Metabolic engineering of Saccharomyces cerevisiae for 2,3-butanediol production

Kim, Soojung; Kim, Jin-Woo; Lee, Ye‐Gi; Park, Yong-Cheol; Seo, Jin‐Ho

doi:10.1007/s00253-017-8172-1

Cited by 46 publications

(30 citation statements)

References 58 publications

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“…Nevertheless, novel strains capable of producing high concentrations of 2,3-BD using low-cost carbon sources with minimal production of by-products are highly desirable [219]. After high-throughput screening for safe efficient microorganisms able to produce 2,3-BD [220], metabolic engineering can be applied [221,222]. Finally, genetic modifications have been performed for improvement of 2,3-BD productivity and yield, elimination of byproducts synthesis, enhancement of the ability to utilize complex substrates (most preferably without pretreatment) and resistance to environmental stresses [223,224].…”

Section: Chemicals From Hemicellulosementioning

confidence: 99%

A Bibliometric Study of Scientific Publications regarding Hemicellulose Valorization during the 2000–2016 Period: Identification of Alternatives and Hot Topics

Abejón

2018

ChemEngineering

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A bibliometric analysis of the Scopus database was carried out to identify the research trends related to hemicellulose valorization from 2000 to 2016. The results from the analysis revealed an increasing number of annual publications, a high degree of transdisciplinary collaboration and prolific contributions by European researchers on this topic. The importance of a holistic approach to consider the simultaneous valorization of the three main components of lignocellulosic biomass (cellulose, hemicellulose and lignin) must be highlighted. Optimal pretreatment processes are critical for the correct fractionation of the biomass and the subsequent valorization. On the one hand, biological conversion of sugars derived from hemicellulose can be employed for the production of biofuel (ethanol) or chemicals such as 2,3-butadiene, xylitol and lactic acid. On the other hand, the chemical transformation of these sugars produces furfural, 5-hydroxyfurfural and levulinic acid, which must be considered very important starting blocks for the synthesis of organic derivatives.

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Section: Chemicals From Hemicellulosementioning

confidence: 99%

A Bibliometric Study of Scientific Publications regarding Hemicellulose Valorization during the 2000–2016 Period: Identification of Alternatives and Hot Topics

Abejón

2018

ChemEngineering

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“…2,3-BDO can be directly used as an anti-freeze agent or chemically converted to other chemicals (Fig. 1 d) [ 10 , 12 ]. For example, 1,3-butadiene (1,3-BD) can be produced by the two-step dehydration of 2,3-BDO [ 13 , 14 ].…”

Section: Introductionmentioning

confidence: 99%

A pyruvate carbon flux tugging strategy for increasing 2,3-butanediol production and reducing ethanol subgeneration in the yeast Saccharomyces cerevisiae

