Design and construction of acetyl-CoA overproducing Saccharomyces cerevisiae strains

Saccharomyces cerevisiae has increasingly been engineered as a cell factory for efficient and economic production of fuels and chemicals from renewable resources. Notably, a wide variety of industrially important products are derived from the same precursor metabolite, acetyl-CoA. However, the limited supply of acetyl-CoA in the cytosol, where biosynthesis generally happens, often leads to low titer and yield of the desired products in yeast. In the present work, combined strategies of disrupting competing pathways and introducing heterologous biosynthetic pathways were carried out to increase acetyl-CoA levels by using the CoA-dependent n-butanol production as a reporter. By inactivating ADH1 and ADH4 for ethanol formation and GPD1 and GPD2 for glycerol production, the glycolytic flux was redirected towards acetyl-CoA, resulting in 4-fold improvement in n-butanol production. Subsequent introduction of heterologous acetyl-CoA biosynthetic pathways, including pyruvate dehydrogenase (PDH), ATP-dependent citrate lyase (ACL), and PDH-bypass, further increased n-butanol production. Recombinant PDHs localized in the cytosol (cytoPDHs) were found to be the most efficient, which increased n-butanol production by additional 3 fold. In total, n-butanol titer and acetyl-CoA concentration were increased more than 12 fold and 3 fold, respectively. By combining the most effective and complementary acetyl-CoA pathways, more than 100 mg/L n-butanol could be produced using high cell density fermentation, which represents the highest titer ever reported in yeast using the clostridial CoA-dependent pathway.     

Introduction of a bacterial acetyl-CoA synthesis pathway improves lactic acid production in Saccharomyces cerevisiae

Acid-tolerant Saccharomyces cerevisiae was engineered to produce lactic acid by expressing heterologous lactate dehydrogenase (LDH) genes, while attenuating several key pathway genes, including glycerol-3-phosphate dehydrogenase1 (GPD1) and cytochrome-c oxidoreductase2 (CYB2). In order to increase the yield of lactic acid further, the ethanol production pathway was attenuated by disrupting the pyruvate decarboxylase1 (PDC1) and alcohol dehydrogenase1 (ADH1) genes. Despite an increase in lactic acid yield, severe reduction of the growth rate and glucose consumption rate owing to the absence of ADH1 caused a considerable decrease in the overall productivity. In Δadh1 cells, the levels of acetyl-CoA, a key precursor for biologically applicable components, could be insufficient for normal cell growth. To increase the cellular supply of acetyl-CoA, we introduced bacterial acetylating acetaldehyde dehydrogenase (A-ALD) enzyme (EC 1.2.1.10) genes into the lactic acid-producing S. cerevisiae. Escherichia coli-derived A-ALD genes, mhpF and eutE, were expressed and effectively complemented the attenuated acetaldehyde dehydrogenase (ALD)/acetyl-CoA synthetase (ACS) pathway in the yeast. The engineered strain, possessing a heterologous acetyl-CoA synthetic pathway, showed an increased glucose consumption rate and higher productivity of lactic acid fermentation. The production of lactic acid was reached at 142 g/L with production yield of 0.89 g/g and productivity of 3.55 g L⁻¹ h⁻¹ under fed-batch fermentation in bioreactor. This study demonstrates a novel approach that improves productivity of lactic acid by metabolic engineering of the acetyl-CoA biosynthetic pathway in yeast.     

Metabolic engineering of Saccharomyces cerevisiae for the production of triacetic acid lactone

