Cytosolic re-localization and optimization of valine synthesis and catabolism enables increased isobutanol production with the yeast Saccharomyces cerevisiae

ABSTRACT: BACKGROUND: The branched chain alcohol isobutanol exhibits superior physicochemical properties as an alternative biofuel. The yeast Saccharomyces cerevisiae naturally produces low amounts of isobutanol as a by-product during fermentations, resulting from the catabolism of valine. As S. cerevisiae is widely used in industrial applications and can easily be modified by genetic engineering, this microorganism is a promising host for the fermentative production of higher amounts of isobutanol. RESULTS: Isobutanol production could be improved by re-locating the valine biosynthesis enzymes Ilv2, Ilv5 and Ilv3 from the mitochondrial matrix into the cytosol. To prevent the import of the three enzymes into yeast mitochondria, N-terminally shortened Ilv2, Ilv5 and Ilv3 versions were constructed lacking their mitochondrial targeting sequences. SDS-PAGE and immunofluorescence analyses confirmed expression and re-localization of the truncated enzymes. Growth tests or enzyme assays confirmed enzymatic activities. Isobutanol production was only increased in the absence of valine and the simultaneous blockage of the mitochondrial valine synthesis pathway. Isobutanol production could be even more enhanced after adapting the codon usage of the truncated valine biosynthesis genes to the codon usage of highly expressed glycolytic genes. Finally, a suitable ketoisovalerate decarboxylase, Aro10, and alcohol dehydrogenase, Adh2, were selected and overexpressed. The highest isobutanol titer was 0.63 g/L at a yield of nearly 15 mg per g glucose. CONCLUSION: A cytosolic isobutanol production pathway was successfully established in yeast by re-localization and optimization of mitochondrial valine synthesis enzymes together with overexpression of Aro10 decarboxylase and Adh2 alcohol dehydrogenase. Driving forces were generated by blocking competition with the mitochondrial valine pathway and by omitting valine from the fermentation medium. Additional deletion of pyruvate decarboxylase genes and engineering of co-factor imbalances should lead to even higher isobutanol production.     

Cytosolic re-localization and optimization of valine synthesis and catabolism enables inseased isobutanol production with the yeast Saccharomyces cerevisiae

Background

The branched chain alcohol isobutanol exhibits superior physicochemical properties as an alternative biofuel. The yeast Saccharomyces cerevisiae naturally produces low amounts of isobutanol as a by-product during fermentations, resulting from the catabolism of valine. As S. cerevisiae is widely used in industrial applications and can easily be modified by genetic engineering, this microorganism is a promising host for the fermentative production of higher amounts of isobutanol.

Results

Isobutanol production could be improved by re-locating the valine biosynthesis enzymes Ilv2, Ilv5 and Ilv3 from the mitochondrial matrix into the cytosol. To prevent the import of the three enzymes into yeast mitochondria, N-terminally shortened Ilv2, Ilv5 and Ilv3 versions were constructed lacking their mitochondrial targeting sequences. SDS-PAGE and immunofluorescence analyses confirmed expression and re-localization of the truncated enzymes. Growth tests or enzyme assays confirmed enzymatic activities. Isobutanol production was only increased in the absence of valine and the simultaneous blockage of the mitochondrial valine synthesis pathway. Isobutanol production could be even more enhanced after adapting the codon usage of the truncated valine biosynthesis genes to the codon usage of highly expressed glycolytic genes. Finally, a suitable ketoisovalerate decarboxylase, Aro10, and alcohol dehydrogenase, Adh2, were selected and overexpressed. The highest isobutanol titer was 0.63?g/L at a yield of nearly 15?mg per g glucose.

Conclusion

A cytosolic isobutanol production pathway was successfully established in yeast by re-localization and optimization of mitochondrial valine synthesis enzymes together with overexpression of Aro10 decarboxylase and Adh2 alcohol dehydrogenase. Driving forces were generated by blocking competition with the mitochondrial valine pathway and by omitting valine from the fermentation medium. Additional deletion of pyruvate decarboxylase genes and engineering of co-factor imbalances should lead to even higher isobutanol production.     

