Pyruvate decarboxylase: An indispensable enzyme for growth of Saccharomyces cerevisiae on glucose

In Saccharomyces cerevisiae, the structural genes PDC1, PDC5 and PDC6 each encode an active pyruvate decarboxylase. Replacement mutations in these genes were introduced in a homothallic wild-type strain, using the dominant marker genes APT1 and Tn5ble. A pyruvate-decarboxylase-negative (Pdc⁻) mutant lacking all three PDC genes exhibited a three-fold lower growth rate in complex medium with glucose than the isogenic wild-type strain. Growth in batch cultures on complex and defined media with ethanol was not impaired in Pdc⁻ strains. Furthermore, in ethanol-limited chemostat cultures, the biomass yield of Pdc⁻ and wild-type S. cerevisiae were identical. However, Pdc⁻ S. cerevisiae was unable to grow in batch cultures on a defined mineral medium with glucose as the sole carbon source. When aerobic, ethanol-limited chemostat cultures (D = 0·10 h⁻¹) were switched to a feed containing glucose as the sole carbon source, growth ceased after approximately 4 h and, consequently, the cultures washed out. The mutant was, however, able to grow in chemostat cultures on mixtures of glucose and small amounts of ethanol or acetate (5% on a carbon basis). No growth was observed when such cultures were used to inoculate batch cultures on glucose. Furthermore, when the mixed-substrate cultures were switched to a feed containing glucose as the sole carbon source, wash-out occurred. It is concluded that the mitochondrial pyruvate dehydrogenase complex cannot function as the sole source of acetyl-CoA during growth of S. cerevisiae on glucose, neither in batch cultures nor in glucose-limited chemostat cultures.     

The Pichia pastoris dihydroxyacetone kinase is a PTS1-containing,but cytosolic,protein that is essential for growth on methanol

Dihydroxyacetone kinase (DAK) is essential for methanol assimilation in methylotrophic yeasts. We have cloned the DAK gene from Pichia pastoris by functional complementation of a mutant that was unable to grow on methanol. An open reading frame of 1824 bp was identified that encodes a 65·3 kDa protein with high homology to DAK from Saccharomyces cerevisiae. Although DAK from P. pastoris contained a C-terminal tripeptide, TKL, which we showed can act as a peroxisomal targeting signal when fused to the green fluorescent protein, the enzyme was primarily cytosolic. The TKL tripeptide was not required for the biochemical function of DAK because a deletion construct lacking the DNA encoding this tripeptide was able to complement the P. pastoris dakΔ mutant. Peroxisomes, which are essential for growth of P. pastoris on methanol, were present in the dakΔ mutant and the import of peroxisomal proteins was not disturbed. The dakΔ mutant grew at normal rates on glycerol and oleate media. However, unlike the wild-type cells, the dakΔ mutant was unable to grow on methanol as the sole carbon source but was able to grow on dihydroxyacetone at a much slower rate. The metabolic pathway explaining the reduced growth rate of the dakΔ mutant on dihydroxyacetone is discussed. The nucleotide sequence reported in this paper has been submitted to GenBank with Accession Number AF019198. © 1998 John Wiley & Sons, Ltd.     

Isolation and characterization of Kluyveromyces marxianus mutants deficient in malate transport

In malic acid-grown cells of the strains ATCC 10022 and KMS3 of Kluyveromyces marxianus the transport of malic acid occurred by a malate-proton symport, which accepted l-malic, d-malic, succinic and fumaric acids, but not tartaric, malonic or maleic acids. The system was inducible and subjected to glucose repression. Mutants of the strain KMS3, unable to grow in a medium with malic acid, were isolated and checked for their capacity to utilize several carbon sources and to transport dicarboxylic acids by the malate-proton symport. Two distinct clones affected on malate transport were obtained. Both were able to grow on a medium with glycerol or ethanol but not with dl-malic, succinic, oxoglutaric and oxaloacetic acids as the sole carbon and energy sources. However, while one of the mutants (Mal7) displayed activity levels for the enzymes malate dehydrogenase, isocitrate lyase, and phosphoenolpyruvate carboxykinase similar to those of the wild strain, in the other mutant type (Mal6) the activities for the same enzymes were significantly reduced. Plasma membranes from derepressed cells of the wild strain and of the mutants Mal6 and Mal7 were isolated and the protein analysed by SDS–PAGE. The electrophoretic patterns of these preparations differed in a polypeptide with an apparent molecular mass of about 28 kDa, which was absent only in the mutant Mal7. The results indicated that Mal7 can be affected in a gene that encodes a malate carrier in K. marxianus. © 1998 John Wiley & Sons, Ltd.     