Biobased chemicals have become attractive replacements for their fossil-fuel counterparts. Recent studies have shown triacetic acid lactone (TAL) to be a promising candidate, capable of undergoing chemical conversion to sorbic acid and other valuable intermediates. In this study, Saccharomyces cerevisiae was engineered for the high-level production of TAL by overexpression of the Gerbera hybrida 2-pyrone synthase (2-PS) and systematic engineering of the yeast metabolic pathways. Pathway analysis and a computational approach were employed to target increases in cofactor and precursor pools to improve TAL synthesis. The pathways engineered include those for energy storage and generation, pentose biosynthesis, gluconeogenesis, lipid biosynthesis and regulation, cofactor transport, and fermentative capacity. Seventeen genes were selected for disruption and independently screened for their effect on TAL production; combinations of knockouts were then evaluated. A combination of the pathway engineering and optimal culture parameters led to a 37-fold increase in titer to 2.2 g/L and a 50-fold increase in yield to 0.13 (g/g glucose). These values are the highest reported in the literature, and provide a 3-fold improvement in yield over previous reports using S. cerevisiae. Identification of these metabolic bottlenecks provides a strategy for overproduction of other acetyl-CoA-dependent products in yeast.     

Metabolic pathway engineering for complex polyketide biosynthesis in Saccharomyces cerevisiae

Polyketides are a diverse group of natural products with significance in human and veterinary medicine. Because polyketides are structurally complex molecules and fermentation is the most commercially viable route of production, a generic heterologous host system for high-level polyketide production is desirable. Saccharomyces cerevisiae has been shown to be an excellent production host for a simple polyketide, yielding 1.7 g of 6-methylsalicylic acid per liter of culture in un-optimized shake-flask fermentations. However, a barrier to the heterologous production of more complex 'modular' polyketides in S. cerevisiae is the lack of required polyketide precursor pathways. In this work, we describe the introduction into S. cerevisiae of pathways for the production of methylmalonyl-coenzyme A (CoA), a precursor for complex polyketides, by both propionyl-CoA-dependent and propionyl-CoA-independent routes. Furthermore, we demonstrate that the methylmalonyl-CoA produced in the engineered yeast strains is used in vivo for the production of a polyketide product, a triketide lactone.     

Engineering cytosolic acetyl-coenzyme A supply in Saccharomyces cerevisiae: Pathway stoichiometry,free-energy conservation and redox-cofactor balancing

Saccharomyces cerevisiae is an important industrial cell factory and an attractive experimental model for evaluating novel metabolic engineering strategies. Many current and potential products of this yeast require acetyl coenzyme A (acetyl-CoA) as a precursor and pathways towards these products are generally expressed in its cytosol. The native S. cerevisiae pathway for production of cytosolic acetyl-CoA consumes 2 ATP equivalents in the acetyl-CoA synthetase reaction. Catabolism of additional sugar substrate, which may be required to generate this ATP, negatively affects product yields. Here, we review alternative pathways that can be engineered into yeast to optimize supply of cytosolic acetyl-CoA as a precursor for product formation. Particular attention is paid to reaction stoichiometry, free-energy conservation and redox-cofactor balancing of alternative pathways for acetyl-CoA synthesis from glucose. A theoretical analysis of maximally attainable yields on glucose of four compounds (n-butanol, citric acid, palmitic acid and farnesene) showed a strong product dependency of the optimal pathway configuration for acetyl-CoA synthesis. Moreover, this analysis showed that combination of different acetyl-CoA production pathways may be required to achieve optimal product yields. This review underlines that an integral analysis of energy coupling and redox-cofactor balancing in precursor-supply and product-formation pathways is crucial for the design of efficient cell factories.     

A mutation affecting lipoamide dehydrogenase, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase activities in Saccharomyces cerevisiae

Summary In Saccharomyces cerevisiae a nuclear recessive mutation, lpd1, which simultaneously abolishes the activities of lipoamide dehydrogenase, 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase has been identified. Strains carrying this mutation can grow on glucose or poorly on ethanol, but are unable to grow on media with glycerol or acetate as carbon source. The mutation does not prevent the formation of other tricarboxylic acid cycle enzymes such as fumarase, NAD⁺-linked isocitrate dehydrogenase or succinate-cytochrome c oxidoreductase, but these are produced at about 50%–70% of the wild-type levels. The mutation probably affects the structural gene for lipoamide dehydrogenase since the amount of this enzyme in the cell is subject to a gene dosage effect; heterozygous lpd1 diploids produce half the amount of a homozygous wild-type strain. Moreover, a yeast sequence complementing this mutation when present in the cell on a multicopy plasmid leads to marked overproduction of lipoamide dehydrogenase. Homozygous lpd1 diploids were unable to sporulate indicating that some lipoamide dehydrogenase activity is essential for sporulation to occur on acetate.     

