Bingyin Peng

Improvement of xylose fermentation in respiratory-deficient xylose-fermenting Saccharomyces cerevisiae

Effective conversion of xylose in lignocelluloses is expected to reduce the production cost of second-generation biofuels significantly. The factors affecting xylose fermentation in Saccharomyces cerevisiae that express xylose reductase-xylitol dehydrogenase (XR-XDH) are studied. Although overproduction of non-oxidative pentose phosphate pathway significantly increased the aerobic-specific growth rate on xylose and slightly improved conversion of xylose to ethanol under oxygen-limited conditions, the elimination of respiration by deleting cytochrome C oxidase subunit IV gene impeded aerobic growth on xylose. However, the adaptive evolution of the respiratory-deficient strain with an NADP(+)-preferring XDH mutant in xylose media dramatically improved its xylose-fermenting ability. The specific growth rate, ethanol yield, and xylitol yield of the evolved strain on xylose were 0.06h(-1), 0.39gg(-1), and 0.13gg(-1) consumed xylose, respectively. Similar to anaerobic fermentation, the evolved strain exhibited accumulated ethanol rather than recycled it under aerobic conditions.

Controlling heterologous gene expression in yeast cell factories on different carbon substrates and across the diauxic shift: a comparison of yeast promoter activities

AbstractBackgroundPredictable control of gene expression is necessary for the rational design and optimization of cell factories. In the yeast Saccharomyces cerevisiae, the promoter is one of the most important tools available for controlling gene expression. However, the complex expression patterns of yeast promoters have not been fully characterised and compared on different carbon sources (glucose, sucrose, galactose and ethanol) and across the diauxic shift in glucose batch cultivation. These conditions are of importance to yeast cell factory design because they are commonly used and encountered in industrial processes. Here, the activities of a series of “constitutive” and inducible promoters were characterised in single cells throughout the fermentation using green fluorescent protein (GFP) as a reporter.ResultsThe “constitutive” promoters, including glycolytic promoters, transcription elongation factor promoters and ribosomal promoters, differed in their response patterns to different carbon sources; however, in glucose batch cultivation, expression driven by these promoters decreased sharply as glucose was depleted and cells moved towards the diauxic shift. Promoters induced at low-glucose levels (PHXT7, PSSA1 and PADH2) varied in induction strength on non-glucose carbon sources (sucrose, galactose and ethanol); in contrast to the “constitutive” promoters, GFP expression increased as glucose decreased and cells moved towards the diauxic shift. While lower than several “constitutive” promoters during the exponential phase, expression from the SSA1 promoter was higher in the post-diauxic phase than the commonly-used TEF1 promoter. The galactose-inducible GAL1 promoter provided the highest GFP expression on galactose, and the copper-inducible CUP1 promoter provided the highest induced GFP expression following the diauxic shift.ConclusionsThe data provides a foundation for predictable and optimised control of gene expression levels on different carbon sources and throughout batch fermentation, including during and after the diauxic shift. This information can be applied for designing expression approaches to improve yields, rates and titres in yeast cell factories.

Secretory pathway engineering enhances secretion of cellobiohydrolase I from Trichoderma reesei in Saccharomyces cerevisiae

Improving the cellulase secretion is beneficial for Saccharomyces cerevisiae used in consolidated bioprocessing (CBP) of cellulosic ethanol. In this study, protein secretory pathway, including protein folding, disulfide bond formation, and protein trafficking and sorting, was modified in S. cerevisiae. The effects of these modifications on the secretion of cellobiohydrolase I (Tr-Cel7A) with its native signal peptide from Trichoderma reesei were investigated. The results showed that overexpression of the protein disulfide isomerase Sc-PDI1 and the plasma membrane targeting soluble N-ethylmaleimide-sensitive factor attachment protein receptor Sc-SSO1, and disruption of the sorting receptor Sc-VPS10 and a Ca(2+)/Mn(2+) ATPase Sc-PMR1, improved respectively the extracellular Tr-Cel7A activities. Among them, disruption of Sc-PMR1 showed better improvement of 162% in the extracellular activity and decreased the glycosylation of Tr-Cel7A. Multiple modifications generally resulted in higher activities. The extracellular activities of the quadruple-modified strain (vps10Δ/pmr1Δ/SSO1/PDI1/cel7AF) using p-nitrophenyl-β-d-cellobioside (pNPC) and phosphoric acid swollen cellulose (PASC) as the substrates, respectively, were 3.9-fold and 1.3-fold higher than that of the reference strain cel7AF. The results indicated that engineering of the protein secretory pathway is an effective approach to improve the Tr-Cel7A secretion in S. cerevisiae.

