Engineering of the pyruvate dehydrogenase bypass in Saccharomyces cerevisiae for high-level production of isoprenoids

Shiba, Yoichiro; Paradise, Eric M.; Kirby, James; Ro, Dae‐Kyun; Keasling, Jay D.

doi:10.1016/j.ymben.2006.10.005

Cited by 310 publications

(207 citation statements)

References 33 publications

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“…In the engineering of E. coli hosts, the existence of two separate biosynthetic routes has been exploited by introducing the non-native mevalonate path-way. In yeast, the native mevalonate pathway has been improved by several approaches [50,[54][55][56].…”

Section: The Isoprenoid Pathwaymentioning

confidence: 99%

Biofuel alternatives to ethanol: pumping the microbial well

Fortman

Chhabra

Mukhopadhyay

et al. 2008

Trends in Biotechnology

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338

209

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Section: The Isoprenoid Pathwaymentioning

confidence: 99%

Biofuel alternatives to ethanol: pumping the microbial well

Fortman

Chhabra

Mukhopadhyay

et al. 2008

Trends in Biotechnology

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338

209

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“…Consequently, microbial fermentation from sugars using microbial cell factories is being pursued as a competitive approach for their bulk production (Ajikumar et al, 2010;Scalcinati et al, 2012b;Shiba et al, 2007;Vickers et al, 2015a).…”

Section: Introductionmentioning

confidence: 99%

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

Peng

Plan

Chrysanthopoulos

et al. 2017

Metabolic Engineering

128

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A squalene synthase protein degradation method for improved sesquiterpene production in Saccharomyces cerevisiae, Metabolic Engineering, http://dx.doi.org/10. 1016/j.ymben.2016.12.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. AbstractSesquiterpenes 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 redirect 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. 2A 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 ~100 mg L -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.

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“…These include lactate (van Maris et al 2004;Ishida et al 2006), malate (Zelle et al 2008), isoprenoids (Shiba et al 2007;Herrero et al 2008;Kizer et al 2008), glycerol (Geertman et al 2006;Cordier et al 2007), and ethanol (Alper et al 2006;Bro et al 2006), among others. Although nonfermentative by-products represent a class of biologically interesting and commercially attractive small molecules, efforts aimed at engineering microbes for increased production of these metabolites are comparatively infrequent.…”

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confidence: 99%

Systems-Level Engineering of Nonfermentative Metabolism in Yeast

Kennedy

Boyle²,

Waks

et al. 2009

Genetics

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We designed and experimentally validated an in silico gene deletion strategy for engineering endogenous one-carbon (C1) metabolism in yeast. We used constraint-based metabolic modeling and computer-aided gene knockout simulations to identify five genes (ALT2, FDH1, FDH2, FUM1, and ZWF1), which, when deleted in combination, predicted formic acid secretion in Saccharomyces cerevisiae under aerobic growth conditions. Once constructed, the quintuple mutant strain showed the predicted increase in formic acid secretion relative to a formate dehydrogenase mutant ( fdh1 fdh2), while formic acid secretion in wild-type yeast was undetectable. Gene expression and physiological data generated post hoc identified a retrograde response to mitochondrial deficiency, which was confirmed by showing Rtg1-dependent NADH accumulation in the engineered yeast strain. Formal pathway analysis combined with gene expression data suggested specific modes of regulation that govern C1 metabolic flux in yeast. Specifically, we identified coordinated transcriptional regulation of C1 pathway enzymes and a positive flux control coefficient for the branch point enzyme 3-phosphoglycerate dehydrogenase (PGDH).Together, these results demonstrate that constraint-based models can identify seemingly unrelated mutations, which interact at a systems level across subcellular compartments to modulate flux through nonfermentative metabolic pathways.

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Engineering of the pyruvate dehydrogenase bypass in Saccharomyces cerevisiae for high-level production of isoprenoids

Cited by 310 publications

References 33 publications

Biofuel alternatives to ethanol: pumping the microbial well

Biofuel alternatives to ethanol: pumping the microbial well

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

Systems-Level Engineering of Nonfermentative Metabolism in Yeast

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