Lactic acid production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing a <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> lactate dehydrogenase gene

This work demonstrates the first example of a fungal lactate dehydrogenase (LDH) expressed in yeast. A L(+)-LDH gene, ldhA, from the filamentous fungus Rhizopus oryzae was modified to be expressed under control of the Saccharomyces cerevisiae adh1 promoter and terminator and then placed in a 2μ-containing yeast-replicating plasmid. The resulting construct, pLdhA68X, was transformed and tested by fermentation analyses in haploid and diploid yeast containing similar genetic backgrounds. Both recombinant strains utilized 92 g glucose/l in approximately 30 h. The diploid isolate accumulated approximately 40% more lactic acid with a final concentration of 38 g lactic acid/l and a yield of 0.44 g lactic acid/g glucose. The optimal pH for lactic acid production by the diploid strain was pH 5. LDH activity in this strain remained relatively constant at 1.5 units/mg protein throughout the fermentation. The majority of carbon was still diverted to the ethanol fermentation pathway, as indicated by ethanol yields between 0.25–0.33 g/g glucose. S. cerevisiae mutants impaired in ethanol production were transformed with pLdhA68X in an attempt to increase the lactic acid yield by minimizing the conversion of pyruvate to ethanol. Mutants with diminished pyruvate decarboxylase activity and mutants with disrupted alcohol dehydrogenase activity did result in transformants with diminished ethanol production. However, the efficiency of lactic acid production also decreased. Electronic Publication     

Utilizing an endogenous pathway for 1-butanol production in Saccharomyces cerevisiae

Microbial production of higher alcohols from renewable feedstock has attracted intensive attention thanks to its potential as a source for next-generation gasoline substitutes. Here we report the discovery, characterization and engineering of an endogenous 1-butanol pathway in Saccharomyces cerevisiae. Upon introduction of a single gene deletion adh1Δ, S. cerevisiae was able to accumulate more than 120 mg/L 1-butanol from glucose in rich medium. Precursor feeding, ¹³C-isotope labeling and gene deletion experiments demonstrated that the endogenous 1-butanol production was dependent on catabolism of threonine in a manner similar to fusel alcohol production by the Ehrlich pathway. Specifically, the leucine biosynthesis pathway was engaged in the conversion of key 2-keto acid intermediates. Overexpression of the pathway enzymes and elimination of competing pathways achieved the highest reported 1-butanol titer in S. cerevisiae (242.8 mg/L).     

Pyruvate Decarboxylase Catalyzes Decarboxylation of Branched-Chain 2-Oxo Acids but Is Not Essential for Fusel Alcohol Production by Saccharomyces cerevisiae