Recent advances in synthetic biology for engineering isoprenoid production in yeast

Isoprenoids (terpenes/terpenoids) have many useful industrial applications, but are often not produced at industrially viable level in their natural sources. Synthetic biology approaches have been used extensively to reconstruct metabolic pathways in tractable microbial hosts such as yeast and re-engineer pathways and networks to increase yields. Here we review recent advances in this field, focusing on central carbon metabolism engineering to increase precursor supply, re-directing carbon flux for production of C10, C15, or C20 isoprenoids, and chemical decoration of high value diterpenoids (C20). We also overview other novel synthetic biology strategies that have potential utility in yeast isoprenoid pathway engineering. Finally, we address the question of what is required in the future to move the field forwards.Isoprenoids (terpenes/terpenoids) have many useful industrial applications, but are often not produced at industrially viable level in their natural sources. Synthetic biology approaches have been used extensively to reconstruct metabolic pathways in tractable microbial hosts such as yeast and re-engineer pathways and networks to increase yields. Here we review recent advances in this field, focusing on central carbon metabolism engineering to increase precursor supply, redirecting carbon flux for production of C10, C15, or C20 isoprenoids, and chemical decoration of high value diterpenoids (C20). We also overview other novel synthetic biology strategies that have potential utility in yeast isoprenoid pathway engineering. Finally, we address the question of what is required in the future to move the field forwards.

Enhanced resistance of Saccharomyces cerevisiae to vanillin by expression of lacA from Trametes sp. AH28-2.

Saccharomyces cerevisiae is affected by the presence of certain phenolic compounds such as vanillin during fermentation of pretreated lignocellulosic hydrolysates. Since vanillin can be polymerized in the presence of laccase into compounds with lower toxicity, the laccase gene, lacA, from Trametes sp. AH28-2 was fused to the α-factor signal sequence and transferred into S. cerevisiae CEN.PK strains for secretory expression. Furthermore, the chaperone gene, KAR2, was overexpressed to promote the translocation of laccase. In the presence of 8 mmol/L vanillin, a shorter lag phase was observed in the lacA gene expressing strains. The vanillin-specific conversion rate of the lacA-expressing strain BSJX0A2 was 0.069 g g(-1)biomass h(-1), while it was 0.065 g g(-1)biomass h(-1) in the reference strain.

A squalene synthase protein degradation method for improved sesquiterpene production in Saccharomyces cerevisiae

Sesquiterpenes are C15 isoprenoids with utility as fragrances, flavours, pharmaceuticals, and potential biofuels. Microbial fermentation is being examined as a competitive approach for bulk production of these compounds. Competition for carbon allocation between synthesis of endogenous sterols and production of the introduced sesquiterpene limits yields. Achieving balance between endogenous sterols and heterologous sesquiterpenes is therefore required to achieve economical yields. In the current study, the yeast Saccharomyces cerevisiae was used to produce the acyclic sesquiterpene alcohol, trans-nerolidol. Nerolidol production was first improved by enhancing the upstream mevalonate pathway for the synthesis of the precursor farnesyl pyrophosphate (FPP). However, excess FPP was partially directed towards squalene by squalene synthase (Erg9p), resulting in squalene accumulation to 1% biomass; moreover, the specific growth rate declined. In order to re-direct carbon away from sterol production and towards the desired heterologous sesquiterpene, a novel protein destabilisation approach was developed for Erg9p. It was shown that Erg9p is located on endoplasmic reticulum and lipid droplets through a C-terminal ER-targeted transmembrane peptide. A PEST (rich in Pro, Glu/Asp, Ser, and Thr) sequence-dependent endoplasmic reticulum-associated protein degradation (ERAD) mechanism was established to decrease cellular levels of Erg9p without relying on inducers, repressors or specific repressing conditions. This improved nerolidol titre by 86% to ~100mgL-1. In this strain, squalene levels were similar to the wild-type control strain, and downstream ergosterol levels were slightly decreased relative to the control, indicating redirection of carbon away from sterols and towards sesquiterpene production. There was no negative effect on cell growth under these conditions. Protein degradation is an efficient mechanism to control carbon allocation at flux-competing nodes in metabolic engineering applications. This study demonstrates that an engineered ERAD mechanism can be used to balance flux competition between the endogenous sterol pathway and an introduced bio-product pathways at the FPP node. The approach of protein degradation in general might be more widely applied to improve metabolic engineering outcomes.

