters can be selected based on expression data from Lee et al [13], or from [14] or PCT Publication No. W02017100376A2 (for Corynebacterium glutamicum promoters). Reduced enzyme activity in the cell can also be engineered by modifying the nucleotide and/or amino acid sequence of the enzyme with one or more nucleotide or amino acid substitution(s), insertion(s), deletion(s), truncations, or addition(s) that that decrease enzyme activity and/or the lifetime of the corresponding mRNA or protein or by modifying the post-translational processing of an enzyme so as to decrease the enzyme activity or lifetime.

[0124] Target enzymes for this approach can be selected to redirect flux supply of precursors to tyrosine, the precursor of p-coumaric acid, or enzymes that degrade p- coumaric acid. Specific examples include anthranilate synthase, indole-3-glycerol phosphate synthase, prephenate dehydratase, phenylpyruvate decarboxylase, SIT4 phosphatase, pyruvate decarboxylase, pyruvate dehydrogenase, citrate synthase, alcohol dehydrogenase, aldehyde oxidase, ferulic acid decarboxylase, phenylacrylic acid decarboxylase, and alcohol acetyl transferase.

[0125] In various embodiments, the engineering of a p-coumaric acid-producing microbial cell to reduce precursor consumption by one or more side pathways increases the -coumaric acid titer by at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 percent or by at least 2- fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5- fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 30- fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400- fold, 450-fold, 500-fold, 550-fold, 600-fold, 650-fold, 700-fold, 750-fold, 800-fold, 850- fold, 900-fold, 950-fold, or 1000-fold. In various embodiments, the increase in p-coumaric acid titer is in the range of 10-fold to 1000-fold, 20-fold to 500-fold, 50-fold to 400-fold, 10-fold to 300-fold, or any range bounded by any of the values listed above. These increases are determined relative to the p-coumaric acid titer observed in a p-coumaric acidproducing microbial cell that does not include genetic alterations to reduce precursor consumption. This reference cell may (but need not) have other genetic alterations aimed at increasing p-coumaric acid production, i.e., the cell may have increased activity of an upstream pathway enzyme. [0126] In various embodiments, the p-coumaric acid titers achieved by reducing precursor consumption, or consumption of p-coumaric acid itself, are at least 10, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, or 900 μg/L or at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg/L. In various embodiments, the titer is in the range of 10 μg/L to 100 mg/L, 100 μg/L to 75 mg/L, 200 μg/L to 50 mg/L, 300 μg/L to 40 mg/L, or any range bounded by any of the values listed above.

Increasing the NADPH Supply

[0127] Another approach to increasing p-coumaric acid production in a microbial cell that is capable of such production is to increase the supply of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), which provides the reducing equivalents for biosynthetic reactions. For example, the activity of one or more enzymes that increase the NADPH supply can be increased by means similar to those described above for upstream pathway enzymes, e.g., by modulating the expression or activity of the native enzyme(s), replacing the native promoter(s) with a stronger and/or constitutive promoter, and/or introducing one or more gene(s) encoding enzymes that increase the NADPH supply.

[0128] Illustrative enzymes, for this purpose, include, but are not limited to, pentose phosphate pathway enzymes, NADP+-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and NADP+-dependent glutamate dehydrogenase. Such enzymes may be derived from any available source, including, for example, any of those described herein with respect to other enzymes. Examples include the GAPDH encoded by gapC from Clostridium acetobutylicum, the GAPDH encoded by gapB from Bacillus subtilis, and the non-phosphorylating GAPDH encoded by gapN from Streptococcus mutans.

[0129] In various embodiments, the engineering of a p-coumaric acid-producing microbial cell to increase the activity of one or more of such enzymes increases the p- coumaric acid titer by at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 percent or by at least 2- fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5- fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 30- fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400- fold, 450-fold, 500-fold, 550-fold, 600-fold, 650-fold, 700-fold, 750-fold, 800-fold, 850- fold, 900-fold, 950-fold, or 1000-fold. In various embodiments, the increase in p-coumaric acid titer is in the range of 10-fold to 1000-fold, 20-fold to 500-fold, 50-fold to 400-fold, 10-fold to 300-fold, or any range bounded by any of the values listed above. (Ranges herein include their endpoints.) These increases are determined relative to the p-coumaric acid titer observed in a p-coumaric acid-producing microbial cell that lacks any increase in activity of such enzymes. This reference cell may have one or more other genetic alterations aimed at increasing p-coumaric acid production.

[0130] In various embodiments, the p-coumaric acid titers achieved by increasing the activity of one or more enzymes that increase the NADPH supply are at least 10, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, or 900 μg/L or at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg/L. In various embodiments, the titer is in the range of 10 μg/L to 100 mg/L, 100 μg/L to 75 mg/L, 200 μg/L to 50 mg/L, 300 μg/L to 40 mg/L, or any range bounded by any of the values listed above.

[0131] In some embodiments, NADPH can be generated for shikimate dehydrogenase and prephenate dehydrogenase in the p-coumaric acid pathway by expressing one or more NADP⁺-dependent D-glyceraldehyde-3-phosphate dehydrogenase(s), while reducing the expression of one or more native NAD⁺-dependent D- glyceraldehyde-3-phosphate dehydrogenase, such as those encoded by tdhl, tdh2, and tdh3 in yeast.

Deleting the PEP:Sugar Phosphotransferase System

[0132] Some organisms use a PEP:sugar phosphotransferase system (PTS) to import sugar into the cell. Deletion of the PTS decouples sugar uptake from the conversion of phosphoenolpyruvate (PEP) to pyruvate and has been shown to improve the availability of the key aromatic biosynthesis pathway precursor PEP used in shikimate biosynthesis [26], Such a genetic modification can improve production of p-coumaric acid. In Corynebacterium glutamicum, sugar consumption in the APTS strain occurs through overexpression of the endogenous myo-inositol transporter lolTl and glucokinases [26], Glucose utilization in Bacillus subtilis is dependent on the uptake of the sugar by the glucose PTS system [27, 28], Deletion of the PTS system in both C. glutamicum and B. subtilis, optionally combined with over-expression of one or more glucokinase(s) can have a similar benefit to redirecting flux of PEP towards the shikimate pathway and p-coumaric acid production.

[0133] In various embodiments, the engineering of a p-coumaric acid-producing microbial cell to increase the activity of one or more of such enzymes increases the p- coumaric acid titer by at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 percent or by at least 2- fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5- fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 30- fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400- fold, 450-fold, 500-fold, 550-fold, 600-fold, 650-fold, 700-fold, 750-fold, 800-fold, 850- fold, 900-fold, 950-fold, or 1000-fold. In various embodiments, the increase in p-coumaric acid titer is in the range of 10-fold to 1000-fold, 20-fold to 500-fold, 50-fold to 400-fold, 10-fold to 300-fold, or any range bounded by any of the values listed above. (Ranges herein include their endpoints.) These increases are determined relative to the p-coumaric acid titer observed in a p-coumaric acid-producing microbial cell that lacks any increase in activity of such enzymes. This reference cell may have one or more other genetic alterations aimed at increasing p-coumaric acid production.

[0134] In various embodiments, the p-coumaric acid titers achieved by increasing the activity of one or more enzymes that increase the NADPH supply are at least 10, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, or 900 μg/L or at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg/L. In various embodiments, the titer is in the range of 10 μg/L to 100 mg/L, 100 μg/L to 75 mg/L, 200 μg/L to 50 mg/L, 300 μg/L to 40 mg/L, or any range bounded by any of the values listed above.

[0135] Any of the approaches for increasing p-coumaric acid production described above can be combined, in any combination, to achieve even higher p-coumaric acid production levels.

Illustrative Amino Acid and Nucleotide Sequences

[0136] The following table identifies amino acid and nucleotide sequences used in Examples 1-3. The corresponding sequences are shown in the Sequence Listing. SEO ID NO Cross-Reference Table

Microbial Host Cells

[0137] Any microbe that can be used to express introduced genes can be engineered for fermentative production of p-coumaric acid as described above. In certain embodiments, the microbe is one that is naturally incapable of fermentative production of p- coumaric acid. In some embodiments, the microbe is one that is readily cultured, such as, for example, a microbe known to be useful as a host cell in fermentative production of compounds of interest. Bacteria cells, including gram-positive or gram-negative bacteria can be engineered as described above. Examples include, in addition to C. glutamicum cells, P. citrea, B. subtilis, B. licheniformis , B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, B. thuringiensis, S. albus, S. lividans, S. coelicolor, S. griseus, Pseudomonas sp., P. alcaligenes, Lactobacilis spp. (such as L. lactis, L. plantarum), L. grayi, E. coli, E. faecium, E. gallinarum, E. casseliflavus, and/or E. faecalis cells.

[0138] There are numerous types of anaerobic cells that can be used as microbial host cells in the methods described herein. In some embodiments, the microbial cells are obligate anaerobic cells. Obligate anaerobes typically do not grow well, if at all, in conditions where oxygen is present. It is to be understood that a small amount of oxygen may be present, that is, there is some level of tolerance level that obligate anaerobes have for a low level of oxygen. Obligate anaerobes engineered as described above can be grown under substantially oxygen-free conditions, wherein the amount of oxygen present is not harmful to the growth, maintenance, and/or fermentation of the anaerobes.

[0139] Alternatively, the microbial host cells used in the methods described herein can be facultative anaerobic cells. Facultative anaerobes can generate cellular ATP by aerobic respiration (e.g., utilization of the TCA cycle) if oxygen is present. However, facultative anaerobes can also grow in the absence of oxygen. Facultative anaerobes engineered as described above can be grown under substantially oxygen-free conditions, wherein the amount of oxygen present is not harmful to the growth, maintenance, and/or fermentation of the anaerobes, or can be alternatively grown in the presence of greater amounts of oxygen.