Effect of growth rate reduction and genetic modifications on acetate accumulation and biomass yields in Escherichia coli

Although acetate biosynthesis in Escherichia coli provides an important intermediary for ATP synthesis, its accumulation inhibits both cell growth and protein production. Since pyruvate provides the largest flux to acetate and is central to the problem of acetate production, acetate accumulation could be reduced or abolished if the pyruvate pool for the TCA cycle was reduced. To examine this possibility, various pyruvate kinase (pyk) and phosphotransferase system (pts) mutants were tested for acetate production in batch cultures with glucose as the only carbon source. The pykA pykF mutant exhibited significant reductions in the specific growth rate and acetate production compared with the wild-type strain. Interestingly, in the case of pts and pts pyk mutants in which increased biomass yields were observed in comparison with the wild-type strain, no acetate production was detected. Therefore, these mutants are potentially useful for higher production of recombinant proteins. The results from the continuous cultivation performed using the wild-type strain at various dilution rates, suggest acetate reduction as a consequence of both genetic changes and growth rate diminutions.     

Evaluation of Brazilian ethanol production yeasts for maltose fermentation in media containing structurally complex nitrogen sources

Maltose and glucose fermentations are strongly affected by the structural complexity of the nitrogen source and by the presence of oxygen. In this study five industrial Saccharomyces cerevisiae strains were grown in synthetic medium, containing maltose or glucose, supplemented with different nitrogen sources, with or without agitation. All strains were able to grow and efficiently ferment glucose, but not all strains were able to grow and ferment maltose well. Peptone and ammonium sulfate induced improved fermentation for all strains and conditions. Under agitation, as expected, higher biomass accumulation was detected. Casamino acids supplementation induced efficient maltose fermentation for all of the strains under aerated conditions, but differing maltose utilization patterns were observed for the static cultures. Copyright © 2012 The Institute of Brewing & Distilling     

Partial biochemical characterization of tetrazolium red‐reactive Lactobacillus plantarum mutants

One thousand colonies derived from Lactobacillus plantarum ATCC 8014 cells that survived 34–43 × 10³ ergs cm^‐2 ultraviolet irradiation were screened on media containing tetrazolium red to detect fermentative mutants. Fermentation end‐products formed from pyruvate, glucose, or lactose catabolism were determined. All 37 stable tetrazolium red‐reactive mutants had increased pyruvate utilization compared to the wild‐type strain. Only two did not produce lactate from exogenous pyruvate. When glucose or lactose were substrates, these two mutants and six other representative mutants produced lactate at levels similar to the parent strain. Although the average lactate and acetoin production from pyruvate by the mutants and wild‐type strains were similar, 25% of the mutants had increased acetoin production.     

SHM1: A multicopy suppressor of a temperature-sensitive null mutation in the HMG1-like abf2 gene

HM, an HMG1-like mitochondrial DNA-binding protein, is required for maintenance of the yeast mitochondrial genome when cells are grown in glucose. To better understand the role of HM in mitochondria, we have isolated several multicopy suppressors of the temperature-sensitive defect associated with an abf2 null mutation (lacking HM protein). One of these suppressors, SHM1, has been characterized at the molecular level and is described herein. SHM1 encodes a protein (SHM1p) that shares sequence similarity to a family of mitochondrial carrier proteins. On glycerol medium, where mitochondrial function is required for growth, shm1 deletion mutants are able to grow, whereas shm1 abf2 double mutants are severely inhibited. These results suggest that SHM1p plays an accessory role to HM in the mitochondrion. The GenBank Accession Number for the SHM1 sequence is U08352.     

高产γ-氨基丁酸的富锌乳酸菌培养基优化及GAD基因的克隆和序列分析

为了检测和提高乳酸菌γ- 氨基丁酸(GABA)产量,本研究采用纸层析预染法分析Lactobacillus brevis BS2最佳发酵产GABA 条件为：pH5.0,以葡萄糖为碳源,以大豆蛋白胨和牛肉膏为复合氮源,碳氮比为1:1,底物质量分数1%,GABA 产量可达到6.32g/L。采用CTAB 法提取该菌株基因组,并以此基因组为模板应用降落PCR扩增出1407bp 的谷氨酸脱羧酶基因gad,将gad 克隆到T 载体后经测序分析发现与L. brevis strain BH2 的gad 具有高度的同源性,同源性达98%,证明该菌株为高产GABA 的富锌短小乳酸杆菌,在工业应用方面具有很大潜力。     