The Saccharomyces cerevisiae pheromone-response is a metabolically active stationary phase for bio-production

The growth characteristics and underlying metabolism of microbial production hosts are critical to the productivity of metabolically engineered pathways. Production in parallel with growth often leads to biomass/bio-product competition for carbon. The growth arrest phenotype associated with the Saccharomyces cerevisiae pheromone-response is potentially an attractive production phase because it offers the possibility of decoupling production from population growth. However, little is known about the metabolic phenotype associated with the pheromone-response, which has not been tested for suitability as a production phase. Analysis of extracellular metabolite fluxes, available transcriptomic data, and heterologous compound production (para-hydroxybenzoic acid) demonstrate that a highly active and distinct metabolism underlies the pheromone-response. These results indicate that the pheromone-response is a suitable production phase, and that it may be useful for informing synthetic biology design principles for engineering productive stationary phase phenotypes.

Expression of Aspergillus niger glucose oxidase in yeast Pichia pastoris SMD1168

ABSTRACT The aim of this study was to establish a system for high expression of Aspergillus niger glucose oxidase (GOD) with glyceraldehyde-3-phosphate dehydrogenase gene promoter (pGAP) in Pichia SMD1168. The GOD gene from A. niger accc30161 was inserted into pGAPZαA plasmid carrying a pGAP promoter and was expressed in yeast Pichia pastoris. The expression vector, pGAPZαA-GOD, was validated by colony polymerase chain reaction (PCR), agarose gel electrophoresis, restriction enzyme analysis and sequencing methods. The pGAPZαA-GOD vector was transformed into yeast P. pastoris SMD1168 by electroporation and the positive strain was validated by PCR. The expression and enzyme activity of the recombinant GOD were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and enzymatic detection. The recombinant GOD in yeast was purified by ion exchange chromatography, and its biochemical properties (dynamics, thermal and pH stability) were analysed. The obtained results showed that the expression vector, pGAPZαA-GOD, was successfully constructed to express A. niger accc30161 GOD protein. After transformation, the pGAPZαA-GOD DNA fragment had been integrated into the P. pastoris SMD1168-GOD genome. High expression of GOD was achieved in SMD1168-GOD, and the enzyme activity of GOD reached 107.18 U/mL in the supernatant of the culture medium at 30 °C and pH 6. The activity of recombinant GOD protein in yeast was 1.35-fold higher than the commercial A. niger GOD, and its affinity to glucose, thermal stability and pH stability are similar to commercial GOD. Using pGAP promoter, A. niger GOD protein is efficiently expressed in recombinant P. pastoris SMD1168-GOD with defective protease.

Engineered protein degradation of farnesyl pyrophosphate synthase is an effective regulatory mechanism to increase monoterpene production in Saccharomyces cerevisiae

Monoterpene production in Saccharomyces cerevisae requires the introduction of heterologous monoterpene synthases (MTSs). The endogenous farnesyl pyrosphosphate synthase (FPPS; Erg20p) competes with MTSs for the precursor geranyl pyrophosphate (GPP), which limits the production of monoterpenes. ERG20 is an essential gene that cannot be deleted and transcriptional down-regulation of ERG20 has failed to improve monoterpene production. Here, we investigated an N-degron-dependent protein degradation strategy to down-regulate Erg20p activity. Degron tagging decreased GFP protein half-life drastically to 1 h (degron K3K15) or 15 min (degrons KN113 and KN119). Degron tagging of ERG20 was therefore paired with a sterol responsive promoter to ensure sufficient metabolic flux to essential downstream sterols despite the severe destabilisation effect of degron tagging. A dual monoterpene/sesquiterpene (linalool/nerolidol) synthase, AcNES1, was used as a reporter of intracellular GPP and FPP production. Transcription of the synthetic pathway was controlled by either constitutive or diauxie-inducible promoters. A combination of degron K3K15 and the ERG1 promoter increased linalool titre by 27-fold to 11 mg L-1 in the strain with constitutive promoter constructs, and by 17-fold to 18 mg L-1 in the strain with diauxie-inducible promoter constructs. The sesquiterpene nerolidol remained the major product in both strains. The same strategies were applied to construct a limonene-producing strain, which produced 76 mg L-1 in batch cultivation. The FPPS regulation method developed here successfully redirected metabolic flux toward monoterpene production. Examination of growth defects in various strains suggested that the intracellular FPP concentration had a significant effect on growth rate. Further strategies are required to balance intracellular production of FPP and GPP so as to maximise monoterpene production without impacting on cellular growth.