[0140] In some embodiments, the microbial host cells used in the methods described herein are filamentous fungal cells. (See, e.g., Berka & Barnett, Biotechnology Advances, (1989), 7(2): 127-154). Examples include Trichoderma longibrachiatum, T. viride, T. koningii, T. harzianum, Penicillium sp., Humicola insolens, H. lanuginose, H. grisea, Chrysosporium sp., C. lucknowense, Gliocladium sp., Aspergillus sp. (such as A. oryzae, A. niger, A. sojae, A. japonicus, A. nidulans, or A. awamori), Fusarium sp. (such as F. roseum, F. graminum F. cerealis, F. oxysporuim, or F. venenatum), Neurospora sp. (such as N. crassa or Hypocrea sp.), Mucor sp. (such as M. miehei), Rhizopus sp. , and Emericella sp. cells. In particular embodiments, the fungal cell engineered as described above is A. nidulans, A. awamori, A. oryzae, A. aculeatus, A. niger, A. japonicus, T. reesei, T. viride, F. oxysporum, or F. solani. Illustrative plasmids or plasmid components for use with such hosts include those described in U.S. Patent Pub. No. 2011/0045563.

[0141] Yeasts can also be used as the microbial host cell in the methods described herein. Examples include: Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., Hansenula polymorpha, Pichia stipites, Kluyveromyces marxianus, Kluyveromyces spp., Yarrowia lipolytica and Candida sp. In some embodiments, the Saccharomyces sp. is S. cerevisiae (See, e.g., Romanos et al., Yeast, (1992), 8(6):423-488). Illustrative plasmids or plasmid components for use with such hosts include those described in U.S. Pat. No. 7,659,097 and U.S. Patent Pub. No. 2011/0045563.

[0142] In some embodiments, the host cell can be an algal cell derived, e.g., from a green algae, red algae, a glaucophyte, a chlorarachniophyte, a euglenid, a chromista, or a dinoflagellate. (See, e.g., Saunders & Warmbrodt, “Gene Expression in Algae and Fungi, Including Yeast,” (1993), National Agricultural Library, Beltsville, Md.). Illustrative plasmids or plasmid components for use in algal cells include those described in U.S. Patent Pub. No. 2011/0045563.

[0143] In other embodiments, the host cell is a cyanobacterium, such as cyanobacterium classified into any of the following groups based on morphology: Chlorococcales, Pleurocapsales, Oscillatoriales, Nostocales, Synechosystic or Stigonematales (See, e.g., Lindberg et al., Metab. Eng., (2010) 12(l):70-79). Illustrative plasmids or plasmid components for use in cyanobacterial cells include those described in U.S. Patent Pub. Nos. 2010/0297749 and 2009/0282545 and in Inti. Pat. Pub. No. WO 2011/034863.

Genetic Engineering Methods

[0144] Microbial cells can be engineered for fermentative p-coumaric acid production using conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such techniques are explained fully in the literature, see e.g., “Molecular Cloning: A Laboratory Manual,” fourth edition (Sambrook et al., 2012); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications” (R. I. Freshney, ed., 6th Edition, 2010); “Methods in Enzymology” (Academic Press, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987, and periodic updates); “PCR: The Polymerase Chain Reaction,” (Mullis et al., eds., 1994); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994).

[0145] Vectors are polynucleotide vehicles used to introduce genetic material into a cell. Vectors useful in the methods described herein can be linear or circular. Vectors can integrate into a target genome of a host cell or replicate independently in a host cell. For many applications, integrating vectors that produced stable transformants are preferred. Vectors can include, for example, an origin of replication, a multiple cloning site (MCS), and/or a selectable marker. An expression vector typically includes an expression cassette containing regulatory elements that facilitate expression of a polynucleotide sequence (often a coding sequence) in a particular host cell. Vectors include, but are not limited to, integrating vectors, prokaryotic plasmids, episomes, viral vectors, cosmids, and artificial chromosomes.

[0146] Illustrative regulatory elements that may be used in expression cassettes include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g,. transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, Gene Expression Technology: Methods In Enzymology 185, Academic Press, San Diego, Calif (1990).

[0147] In some embodiments, vectors may be used to introduce systems that can carry out genome editing, such as CRISPR systems. See U.S. Patent Pub.

No. 2014/0068797, published 6 March 2014; see also Jinek M., et al., “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science 337:816- 21, 2012). In Type II CRISPR-Cas9 systems, Cas9 is a site-directed endonuclease, namely an enzyme that is, or can be, directed to cleave a polynucleotide at a particular target sequence using two distinct endonuclease domains (HNH and RuvC/RNase H-like domains). Cas9 can be engineered to cleave DNA at any desired site because Cas9 is directed to its cleavage site by RNA. Cas9 is therefore also described as an “RNA-guided nuclease.” More specifically, Cas9 becomes associated with one or more RNA molecules, which guide Cas9 to a specific polynucleotide target based on hybridization of at least a portion of the RNA molecule(s) to a specific sequence in the target polynucleotide. Ran, F.A., et al., ("In vivo genome editing using Staphylococcus aureus Cas9." Nature 520(7546): 186-91, 2015, Apr 9], including all extended data) present the crRNA/tracrRNA sequences and secondary structures of eight Type II CRISPR-Cas9 systems. Cas9-like synthetic proteins are also known in the art (see U.S. Published Patent Application No. 2014-0315985, published 23 October 2014).

[0148] Example 1 describes two illustrative integration approaches for introducing polynucleotides into the genomes of S. cerevisiae and C. glutamicum cells.

[0149] Vectors or other polynucleotides can be introduced into microbial cells by any of a variety of standard methods, such as transformation, conjugation, electroporation, nuclear microinjection, transduction, transfection (e.g., lipofection mediated or DEAE- Dextrin mediated transfection or transfection using a recombinant phage virus), incubation with calcium phosphate DNA precipitate, high velocity bombardment with DNA-coated microprojectiles, and protoplast fusion. Transformants can be selected by any method known in the art. Suitable methods for selecting transformants are described in U.S. Patent Pub. Nos. 2009/0203102, 2010/0048964, and 2010/0003716, and International Publication Nos. WO 2009/076676, WO 2010/003007, and WO 2009/132220.

Engineered Microbial Cells

[0150] The above-described methods can be used to produce engineered microbial cells that produce, and in certain embodiments, overproduce, p-coumaric acid. Engineered microbial cells can have at least 1, 2, 3, 4, 5, 6 ,7, 8, 9, 10, or more genetic alterations, such as 30-40 alterations, as compared to a wild-type microbial cell, such as any of the microbial host cells described herein. Engineered microbial cells described in the Example below have one, two, or three genetic alterations, but those of skill in the art can, following the guidance set forth herein, design microbial cells with additional alterations. In some embodiments, the engineered microbial cells have not more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 genetic alterations, as compared to a wild-type microbial cell. In various embodiments, microbial cells engineered for p-coumaric acid production can have a number of genetic alterations falling within the any of the following illustrative ranges: 1-10, 1-9, 1-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-7, 3-6, 3-5, 3-4, etc. [0151] In some embodiments, an engineered microbial cell expresses at least one heterologous tyrosine decarboxylase (TAL). This is necessary in the case of a microbial host cell that does not naturally produce p-coumaric acid. In various embodiments, the microbial cell can include and express, for example: (1) a single heterologous TAL gene, (2) two or more heterologous TAL genes, which can be the same or different (in other words, multiple copies of the same heterologous TAL genes can be introduced or multiple, different heterologous TAL genes can be introduced), (3) a single heterologous TAL gene and one or more additional copies of an endogenous TAL gene, or (4) two or more heterologous TAL genes, which can be the same or different, and one or more additional copies of an endogenous TAL gene.

[0152] This engineered host cell can include at least one additional genetic alteration that increases flux through the pathway leading to the production of tyrosine (the immediate precursor of p-coumaric acid). These “upstream” enzymes in the pathway include those described above, including any isoforms, paralogs, or orthologs having these enzymatic activities (which as those of skill in the art readily appreciate may be known by different names). The at least one additional alteration can increase the activity of the upstream pathway enzyme(s) by any available means, e.g., by: (1) modulating the expression or activity of the endogenous enzyme(s), (2) expressing one or more additional copies of the genes for the endogenous enzymes, or (3) expressing one or more copies of the genes for one or more heterologous enzymes.

[0153] In some embodiments, increased flux through the pathway can be achieved by expressing one or more genes encoding a feedback-deregulated enzyme, as discussed above. For example, the engineered host cell can include and express two or more genes encoding feed-back deregulated enzymes wherein at least two of the enzymes are the same (described herein as two “copies” of the same gene or enzyme) or two or more (or all) of the enzymes are different.. Thus, an engineered microbial cell having any of these genetic alterations can also include at least one heterologous TAL and, optionally, one more genetic alterations that increase the activity of one or more upstream pathway enzymes.

[0154] Similar considerations apply to the other means of engineering for increased p-coumaric acid production described above.

[0155] The engineered microbial cells can contain introduced genes that have a wild-type nucleotide sequence or that differ from wild-type. For example, the wild-type nucleotide sequence can be codon-optimized for expression in a particular host cell. The amino acid sequences encoded by any of these introduced genes can be wild-type or can differ from wild-type. In various embodiments, the amino acid sequences have at least 0 percent, 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity with a wild-type amino acid sequence.

[0156] The engineered microbial cells can, in various embodiments, be capable of producing p-coumaric acid at high titer, as described above. In some embodiments, the engineered microbial cell can produce p-coumaric acid by fermentation of a substrate, wherein at least 20 percent of the substrate is not derived from protein or amino acid sources. In various embodiments, at least 25 percent, 30 percent, 35 percent, 40 percent, 45 percent, 50 percent, 55 percent, 60 percent, 65 percent, 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent of the substrate is not derived from protein or amino acid sources. In some embodiments, the percentage of the fermentation substrate that is not derived from protein or amino acid sources falls within any of the following illustrative ranges: 40-100 percent, 40-90 percent, 40-80 percent, 50-100 percent, 50-90 percent, 50-80 percent, 60-100 percent, 60-90 percent, 60-80 percent, etc.

[0157] The approach described herein has been carried out in fungal cells, namely the yeast S. cerevisiae (a eukaryote), and in bacterial cells, namely C. glutamicum (a prokaryote). (See Example 1.)

Illustrative Engineered Fungal Cells

Illustrative Engineered Saccharomyces cerevisiae Cells

[0158] In certain embodiments an engineered yeast (e.g., S. cerevisiae) cell expresses a heterologous L-tyrosine ammonia lyase (TAL) having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a TAL from Acremonium chrysogenum strain ATCC 11550. In various embodiments, the A. chrysogenum strain ATCC 11550 TAL can include SEQ ID NO: 1. This may be the only genetic alteration of the engineered yeast cell, or the yeast cell can include one or more additional genetic alterations, as discussed more generally above.