Atan1p—an extracellular tannase from the dimorphic yeast Arxula adeninivorans: molecular cloning of the ATAN1 gene and characterization of the recombinant enzyme

The tannase‐encoding Arxula adeninivorans gene ATAN1 was isolated from genomic DNA by PCR, using as primers oligonucleotide sequences derived from peptides obtained after tryptic digestion of the purified tannase protein. The gene harbours an ORF of 1764 bp, encoding a 587‐amino acid protein, preceded by an N‐terminal secretion sequence comprising 28 residues. The deduced amino acid sequence was similar to those of tannases from Aspergillus oryzae (50% identity), A. niger (48%) and putative tannases from A. fumigatus (52%) and A. nidulans (50%). The sequence contains the consensus pentapeptide motif (–Gly–X–Ser–X–Gly–) which forms part of the catalytic centre of serine hydrolases. Expression of ATAN1 is regulated by the carbon source. Supplementation with tannic acid or gallic acid leads to induction of ATAN1, and accumulation of the native tannase enzyme in the medium. The enzymes recovered from both wild‐type and recombinant strains were essentially indistinguishable. A molecular mass of ～320 kDa was determined, indicating that the native, glycosylated tannase consists of four identical subunits. The enzyme has a temperature optimum at 35–40 °C and a pH optimum at ～6.0. The enzyme is able to remove gallic acid from both condensed and hydrolysable tannins. The wild‐type strain LS3 secreted amounts of tannase equivalent to 100 U/l under inducing conditions, while the transformant strain, which overexpresses the ATAN1 gene from the strong, constitutively active A. adeninivorans TEF1 promoter, produced levels of up to 400 U/l when grown in glucose medium in shake flasks. Copyright © 2009 John Wiley & Sons, Ltd.     

Disruption of the KEX1 gene in Pichia pastoris allows expression of full‐length murine and human endostatin

Endostatin is a potent angiogenesis inhibitor. In order to isolate sufficient quantities of soluble protein for in vivo studies in mice, we expressed murine endostatin in Pichia pastoris. Analysis of the expressed protein by mass spectrometry indicated that the protein was truncated. N‐terminal sequence analysis determined that the N‐terminus was intact, suggesting that the C‐terminal lysine was missing. In Saccharomyces cerevisiae, Kex1p can cleave lysine and arginine residues from the C‐terminus of peptides and proteins. We hypothesized that the KEX1 homologue in P. pastoris is responsible for the loss of the C‐terminal lysine of endostatin. To test this hypothesis, we cloned and disrupted the P. pastoris KEX1 gene. Although the overall amino acid identity between the P. pastoris and the S. cerevisae Kex1p is only 36%, the amino acid residues involved in the catalytic activity or close to the active residues are highly conserved. Disruption of the KEX1 reading frame allowed expression of murine and human endostatin with the C‐terminal lysine. The KEX1 disruption strain may be a useful tool for the expression of other proteins with a C‐terminal basic amino acid. Addition of a lysine to the C‐terminus of recombinant proteins may protect the C‐terminus from degradation by other carboxypeptidases. 3·5 kb of the P. pastoris KEX1 gene locus have been deposited in the GeneBank database and are available under Accession No. AF095574. Copyright © 1999 John Wiley & Sons, Ltd.     

Identification of major ADH genes in ethanol metabolism of Pichia pastoris

The methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii) is a successful host widely used in recombinant protein production. The widespread use of a methanol-regulated alcohol oxidase 1 (AOX1) promoter for recombinant protein production has directed studies particularly about methanol metabolism in this yeast. Although there is comprehensive knowledge about methanol metabolism, there are other mechanisms in P. pastoris that have not been investigated yet, such as ethanol metabolism. The gene responsible for the consumption of ethanol ADH2 (XM_002491337, known as ADH3) was identified and characterized in our previous study. In this study, the ADH genes (XM_002489969, XM_002491163, XM_002493969) in P. pastoris genome were investigated to determine their roles in ethanol production by gene disruption analysis. We report that the ADH900 (XM_002491163) is the main gene responsible for ethanol production in P. pastoris. The ADH2 gene, previously identified as the only gene responsible for ethanol consumption, also plays a minor role in ethanol production in the absence of the ADH900 gene. The investigation of the carbon source regulation mechanism has also revealed that the ADH2 gene exhibit similar expression behaviours with ADH900 on glucose, glycerol, and methanol, however, it is strongly induced by ethanol.     