Engineering oxygen-independent biotin biosynthesis in Saccharomyces cerevisiae

An oxygen requirement for de novo biotin synthesis in Saccharomyces cerevisiae precludes the application of biotin-prototrophic strains in anoxic processes that use biotin-free media. To overcome this issue, this study explores introduction of the oxygen-independent Escherichia coli biotin-biosynthesis pathway in S. cerevisiae. Implementation of this pathway required expression of seven E. coli genes involved in fatty-acid synthesis and three E. coli genes essential for the formation of a pimelate thioester, key precursor of biotin synthesis. A yeast strain expressing these genes readily grew in biotin-free medium, irrespective of the presence of oxygen. However, the engineered strain exhibited specific growth rates 25% lower in biotin-free media than in biotin-supplemented media. Following adaptive laboratory evolution in anoxic cultures, evolved cell lines that no longer showed this growth difference in controlled bioreactors, were characterized by genome sequencing and proteome analyses. The evolved isolates exhibited a whole-genome duplication accompanied with an alteration in the relative gene dosages of biosynthetic pathway genes. These alterations resulted in a reduced abundance of the enzymes catalyzing the first three steps of the E. coli biotin pathway. The evolved pathway configuration was reverse engineered in the diploid industrial S. cerevisiae strain Ethanol Red. The resulting strain grew at nearly the same rate in biotin-supplemented and biotin-free media non-controlled batches performed in an anaerobic chamber. This study established an unique genetic engineering strategy to enable biotin-independent anoxic growth of S. cerevisiae and demonstrated its portability in industrial strain backgrounds.     

Engineering redox cofactor regeneration for improved pentose fermentation in Saccharomyces cerevisiae

Pentose fermentation to ethanol with recombinant Saccharomyces cerevisiae is slow and has a low yield. A likely reason for this is that the catabolism of the pentoses D-xylose and L-arabinose through the corresponding fungal pathways creates an imbalance of redox cofactors. The process, although redox neutral, requires NADPH and NAD+, which have to be regenerated in separate processes. NADPH is normally generated through the oxidative part of the pentose phosphate pathway by the action of glucose-6-phosphate dehydrogenase (ZWF1). To facilitate NADPH regeneration, we expressed the recently discovered gene GDP1, which codes for a fungal NADP+-dependent D-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH) (EC 1.2.1.13), in an S. cerevisiae strain with the D-xylose pathway. NADPH regeneration through an NADP-GAPDH is not linked to CO2 production. The resulting strain fermented D-xylose to ethanol with a higher rate and yield than the corresponding strain without GDP1; i.e., the levels of the unwanted side products xylitol and CO2 were lowered. The oxidative part of the pentose phosphate pathway is the main natural path for NADPH regeneration. However, use of this pathway causes wasteful CO2 production and creates a redox imbalance on the path of anaerobic pentose fermentation to ethanol because it does not regenerate NAD+. The deletion of the gene ZWF1 (which codes for glucose-6-phosphate dehydrogenase), in combination with overexpression of GDP1 further stimulated D-xylose fermentation with respect to rate and yield. Through genetic engineering of the redox reactions, the yeast strain was converted from a strain that produced mainly xylitol and CO2 from D-xylose to a strain that produced mainly ethanol under anaerobic conditions.     