[0159] In particular embodiments, the above described TAL-expressing engineered yeast (e.g., S. cerevisiae) cell expresses at least the following three additional enzymes:

[0160] (1) a shikimate kinase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a shikimate kinase from Corynebacterium glutamicum ATCC 13032. In some embodiments, the shikimate kinase can include or have SEQ ID NO: 2.

[0161] (2) a 3-deydroquinate synthase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a 3-deydroquinate synthase from Corynebacterium glutamicum ATCC 13032. In some embodiments, the 3-deydroquinate synthase can include or have SEQ ID NO:3. In some embodiments, this 3-deydroquinate synthase is replaced by a chorismate sythase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a chorismate sythase from Corynebacterium glutamicum ATCC 13032. In some embodiments, the chorismate synthase can include or have SEQ ID NO:4.

[0162] (3) a feedback-deregulated DAHP synthase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a feedback-deregulated DAHP synthase from Saccharomyces cerevisiae, wherein the DAHP synthase includes a K229L substitution. In some embodiments, the feedback- deregulated DAHP synthase can include or have SEQ ID NO:5.

[0163] For ease of discussion, the above-described cell, with these four genetic alterations, is referred to as “the improved yeast cell.” These may be the only genetic alterations of the engineered yeast cell, or the yeast cell can include one or more additional genetic alterations, such as any of those discussed more generally above. Example 1 discloses the following further improved yeast cells, which contain further genetic alterations, in addition to the four genetic alterations in the improved yeast cell.

[0164] A first further improved yeast cell additionally expresses the following three enzymes:

[0165] (1) a second copy of the shikimate kinase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a shikimate kinase from Corynebacterium glutamicum ATCC 13032. In some embodiments, the shikimate kinase can include or have SEQ ID NO:2.

[0166] (2) a chorismate synthase having at least 70 percent, 75 percent, 80 percent,

85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a chorismate synthase from Corynebacterium glutamicum ATCC 13032. In some embodiments, the chorismate synthase can include or have SEQ ID NO:4. [0167] (3) a transaldolase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a transaldolase from Corynebacterium glutamicum ATCC 13032. In some embodiments, the transaldolase can include or have SEQ ID NO:6.

[0168] A second further improved yeast cell additionally expresses two copies of a feedback-deregulated DAHP synthase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a feedback- deregulated DAHP synthase from Escherichia coli, wherein the DAHP synthase includes a D146N substitution. In some embodiments, the feedback-deregulated DAHP synthase can include or have SEQ ID NO:7.

[0169] A third further improved yeast cell additionally expresses the following three enzymes:

[0170] (1) a second copy of the shikimate kinase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a shikimate kinase from Corynebacterium glutamicum ATCC 13032. In some embodiments, the shikimate kinase can include or have SEQ ID NO:2.

[0171] (2) a glucose-6-phosphate dehydrogenase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a glucose-6-phosphate dehydrogenase from Corynebacterium glutamicum R, wherein the glucose-6-phosphate dehydrogenase includes an A243T substitution. In some embodiments, the glucose-6-phosphate dehydrogenase can include or have SEQ ID NO: 8.

[0172] (3) a prephenate dehydrogenase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a prephenate dehydrogenase from Corynebacterium glutamicum ATCC 13032. In some embodiments, the prephenate dehydrogenase can include or have SEQ ID NO:9.

[0173] A fourth further improved yeast cell additionally expresses the following three enzymes:

[0174] (1) a second copy of the shikimate kinase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a shikimate kinase from Corynebacterium glutamicum ATCC 13032. In some embodiments, the shikimate kinase can include or have SEQ ID NO:2. [0175] (2) a 6-phosphogluconate dehydrogenase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a 6-phosphogluconate dehydrogenase from Corynebacterium glutamicum ATCC 1303, wherein the 6-phosphogluconate dehydrogenase includes an S361F substitution. In some embodiments, the 6-phosphogluconate dehydrogenase can include or have SEQ ID NO: 10.

[0176] (3) a prephenate dehydrogenase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a prephenate dehydrogenase from Corynebacterium glutamicum ATCC 13032. In some embodiments, the prephenate dehydrogenase can include or have SEQ ID NO:9.

[0177] A fifth further improved yeast cell additionally expresses the following two enzymes (with one present in two copies):

[0178] (1) a 3-deydroquinate dehydratase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a 3-deydroquinate synthase from Corynebacterium glutamicum ATCC 13032. In some embodiments, the 3-deydroquinate synthase can include or have SEQ ID NO: 11.

[0179] (2) two copies of a transaldolase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a transaldolase from Corynebacterium glutamicum ATCC 13032. In some embodiments, the transaldolase can include or have SEQ ID NO:6.

[0180] The first through fifth further improved cells produced p-coumaric acid at titers of 37 mg/L, 36 mg/L, 35 mg/L, 35 mg/L, and 34 37 mg/L of culture medium (Example 1). These may be the only genetic alterations of the engineered yeast cell, or the yeast cell can include one or more additional genetic alterations, such as any of those discussed more generally above.

Illustrative Engineered Bacterial Cells

Illustrative Engineered Corynebacterium glutamicum Cells

[0181] In certain embodiments an engineered bacterial (e.g., C. glutamicum) cell expresses a heterologous L-tyrosine ammonia lyase (TAL) having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a TAL from Amycolatopsis orientalis HCCB 10007. In some embodiments, the A. orientalis HCCB10007 can include or have SEQ ID NO: 12. This may be the only genetic alteration of the engineered yeast cell, or the yeast cell can include one or more additional genetic alterations, as discussed more generally above.

[0182] In particular embodiments, the above-described TAL-expressing engineered bacterial (e.g., C. glutamicum) cell has increased chorismate mutase activity, compared to a control cell, e.g., produced by replacing the native promoter with a strong, constitutive promoter to produce an improved engineered bacterial cells. These may be the only genetic alterations of the improved engineered bacterial cell, or the cell can include one or more additional genetic alterations, such as any of those discussed more generally above.

[0183] In an illustrative embodiment, the improved engineered bacterial cell can be further improved by expressing a feedback-deregulated DAHP synthase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a feedback-deregulated DAHP synthase from Escherichia coli, wherein the DAHP synthase includes a P150L substitution. In some embodiments, the feedback-deregulated DAHP synthase can include or have SEQ ID NO: 13. This further improved bacterial cell produced p-coumaric acid at a titer of 140-170 mg/L of culture medium (Example 1).

Culturing of Engineered Microbial Cells

[0184] Any of the microbial cells described herein can be cultured, e.g., for maintenance, growth, and/or p-coumaric acid production. Generally, p-coumaric acid is produced from fermentation of a substrate wherein at least 20% of the substrate is not derived from protein or amino acid sources. Accordingly, cultures of the engineered microbial cells described herein include a fermentation substrate, wherein at least 20 percent of the substrate is not derived from protein or amino acid sources. In various embodiments, at least 25 percent, 30 percent, 35 percent, 40 percent, 45 percent, 50 percent, 55 percent, 60 percent, 65 percent, 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent of the substrate is not derived from protein or amino acid sources. In some embodiments, the percentage of the fermentation substrate that is not derived from protein or amino acid sources falls within any of the following illustrative ranges: 40-100 percent, 40-90 percent, 40-80 percent, 50-100 percent, 50-90 percent, 50-80 percent, 60-100 percent, 60-90 percent, 60-80 percent, etc. [0185] In some embodiments, the cultures are grown to an optical density at 600 nm of 10-500, such as an optical density of 50-150.

[0186] In various embodiments, the cultures include produced p-coumaric acid at titers of at least 10, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, or 900 mg/L or at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, or 10 gm/L. In various embodiments, the titer is in the range of 10 mg/L to 10 gm/L, 100 mg/L to 5 gm/L, 200 mg/L to 4 gm/L, 300 mg/L to 3 gm/L, or any range bounded by any of the values listed above.

Culture Media

[0187] Microbial cells can be cultured in any suitable medium including, but not limited to, a minimal medium, i.e., one containing the minimum nutrients possible for cell growth. Minimal medium typically contains: (1) a carbon source for microbial growth; (2) salts, which may depend on the particular microbial cell and growing conditions; and (3) water. Suitable media can also include any combination of the following: a nitrogen source for growth and product formation, a sulfur source for growth, a phosphate source for growth, metal salts for growth, vitamins for growth, and other cofactors for growth.

[0188] Any suitable carbon source can be used to cultivate the host cells. The term “carbon source” refers to one or more carbon-containing compounds capable of being metabolized by a microbial cell. In various embodiments, the carbon source is a carbohydrate (such as a monosaccharide, a disaccharide, an oligosaccharide, or a polysaccharide), or an invert sugar (e.g., enzymatically treated sucrose syrup). Illustrative monosaccharides include glucose (dextrose), fructose (levulose), and galactose; illustrative oligosaccharides include dextran or glucan, and illustrative polysaccharides include starch and cellulose. Suitable sugars include C6 sugars (e.g., fructose, mannose, galactose, or glucose) and C5 sugars (e.g., xylose or arabinose). Other, less expensive carbon sources include sugar cane juice, beet juice, sorghum juice, and the like, any of which may, but need not be, fully or partially deionized.

[0189] The salts in a culture medium generally provide essential elements, such as magnesium, nitrogen, phosphorus, and sulfur to allow the cells to synthesize proteins and nucleic acids.

[0190] Minimal medium can be supplemented with one or more selective agents, such as antibiotics. [0191] To produce p-coumaric acid, the culture medium can include, and/or is supplemented during culture with, glucose and/or a nitrogen source such as urea, an ammonium salt, ammonia, or any combination thereof.

Culture Conditions

[0192] Materials and methods suitable for the maintenance and growth of microbial cells are well known in the art. See, for example, U.S. Pub. Nos. 2009/0203102, 2010/0003716, and 2010/0048964, and International Pub. Nos. WO 2004/033646, WO 2009/076676, WO 2009/132220, and WO 2010/003007, Manual of Methods for General Bacteriology Gerhardt et al., eds), American Society for Microbiology, Washington, D.C. (1994) or Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass.