Development of the methylotrophic yeast Pichia methanolica for the expression of the 65 kilodalton isoform of human glutamate decarboxylase

We describe a protein expression system in the methylotrophic yeast, Pichia methanolica. Methods for transformation and genetic manipulation of the organism were developed using an ade2 strain and the wild-type ADE2 gene. A vacuolar protease-deficient strain was constructed. Two genes encoding alcohol oxidases were found, yet a single isoform of alcohol oxidase was produced during methanol-fed fermentations. The promoter from this gene was used to drive expression. An integrating plasmid for the cytoplasmic expression of the 65 kDa isoform of human glutamate decarboxylase (human GAD₆₅) was assembled. A strain harboring eight copies of this plasmid expressed enzymatically active human GAD₆₅ at levels approaching 0·5 g/l. Identical amounts were made in Pichia pastoris. The recombinant GAD₆₅ was purified to greater than 90% purity. © 1998 John Wiley & Sons, Ltd.     

Physiological role of the D-amino acid oxidase gene, DAO1, in carbon and nitrogen metabolism in the methylotrophic yeast Candida boidinii

A methylotrophic yeast, Candida boidinii, exhibits D-amino acid oxidase activity (DAO, EC 1.4.3.3) during its growth on D-alanine as a sole carbon or a nitrogen source. The structural gene (DAO1), encoding DAO, was cloned from a genomic library of C. boidinii. The 1035-bp gene encoded 345 amino acids and the predicted amino acid sequence showed significant similarity to those of DAOs from other organisms. The DAO1 gene was disrupted in the C. boidinii genome by one-step gene disruption. The DAO1-deleted strain did not grow on D-alanine as a carbon source but did grow on D-alanine as a sole nitrogen source (with glucose as the carbon source). These results suggested that, while DAO is critically involved in growth on D-alanine as a carbon source, there should be another enzyme system which metabolizes D-alanine as a nitrogen source in C. boidinii. We also showed that the three C-terminal amino acid sequence of DAO, -AKL was necessary and sufficient for the import of DAO into peroxisomes.     

Control of Saccharomyces cerevisiae carboxypeptidase S (CPS1) gene expression under nutrient limitation

Expression of the vacuolar carboxypeptidase S (CPS1) gene in Saccharomyces cerevisiae is regulated by the availability of nutrients. Enzyme production is sensitive to nitrogen catabolite repression; i.e. the presence of ammonium ions maintains expression of the gene at a low level. Transfer of ammonium–glucose pre-grown cells to a medium deprived of nitrogen causes a drastic increase in CPS1 RNA level provided that a readily usable carbon source, such as glucose or fructose, is available to the cells. Derepression of the gene by nitrogen limitation is cycloheximide-insensitive. Neither glycerol, ethanol, acetate nor galactose support derepression of CPS1 expression under nitrogen starvation conditions. Non-metabolizable sugar analogs (2-deoxyglucose, 6-methyl-glucose or glucosamine) do not allow derepression of CPS1, showing that the process is energy-dependent. Production of carboxypeptidase yscS also increases several-fold when ammonium-pregrown cells are transferred to media containing glucose and a non-readily metabolizable nitrogen source such as proline, leucine, valine or leucyl-glycine. Analysis of CPS1 expression in RAS2⁺ (high cAMP) and ras2 mutant (low cAMP) strains and in cells grown at low temperature (23°C) and in heat-shocked cells (38°C) shows that steady-state levels of CPS1 mRNA are not controlled by a low cAMP level-signalling pathway.     

GAP1, a novel selection and counter-selection marker for multiple gene disruptions in Saccharomyces cerevisiae

We report on the use of a new homologous marker for use in multiple gene deletions in S. cerevisiae, the general amino acid permease gene (GAP1). A GAP1 strain can utilize L-citrulline as the sole nitrogen source but cannot grow in the presence of the toxic amino acid D-histidine. L-citrulline as well as D-histidine uptake is mediated solely by the general amino acid permease, and a gap1 strain is therefore able to grow in the presence of D-histidine but cannot utilize L-citrulline. Gene disruption is effected by transforming a gap1 strain with a gene cassette generated by PCR, containing GAP1 flanked by short (60 bp) stretches of the gene in question. Through homologous recombination, the cassette will integrate into the target gene, which is thus replaced by GAP1, and mutants are selected for on minimal L-citrulline medium. When propagated under non-selective conditions, some cells will lose the GAP1 gene. This is caused by recombination between two Ashbya gossypii AgLEU2 [corrected] direct repeats embracing GAP1, and will result in a sub-population of gap1 cells. Such cells are selected on a medium containing D-histidine, and may subsequently be used for a second gene disruption. Hence, multiple gene disruptions can be made fast, cheaply and easily in a gap1 strain, with two positive selection steps for each disruption.     

