Increasing anaerobic acetate consumption and ethanol yields in Saccharomyces cerevisiae with NADPH-specific alcohol dehydrogenase.

Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter(-1) acetate during fermentation of 114 g liter(-1) glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter(-1), this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter(-1) and raised the ethanol yield to 7% above the wild-type level.

Anaplerotic role for cytosolic malic enzyme in engineered Saccharomyces cerevisiae strains.

Malic enzyme catalyzes the reversible oxidative decarboxylation of malate to pyruvate and CO(2). The Saccharomyces cerevisiae MAE1 gene encodes a mitochondrial malic enzyme whose proposed physiological roles are related to the oxidative, malate-decarboxylating reaction. Hitherto, the inability of pyruvate carboxylase-negative (Pyc(-)) S. cerevisiae strains to grow on glucose suggested that Mae1p cannot act as a pyruvate-carboxylating, anaplerotic enzyme. In this study, relocation of malic enzyme to the cytosol and creation of thermodynamically favorable conditions for pyruvate carboxylation by metabolic engineering, process design, and adaptive evolution, enabled malic enzyme to act as the sole anaplerotic enzyme in S. cerevisiae. The Escherichia coli NADH-dependent sfcA malic enzyme was expressed in a Pyc(-) S. cerevisiae background. When PDC2, a transcriptional regulator of pyruvate decarboxylase genes, was deleted to increase intracellular pyruvate levels and cells were grown under a CO(2) atmosphere to favor carboxylation, adaptive evolution yielded a strain that grew on glucose (specific growth rate, 0.06 ± 0.01 h(-1)). Growth of the evolved strain was enabled by a single point mutation (Asp336Gly) that switched the cofactor preference of E. coli malic enzyme from NADH to NADPH. Consistently, cytosolic relocalization of the native Mae1p, which can use both NADH and NADPH, in a pyc1,2Δ pdc2Δ strain grown under a CO(2) atmosphere, also enabled slow-growth on glucose. Although growth rates of these strains are still low, the higher ATP efficiency of carboxylation via malic enzyme, compared to the pyruvate carboxylase pathway, may contribute to metabolic engineering of S. cerevisiae for anaerobic, high-yield C(4)-dicarboxylic acid production.

Phosphoenolpyruvate carboxykinase as the sole anaplerotic enzyme in Saccharomyces cerevisiae.

Pyruvate carboxylase is the sole anaplerotic enzyme in glucose-grown cultures of wild-type Saccharomyces cerevisiae. Pyruvate carboxylase-negative (Pyc(-)) S. cerevisiae strains cannot grow on glucose unless media are supplemented with C(4) compounds, such as aspartic acid. In several succinate-producing prokaryotes, phosphoenolpyruvate carboxykinase (PEPCK) fulfills this anaplerotic role. However, the S. cerevisiae PEPCK encoded by PCK1 is repressed by glucose and is considered to have a purely decarboxylating and gluconeogenic function. This study investigates whether and under which conditions PEPCK can replace the anaplerotic function of pyruvate carboxylase in S. cerevisiae. Pyc(-) S. cerevisiae strains constitutively overexpressing the PEPCK either from S. cerevisiae or from Actinobacillus succinogenes did not grow on glucose as the sole carbon source. However, evolutionary engineering yielded mutants able to grow on glucose as the sole carbon source at a maximum specific growth rate of ca. 0.14 h(-1), one-half that of the (pyruvate carboxylase-positive) reference strain grown under the same conditions. Growth was dependent on high carbon dioxide concentrations, indicating that the reaction catalyzed by PEPCK operates near thermodynamic equilibrium. Analysis and reverse engineering of two independently evolved strains showed that single point mutations in pyruvate kinase, which competes with PEPCK for phosphoenolpyruvate, were sufficient to enable the use of PEPCK as the sole anaplerotic enzyme. The PEPCK reaction produces one ATP per carboxylation event, whereas the original route through pyruvate kinase and pyruvate carboxylase is ATP neutral. This increased ATP yield may prove crucial for engineering of efficient and low-cost anaerobic production of C(4) dicarboxylic acids in S. cerevisiae.

Key process conditions for production of C(4) dicarboxylic acids in bioreactor batch cultures of an engineered Saccharomyces cerevisiae strain.

A recent effort to improve malic acid production by Saccharomyces cerevisiae by means of metabolic engineering resulted in a strain that produced up to 59 g liter(-1) of malate at a yield of 0.42 mol (mol glucose)(-1) in calcium carbonate-buffered shake flask cultures. With shake flasks, process parameters that are important for scaling up this process cannot be controlled independently. In this study, growth and product formation by the engineered strain were studied in bioreactors in order to separately analyze the effects of pH, calcium, and carbon dioxide and oxygen availability. A near-neutral pH, which in shake flasks was achieved by adding CaCO(3), was required for efficient C(4) dicarboxylic acid production. Increased calcium concentrations, a side effect of CaCO(3) dissolution, had a small positive effect on malate formation. Carbon dioxide enrichment of the sparging gas (up to 15% [vol/vol]) improved production of both malate and succinate. At higher concentrations, succinate titers further increased, reaching 0.29 mol (mol glucose)(-1), whereas malate formation strongly decreased. Although fully aerobic conditions could be achieved, it was found that moderate oxygen limitation benefitted malate production. In conclusion, malic acid production with the engineered S. cerevisiae strain could be successfully transferred from shake flasks to 1-liter batch bioreactors by simultaneous optimization of four process parameters (pH and concentrations of CO(2), calcium, and O(2)). Under optimized conditions, a malate yield of 0.48 +/- 0.01 mol (mol glucose)(-1) was obtained in bioreactors, a 19% increase over yields in shake flask experiments.

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

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.

Malic acid production by Saccharomyces cerevisiae: engineering of pyruvate carboxylation, oxaloacetate reduction, and malate export.

Malic acid is a potential biomass-derivable "building block" for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production from glucose proceeds via carboxylation of pyruvate, followed by reduction of oxaloacetate to malate. This redox- and ATP-neutral, CO(2)-fixing pathway has a theoretical maximum yield of 2 mol malate (mol glucose)(-1). A previously engineered glucose-tolerant, C(2)-independent pyruvate decarboxylase-negative S. cerevisiae strain was used as the platform to evaluate the impact of individual and combined introduction of three genetic modifications: (i) overexpression of the native pyruvate carboxylase encoded by PYC2, (ii) high-level expression of an allele of the MDH3 gene, of which the encoded malate dehydrogenase was retargeted to the cytosol by deletion of the C-terminal peroxisomal targeting sequence, and (iii) functional expression of the Schizosaccharomyces pombe malate transporter gene SpMAE1. While single or double modifications improved malate production, the highest malate yields and titers were obtained with the simultaneous introduction of all three modifications. In glucose-grown batch cultures, the resulting engineered strain produced malate at titers of up to 59 g liter(-1) at a malate yield of 0.42 mol (mol glucose)(-1). Metabolic flux analysis showed that metabolite labeling patterns observed upon nuclear magnetic resonance analyses of cultures grown on (13)C-labeled glucose were consistent with the envisaged nonoxidative, fermentative pathway for malate production. The engineered strains still produced substantial amounts of pyruvate, indicating that the pathway efficiency can be further improved.