Ishii

Morita

Ida

et al. 2018

Biotechnol Biofuels

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BackgroundThe yeast Saccharomyces cerevisiae is a promising host cell for producing a wide range of chemicals. However, attempts to metabolically engineer Crabtree-positive S. cerevisiae invariably face a common issue: how to reduce dominant ethanol production. Here, we propose a yeast metabolic engineering strategy for decreasing ethanol subgeneration involving tugging the carbon flux at an important hub branching point (e.g., pyruvate). Tugging flux at a central glycolytic overflow metabolism point arising from high glycolytic activity may substantially increase higher alcohol production in S. cerevisiae. We validated this possibility by testing 2,3-butanediol (2,3-BDO) production, which is routed via pyruvate as the important hub compound.ResultsBy searching for high-activity acetolactate synthase (ALS) enzymes that catalyze the important first-step reaction in 2,3-BDO biosynthesis, and tuning several fermentation conditions, we demonstrated that a stronger pyruvate pulling effect (tugging of pyruvate carbon flux) is very effective for increasing 2,3-BDO production and reducing ethanol subgeneration by S. cerevisiae. To further confirm the validity of the pyruvate carbon flux tugging strategy, we constructed an evolved pyruvate decarboxylase (PDC)-deficient yeast (PDCΔ) strain that lacked three isozymes of PDC. In parallel with re-sequencing to identify genomic mutations, liquid chromatography–tandem mass spectrometry analysis of intermediate metabolites revealed significant accumulation of pyruvate and NADH in the evolved PDCΔ strain. Harnessing the high-activity ALS and additional downstream enzymes in the evolved PDCΔ strain resulted in a high yield of 2,3-BDO (a maximum of 0.41 g g−1 glucose consumed) and no ethanol subgeneration, thereby confirming the utility of our strategy. Using this engineered strain, we demonstrated a high 2,3-BDO titer (81.0 g L−1) in a fed-batch fermentation using a high concentration of glucose as the sole carbon source.ConclusionsWe demonstrated that the pyruvate carbon flux tugging strategy is very effective for increasing 2,3-BDO production and decreasing ethanol subgeneration in Crabtree-positive S. cerevisiae. High activity of the common first-step enzyme for the conversion of pyruvate, which links to both the TCA cycle and amino acid biosynthesis, is likely important for the production of various chemicals by S. cerevisiae.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1176-y) contains supplementary material, which is available to authorized users.

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“…As an example of a case study to demonstrate this premise, previous exploration of 2,3‐butanediol (BDO) production identified that disruptions to ADH1‐5 and GPD1‐2 genes controlling ethanol and glycerol production (respectively) greatly enhanced BDO production. This rewiring is due to the removal of NADH sinks in the cell, forcing flux from acetoin to BDO through the NADH‐dependent BDH1 enzyme (Figure A). To implement this approach, researchers needed to modulate these major NADH consuming pathways simultaneously.…”

Section: Resultsmentioning

confidence: 99%

Modular Ligation Extension of Guide RNA Operons (LEGO) for Multiplexed dCas9 Regulation of Metabolic Pathways in Saccharomyces cerevisiae

Deaner

Holzman

Alper

2018

Biotechnology Journal

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Metabolic engineering typically utilizes a suboptimal step-wise gene target optimization approach to parse a highly connected and regulated cellular metabolism. While the endonuclease-null CRISPR/Cas system has enabled gene expression perturbations without genetic modification, it has been mostly limited to small sets of gene targets in eukaryotes due to inefficient methods to assemble and express large sgRNA operons. In this work, we develop a TEF1p-tRNA expression system and demonstrate that the use of tRNAs as splicing elements flanking sgRNAs provides higher efficiency than both Pol III and ribozyme-based expression across a variety of single sgRNA and multiplexed contexts. Next, we devise and validate a scheme to allow modular construction of tRNA-sgRNA (TST) operons using an iterative Type IIs digestion/ligation extension approach, termed CRISPR-Ligation Extension of sgRNA Operons (LEGO). This approach enables facile construction of large TST operons. We demonstrate this utility by constructing a metabolic rewiring prototype for 2,3-butanediol production in 2 distinct yeast strain backgrounds. These results demonstrate that our approach can act as a surrogate for traditional genetic modification on a much shorter design-cycle timescale.

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Metabolic engineering of Saccharomyces cerevisiae for 2,3-butanediol production

Cited by 46 publications

References 58 publications

A Bibliometric Study of Scientific Publications regarding Hemicellulose Valorization during the 2000–2016 Period: Identification of Alternatives and Hot Topics

A Bibliometric Study of Scientific Publications regarding Hemicellulose Valorization during the 2000–2016 Period: Identification of Alternatives and Hot Topics

A pyruvate carbon flux tugging strategy for increasing 2,3-butanediol production and reducing ethanol subgeneration in the yeast Saccharomyces cerevisiae

Modular Ligation Extension of Guide RNA Operons (LEGO) for Multiplexed dCas9 Regulation of Metabolic Pathways in Saccharomyces cerevisiae

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