The fusel alcohols 3-methyl-1-butanol, 2-methyl-1-butanol, and 2-methyl-propanol are important flavor compounds in yeast-derived food products and beverages. The formation of these compounds from branched-chain amino acids is generally assumed to occur via the Ehrlich pathway, which involves the concerted action of a branched-chain transaminase, a decarboxylase, and an alcohol dehydrogenase. Partially purified preparations of pyruvate decarboxylase (EC 4.1.1.1) have been reported to catalyze the decarboxylation of the branched-chain 2-oxo acids formed upon transamination of leucine, isoleucine, and valine. Indeed, in a coupled enzymatic assay with horse liver alcohol dehydrogenase, cell extracts of a wild-type Saccharomyces cerevisiae strain exhibited significant decarboxylation rates with these branched-chain 2-oxo acids. Decarboxylation of branched-chain 2-oxo acids was not detectable in cell extracts of an isogenic strain in which all three PDC genes had been disrupted. Experiments with cell extracts from S. cerevisiae mutants expressing a single PDC gene demonstrated that both PDC1- and PDC5-encoded isoenzymes can decarboxylate branched-chain 2-oxo acids. To investigate whether pyruvate decarboxylase is essential for fusel alcohol production by whole cells, wild-type S. cerevisiae and an isogenic pyruvate decarboxylase-negative strain were grown on ethanol with a mixture of leucine, isoleucine, and valine as the nitrogen source. Surprisingly, the three corresponding fusel alcohols were produced in both strains. This result proves that decarboxylation of branched-chain 2-oxo acids via pyruvate decarboxylase is not an essential step in fusel alcohol production.Saccharomyces cerevisiae has been used for centuries in the production of bread and alcoholic beverages. Along with ethanol and carbon dioxide, fermenting cultures of this yeast produce a variety of low-molecular-weight flavor compounds (including alcohols, diacetyl, esters, organic acids, organic sulfides, and carbonyl compounds). The compounds 3-methyl-1-butanol, 2-methyl-1-butanol, and 2-methyl-1-propanol, commonly known as fusel alcohols, and their esters make an important contribution to the flavor of alcoholic beverages and bread (1, 14).A metabolic pathway for production of fusel alcohols by yeast was first proposed by Ehrlich (6). The Ehrlich pathway starts with the enzyme-catalyzed decarboxylation of branched-chain 2-oxo acids to the corresponding aldehydes. Subsequently, the aldehyde is reduced to the corresponding fusel alcohol by an alcohol dehydrogenase (11, 16, 24). The branched-chain 2-oxo acid substrates for the Ehrlich pathway can be produced by the deamination of l-leucine, l-isoleucine, or l-valine. Growth of S. cerevisiae with any of these three amino acids as the nitrogen source results in the accumulation of the corresponding fusel alcohol (2, 3, 21). Alternatively, branched-chain 2-oxo acids may be synthesized de novo from carbohydrates as intermediates of branched-chain amino acid synthesis (13).The conversion of branched-chain oxo acids into their respective aldehydes and alcohols via the Ehrlich pathway resembles the fermentative metabolism of pyruvate, which yields ethanol and carbon dioxide. In both cases, the decarboxylation of a 2-oxo acid is followed by the reduction of the resulting aldehyde. Partially purified preparations of yeast pyruvate decarboxylase have been shown to catalyze the decarboxylation of various 2-oxo acids, including the putative intermediates of the Ehrlich pathway (8, 12, 16, 21). However, it has not been conclusively proven that pyruvate decarboxylase is essential for or even involved in fusel alcohol production by S. cerevisiae.Dickinson and Dawes (4) have reported that, at least under some conditions, oxidative decarboxylation by a mitochondrial branched-chain oxo acid dehydrogenase complex (17) is involved in the catabolism of branched-chain 2-oxo acids. Mutants that did not express the lipoamide dehydrogenase subunit of this enzyme complex accumulated branched-chain oxo acids in batch cultures grown on media containing leucine, isoleucine, or valine (4), thus casting some doubt on the exclusive role of pyruvate decarboxylase in the decarboxylation of branched-chain oxo acids.The aim of this study was to reinvestigate the role of pyruvate decarboxylase in the production of fusel alcohols by S. cerevisiae. The S. cerevisiae genome harbors three structural genes (PDC1, PDC5, and PDC6) that can each encode an active pyruvate decarboxylase (9). In wild-type yeast strains, PDC6 expression is either very low or absent (7, 9). However, revertants of pdc1-pdc5 double mutants, in which a recombination event has caused a fusion of the PDC1 promoter and the PDC6 open reading frame, express a functional enzyme (10). Therefore, studies on the physiological effects of pyruvate decarboxylase deficiency are most easily interpreted when they are performed with strains in which all three PDC genes are disrupted.In the present study, the decarboxylation of branched-chain 2-oxo acids was studied in cell extracts of wild-type S. cerevisiae and in extracts of an isogenic pyruvate decarboxylase-negative mutant. Furthermore, conversion of branched-chain amino acids to the corresponding fusel alcohols by intact cells was analyzed in ethanol-grown cultures of a wild-type S. cerevisiae strain and in those of the Pdc⁻ mutant.     