酿酒酵母产苹果酸的还原TCA路径构建及发酵调控

苹果酸广泛应用于食品、化工行业。文中通过在酿酒酵母内敲除丙酮酸脱羧酶PDC1,并通过构建胞质内还原TCA的路径,即超表达丙酮酸羧化酶和苹果酸脱氢酶,成功地实现了苹果酸的生产。在野生型菌株中基本检测不到苹果酸的生成,而在工程菌株,苹果酸发酵浓度达到了45 mmol /L,同时副产物乙醇的产量也降低了18%。进一步通过发酵调控提高第二信使Ca2+的浓度使苹果酸的产量提高了7 %,在此基础上提高丙酮酸羧化酶的辅酶生物素浓度,使苹果酸的产量达到52.5 mmol /L,较原始菌株提高了16%。     

Uncovering the role of branched-chain amino acid transaminases in Saccharomyces cerevisiae isobutanol biosynthesis

Isobutanol and other branched-chain higher alcohols (BCHAs) are promising advanced biofuels derived from the degradation of branched-chain amino acids (BCAAs). The yeast Saccharomyces cerevisiae is a particularly attractive host for the production of BCHAs due to its high tolerance to alcohols and prevalent use in the bioethanol industry. Degradation of BCAAs begins with transamination reactions, catalyzed by branched-chain amino acid transaminases (BCATs) located in the mitochondria (Bat1p) and cytosol (Bat2p). However, the roles that these transaminases play in isobutanol production remain poorly understood and obscured by conflicting reports in the literature. In this work, we elucidate the influence of BCATs on isobutanol production in two genetic backgrounds (CEN.PK2-1C and BY4741). In the process, we uncover and characterize two competing isobutanol pathways, which can be manipulated by overexpressing or deleting BAT1 or BAT2, and adding or removing valine from the fermentation media. We show that deletion of BAT1 alone increases isobutanol production by 14.2-fold over wild type strains in media lacking valine, and examine how interactions between valine and the regulatory protein Ilv6p affect isobutanol production. Compartmentalizing the five-gene isobutanol biosynthetic pathway in mitochondria of BAT1 deletion strains results in an additional 2.1-fold increase in isobutanol production in the absence of valine. While valine inhibits isobutanol production, it boosts 2-methyl-1-butanol production. This work clarifies the role of transamination activity in BCHA biosynthesis, and develops valuable strategies and strains for future optimization of isobutanol production.     

Compartmentalizing metabolic pathway in Candida glabrata for acetoin production

Acetoin, a valuable compound, has high potential as a biochemical building block. In this study, subcellular metabolic engineering was applied to engineer the mitochondrion of Candida glabrata for acetoin production. With the aid of mitochondrial targeting sequences, a heterologous acetoin pathway was targeted into the mitochondria to increase the enzyme concentrations and level of intermediate, followed by coupling with the mitochondrial pyruvate carrier (MPC) to increase the availability of mitochondrial pyruvate. As a result, the strain comprising the combination of the mitochondrial pathway and MPC could yield approximately 3.26 g/L of acetoin, which was about 59.8% higher than that produced by the cytoplasmic pathway. These results provided a new insight into the metabolic engineering of C. glabrata for acetoin production, and offered a potential platform to improve the performance of engineered pathways.     

Heterologous biosynthesis of artemisinic acid in Saccharomyces cerevisiae

Artemisinic acid is a precursor of antimalarial compound artemisinin. The titre of biosynthesis of artemisinic acid using Saccharomyces cerevisiae platform has been achieved up to 25 g l^?1; however, the performance of platform cells is still industrial unsatisfied. Many strategies have been proposed to improve the titre of artemisinic acid. The traditional strategies mainly focused on partial target sites, simple up‐regulation key genes or repression competing pathways in the total synthesis route. However, this may result in unbalance of carbon fluxes and dysfunction of metabolism. In this review, the recent advances on the promising methods in silico and in vivo for biosynthesis of artemisinic acid have been discussed. The bioinformatics and omics techniques have brought a great prospect for improving production of artemisinin and other pharmacal compounds in heterologous platform.     

The isolation and characterisation of a Saccharomyces cerevisiae gene (LIP2) involved in the attachment of lipoic acid groups to mitochondrial enzymes

Lipoic acid is an essential cofactor for a variety of mitochondrial enzymes. We have characterised a gene from Saccharomyces cerevisiae which appears to encode a protein involved in the attachment of lipoic acid groups to the pyruvate dehydrogenase and glycine decarboxylase complexes. The predicted protein product of this gene has significant identity to the lipoyl ligase B of both Escherichia coli and Kluyveromyces lactis. A strain harbouring a null allele of this S. cerevisiae gene is respiratory deficient due to inactive pyruvate dehydrogenase, and is unable to utilise glycine as a sole nitrogen source.     

