Redesigned Pathway for De Novo Synthesis of Vanillin and Co-conversion of Multiple Renewable Substrates in Saccharomyces cerevisiae

Abstract

Vanillin, known as the "queen of flavors", is an extensively used and important aromatic compound with multiple functions, including aldehyde, ether, and hydroxyl. Converting lignocellulosic biomass-derived substrates to natural vanillin is sustainable and economical through artificial cell factories. However, the inefficiency of exogenous enzymes and the toxic effect of vanillin on cells limit its production. In this study, pathway reconstruction and integration site optimization of Saccharomyces cerevisiae enabled the production of 363.0 mg/L of vanillin in just four steps, with chorismate serving as an intermediate, by enhancing the endogenous shikimate pathway. A feeding strategy further yielded a record titer of 533.0 mg/L vanillin using engineered S. cerevisiae in flask shake fermentation. Promoter optimization enabled module adaptation for co-conversion of lignocellulose-derived monomers, including glucose, xylose, vanillic acid, p-coumaric acid, and ferulic acid, toward vanillin. Glycosylation of vanillin enabled the removal of product feedback inhibition, achieving a titer of 1745.5 mg/L glucovanillin through the co-conversion of multisubstrate. As a result, the artificial cell factories achieved the de novo production of vanillin and synthesized 1339.5 and 7476.5 mg/L glucovanillin (equivalent to 3619.4 mg/L vanillin) from glucose in shake flasks and 5-L bioreactors, respectively. The designed artificial cell factories of vanillin provided an alternative strategy for the industrial production of vanillin from sustainable resources.

Figure 1

Multisubstrate co-conversion and glycosylation removal product for enhanced vanillin synthesis. G6P, glucose-6-phosphate; G1P, glucose-1-phosphate; PEP, phosphoenolpyruvate; E4P, erythrose-4-phosphate; DAHP, 3-deoxy-d-arabino-2-heptulosonic acid 7-phosphate; SHIK, shikimate; EPSP, 5-enolpyruvyl-shikimate-3-phosphate; CHA, chorismic acid; PPA, prephenate; p-HBA, p-hydroxybenzoic acid; p-HBDE, p-hydroxybenzaldehyde; PADE, protocatechualdehyde, FA, ferulic acid; p-CA, p-coumaric acid; VA, vanillic acid.

Figure 2

Establishment and optimization of a de novo biosynthetic pathway for p-hydroxybenzoic acid. (a) Overview of biosynthesis pathway for p-hydroxybenzoic acid. G6P, glucose-6-phosphate; G1P, glucose 1-phosphate; E4P, erythrose-4-phosphate; PEP, phosphoenolpyruvate; DAHP, 3-deoxy-d-arabino-2-heptulosonic acid 7-phosphate; DHS, 3-dehydro-shikimate; CHA, chorismic acid; PPA, prephenate. PGM1, phosphoglycerate mutase; TktA, transketolase from E. coli; PpsA, phosphoenolpyruvate synthase from E. coli; Aro3^K222L, l-tyrosine-feedback-insensitive DAHP synthase; Aro4^K229L, l-tyrosine-feedback-insensitive DAHP synthase; Aro7, chorismate mutase; EcoAroL, shikimate kinase from E. coli; Aro2, chorismate synthase; UbiC, chorismate pyruvate-lyase from E. coli. (b) Genes knockout and overexpression of the genome for p-hydroxybenzoic acid biosynthesis. (c) The concentrations of p-hydroxybenzoic acid produced by 24-deep well plates’ fermentation. (d) The p-hydroxybenzoic acid concentration and OD₆₀₀ of yScXIN349 strain in flasks fermentation. (e) The results of glucose-feed fermentation by the yScXIN349 strain.