[0193] In general, cells are grown and maintained at an appropriate temperature, gas mixture, and pH (such as about 20°C to about 37°C, about 6% to about 84% CO₂, and a pH between about 5 to about 9). In some aspects, cells are grown at 35°C. In certain embodiments, such as where thermophilic bacteria are used as the host cells, higher temperatures (e.g., 50°C -75°C) may be used. In some aspects, the pH ranges for fermentation are between about pH 5.0 to about pH 9.0 (such as about pH 6.0 to about pH 8.0 or about 6.5 to about 7.0). Cells can be grown under aerobic, anoxic, or anaerobic conditions based on the requirements of the particular cell.

[0194] Standard culture conditions and modes of fermentation, such as batch, fed- batch, or continuous fermentation that can be used are described in U.S. Publ. Nos. 2009/0203102, 2010/0003716, and 2010/0048964, and International Pub. Nos. WO 2009/076676, WO 2009/132220, and WO 2010/003007. Batch and Fed-Batch fermentations are common and well known in the art, and examples can be found in Brock, Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc.

[0195] In some embodiments, the cells are cultured under limited sugar (e.g., glucose) conditions. In various embodiments, the amount of sugar that is added is less than or about 105% (such as about 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%) of the amount of sugar that can be consumed by the cells. In particular embodiments, the amount of sugar that is added to the culture medium is approximately the same as the amount of sugar that is consumed by the cells during a specific period of time. In some embodiments, the rate of cell growth is controlled by limiting the amount of added sugar such that the cells grow at the rate that can be supported by the amount of sugar in the cell medium. In some embodiments, sugar does not accumulate during the time the cells are cultured. In various embodiments, the cells are cultured under limited sugar conditions for times greater than or about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, or 70 hours or even up to about 5-10 days. In various embodiments, the cells are cultured under limited sugar conditions for greater than or about 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 95, or 100% of the total length of time the cells are cultured. While not intending to be bound by any particular theory, it is believed that limited sugar conditions can allow more favorable regulation of the cells.

[0196] In some aspects, the cells are grown in batch culture. The cells can also be grown in fed-batch culture or in continuous culture. Additionally, the cells can be cultured in minimal medium, including, but not limited to, any of the minimal media described above. The minimal medium can be further supplemented with 1.0% (w/v) glucose (or any other six-carbon sugar) or less. Specifically, the minimal medium can be supplemented with 1% (w/v), 0.9% (w/v), 0.8% (w/v), 0.7% (w/v), 0.6% (w/v), 0.5% (w/v), 0.4% (w/v), 0.3% (w/v), 0.2% (w/v), or 0.1% (w/v) glucose. In some cultures, significantly higher levels of sugar (e.g., glucose) are used, e.g., at least 10% (w/v), 20% (w/v), 30% (w/v), 40 % (w/v), 50% (w/v), 60% (w/v), 70% (w/v), or up to the solubility limit for the sugar in the medium. In some embodiments, the sugar levels falls within a range of any two of the above values, e.g.: 0.1-10% (w/v), 1.0-20% (w/v), 10-70 % (w/v), 20-60 % (w/v), or SOSO % (w/v). Furthermore, different sugar levels can be used for different phases of culturing. For fed-batch culture (e.g., of S. cerevisiae or C. glutamicum), the sugar level can be about 100-200 g/L (10-20 % (w/v)) in the batch phase and then up to about 500-700 g/L (50-70 % in the feed).

[0197] Additionally, the minimal medium can be supplemented 0.1% (w/v) or less yeast extract. Specifically, the minimal medium can be supplemented with 0.1% (w/v), 0.09% (w/v), 0.08% (w/v), 0.07% (w/v), 0.06% (w/v), 0.05% (w/v), 0.04% (w/v), 0.03% (w/v), 0.02% (w/v), or 0.01% (w/v) yeast extract. Alternatively, the minimal medium can be supplemented with 1% (w/v), 0.9% (w/v), 0.8% (w/v), 0.7% (w/v), 0.6% (w/v), 0.5% (w/v), 0.4% (w/v), 0.3% (w/v), 0.2% (w/v), or 0.1% (w/v) glucose and with 0.1% (w/v), 0.09% (w/v), 0.08% (w/v), 0.07% (w/v), 0.06% (w/v), 0.05% (w/v), 0.04% (w/v), 0.03% (w/v), or 0.02% (w/v) yeast extract. In some cultures, significantly higher levels of yeast extract can be used, e.g., at least 1.5% (w/v), 2.0% (w/v), 2.5% (w/v), or 3 % (w/v). In some cultures (e.g., of S. cerevisiae or C. glutamicum), the yeast extract level falls within a range of any two of the above values, e.g.: 0.5-3.0% (w/v), 1.0-2.5% (w/v), or 1.5-2.0% (w/v).

[0198] Illustrative materials and methods suitable for the maintenance and growth of the engineered microbial cells described herein can be found below in Example 1. p-Coumaric acid Production and Recovery

[0199] Any of the methods described herein may further include a step of recovering p-coumaric acid. In some embodiments, the produced p-coumaric acid contained in a so-called harvest stream is recovered/harvested from the production vessel. The harvest stream may include, for instance, cell-free or cell-containing aqueous solution coming from the production vessel, which contains p-coumaric acid as a result of the conversion of production substrate by the resting cells in the production vessel. Cells still present in the harvest stream may be separated from the p-coumaric acid by any operations known in the art, such as for instance filtration, centrifugation, decantation, membrane crossflow ultrafiltration or microfiltration, tangential flow ultrafiltration or microfiltration or dead end filtration. After this cell separation operation, the harvest stream is essentially free of cells.

[0200] Further steps of separation and/or purification of the produced p-coumaric acid from other components contained in the harvest stream, i.e., so-called downstream processing steps may optionally be carried out. These steps may include any means known to a skilled person, such as, for instance, concentration, extraction, crystallization, precipitation, adsorption, ion exchange, chromatography, distillation, electrodialysis, bipolar membrane electrodialysis and/or reverse osmosis. Any of these procedures can be used alone or in combination to purify p-coumaric acid. Further purification steps can include one or more of, e.g., concentration, crystallization, precipitation, washing and drying, treatment with activated carbon, ion exchange and/or re-crystallization. The design of a suitable purification protocol may depend on the cells, the culture medium, the size of the culture, the production vessel, etc. and is within the level of skill in the art.

[0201] The following examples are given for the purpose of illustrating various embodiments of the disclosure and is not meant to limit the present disclosure in any fashion. Changes therein and other uses which are encompassed within the spirit of the disclosure, as defined by the scope of the claims, will be identifiable to those skilled in the art.

EXAMPLE 1 - Construction and Selection of Strains of Saccharomyces cerevisiae and Corynebacterium slutamicum Engineered to Produce p-Coumaric Acid

Abstract

[0202] We conducted a search of metabolism [1] to identify enzymes that enable a metabolic pathway to produce p-coumaric acid in industrial host organisms from simple carbon and nitrogen sources (see Table 1). To engineer production of p-coumaric acid in an industrial microorganism, we employed genetic engineering tools and methods to manipulate DNA sequences, such as those shown in Figs. 11-14). We systematically, reengineered the microbial metabolism to produce p-coumaric acid (including in industrial hosts for which not all biochemical reactions or modes of metabolic regulation have been characterized) by iterative high-throughput (HTP) strain engineering using single-gene and multiple-gene modifications (see, e.g., US20170159045A1, which is hereby incorporated by reference herein for its description of HTP strain engineering). More specifically, to produce p-coumaric acid in an industrial fermentation we assembled pathways with improved biosynthesis of the amino acid precursor tyrosine and a highly active tyrosine ammonia lyase. We have identified active tyrosine ammonia lyases and found that feedback-deregulated DAHP synthases and constitutive expression of shikimate pathway enzymes improve the titers of p-coumaric acid.

Plasmid/DNA Design

[0203] All strains tested for this work were transformed with plasmid DNA designed using proprietary software. Plasmid designs were specific to one of the two host organisms engineered in this work. The plasmid DNA was physically constructed by a standard DNA assembly method. This plasmid DNA was then used to integrate metabolic pathway inserts by one of two host-specific methods, each described below.

S. cerevisiae and Y. lipolitica Pathway Integration

[0204] A “split-marker, double-crossover” genomic integration strategy has been developed to engineer S. cerevisiae strains. Fig. 11 illustrates genomic integration of complementary, split-marker plasmids and verification of correct genomic integration via colony PCR in S. cerevisiae, or Y. lipolytica. Two plasmids with complementary 5’ and 3’ homology arms and overlapping halves of a URA3 selectable marker (direct repeats shown by the hashed bars) were digested with meganucleases and transformed as linear fragments. A triple-crossover event integrated the desired heterologous genes into the targeted locus and re-constituted the full URA3 gene. Colonies derived from this integration event were assayed using two 3-primer reactions to confirm both the 5’ and 3’ junctions (UF/IF/wt-R and DR/IF/wt-F). For strains in which further engineering is desired, the strains can be plated on 5-FOA plates to select for the removal of URA3, leaving behind a small single copy of the original direct repeat. This genomic integration strategy can be used for gene knock-out (Fig. 13), gene knock-in (Fig. 11), and promoter titration (Fig. 12) in the same workflow.

C. slutamicum Pathway Integration

[0205] A “loop-in, single-crossover” genomic integration strategy has been developed to engineer C. glutamicum strains. Fig. 14 illustrates genomic integration of loop-in only and loop-in/loop-out constructs and verification of correct integration via colony PCR. Loop-in only constructs (shown under the heading “Loop-in”) contained a single 2-kb homology arm (denoted as “integration locus”), a positive selection marker (denoted as “Marker”)), and gene(s) of interest (denoted as “promoter-gene-terminator”). A single crossover event integrated the plasmid into the C. glutamicum chromosome. Integration events are stably maintained in the genome by growth in the presence of antibiotic (25μg/ml kanamycin). Correct genomic integration in colonies derived from loop-in integration were confirmed by colony PCR with UF/IR and DR/IF PCR primers.