Substrate Specificity of Thiamine Pyrophosphate-Dependent 2-Oxo-Acid Decarboxylases in Saccharomyces cerevisiae

Fusel alcohols are precursors and contributors to flavor and aroma compounds in fermented beverages, and some are under investigation as biofuels. The decarboxylation of 2-oxo acids is a key step in the Ehrlich pathway for fusel alcohol production. In Saccharomyces cerevisiae, five genes share sequence similarity with genes encoding thiamine pyrophosphate-dependent 2-oxo-acid decarboxylases (2ODCs). PDC1, PDC5, and PDC6 encode differentially regulated pyruvate decarboxylase isoenzymes; ARO10 encodes a 2-oxo-acid decarboxylase with broad substrate specificity, and THI3 has not yet been shown to encode an active decarboxylase. Despite the importance of fusel alcohol production in S. cerevisiae, the substrate specificities of these five 2ODCs have not been systematically compared. When the five 2ODCs were individually overexpressed in a pdc1Δ pdc5Δ pdc6Δ aro10Δ thi3Δ strain, only Pdc1, Pdc5, and Pdc6 catalyzed the decarboxylation of the linear-chain 2-oxo acids pyruvate, 2-oxo-butanoate, and 2-oxo-pentanoate in cell extracts. The presence of a Pdc isoenzyme was also required for the production of n-propanol and n-butanol in cultures grown on threonine and norvaline, respectively, as nitrogen sources. These results demonstrate the importance of pyruvate decarboxylases in the natural production of n-propanol and n-butanol by S. cerevisiae. No decarboxylation activity was found for Thi3 with any of the substrates tested. Only Aro10 and Pdc5 catalyzed the decarboxylation of the aromatic substrate phenylpyruvate, with Aro10 showing superior kinetic properties. Aro10, Pdc1, Pdc5, and Pdc6 exhibited activity with all branched-chain and sulfur-containing 2-oxo acids tested but with markedly different decarboxylation kinetics. The high affinity of Aro10 identified it as a key contributor to the production of branched-chain and sulfur-containing fusel alcohols.     

Engineering of carboligase activity reaction in Candida glabrata for acetoin production

Utilization of Candida glabrata overproducing pyruvate is a promising strategy for high-level acetoin production. Based on the known regulatory and metabolic information, acetaldehyde and thiamine were fed to identify the key nodes of carboligase activity reaction (CAR) pathway and provide a direction for engineering C. glabrata. Accordingly, alcohol dehydrogenase, acetaldehyde dehydrogenase, pyruvate decarboxylase, and butanediol dehydrogenase were selected to be manipulated for strengthening the CAR pathway. Following the rational metabolic engineering, the engineered strain exhibited increased acetoin biosynthesis (2.24 g/L). In addition, through in silico simulation and redox balance analysis, NADH was identified as the key factor restricting higher acetoin production. Correspondingly, after introduction of NADH oxidase, the final acetoin production was further increased to 7.33 g/L. By combining the rational metabolic engineering and cofactor engineering, the acetoin-producing C. glabrata was improved stepwise, opening a novel pathway for rational development of microorganisms for bioproduction.     

Current knowledge on isobutanol production with Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum

Due to steadily rising crude oil prices great efforts have been made to develop designer bugs for the fermentative production of higher alcohols, such as 2-methyl-1-butanol, 3-methyl-1-butanol and 2-Methyl-1-propanol (isobutanol), which all possess quality characteristics comparable to traditional oil based fuels. The common metabolic engineering approach uses the last two steps of the Ehrlich pathway, catalyzed by 2-ketoacid decarboxylase and an alcohol dehydrogenase converting the branched chain 2-ketoacids of L-isoleucine, L-leucine, and L-valine into the respective alcohols. This strategy was successfully used to engineer well suited and industrially employed bacteria, such as Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum for the production of higher alcohols. Among these alcohols, isobutanol is currently the most promising one regarding final titer and yield. This article summarizes the current knowledge and achievements on isobutanol production with E. coli, B. subtilis and C. glutamicum regarding the metabolic engineering approaches and process conditions.     

