Progress in metabolic engineering of Saccharomyces cerevisiae

Abstract

The traditional use of the yeast Saccharomyces cerevisiae in alcoholic fermentation has, over time, resulted in substantial accumulated knowledge concerning genetics, physiology, and biochemistry as well as genetic engineering and fermentation technologies. S. cerevisiae has become a platform organism for developing metabolic engineering strategies, methods, and tools. The current review discusses the relevance of several engineering strategies, such as rational and inverse metabolic engineering, evolutionary engineering, and global transcription machinery engineering, in yeast strain improvement. It also summarizes existing tools for fine-tuning and regulating enzyme activities and thus metabolic pathways. Recent examples of yeast metabolic engineering for food, beverage, and industrial biotechnology (bioethanol and bulk and fine chemicals) follow. S. cerevisiae currently enjoys increasing popularity as a production organism in industrial ("white") biotechnology due to its inherent tolerance of low pH values and high ethanol and inhibitor concentrations and its ability to grow anaerobically. Attention is paid to utilizing lignocellulosic biomass as a potential substrate.

FIG. 1.

Microbial utilization pathways for d-xylose and l-arabinose transferred to S. cerevisiae. S.c., S. cerevisiae; P.s., Pichia stipitis; T.r., Trichoderma reesei (Hypocrea jecorina).

FIG. 2.

Glycerol biosynthesis and other major pathways contributing to cytosolic NAD⁺/NADH balance in S. cerevisiae. All genes with roles in either increasing or abolishing glycerol formation as well as accumulating l-G3P are included. (A) Glycolytic pathway linked to the formation of glycerol, ethanol, and acetate. (B) Redox factor utilization during glutamate formation from 2-oxoglutarate modified per data from Nissen et al. (250) to reduce cytosolic NADH generation and glycerol formation. (C) Glycerol overproduction in S. cerevisiae, per data from Geertman et al. (95), with formate dehydrogenase (FDH1) overexpression and formate feeding to generate an additional cytosolic NADH source. Gene names: ADH1, alcohol dehydrogenase; ALD4/5/6, cytosolic and mitochondrial acetaldehyde dehydrogenases; GPD1/2, cytosolic l-G3P dehydrogenase; GPP1/2, l-glycerol 3-phosphatase; GUT2, mitochondrial FAD⁺-dependent G3P dehydrogenase; FDH1/2, formate dehydrogenase; NDE1/2, external mitochondrial NADH dehydrogenase; PDC1/2/5/6, PDC; TPI1, triosephosphate isomerase; GLT1, glutamate synthase (NADH); GLN1, glutamine synthetase; GDH1, glutamate dehydrogenase (NADP⁺); GDH2, glutamate dehydrogenase (NAD⁺); FPS1, glycerol facilitator (glycerol export). FBP, fructose-1,6-bisphosphate; GAP, glyceraldehyde phosphate; P_i, inorganic phosphate; NH₄⁺, ammonium ions.

FIG. 3.

Pathways for 1,2-PD and 1,3-PD formation from glycolytic DHAP. E. coli genes encoding methylglyoxal synthase (mgs) and glycerol dehydrogenase (gldA) were introduced into S. cerevisiae per Lee and DaSilva (196) to enhance 1,2-PD production. In attempts to produce 1,3-PD in S. cerevisiae, bacterial genes encoding glycerol dehydratase and 1,3-PD oxidoreductase were expressed (42, 212) (see “Propanediol” in text).

FIG. 4.

Pathways, enzymes, and genes for engineering S. cerevisiae isoprenoid biosynthesis. Gene names and mutations: ADH6, alcohol dehydrogenase (NADP⁺); BTS1, GGPP synthase; DPP1, DGPP phosphatase; ERG9, squalene synthase; ERG20, farnesyl-diphosphate synthase; HMG1/2, 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase; upc2-1, mutation in the transcriptional regulator of anoxic genes (Upc2p) responsible for the aerobic uptake of sterol (64); sue, unknown mutations selected for aerobic sterol uptake (342). Other abbreviations: IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; FOH, farnesol.