[0206] Loop-in, loop-out constructs (shown under the heading “Loop-in, loop-out) contained two 2-kb homology arms (5’ and 3’ arms), gene(s) of interest (arrows), a positive selection marker (denoted “Marker”), and a counter-selection marker. Similar to “loop-in” only constructs, a single crossover event integrated the plasmid into the chromosome of C. glutamicum. Note: only one of two possible integrations is shown here. Correct genomic integration was confirmed by colony PCR and counter-selection was applied so that the plasmid backbone and counter-selection marker could be excised. This results in one of two possibilities: reversion to wild-type (lower left box) or the desired pathway integration (lower right box). Again, correct genomic loop-out is confirmed by colony PCR.

(Abbreviations: Primers: UF = upstream forward, DR = downstream reverse, IR = internal reverse, IF = internal forward.) Cell Culture

[0207] Separate workflows were established for C. glutamicum and S. cerevisiae due to differences in media requirements and growth. Both processes involved a hit-picking step that consolidated successfully built strains using an automated workflow that randomized strains across the plate. For each strain that was successfully built, up to four replicates were tested from distinct colonies to test colony -to-colony variation and other process variation. If fewer than four colonies were obtained, the existing colonies were replicated so that at least four wells were tested from each desired genotype.

[0208] The colonies were consolidated into 96-well plates with selective medium (BHI for C. glutamicum, SD-ura for S. cerevisiae) and cultivated for two days until saturation and then frozen with 16.6% glycerol at -80°C for storage. The frozen glycerol stocks were then used to inoculate a seed stage in minimal media with a low level of amino acids to help with growth and recovery from freezing. The seed plates were grown at 30°C for 1-2 days. The seed plates were then used to inoculate a main cultivation plate with minimal medium and grown for 48-88 hours. Plates were removed at the desired time points and tested for cell density (OD600), viability and glucose, supernatant samples stored for LC-MS analysis for product of interest.

Cell Density

[0209] Cell density was measured using a spectrophotometric assay detecting absorbance of each well at 600nm. Robotics were used to transfer fixed amounts of culture from each cultivation plate into an assay plate, followed by mixing with 175mM sodium phosphate (pH 7.0) to generate a 10-fold dilution. The assay plates were measured using a Tecan Ml 000 spectrophotometer and assay data uploaded to a LIMS database. A noninoculated control was used to subtract background absorbance. Cell growth was monitored by inoculating multiple plates at each stage, and then sacrificing an entire plate at each time point.

[0210] To minimize settling of cells while handling large number of plates (which could result in a non-representative sample during measurement) each plate was shaken for 10-15 seconds before each read. Wide variations in cell density within a plate may also lead to absorbance measurements outside of the linear range of detection, resulting in underestimate of higher OD cultures. In general, the tested strains so far have not varied significantly enough for this be a concern. Cell Viability

[0211] Two methods were used to measure cell viability. The first assay utilized a single stain, propidium iodide, to assess cell viability. Propidium iodide binds to DNA and is permeable to cells with compromised cell membranes. Cells that take up the propidium iodide are considered non-viable. A dead cell control was used to normalize to total number of cells, by incubating a cell sample of control culture at 95°C for 10 minutes. These control samples and test samples were incubated with the propidium iodide stain for 5 minutes, washed twice with 175mM phosphate buffer, and fluorescence measured in black solid-bottom 96-well plates at 617nm.

Glucose

[0212] Glucose is measured using an enzymatic assay with 16U/mL glucose oxidase (Sigma) with 0.2 U/mL horseradish peroxidase (Sigma) and 0.2mM Amplex red in 175mM sodium phosphate buffer, pH 7. Oxidation of glucose generates hydrogen peroxide, which is then oxidized to reduce Amplex red, which changes absorbance at 560nm. The change is absorbance is correlated to the glucose concentration in the sample using standards of known concentration.

Liquid-Solid Separation

[0213] To harvest extracellular samples for analysis by LC-MS, liquid and solid phases were separated via centrifugation. Cultivation plates were centrifuged at 2000 rpm for 4 minutes, and the supernatant was transferred to destination plates using robotics.

75 pL of supernatant was transferred to each plate, with one stored at 4°C, and the second stored at 80°C for long-term storage.

Genetic Engineering Approach and Results

First Round of Genetic Engineering

[0214] A library approach was taken to screen heterologous pathway enzymes to establish a p-coumaric acid pathway. In Corynebacterium glutamicum, 13 tyrosine ammonia lyases (EC 4.3.1.23) were tested from 11 bacterial and two fungal sources listed in Table 1 (below). In Saccharomyces cerevisiae, 16 tyrosine ammonia lyases were tested from 11 bacterial, four fungal and one Viridiplantae sources, also listed in Table 1. The best tyrosine ammonia lyase in C. glutamicum was from Amycolatopsis orientalis HCCB10007 (UniProt ID R4TC14). In S. cerevisiae, the best tyrosine ammonia lyase was from Acremonium chrysogenum strain ATCC 11550 (UniProt ID A0A086SVQ5). A C. glutamicum strain expressing the Amycolatops is orientalis HCCB 10007 (UniProt ID R4TC14) tyrosine ammonia lyase and an S. cerevisiae strain expressing Acremonium chrysogenum strain ATCC 11550 (UniProt ID A0A086SVQ5) tyrosine ammonia lyase were selected for a second round of genetic engineering.

Second Round of Genetic Engineering

[0215] A combinatorial library approach was taken to screen for the rate-limiting step in the second round of genetic engineering to improve p-coumaric acid production in C. glutamicum by separately expressing upstream pathway enzymes with a constitutive promoter (Table 2A). Native promoters were swapped with strong constitutive promoters for the enzymes: prephenate dehydrogenase, chorismate mutase, 3-deoxy-D-arabino- heptulosonate 7-phosphate synthase (DAHP synthase) (EC 2.5.1.54), 3-dehydroquinate dehydratase (EC 4.2.1.10), shikimate 5-dehydrogenase (EC 1.1.1.25) and chorismate synthase (CS) (EC 4.2.3.5). Promoter swap (PROSWP) engineering in C. glutamicum was accomplished by the single cross-over method (Fig. 14).

[0216] In C. glutamicum, the largest improvement in p-coumaric acid titer occurred in a strain having constitutive expression of the native chorismate mutase (NCglO819, UniProt ID Q8NS29 (Table 2(A); Figure 4).

[0217] In S. cerevisiae, a combinatorial library approach was used to design strains constructed to test additional upstream enzymes in a second round of genetic engineering (Table 2(B); Fig. 5). Each integrating plasmid was designed to constitutively express three enzymes from C. glutamicum or S. cerevisiae in a strain selected from the list: DAHP synthase (EC 2.5.1.54), shikimate 5-dehydrogenase (EC 1.1.1.25), 3-dehydroquinate synthase (EC 4.2.3.4), shikimate kinase (EC 2.7.1.71), transaldolase (EC 2.2.1.2), enolase (4.2.1.11), chorismate mutase (EC 5.4.99.5), chorismate synthase (EC 4.2.3.5), 3- phosphoshikimate 1 -carboxy vinyltransferase (EC 2.5.1.19 ), 3-dehydroquinate dehydratase (EC 4.2.1.10), aspartate transaminase (EC 2.6.1), prephenate dehydrogenase (EC 1.3.1.12). In some cases the feedback-deregulated variant of DAHP synthase (UniProt ID P32449) from S. cerevisiae harboring K229L was tested [4] .

[0218] In S. cerevisiae the largest improvement in p-coumaric acid titer for the second round of genetic engineering occurred in a strain harboring constitutive expression of shikimate kinase (UniProt ID Q9X5D1) from C. glutamicum and feedback-deregulated DAHP synthase (UniProt ID P32449) harboring the amino acid substitution K229L, together with either 3-dehydroquinate synthase (UniProt ID Q9X5D2) from C. glutamicum or chorismate synthase (UnitProt ID Q9X5D0) from C. glutamicum. (Table 2(B); Fig. 5).

[0219] The best-preforming stains from the second round of genetic engineering were used as the starting strains for the third round of genetic engineering.

Third Round of Genetic Engineering

[0220] A combinatorial library approach was taken to screen for the rate-limiting step(s) in the third round of genetic engineering to improve p-coumaric acid production in C. glutamicum by additionally expressing one, two, or three of the following upstream pathway enzymes with a constitutive promoter: enolase (EC 4.2.1.11), shikimate kinase (EC 2.7.1.71), 3-phosphoshikimate 1 -carboxy vinyltransferase (EC 2.5.1.19 ), DAHP synthase (EC 2.5.1.54), 3-dehydroquinate dehydratase (EC 4.2.1.10), 3-dehydroquinate synthase (EC 4.2.3.4), chorismate synthase (EC 4.2.3.5), shikimate 5 -dehydrogenase (EC 1.1.1.25), transaldolase (EC 2.2.1.2), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), and prephenate dehydrogenase (EC 1.3.1.12) (Table 3A). In some cases, feedback-deregulated variants of the enzymes were tested, such as DAHP synthase (UniProt ID P00888) from Escherichia coli containing the amino acid substitution N8K, DAHP synthase (UniProt ID P0AB91) from E. coli containing the amino acid substitution P150L, 6-phosphogluconate dehydrogenase (UniProt ID Q8NQI2) from C. glutamicum with the amino acid substitution S361F, glucose-6- phosphate dehydrogenase (UniProt ID A4QEF2) from C. glutamicum with the amino acid substitution A243T.

[0221] In C. glutamicum the best-performing strain produced 140-170 microgram/L, and this strain harbored the addition of DHAP synthase (UniProt ID P0AB91) from E. coli containing the amino acid substitution P150L [5] (Table 3(A); Fig. 6).

