Metabolic engineering of Saccharomyces cerevisiae for production of carboxylic acids: Current status and challenges

Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands
FEMS Yeast Research (Impact Factor: 2.82). 11/2009; 9(8):1123 - 1136. DOI: 10.1111/j.1567-1364.2009.00537.x


To meet the demands of future generations for chemicals and energy and to reduce the environmental footprint of the chemical industry, alternatives for petrochemistry are required. Microbial conversion of renewable feedstocks has a huge potential for cleaner, sustainable industrial production of fuels and chemicals. Microbial production of organic acids is a promising approach for production of chemical building blocks that can replace their petrochemically derived equivalents. Although Saccharomyces cerevisiae does not naturally produce organic acids in large quantities, its robustness, pH tolerance, simple nutrient requirements and long history as an industrial workhorse make it an excellent candidate biocatalyst for such processes. Genetic engineering, along with evolution and selection, has been successfully used to divert carbon from ethanol, the natural endproduct of S. cerevisiae, to pyruvate. Further engineering, which included expression of heterologous enzymes and transporters, yielded strains capable of producing lactate and malate from pyruvate. Besides these metabolic engineering strategies, this review discusses the impact of transport and energetics as well as the tolerance towards these organic acids. In addition to recent progress in engineering S. cerevisiae for organic acid production, the key limitations and challenges are discussed in the context of sustainable industrial production of organic acids from renewable feedstocks.

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    • "The yeast Saccharomyces cerevisiae is a popular microorganism in industrial biotechnology, mainly due to its robustness in industrial processes and the extensive toolbox available for engineering this organism (Nevoigt, 2008; Hong & Nielsen, 2012). Although S. cerevisiae has an innate tolerance to moderate concentrations of acetic acid (Abbott et al., 2009), this is not sufficient for processes based on crude lignocellulosic hydrolysates that contain substantially higher concentrations (Chandel et al., 2011; Zha et al., 2012; Demeke et al., 2013). Acetic acid is a weak acid (pK a = 4.76) and is therefore mainly present in an undissociated state at the relatively low pH values of typical industrial batch fermentations using yeast without pH control. "
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    ABSTRACT: High acetic acid tolerance of Saccharomyces cerevisiae is a relevant phenotype in industrial biotechnology when using lignocellulosic hydrolysates as feedstock. A screening of 38 S. cerevisiae strains for tolerance to acetic acid revealed considerable differences, particularly with regard to the duration of the latency phase. In order to understand how this phenotype is quantitatively manifested, four strains exhibiting significant differences were studied in more detail. Our data show that the duration of the latency phase is primarily determined by the fraction of cells within the population that resume growth. Only this fraction contributed to the exponential growth observed after the latency phase, while all other cells persisted in a viable but non-proliferating state. A remarkable variation in the size of the fraction was observed among the tested strains differing by several orders of magnitude. In fact, only 11 out of 10(7) cells of the industrial bioethanol production strain Ethanol Red resumed growth after exposure to 157 mM acetic acid at pH 4.5, while this fraction was 3.6 x 10(6) (out of 10(7) cells) in the highly acetic acid tolerant isolate ATCC 96581. These strain-specific differences are genetically determined, and represent a valuable starting point to identify genetic targets for future strain improvement. This article is protected by copyright. All rights reserved.
    Full-text · Article · Mar 2014 · FEMS Yeast Research
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    • "Carboxylic acids are useful biorenewable chemicals that can serve as precursors for drop-in replacements for petroleum-derived industrial chemicals (Mäki-Arvela et al., 2007; Lennen et al., 2010; Shanks, 2010; Carlos Serrano-Ruiz et al., 2012) and biologically-produced polymers (Wang et al., 2011) and alcohols (Perez et al., 2013). Much progress has been made in recent years in engineering workhorse biocatalysts, such as Escherichia coli and S. cerevisiae, for production of carboxylic acids (Lennen et al., 2010; Ranganathan et al., 2012; Zhang et al., 2012a,b), including recent reviews for both of these species (Abbott et al., 2009; Lennen and Pfleger, 2012; Liu and Jarboe, 2012). "
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    ABSTRACT: Carboxylic acids are an attractive biorenewable chemical in terms of their flexibility and usage as precursors for a variety of industrial chemicals. It has been demonstrated that such carboxylic acids can be fermentatively produced using engineered microbes, such as Escherichia coli and Saccharomyces cerevisiae. However, like many other attractive biorenewable fuels and chemicals, carboxylic acids become inhibitory to these microbes at concentrations below the desired yield and titer. In fact, their potency as microbial inhibitors is highlighted by the fact that many of these carboxylic acids are routinely used as food preservatives. This review highlights the current knowledge regarding the impact that saturated, straight-chain carboxylic acids, such as hexanoic, octanoic, decanoic, and lauric acids can have on E. coli and S. cerevisiae, with the goal of identifying metabolic engineering strategies to increase robustness. Key effects of these carboxylic acids include damage to the cell membrane and a decrease of the microbial internal pH. Certain changes in cell membrane properties, such as composition, fluidity, integrity, and hydrophobicity, and intracellular pH are often associated with increased tolerance. The availability of appropriate exporters, such as Pdr12, can also increase tolerance. The effect on metabolic processes, such as maintaining appropriate respiratory function, regulation of Lrp activity and inhibition of production of key metabolites such as methionine, are also considered. Understanding the mechanisms of biocatalyst inhibition by these desirable products can aid in the engineering of robust strains with improved industrial performance.
    Full-text · Article · Sep 2013 · Frontiers in Microbiology
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    • "These features, in conjunction with the sophisticated toolbox for genetic engineering, make S. cerevisiae particularly suitable for the production of carboxylic acids such as lactic acid (Pacheco et al., 2012), malic acid (Zelle et al., 2008, 2010), and succinic acid (Otero et al., 2013; Raab et al., 2010). It was proposed that the challenge in metabolic engineering of S. cerevisiae for the production of carboxylic acids involves at least four levels, one of which is engineering fast and efficient metabolic pathways that link the high-capacity glycolytic pathway in S. cerevisiae to the target compound, taking into account the redox and free-energy constraints (Abbott et al., 2009). S. cerevisiae, in its natural state, cannot produce fumaric acid, and thus, an efficient synthetic pathway of this chemical needs to be constructed. "
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    ABSTRACT: In this study, the simultaneous use of reductive and oxidative routes to produce fumaric acid was explored. The strain FMME003 (Saccharomyces cerevisiae CEN.PK2-1CΔTHI2) exhibited capability to accumulate pyruvate and was used for fumaric acid production. The fum1 mutant FMME004 could produce fumaric acid via oxidative route, but the introduction of reductive route derived from Rhizopus oryzae NRRL 1526 led to lower fumaric acid production. Analysis of the key factors associated with fumaric acid production revealed that pyruvate carboxylase had a low degree of control over the carbon flow to malic acid. The fumaric acid titer was improved dramatically when the heterologous gene RoPYC was overexpressed and 32μg/L of biotin was added. Furthermore, under the optimal carbon/nitrogen ratio, the engineered strain FMME004-6 could produce up to 5.64±0.16g/L of fumaric acid. These results demonstrated that the proposed fermentative method is efficient for fumaric acid production.
    Full-text · Article · Aug 2013 · Bioresource Technology
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