Figure 3

Construction of downstream pathway for vanillin synthesis. (a) Pathway from p-hydroxybenzoic acid to vanillin. HFD1, aldehyde dehydrogenase; CAR, carboxylic acid reductase; Pcg-1, phosphopantetheinyl transferase; HpaC-(GGGGS)₃-HpaB, the fusion of HpaC (NADH-flavin oxidoreductase) and HpaB (FAD-dependent 4-hydroxyphenylacetate-3-monooxygenas); NtCOMT, O-methyltransferase; SAM, S-adenosylmethionine; SAH, S-adenosyl-l-homocysteine. (b) Protocatechualdehyde production from p-hydroxybenzaldehyde driven by HpaC-(GGGGS)₃-HpaB. (c) Screening of PPTases needed for active carboxylic acid reduction. (d) Screening of O-methyltransferase for vanillin synthesis from protocatechualdehyde. (e) Vanillin production from protocatechualdehyde driven by two copies of NtCOMT. (f,g) Enhanced vanillin production coupled with SAM metabolism. SAM, S-adenosylmethionine; SAH, S-adenosyl-l-homocysteine; 5,10-CH₃-THF, 5,10-methylenetetrahydrofolic acid; 5-CH₃-THF, 5-methylenetetrahydrofolic acid; THF, tetrahydrofolic acid. MET13atsc, methylenetetrahydrofolate reductase; MET6, homocysteine S-methyltransferase; SAH1, adenosylhomocysteinase.

Figure 4

Enhanced vanillin synthesis by integration site reassignment. (a) Integration sites for the vanillin synthesis pathway of the strain yScXIN361. (b) Integration sites for the vanillin synthesis pathway of the strain yScXIN392. (c) The fermentation results of the strain yScXIN361 in YPD medium. (d) The fermentation results of the strain yScXIN392 in YPD medium. (e) The log₂ fold change of vanillin biosynthesis-related genes of strain yScXIN361 and yScXIN392. PEP, phosphoenolpyruvate; E4P, erythrose-4-phosphate; DAHP, 3-deoxy-d-arabino-2-heptulosonic acid 7-phosphate; DHQ, dehydroquinate; DHS, dehydroshikimate; SHIK, shikimate; SHIK-3P, shikimate-3-phosphate; CHA, chorismic acid; PPA, prephenate; p-HBA, p-hydroxybenzoic acid; p-HBDE, p-hydroxybenzaldehyde; PADE, protocatechualdehyde. The log₂ fold change means log₂(FPKM of yScXIN392/yScXIN361).

Figure 5

Analysis of aromatic acid conversion with the yScXIN392 strain (a–e) and synthesis of p-hydroxybenzoic acid and vanillin with the yScXIN401 and yScXIN402 strains, respectively (f). XI, xylose isomerase; XKS1, xylulose kinase.

Figure 6

Promoter optimization to achieve multiple substrates conversion for vanillin synthesis. (a) Design of the vanillin synthesis pathways from glucose, xylose, p-hydroxybenzoic acid, vanillic acid, p-coumaric acid, and ferulic acid. (b–d) Promoter optimization and fermentation optimization for the synthesis of vanillin by conversion of p-coumaric acid and ferulic acid. 4CL, 4-coumarate CoA ligase; Ech, hydratase/aldolase; p-CA, p-coumaric acid; FA, ferulic acid.

Figure 7

Glycosylation removal products to enhance vanillin production. (a) Screening of glycosyltransferases. (b) Stable accumulation of glucovanillin by knocking out endogenous hydrolases. (c) Schematic representation of the vanillin glycosylation process. (d) Flask shakes fermentation of the yScXIN428 strain in YPDX medium. (e) Glycosylation removal products to enhance vanillin production during the fermentation in YPDX medium with p-coumaric acid, ferulic acid, and vanillic acid added at 48 h using yScXIN428 strain. (f) Glycosylation removal products to enhance vanillin production during glucose feeding fermentation of yScXIN427 strain. (g) The glucovanillin production of yScXIN427 in a 5-L bioreactor using the fed-batch strategies.