[0222] A combinatorial library approach was taken to screen for the rate-limiting step in the third round of genetic engineering to improve p-coumaric acid production in S. cerevisiae by expressing two or three of the following upstream pathway enzymes with a constitutive promoter: 3-dehydroquinate synthase (EC 4.2.3.4), 3-dehydroquinate dehydratase (EC 4.2.1.10), enolase (4.2.1.11), L-tyrosine ammonia lyase (EC 4.3.1.23), shikimate kinase (EC 2.7.1.71), chorismate mutase (EC 5.4.99.5), prephenate dehydrogenase (EC 1.3.1.12), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), DAHP synthase (EC 2.5.1.54), glucose-6-phosphate dehydrogenase (zwl) (EC 1.1.1.49), hydroxy phenylpyruvate synthase (EC 1.3.1.12), phospho-2-dehydro-3-deoxyheptonate aldolase (EC 2.5.1.54), transaldolase (EC 2.2.1.2), aspartate transaminase (EC 2.6.1), shikimate 5-dehydrogenase (EC 1.1.1.25), chorismate synthase (EC 4.2.3.5), and 3- phosphoshikimate 1 -carboxy vinyltransferase (EC 2.5.1.19 ). In some cases, feedback- deregulated variants of the following enzymes were tested, e.g., chorismate mutase/prephenate dehydrogenase (UniProt ID P0A9J8) from E. coli containing the amino acid substitutions Q306L and G309C, 6-phosphogluconate dehydrogenase (UniProt ID Q8NQI2) from C. glutamicum containing the amino acid substitution S361F, DAHP synthase (UniProt ID P0AB91) from E. coli containing the amino acid substitution D146N, and glucose-6-phosphate dehydrogenase (UniProt ID A4QEF2) from C. glutamicum with the amino acid substitution A243T.

[0223] In S. cerevisiae, the top five strains produced titers of 34 mg/L or greater and the best-performing strain produced titer of 37 mg/L. The strain harboring the three additional enzymes 3-dehydroquinate dehydratase (UniProt ID 052377), transaldolase (UniProt ID Q8NQ64) and transaldolase (UniProt ID Q8NQ64) produced 34 mg/L.

Another strain harboring two copies of DAHP synthase (UniProt ID P0AB91) from E. coli containing the amino acid substitution D146N produced 36 mg/L. Another strain harboring the three additional C. glutamicum enzymes shikimate kinase (UniProt ID Q9X5D1), glucose-6-phosphate dehydrogenase (UniProt ID A4QEF2) containing the amino acid substitution A243T, and prephenate dehydrogenase (UniProt ID Q8NTS6) produced 35 mg/L. Another strain harboring the three additional C. glutamicum enzymes shikimate kinase (UniProt ID Q9X5D1), 6-phosphogluconate dehydrogenase (UniProt ID Q8NQI2) containing the amino acid substitution S361F, and prephenate dehydrogenase (UniProt ID Q8NTS6) produced 35 mg/L. Another strain harboring the three additional enzymes shikimate kinase (UniProt ID Q9X5D1), chorismate synthase (UniProt ID Q9X5D0), and transaldolase (UniProt ID Q8NQ64) produced 37 mg/L.

EXAMPLE 2 - Construction and Selection of Strains of Yarrowia lipolytica Engineered to Produce p-Coumaric Acid and Host Evaluation

[0224] p-Coumaric acid production was tested in an additional host, Yarrowia lipolytica, by expressing the enzymes from the best-performing strains identified in Corynebacterium glutamicum (Cg)and Saccharomyces cerevisiae (Sc). Yarrowia lipolytica is a safe and robust yeast that is used for industrial applications [20, 21], Strain engineering tools for Y. lipolytica [22-24] were employed to engineer p-coumaric acid production.

[0225] The following strain designs for Y. lipolytica were tested: 1) the best tyrosine ammonia lyase from Cg plus a feedback-deregulated DHAP synthase, 2) the best tyrosine ammonia lyase from Sc plus a feedback-deregulated DHAP synthase, and 3) the best tyrosine ammonia lyase from Sc plus a feedback-deregulated DAHP synthase and a shikimate kinase. Shikimate kinase was shown to improve production of p-coumaric acid in the second round of genetic engineering (Fig. 3 and Table 2). Improved flux through the shikimate pathway in Bacillus subtilis, e.g., can be achieved by expressing one or more S. cerevisiae shikimate kinases, such AROI and/or AROD (See U.S. Patent No. 6,436,664.)

[0226] The best performing strain expressed the enzyme L-tyrosine ammonia lyase (UniProt ID A0A086SVQ5) with DAHP synthase (UniProt ID P32449) and produced 10 mg/L (Figure 8, Table 4).

[0227] In a host evaluation round of genetic engineering, the best enzymes identified in S. cerevisiae and C. glutamicum were compared in Y. lipolytica, S. cerevisiae, C. glutamicum, and Bacillus subtilis. In Y. lipolytica, as noted above, the best performing strain expressed the enzyme L-tyrosine ammonia lyase (UniProt ID A0A086SVQ5) with DAHP synthase (UniProt ID P32449) and produced 10 mg/L (Figure 8, Table 4). p- Coumaric acid production was not observed in Bacillus subtilis (possibly because the strains were not successfully built), and titers in C. glutamicum were not improved over the parent strain. In S. cerevisiae the best strain expressed the enzyme L-tyrosine ammonia lyase (UniProt ID A0A086SVQ5) with DAHP synthase (UniProt ID P32449), and produced 35 mg/L (Figure 9, Table 5).

EXAMPLE 3 - Engineering Approach to Improve p-Coumaric acid titer in

Saccharomyces cerevisiae (Improvement Round)

[0228] p-Coumaric acid production was further pursued in Saccharomyces cerevisiae. An expanded search was implemented to test ten additional tyrosine ammonia lyases (TALs) related to the sequences of hits initially identified to be active enzymes in S. cerevisiae and Corynebacterium glutamicum (Table 6).

[0229] In parallel we pursued modulating native gene expression to further improve p-coumaric acid production. We designed plasmids to integrate additional copies of native upstream pathway genes expressed by a strong constitutive promoter to avoid native regulation of a gene (Table 6).

[0230] Various enzymes shown or expected to improve p-coumaric acid production in the engineered strains that produce it are described below.

[0231] ARO4 is an S. cerevisiae DAHP synthase (EC 2.5.1.54). Feedback regulation of DAHP synthase by tyrosine has been shown to inhibit the shikimate pathway. Removing feedback inhibition from DAHP synthase, such as by the amino acid substitution K229L, results in upregulation of endogenous tyrosine biosynthesis. In some embodiments, an additional copy of ARO4 K229L has been introduced into a host organism [4], In some embodiments, an additional copy of DAHP synthase (UniProt ID P0AB91) from Escherichia coli containing the amino acid substitution D146N was expressed.

[0232] ARO7 is an S. cerevisiae chorismate mutase (EC 5.4.99.5), which catalyzes the Claisen rearrangement of chorismate to prephenate and is inhibited by tyrosine. In some embodiments, a feedback-deregulated chorismate mutase from S. cerevisiae ARO7 harboring the amino acid substitution T226I or I225T has been overexpressed by a strong constitutive promoter [6, 7], In some embodiments, a feedback-deregulated chorismate mutase from E. coli harboring the amino acid substitution G141S is over-expressed by a strong constitutive promoter [4], In some embodiments, a feedback-deregulated chorismate mutase from E. coli harboring the amino acid substitutions M53I and A354V is overexpressed by a strong constitutive promoter [8],

[0233] Shikimate kinase (EC 2.7.1.71) activity is part of the pentafunctional AROM polypeptide ARO1 from S. cerevisiae, which can be increased by overepression In some embodiments, shikimate kinase activity is increased by overexpressing AroL from E. coli.

[0234] Aromatic amino acid transaminase (EC 2.6.1.57) or tyrosine transaminase catalyzes the transamination of 4-hydroxyphenyl pyruvate to tyrosine. In some embodiments, the expression of the aromatic amino acid transaminase encoded by S. cerevisiae ARO9 is increased to improve production of p-coumaric acid.

[0235] Chorismate synthase (EC 4.2.3.5) catalyzes the formation of chorismate, and increased chorismate synthase activity improves production of p-coumaric acid.

[0236] In S. cerevisiae, prephenate dehydrogenase (1.3.1.12) TYR1 expression is dependent on phenylalanine levels [9], Strong expression with a constitutive promoter improves p-coumaric acid production. In some embodiments, the cyclohexadienyl dehydrogenase (EC 1.3.1.79) encoded by the gene TYRC from the bacterium Zymomonas mobilis, which is known to be feedback-insensitive to tyrosine, can be overexpressed [10], Zymomonas mobilis TYRC is NADH-dependent while the S. cerevisiae TYR1 is NADPH- dependent.

[0237] S. cerevisiae TKL1, transketolase (EC 2.2.1.1), catalyzes two reactions: first, the reaction of D-xylulose-5-P and D-ribose-5-P to form a sedoheptulose-7-P and glyceraldehyde-3-P, and second, the reaction of fructose 6-phosphate and glyceraldehyde-3- P to produce D-xylulose-5-P and erythrose-4-phosphate. Both reactions are important to supply erythrose-4-phosphate to the shikimate pathway and therefore strong constitutive expression of transketolase can improve p-coumaric acid production [11],

[0238] In some embodiments, strains can be engineered to express multiple copies of glycolysis genes to improve glycolysis flux [12], which produces metabolic precursors to the p-coumaric pathway.

[0239] PEP synthase (EC 2.7.9.2) has been shown to improve shikimate pathway production, therefore overexpression of PEP synthase improves p-coumaric acid production.

[0240] S. cerevisiae ENO2 - enolase (EC 4.2. 1.11) catalyzes the 2- phosphogly cerate (2 -PG) to PEP, a precursor to the p-coumaric acid pathway, and strong constitutive expression improves p-coumaric acid production.

[0241] S. cerevisiae PGK1, 3-phosphoglycerate kinase (EC 2.7.2.3), catalyzes the glycolytic reaction, and strong constitutive expression improves p-coumaric acid production.

[0242] S. cerevisiae TALI- transaldolase catalyzes the reaction of sedoheptulose 7- phosphate and glyceraldehyde 3-phosphate to fructose 6-phosphate and erythrose 4- phosphate, the latter of which is a precursor to the shikimate pathway. In some embodiments, transaldolase can be overexpressed to improve production of p-coumaric acid.

[0243] Illustrative strain designs employing the above-described enzymes include: four designs to test feedback-deregulated chorismate mutases with chorismate synthase and aromatic amino acid transferase (both from S. cerevisiae and E. coli); four designs to test feedback-deregulated chorismate mutases with prephenate dehydrogenase and chorismate synthase from S. cerevisiae; four designs to test feedback-deregulated chorismate mutases with prephenate dehydrogenase and aromatic amino acid transferase from S. cerevisiae; three designs to test aromatic amino acid transaminase (S. cerevisiae, Homo sapiens); shikimate kinase ( E. coli) seven designs to test combinations of transketolase, transaldolase, PEP synthase, and PEP carboxykinase.

