Production of 2,3-butanediol in Saccharomyces cerevisiae by in silico aided metabolic engineering

Department of Chemical & Biological Engineering, Korea University, Seoul, 136-701, Republic of Korea. .
Microbial Cell Factories (Impact Factor: 4.22). 05/2012; 11(1):68. DOI: 10.1186/1475-2859-11-68
Source: PubMed


2,3-Butanediol is a chemical compound of increasing interest due to its wide applications. It can be synthesized via mixed acid fermentation of pathogenic bacteria such as Enterobacter aerogenes and Klebsiella oxytoca. The non-pathogenic Saccharomyces cerevisiae possesses three different 2,3-butanediol biosynthetic pathways, but produces minute amount of 2,3-butanediol. Hence, we attempted to engineer S. cerevisiae strain to enhance 2,3-butanediol production.
We first identified gene deletion strategy by performing in silico genome-scale metabolic analysis. Based on the best in silico strategy, in which disruption of alcohol dehydrogenase (ADH) pathway is required, we then constructed gene deletion mutant strains and performed batch cultivation of the strains. Deletion of three ADH genes, ADH1, ADH3 and ADH5, increased 2,3-butanediol production by 55-fold under microaerobic condition. However, overproduction of glycerol was observed in this triple deletion strain. Additional rational design to reduce glycerol production by GPD2 deletion altered the carbon fluxes back to ethanol and significantly reduced 2,3-butanediol production. Deletion of ALD6 reduced acetate production in strains lacking major ADH isozymes, but it did not favor 2,3-butanediol production. Finally, we introduced 2,3-butanediol biosynthetic pathway from Bacillus subtilis and E. aerogenes to the engineered strain and successfully increased titer and yield. Highest 2,3-butanediol titer (2.29 g·l-1) and yield (0.113 g·g-1) were achieved by Δadh1 Δadh3 Δadh5 strain under anaerobic condition.
With the aid of in silico metabolic engineering, we have successfully designed and constructed S. cerevisiae strains with improved 2,3-butanediol production.

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    • "Following that, a series of in silico metabolic engineering methods were developed for various gene manipulations other than knockout (Pharkya et al., 2004; Pharkya and Maranas, 2006; Choi et al., 2010; Ranganathan et al., 2010; Park et al., 2012; Chowdhury et al., 2014; Mahalik et al., 2014), leading to a marked expansion in the usage of GEMs. Furthermore, many of the in silico metabolic engineering methods were experimentally validated (Fong et al., 2005; Izallalen et al., 2008; Asadollahi et al., 2009; Brochado et al., 2010; Choi et al., 2010; Yim et al., 2011; Xu et al., 2011; Park et al., 2012; Ranganathan et al., 2012; Otero et al., 2013; Kim et al., 2014), which showed the power of GEMbased applications. With the development of systems biology, GEMs were also used as scaffolds for systematic integration of omics data because GEMs could be used to reconstruct the relationship among genes, enzymes, and metabolism. "
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    ABSTRACT: Genome-scale metabolic models (GEMs) have become a popular tool for systems biology, and they have been used in many fields such as industrial biotechnology and systems medicine. Since more and more studies are being conducted using GEMs, they have recently received considerable attention. In this review, we introduce the basic concept of GEMs and provide an overview of their applications in biotechnology, systems medicine, and some other fields. In addition, we describe the general principle of the applications and analyses built on GEMs. The purpose of this review is to introduce the application of GEMs in biological analysis and to promote its wider use by biologists.
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    • "engineering (Nan et al. 2014; Ng et al. 2012). In particular, K. pneumoniae can grow quickly in simple media with a wide variety of sugars. "
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    ABSTRACT: The microbiological production of 2,3-butanediol (2,3-BDO) has attracted considerable attention as an alternative way to produce high-value chemicals from renewable sources. Among the number of 2,3-BDO-producing microorganisms, Klebsiella pneumoniae has been studied most extensively and is known to produce large quantity of 2,3-BDO from a range of substrates. On the other hand, the pathogenic characteristics of the bacteria have limited its industrial applications. In this study, two major virulence traits, outer core LPS and fimbriae, were removed through homologous recombination from 2,3-BDO-producing K. pneumoniae 2242 to expand its uses to the industrial scale. The K. pneumoniae 2242 ∆wabG mutant strain was found to have an impaired capsule, which significantly reduced its ability to bind to the mucous layer and evade the phagocytic activity of macrophage. The association with the human ileocecal epithelial cell, HCT-8, and the bladder epithelial cell, T-24, was also reduced dramatically in the K. pneumoniae 2242 ∆fimA mutant strain that was devoid of fimbriae. However, the growth rate and production yield for 2,3-BDO were unaffected. The K. pneumoniae strains developed in this study, which are devoid of the major virulence factors, have a high potential for the efficient and sustainable production of 2,3-BDO.
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    • "Therefore, both PDC and ADH have been attractive disruption targets for 2,3-butanediol production in S. cerevisiae. In a previous study, deletion of ADH1, ADH3, and ADH5 genes resulted in increased 2,3-butanediol production (2.29 g/L) with a yield of 0.113 g/g glucose under anaerobic condition (Ng et al., 2012). Although ethanol production can be completely eliminated by deleting PDC1 and PDC5 genes or all PDC genes (PDC1, PDC5, and PDC6), the resulting PDC-deficient strains have severe growth defects on glucose as a sole carbon source and "
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    ABSTRACT: 2,3-Butanediol is a promising valuable chemical that can be used in various areas as a liquid fuel and a platform chemical. Here, 2,3-butanediol production in Saccharomyces cerevisiae was improved stepwise by eliminating byproduct formation and redox rebalancing. By introducing heterologous 2,3-butanediol biosynthetic pathway and deleting competing pathways producing ethanol and glycerol, metabolic flux was successfully redirected to 2,3-butanediol. In addition, the resulting redox cofactor imbalance was restored by overexpressing water-forming NADH oxidase (NoxE) from Lactococcus lactis. In a flask fed-batch fermentation with optimized conditions, the engineered adh1Δadh2Δadh3Δadh4Δadh5Δgpd1Δgpd2Δ strain overexpressing Bacillus subtilis α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD), S. cerevisiae 2,3-butanediol dehydrogenase (Bdh1), and L. lactis NoxE from a single multigene-expression vector, produced 72.9g/L 2,3-butanediol with the highest yield (0.41g/g glucose) and productivity (1.43g/(L·h)) ever reported in S. cerevisiae. Copyright © 2015. Published by Elsevier Inc.
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