Isobutanol production in engineered Saccharomyces cerevisiae by overexpression of 2-ketoisovalerate decarboxylase and valine biosynthetic enzymes

Engineering of Saccharomyces cerevisiae to produce advanced biofuels such as isobutanol has received much attention because this yeast has a natural capacity to produce higher alcohols. In this study, construction of isobutanol production systems was attempted by overexpression of effective 2-keto acid decarboxylase (KDC) and combinatorial overexpression of valine biosynthetic enzymes in S. cerevisiae D452-2. Among the six putative KDC enzymes from various microorganisms, 2-ketoisovalerate decarboxylase (Kivd) from L. lactis subsp. lactis KACC 13877 was identified as the most suitable KDC for isobutanol production in the yeast. Isobutanol production by the engineered S. cerevisiae was assessed in micro-aerobic batch fermentations using glucose as a sole carbon source. 93?mg/L isobutanol was produced in the Kivd overexpressing strain, which corresponds to a fourfold improvement as compared with the control strain. Isobutanol production was further enhanced to 151?mg/L by additional overexpression of acetolactate synthase (Ilv2p), acetohydroxyacid reductoisomerase (Ilv5p), and dihydroxyacid dehydratase (Ilv3p) in the cytosol.     

Acetaldehyde production in Saccharomyces cerevisiae wine yeasts

Abstract Eighty-six strains of Saccharomyces cerevisiae were investigated for their ability to produce acetaldehyde in synthetic medium and in grape must. Acetaldehyde production did not differ significantly between the two media, ranging from a few mg/l to about 60 mg/l, and was found to be a strain characteristic. The fermentation temperature of 30°C considerably increased the acetaldehyde produced. This study allowed us to assign the strains to different phenotypes: low, medium and high acetaldehyde producers. The low and high phenotypes differed considerably also in the production of acetic acid, acetoin and higher alcohols and can be useful for studying acetaldehyde production in S. cerevisiae , both from the technological and genetic point of view.     

酿酒酵母高级醇代谢研究进展

高级醇是酿酒酵母在饮料酒酿造过程中产生的主要代谢副产物之一.饮料酒中高级醇含量过高,易导致饮用后产生头痛、口渴等症状,是醉酒较慢、醉酒后较难醒酒的主要原因.文中系统综述了饮料酒中主要高级醇的风味特征、代谢途径及诱变育种技术在酿酒酵母高级醇代谢调控中的应用,特别阐述了代谢工程技术在氨基转移酶编码基因、α-酮酸代谢基因、乙...     

Metabolic responses of pyruvate decarboxylase-negative Saccharomyces cerevisiae to glucose excess.

In Saccharomyces cerevisiae, oxidation of pyruvate to acetyl coenzyme A can occur via two routes. In pyruvate decarboxylase-negative (Pdc-) mutants, the pyruvate dehydrogenase complex is the sole functional link between glycolysis and the tricarboxylic acid (TCA) cycle. Such mutants therefore provide a useful experimental system with which to study regulation of the pyruvate dehydrogenase complex. In this study, a possible in vivo inactivation of the pyruvate dehydrogenase complex was investigated. When respiring, carbon-limited chemostat cultures of wild-type S. cerevisiae were pulsed with excess glucose, an immediate onset of respiro-fermentative metabolism occurred, accompanied by a strong increase of the glycolytic flux. When the same experiment was performed with an isogenic Pdc- mutant, only a small increase of the glycolytic flux was observed and pyruvate was the only major metabolite excreted. This finding supports the hypothesis that reoxidation of cytosolic NADH via pyruvate decarboxylase and alcohol dehydrogenase is a prerequisite for high glycolytic fluxes in S. cerevisiae. In Pdc- cultures, the specific rate of oxygen consumption increased by ca. 40% after a glucose pulse. Calculations showed that pyruvate excretion by the mutant was not due to a decrease of the pyruvate flux into the TCA cycle. We therefore conclude that rapid inactivation of the pyruvate dehydrogenase complex (e.g., by phosphorylation of its E1 alpha subunit, a mechanism demonstrated in many higher organisms) is not a relevant mechanism in the response of respiring S. cerevisiae cells to excess glucose. Consistently, pyruvate dehydrogenase activities in cell extracts did not exhibit a strong decrease after a glucose pulse.     