[0244] Glucose-6-phosphate dehydrogenase catalyzes the first step of the pentose phosphate pathway (PPP); improving PPP expression provides NADPH. In some embodiments, cells are engineered to have higher expression of the native glucose-6- phosphate dehydrogenase. In some embodiments, glucose-6-phosphate dehydrogenase can be overexpressed to increase the supply of NADPH to the p-coumaric acid pathway.

[0245] For a selection of native enzymes, production of p-coumaric acid can be improved when the activity becomes lower than the specific activity in a control strain (i.e., not having reduced activity of the enzyme(s) at issue) or a wild-type organism. The activity can be reduced, e.g., to 50% or less, 30% or less, or 10% or less per microbial cell, as compared with that in the control or wild-type strain. The activity can also be completely eliminated, such as through deletion of the gene.

[0246] For example, S. cerevisiae TRP2, anthranilate synthase, catalyzes the initial step of tryptophan biosynthesis from chorismate to anthranilate, and production of p- coumaric acid can be improved when anthranilate synthase activity is lower than the specific activity in control cells or is eliminated. In S. cerevisiae anthranilate synthase is encoded by Trp2. In C. glutamicum, anthranilate synthase is encoded by Cgl3029. In Bacillus subtilis, anthranilate synthase is encoded by trpE. In Yarrowia lipolytica, anthranilate synthase is encoded by YALI0D11110p.

[0247] S. cerevisiae TRP3, indole-3-glycerol phosphate synthase (EC 4.1.1.48), forms a bifunctional hetero-oligomeric anthranilate synthase:indole-3-glycerol phosphate synthase enzyme complex with TRP2 and consumes p-coumaric pathway precursors. Lower expression of indole-3 -glycerol phosphate synthase can improve p-coumaric acid production.

[0248] S. cerevisiae PHA2, prephenate dehydratase, catalyzes the conversion of prephanate to phenylpyruvate, which is a step in the phenylalanine biosynthesis pathway. Lower expression of prephenate dehydratase can decrease flux diverted from tyrosine.

[0249] S. cerevisiae ARO10, phenylpyruvate decarboxylase, catalyzes decarboxylation of phenylpyruvate to phenylacetaldehyde, which is the first specific step in the Ehrlich pathway. Loss of aromatic carbon has been limited in a host organism by eliminating phenylpyruvate decarboxylase. In S. cerevisiae, phenylpyruvate decarboxylase is encoded by Aro10. Production of p-coumaric acid can be improved when the activity of phenylpyruvate decarboxylase is lower than the specific activity in a control strain.

[0250] Deletion of the S. cerevisiae gene ending the SIT4 phosphatase has been shown to decrease pyruvate decarboxylase activity, which directs glucose flux to ethanol production in S. cerevisiae [15], Lower expression of SIT4 phosphatase is anticipated to improve p-coumaric acid production by reducing production of ethanol byproduct.

[0251] S. cerevisiae PDC5, PDC1, and PDC are pyruvate decarboxylases (EC

4.1.1.1). Pyruvate decarboxylase is key enzyme in alcohol fermentation and decarboxylates pyruvate to acetaldehyde. Lower expression of either PDC5, PDC1, PDC6, or all three, can improve p-coumaric acid production by decreasing production of ethanol byproduct.

[0252] Pyruvate dehydrogenase (EC 1.2.4.1) catalyzes the reaction of pyruvate to acetyl-CoA and CO₂ and depletes the PEP pool supplying the p-coumaric acid pathway. Lower expression of a pyruvate dehydrogenase gene, such as yeast Lpdl can lower pyruvate dehydrogenase activity and flux through the C3/C2 node, which can improve p-coumaric acid production.

[0253] S. cerevisiae CITI, citrate synthase (EC 2.3.3.1), catalyzes the condensation of acetyl coenzyme A and oxaloacetate to form citrate and is the rate-limiting enzyme of the tricarboxylic acid (TCA) cycle. In some embodiments, decreased citrate synthase activity to modify TCA flux can improve p-coumaric acid production.

[0254] Alcohol dehydrogenase (EC 1.1.1.1 ) activity catalyzes the formation of byproducts such as ethanol. In some embodiments, the expression of alcohol dehydrogenase enzyme activity can be reduced to improve yield of p-coumaric acid.

Alcohol dehydrogenase enzymes can be down-regulated in S. cerevisiae by down-regulating and/or deleting one or more of the ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, and SFA1 genes.

[0255] Aldehyde oxidase (EC 1.2.3.1) activity catalyzes formation of byproducts such as ethanol. In some embodiments, the expression of aldehyde oxidase enzymes can be reduced to improve yield of p-coumaric acid. Aldehyde oxidase enzymes can be down- regulated in S. cerevisiae by down-regulating and/or deleting one or more of the ALD2, ALD3, ALD4, ALD5, and ALD6 genes. [0256] S. cerevisiae FDC1, ferulic acid decarboxylase 1 (EC 4.1.1.102), catalyzes decarboxylation of aromatic carboxylic acids to corresponding vinyl derivatives and it also acta on p-coumaric acid [16-18], Production of p-coumaric acid can be increased by downregulating or deleting the gene encoding ferulic acid decarboxylase.

[0257] S. cerevisiae PAD1, phenylacrylic acid decarboxylase (EC 4.1.1.102), catalyzes decarboxylation of aromatic acids. Production of p-coumaric acid is anticipated to be increased upon down-regulating and/or deleting the gene encoding phenylacrylic acid decarboxylase.

[0258] S. cerevisiae ATF1 and ATF2, alcohol acetyl transferases (EC 2.3.1.84), catalyze the esterification of alcohols using acetyl-CoA. Alcohol acetyl transferases have been shown to form aromatic esters in S. cerevisiae [19], In some embodiments, expression or activity of alcohol acetyl transferases are reduced to improve p-coumaric acid production.

[0259] The approaches tested in the fourth round described above showed improvement in p-coumaric acid over the parent strain control. The top three strains produced p-coumaric acid titers of 46 mg/L or greater, and the best-performing strain produced a titer of 53 mg/L. Seventeen ammonia lyase enzymes identified from sequence similarity to the best L-tyrosine ammonia lyase were tested, and each of the top three strains expressed an enzyme known in UniProt as a histidine ammonia lyase. The top three enzymes were histidine ammonia lyases having UniProt ID A0A139WZS3, UniProt ID K9RDZ0, or UniProt ID A0A1U7HBU8.

[0260] A strain expressing transketolase from S. cerevisiae (UniProt ID P23254) and phosphoenolpyruvate carboxykinase from E. coli (UniProt ID P22259) produced 18.8 mg/L. (Table 6.) A strain that expressed an additional copy of DAPH (UniProt ID P32449) produced 19 mg/L. A strain that expressed an additional copy of chorismate mutase from Yarrowia lipolytica (UniProt ID Q6C5J7), prephanate dehydrogenase from S. cerevisiae (UniProt ID P20049), and L-2-aminoadipate:2-oxoglutarate aminotransferase from S. cerevisiae (UniProt ID P53090) produced 20 mg/L, an improvement over the parent strain control production of 16 mg/L. Table 1. Results of first round of engineering Corynebacterium glutamicum and Saccharomyces cerevisiae

Table 2. Results of second round of engineering Corynebacterium slutamicum and Saccharomyces cerevisiae

[0261] (A) The native promoters of the genes encoding the Corynebacterium glutamicum enzymes in the table below were replaced with the indicated promoter. In addition to the promoter replacements, the C. glutamicum strains also contained the best enzyme from first round of genetic engineering round: tyrosine ammonia lyase (UniProt ID R4TC14) from Amycolatopsis orientalis HCCB10007. (B) Saccharomyces cerevisiae strains expressed the enzymes indicated in the table below, in addition to the best enzyme from first round of genetic engineering round: tyrosine ammonia lyase (UniProt ID A0A086SVQ5) from Acremonium chrysogenum ATCC 11550.

(A) Corynebacterium slutamicum

(B) Saccharomyces cerevisiae

Table 3. Results of third round of engineering Corynebacterium glutamicum and Saccharomyces cerevisiae

[0262] Each of the strains in the tables below included the genetic alterations in the best-performing strain from the second round of genetic engineering.

(A) Corynebacterium glutamicum

(B) Saccharomyces cerevisiae

Table 4. Results of host evaluation round of engineering Yarrowia lipolytica

[0263] Each of the Yarrowia lipolytica strains expressed the enzymes indicated in the table below.

Table 5. Results of host evaluation round of engineering Saccharomyces cerevisiae.

[0264] Each of the Saccharomyces cerevisiae strains expressed the enzymes indicated in the table below.

Table 6. Results of improvement-round of engineering, Saccharomyces cerevisiae.

[0265] Each of the strains in the table below included the genetic alterations in the best-performing strain from third round of genetic engineering.

Table 7. Results of further engineering of Saccharomyces cerevisiae.

[0266] Each of the strains in the table below included the genetic alterations in the best-performing strain from the earlier rounds of genetic engineering: ScPCOUMA_217 (Table 6) for Saccharomyces cerevisiae and Y1PCOUMA 05 (Table5) for Yarrowia lipolytica.

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INFORMAL SEQUENCE LISTING

Claims

CLAIMS What is claimed is:

1. An engineered microbial cell, wherein the engineered microbial cell expresses at least one heterologous L-tyrosine ammonia lyase (TAL) that is from: a fungal phylum selected from the group consisting of Ascomycota and Basidiomycota; or a bacterial phylum selected from the group consisting of Actinobacteria and Proteobacteria; wherein the engineered microbial cell produces p-coumaric acid, optionally: wherein the at least one heterologous TAL is from the phylum Ascomycota, optionally from the genus Acremonium, optionally from Acremonium chrysogenunr, and/or wherein the at least one heterologous TAL is from the phylum Basidiomycota, optionally from the genus Rhodosporidium, optionally from Rhodosporidium toruloides,' and/or wherein the at least one heterologous TAL is from the phylum Actinobacteria, optionally from the genus Amycolatopsis and/or Str eptomyces, optionally from Amycolatopsis orientalis and/or Str eptomyces sp. WK-5344; and/or wherein the at least one heterologous TAL is from the phylum Proteobacteria, optionally from a bacterium selected from the group consisting of an alphaproteobacterium, a betaproteobacterium, and a gamma proteobacterium, optionally from alpha proteobacterium Q-l, Rhodobacter sphaeroides, Cupriavidus metallidurans , and/or Halomonas chromatireducens .