A mutation in the COX5 gene of the yeast Scheffersomyces stipitis alters utilization of amino acids as carbon source, ethanol formation and activity of cyanide insensitive respiration

Scheffersomyces stipitis PJH was mutagenized by random integrative mutagenesis and the integrants were screened for lacking the ability to grow with glutamate as sole carbon source. One of the two isolated mutants was damaged in the COX5 gene, which encodes a subunit of the cytochrome c oxidase. BLAST searches in the genome of Sc. stipitis revealed that only one singular COX5 gene exists in Sc. stipitis, in contrast to Saccharomyces cerevisiae, where two homologous genes are present. Mutant cells had lost the ability to grow with the amino acids glutamate, proline or aspartate and other non-fermentable carbon sources, such as acetic acid and ethanol, as sole carbon sources. Biomass formation of the mutant cells in medium containing glucose or xylose as carbon source was lower compared with the wild-type cells. However, yields and specific ethanol formation of the mutant were much higher, especially under conditions of higher aeration. The mutant cells lacked both cytochrome c oxidase activity and cyanide-sensitive respiration, whereas ADH and PDC activities were distinctly enhanced. SHAM-sensitive respiration was obviously essential for the fermentative metabolism, because SHAM completely abolished growth of the mutant cells with both glucose or xylose as carbon source.     

Glucose metabolism and ethanol production in adh multiple and null mutants of Kluyveromyces lactis

Four genes coding for alcohol dehydrogenase (ADH) activities were identified in Kluyveromyces lactis. Due to the presence in this yeast of multiple ADH isozymes, mutants in the individual genes constructed by gene replacement yielded no clear phenotype. We crossed these mutants and developed a screening procedure which allowed us to identify strains lacking several ADH activities. The analysis of the adh triple mutants revealed that each activity confers to the cell the ability to grow on ethanol as the sole carbon source. On the contrary, adh null strains failed to grow on this substrate, indicating that no other important ADH activities are present in K. lactis cells. In the adh null mutants we also found a residual production of ethanol, as has been reported to be the case in Saccharomyces cerevisiae. This production showed a ten-fold increase when the K1ADHI activity was reintroduced in the null mutant and cells were cultivated under oxygen-limiting conditions. Differently from S. cerevisiae, glycerol is poorly accumulated in K. lactis adh null mutants.     

The IRC7 gene encodes cysteine desulphydrase activity and confers on yeast the ability to grow on cysteine as a nitrogen source

Although cysteine desulphydrase activity has been purified and characterized from Saccharomyces cerevisiae, the gene encoding this activity in vivo has never been defined. We show that the full‐length IRC7 gene, encoded by the YFR055W open reading frame, encodes a protein with cysteine desulphydrase activity. Irc7p purified to homogeneity is able to utilize l ‐cysteine as a substrate, producing pyruvate and hydrogen sulphide as products of the reaction. Purified Irc7p also utilized l ‐cystine and some other cysteine conjugates, but not l ‐cystathionine or l ‐methionine, as substrates. We further show that, in vivo, the IRC7 gene is both necessary and sufficient for yeast to grow on l ‐cysteine as a nitrogen source, and that overexpression of the gene results in increased H₂S production. Strains overexpressing IRC7 are also hypersensitive to a toxic analogue, S‐ethyl‐l ‐cysteine. While IRC7 has been identified as playing a critical role in converting cysteine conjugates to volatile thiols that are important in wine aroma, its biological role in yeast cells is likely to involve regulation of cysteine and redox homeostasis. Copyright © 2015 John Wiley & Sons, Ltd.     

通过添加NaHCO_3和调节pH提高谷氨酸得率

研究了在谷氨酸发酵产酸期分别添加NaHCO3、调节pH及两者耦联操作条件下的发酵性能。结果表明:只有在升高pH值、添加NaHCO3同时进行或先升高pH值后添加NaHCO3的情况下,葡萄糖消耗量大幅下降,谷氨酸得率显著提高,分别比对照提高36%和34%,且谷氨酸产量可以达到正常水平(75 g/L以上)。酶学代谢分析表明,仅仅提高丙酮酸羧化酶活性不能提高谷氨酸得率,只有各个关键酶相互协调配合,即适度弱化丙酮酸脱氢酶和α-酮戊二酸脱氢酶活性的同时,适度提高丙酮酸羧化酶和异柠檬酸脱氢酶活性,才能有效提高谷氨酸发酵整体性能。