A biometric study of higher alcohol production in Saccharomyces cerevisiae

A hundred strains of Saccharomyces cerevisiae were examined for the ability to produce higher alcohols. In the strains tested the production of higher alcohols was found to be an individual strain characteristic and, as such, was statistically significant. The characteristics of the strains used (flocculation ability, foaming ability, killer character, and non-H2S production) were found to be uncorrelated to isobutanol and isoamyl alcohol production, whereas the production of high levels of n-propanol was found to be related to inability to produce H2S. This, in turn, suggests a link to methionine biosynthesis.     

Use of the tac promoter and lacIq for the controlled expression of Zymomonas mobilis fermentative genes in Escherichia coli and Zymomonas mobilis.

The Zymomonas mobilis genes encoding alcohol dehydrogenase I (adhA), alcohol dehydrogenase II (adhB), and pyruvate decarboxylase (pdc) were overexpressed in Escherichia coli and Z. mobilis by using a broad-host-range vector containing the tac promoter and the lacIq repressor gene. Maximal IPTG (isopropyl-beta-D-thiogalactopyranoside) induction of these plasmid-borne genes in Z. mobilis resulted in a 35-fold increase in alcohol dehydrogenase I activity, a 16.7-fold increase in alcohol dehydrogenase II activity, and a 6.3-fold increase in pyruvate decarboxylase activity. Small changes in the activities of these enzymes did not affect glycolytic flux in cells which are at maximal metabolic activity, indicating that flux under these conditions is controlled at some other point in metabolism. Expression of adhA, adhB, or pdc at high specific activities (above 8 IU/mg of cell protein) resulted in a decrease in glycolytic flux (negative flux control coefficients), which was most pronounced for pyruvate decarboxylase. Growth rate and flux are imperfectly coupled in this organism. Neither a twofold increase in flux nor a 50% decline from maximal flux caused any immediate change in growth rate. Thus, the rates of biosynthesis and growth in this organism are not limited by energy generation in rich medium.     

Introducing a single secondary alcohol dehydrogenase into butanol-tolerant Clostridium acetobutylicum Rh8 switches ABE fermentation to high level IBE fermentation

ABSTRACT: BACKGROUND: Previously we have developed a butanol tolerant mutant of Clostridium acetobutylicum, Rh8, from the wild type strain DSM 1731. Strain Rh8 can tolerate up to 19 g/L butanol, with solvent titer improved accordingly, thus exhibiting industrial application potential. To test if strain Rh8 can be used for production of high level mixed alcohols, a single secondary alcohol dehydrogenase from Clostridium beijerinckii NRRL B593 was overexpressed in strain Rh8 under the control of constitutive thl promoter. RESULTS: The heterogenous gene sADH was functionally expressed in C. acetobutylicum Rh8. This simple, one-step engineering approach led to the complete conversion of acetone into isopropanol, achieving a total alcohol titer of 23.88 g/l (7.6 g/l isopropanol, 15 g/l butanol, and 1.28 g/l ethanol) with a yield to glucose of 31.42%. The acid (butyrate and acetate) assimilation rate in isopropanol producing strain Rh8(psADH) was increased. CONCLUSIONS: The improved butanol tolerance and the enhanced solvent biosynthesis machinery in strain Rh8 is beneficial for production of high concentration of mixed alcohols. Strain Rh8 thus can be considered as a good host for further engineering of solvent/alcohol production.     

Biosynthesis of Diacetyl in Bacteria and Yeast

Both diacetyl and acetoin were produced by cell-free extracts and cultures of Pseudomonas fluorescens, Aerobacter aerogenes, Lactobacillus brevis, and Saccharomyces cerevisiae 299, whereas only acetoin was produced by cell-free extracts and cultures of Streptococcus lactis, Serratia marcescens, Escherichia coli, and S. cerevisiae strains 513 and 522. Cell-free extracts that produced diacetyl did not produce it from acetoin; they produced it from pyruvate, but only if acetyl-coenzyme A was was added to the reaction mixtures. Production of diacetyl by S. cerevisiae 299 was prevented by valine, inhibited by sodium arsenite, and stimulated by pantothenic acid. Valine did not prevent the production of acetoin. E. coli and the three strains of S. cerevisiae did not decarboxylate alpha-acetolactate but did use acetaldehyde in the production of acetoin from pyruvate. The other organisms produced acetoin from pyruvate via alpha-acetolactate.