ACR1, a gene encoding a protein related to mitochondrial carriers, is essential for acetyl-CoA synthetase activity in Saccharomyces cerevisiae

The utilization of ethanol via acetate by the yeast Saccharomyces cerevisiae requires the presence of the enzyme acetyl-coenzyme A synthetase (acetyl-CoA synthetase), which catalyzes the activation of acetate to acetyl-coenzyme A (acetyl-CoA). We have isolated a mutant, termed acr1, defective for this activity by screening for mutants unable to utilize ethanol as a sole carbon source. Genetic and biochemical characterization show that, in this mutant, the structural gene for acetyl-CoA synthetase is not affected. Cloning and sequencing demonstrated that the ACR1 gene encodes a protein of 321 amino acids with a molecular mass of 35 370 Da. Computer analysis suggested that the ACR1 gene product (ACR1) is an integral membrane protein related to the family of mitochondrial carriers. The expression of the gene is induced by growing yeast cells in media containing ethanol or acetate as sole carbon sources and is repressed by glucose. ACR1 is essential for the utilization of ethanol and acetate since a mutant carrying a disruption in this gene is unable to grow on these compounds.     

The role of nickel in acetyl-CoA synthesis by the bifunctional enzyme CO dehydrogenase/acetyl-CoA synthase: enzymology and model chemistry

 CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) is one of the four known nickel enzymes. It is a bifunctional protein that catalyzes the oxidation of CO to CO₂ at a nickel iron-sulfur cluster (Cluster C) and a remarkable condensation reaction between a methyl group (donated from a methylated corrinoid iron-sulfur protein), carbon monoxide, and coenzyme A to form acetyl-CoA at a separate nickel iron-sulfur cluster (Cluster A). This review focuses on the current understanding of the structure and function of Cluster A and on related model chemistry. It describes studies that uncovered the first example of a biological organometallic reaction sequence. The mechanism of acetyl-CoA synthesis includes enzymebound methylnickel, iron-carbonyl, and acylmetal intermediates. Discovery of the methylnickel species constituted the first example of an alkylnickel species in biology and unveiled a new biological role for nickel. Received: 10 April 1996 / Accepted: 4 July 1996     

Relationship Between Activation State of Pyruvate Dehydrogenase Complex and Rate of Pyruvate Oxidation in Isolated Cerebro-Cortical Mitochondria: Effects of Potassium Ions and Adenine Nucleotides

The relation between the activation (phosphorylation) state of pyruvate dehydrogenase complex (PDHC; EC 1.2.4.1, EC 2.3.1.12, and EC 1.6.4.3) and the rate of pyruvate oxidation has been examined in isolated, metabolically active, and tightly coupled mitochondria from rat cerebral cortex. With pyruvate and malate as the substrates, the activation state of PDHC decreased on addition of ADP, while the rates of oxygen uptake and 14CO2 formation from [1-14C]pyruvate increased. The lack of correlation between the activation state of PDHC and rate of pyruvate oxidation was seen in media containing 5, 30, or 100 mM KCl. Both the activation state of PDHC and pyruvate oxidation increased, however, when KCl was increased from 5 to 100 mM. Although the PDHC is inactivated by an ATP-dependent kinase (EC 2.7.1.99), direct measurement of ATP and ADP failed to show a consistent relationship between the activation state of PDHC and either ATP levels or ATP/ADP ratios. Comparison of the activation state of PDHC in uncoupled or oligomycin-treated mitochondria also failed to correlate PDHC activation state to adenine nucleotides. In brain mitochondria, unlike those from other tissues, the activation state of PDHC does not seem to be related clearly to the rate of pyruvate oxidation, or to the mitochondrial adenylate energy charge.