2. The engineered microbial cell of claim 1, wherein the engineered microbial cell comprises increased activity of one or more upstream enzyme(s) in the tyrosine biosynthesis pathway, said increased activity being increased relative to a control cell, optionally wherein the one or more upstream enzyme(s) comprises one or more enzyme(s) selected from the group consisting of a 3-phosphogly cerate kinase, a transketolase, a transaldolase, an enolase, a phospoenolpyruvate (PEP) synthase, a phospoenolpyruvate (PEP)carboxykinase, a 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, a dehydroquinate synthase, a dehydroquinate dehydratase, a shikimate dehydrogenase, a shikimate kinase, 3-phosphoshikimate 1 -carboxy vinyl transferase, EPSP synthase, aromatic pentafunctional enzyme, a chorismate synthase, a chorismate mutase, a phenyalananine aminotransferase, a prephenate dehydrogenase, a prephenate aminotransferase, an arogenate dehydrogenase, a phenylalanine 4-hydroxylase, and a tyrosine aminotransferase.

3. The engineered microbial cell of claim 1 or claim 2, wherein the engineered microbial cell expresses one or more feedback-deregulated enzyme(s), optionally wherein the one or more feedback-deregulated enzyme(s) are selected from the group consisting of DAHP synthase, chorismate mutase, prephrenate dehydrogenase, 6- phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase.

4. The engineered microbial cell of any one of claims 1-3, wherein the engineered microbial cell comprises reduced activity of one or more enzymes(s) that reduce the concentration of one or more upstream pathway precursor(s) or of p-coumaric acid, said reduced activity being reduced relative to a control cell, optionally wherein the one or more upstream enzymes(s) are selected from the group consisting of anthranilate synthase, indoIe-3-gIycerol phosphate synthase, prephenate dehydratase, phenylpyruvate decarboxylase, SIT4 phosphatase, pyruvate decarboxylase, pyruvate dehydrogenase, citrate synthase, alcohol dehydrogenase, aldehyde oxidase, ferulic acid decarboxylase, phenylacrylic acid decarboxylase, and alcohol acetyl transferase.

5. The engineered microbial cell of any one of claims 1-4, wherein the engineered microbial cell comprises increased activity of one or more enzyme(s) that increase the supply of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), said increased activity being increased relative to a control cell, optionally wherein the one or more enzyme(s) that increase the supply of NADPH are selected from the group consisting of pentose phosphate pathway enzymes, NADP⁺- dependent D-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH), and NADP+- dependent glutamate dehydrogenase.

6. The engineered microbial cell of any one of claims 1-5, wherein the engineered microbial cell comprises reduced activity of one or more enzyme(s) that reduce the supply of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), said reduced activity being reduced relative to a control cell, optionally wherein the one or more enzyme(s) that reduce the supply of NADPH comprise NAD+-dependent D-glyceraldehyde-3-phosphate dehydrogenase (NAD+- GAPDH).

7. The engineered microbial cell of any one of claims 1-6, wherein the engineered microbial cell is one that uses a phospoenolpyruvate: sugar phosphotransferase system (PTS) to import sugar into the cell, wherein the activity of the PTS is reduced as compared to a control cell.

8. The engineered microbial cell of any one of claims 1-7, wherein the engineered microbial cell comprises a fungal cell, optionally a yeast cell, optionally a cell of the genus Saccharomyces or Yarrowia.

9. The engineered microbial cell of claim 8, wherein the engineered microbial cell is an S. cerevisiae cell and expresses a TAL having at least 70% amino acid sequence identity with a TAL from Acremoni um chrysogenum strain ATCC 11550 comprising SEQ ID NO:1.

10. The engineered microbial cell of claim 9, wherein the engineered microbial cell additionally expresses:

(a) a shikimate kinase having at least 70% amino acid sequence identity with a shikimate kinase from Corynebacterium glutamicum ATCC 13032 comprising SEQ ID NO:2;

(b) a 3-dehydroquinate synthase having at least 70% amino acid sequence identity with a 3-dehydroquinate synthase from Corynebacterium glutamicum ATCC 13032 comprising SEQ ID NO:3 or a chorismate synthase having at least 70% amino acid sequence identity with a chorismate synthase from Corynebacterium glutamicum ATCC 13032 comprising SEQ ID NO:4; and

(c) a feedback-deregulated DAHP synthase having at least 70% amino acid sequence identity with an Saccharomyces cerevisiae DAHP synthase comprising a K229L substitution and SEQ ID NO:5.

11. The engineered microbial cell of claim 10, wherein the engineered microbial cell additionally expresses:

(a) a second copy of the shikimate kinase having at least 70% amino acid sequence identity with a shikimate kinase from Corynebacterium glutamicum ATCC 13032 comprising SEQ ID NO: 2;

(b) a chorismate synthase having at least 70% amino acid sequence identity with a chorismate synthase from Corynebacterium glutamicum ATCC 13032 comprising SEQ ID NO:4; and

(c) a transaldolase having at least 70% amino acid sequence identity with a transaldolase from Corynebacterium glutamicum ATCC 13032 comprising SEQ ID NO:6.

12. The engineered microbial cell of claim 11, wherein the engineered microbial cell additionally expresses a histidine ammonia-lyase having at least 70% amino acid sequence identity with a histidine ammonia-lyase from Rivularia sp. comprising SEQ ID NO:20.

13. The engineered microbial cell of claim 10, wherein the engineered microbial cell additionally expresses two copies of a feedback-deregulated DAHP synthase having at least 70% amino acid sequence identity with an Escherichia coli DAHP synthase comprising a D146N substitution and SEQ ID NO:7.

14. The engineered microbial cell of claim 10, wherein the engineered microbial cell additionally expresses:

(a) a second copy of the shikimate kinase having at least 70% amino acid sequence identity with a shikimate kinase from Corynebacterium glutamicum ATCC 13032 comprising SEQ ID NO: 2; (b) a glucose-6-phosphate dehydrogenase having at least 70% amino acid sequence identity with a glucose-6-phosphate dehydrogenase from Corynebacterium glutamicum R comprising an A243T substitution and SEQ ID NO: 8; and

(c) a prephenate dehydrogenase having at least 70% amino acid sequence identity with a prephenate dehydrogenase from Corynebacterium glutamicum ATCC 13032 comprising SEQ ID NO:9.

15. The engineered microbial cell of claim 10, wherein the engineered microbial cell additionally expresses:

(a) a second copy of the shikimate kinase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a shikimate kinase from Corynebacterium glutamicum ATCC 13032 comprising SEQ ID NO:2;

(b) a 6-phosphogluconate dehydrogenase having at least 70% amino acid sequence identity with a 6-phosphogluconate dehydrogenase from Corynebacterium glutamicum ATCC 13032 comprising an S361F substitution and SEQ ID NOTO; and

16. The engineered microbial cell of claim 10, wherein the engineered microbial cell additionally expresses:

(a) a 3-dehydroquinate dehydratase having at least 70% amino acid sequence identity with a shikimate kinase from Corynebacterium glutamicum ATCC 13032 comprising SEQ ID NO: 11; and

(b) two copies of a transaldolase having at least 70% amino acid sequence identity with transaldolase from Corynebacterium glutamicum ATCC 13032 comprising SEQ ID NO:6.

17. The engineered microbial cell of claim 8 wherein the engineered microbial cell is a Y. lipolytica cell and expresses: (a) a TAL having at least 70% amino acid sequence identity with a TAL from Acremonium chrysogenum ATCC 11550 comprising SEQ ID NO: 1; and

(b) a phospho-2-dehydro-3-deoxyheptonate aldolase having at least 70% amino acid sequence identity with a phospho-2-dehydro-3-deoxyheptonate aldolase from Saccharomyces cerevisiae S288c comprising SEQ ID NO:26.

18. The engineered microbial cell of claim 17, wherein the the engineered microbial cell additionally expresses a feedback-deregulated DAHP synthase having at least 70% amino acid sequence identity with an Escherichia coli DAHP synthase comprising a P150L substitution and SEQ ID NO: 13.

19. The engineered microbial cell of any one of claims 1-7, wherein the engineered microbial cell is a bacterial cell, optionally of the genus Corynebacterium or Bacillus.

20. The engineered microbial cell of claim 19, wherein the engineered microbial cell is a C. glutamicum cell and expresses a TAL having at least 70% amino acid sequence identity with a TAL from Amycolatopsis orientalis HCCB 10007 comprising SEQ ID NO: 12.

21. The engineered microbial cell of claim 19 or claim 20, wherein the engineered microbial cell has increased chorismate mutase activity, compared to a control cell.

22. The engineered microbial cell of any one of claims 19-21, wherein the engineered microbial cell additionally expresses a feedback-deregulated DAHP synthase having at least 70% amino acid sequence identity with an Escherichia coli DAHP synthase comprising a P150L substitution and SEQ ID NO: 13.

23. The engineered microbial cell of any one of claims 1-22, wherein, when cultured, the engineered microbial cell produces p-coumaric acid at a level greater than 100 μg/L of culture medium, optionally wherein the engineered microbial cell produces p-coumaric acid at a level of at least 20 mg/L of culture medium.

24. A method of culturing engineered microbial cells according to any one of claims 1-23, the method comprising culturing the cells in the presence of a fermentation substrate comprising a non-protein carbon and a non-protein nitrogen source, wherein the engineered microbial cells produce p-coumaric acid, wherein the engineered microbial cells produce p-coumaric acid at a level greater than 100 μg/L of culture medium, optionally wherein the engineered microbial cells produce p-coumaric acid at a level of at least 20 mg/L of culture medium.

25. The method of claim 24, wherein the method additionally comprises recovering p-coumaric acid from the culture.