Peroxisome-deficient mutants of Hansenula polymorpha

As a first step in a genetic approach towards understanding peroxisome biogenesis and function, we have sought to isolate mutants of the methylotrophic yeast Hansenula polymorpha which are deficient in peroxisomes. A collection of 260 methanol-utilization-defective strains was isolated and screened for the ability to utilize a second compound, ethanol, the metabolism of which involves peroxisomes. Electron microscopical investigations of ultrathin sections of selected pleiotropic mutants revealed two strains which were completely devoid of peroxisomes. In both, different peroxisomal matrix enzymes were active but located in the cytosol; these included catalase, alcohol oxidase, malate synthase and isocitrate lyase. Subsequent backcrossing experiments revealed that for all crosses involving both strains, the methanol- and ethanol utilizing-deficient phenotypes segregated independently of each other, indicating that different gene mutations were responsible for these phenotypes. The phenotype of the backcrossed peroxisome-deficient derivates was identical: defective in the ability to utilize methanol but capable of growth on other carbon sources, including ethanol. The mutations complemented and therefore were recessive mutations in different genes.     

Characterization of a new set of mutants deficient in fermentation-induced loss of stress resistance for use in frozen dough applications

In frozen dough applications a prefermentation period during the preparation of the dough is unavoidable and might also be important to obtain bread with a good texture. A major disadvantage of the prefermentation period is that it is associated with a rapid loss of the freeze resistance of the yeast cells. A major goal for the development of new baker's yeast strains for use in frozen dough applications is the availability of strains that maintain a better freeze resistance during the prefermentation period. We have isolated mutants that retain a better stress resistance during the initiation of fermentation. Some of these showed the same growth rate and fermentation capacity as the wild type cells. These mutants are called 'fil', for deficient infermentation induced loss of stress resistance. First we used laboratory strains and heat stress treatment, given shortly after the initiation of fermentation, as the selection protocol. The first two mutants isolated in this way were affected in the glucose-activation mechanism of the Ras-cAMP pathway. The fil1 mutant had a partially inactivating point mutation in CYR1, the gene encoding adenylate cyclase, while fil2 contained a nonsense mutation in GPR1. GPR1 encodes a member of the G-protein coupled receptor family which acts as a putative glucose receptor for activation of the Ras-cAMP pathway. In a next step we isolated fil mutants directly in industrial strains using repetitive freeze treatment of doughs as selection protocol. Surviving yeast strains were tested individually for maintenance of fermentation capacity after freeze treatment in laboratory conditions and also for the best performing strains in frozen doughs prepared with yeast cultivated on a pilot scale. The most promising mutant, AT25, displayed under all conditions a better maintenance of gassing power during freeze-storage. It was not affected in other commercially important properties and will now be characterised extensively at the biochemical and molecular level.     

Ady2p is essential for the acetate permease activity in the yeast Saccharomyces cerevisiae

To identify new genes involved in acetate uptake in Saccharomyces cerevisiae, an analysis of the gene expression profiles of cells shifted from glucose to acetic acid was performed. The gene expression reprogramming of yeast adapting to a poor non-fermentable carbon source was observed, including dramatic metabolic changes, global activation of translation machinery, mitochondria biogenesis and the induction of known or putative transporters. Among them, the gene ADY2/YCR010c was identified as a new key element for acetate transport, being homologous to the Yarrowia lipolytica GPR1 gene, which has a role in acetic acid sensitivity. Disruption of ADY2 in S. cerevisiae abolished the active transport of acetate. Microarray analyses of ady2Delta strains showed that this gene is not a critical regulator of acetate response and that its role is directly connected to acetate transport. Ady2p is predicted to be a membrane protein and is a valuable acetate transporter candidate.     

Decreasing acetic acid accumulation by a glycerol overproducing strain of Saccharomyces cerevisiae by deleting the ALD6 aldehyde dehydrogenase gene

Glycerol is a major fermentation product of Saccharomyces cerevisiae that contributes to the sensory character of wine. Diverting sugar to glycerol overproduction and away from ethanol production by overexpressing the glycerol 3-phosphate dehydrogenase gene,GPD2, caused S. cerevisiae to produce more than twice as much acetic acid as the wild-type strain (S288C background) in anaerobic cell culture. Deletion of the aldehyde dehydrogenase gene, ALD6, in wild-type and GPD2 overexpressing strains (GPD2-OP) decreased acetic acid production by three- and four-fold, respectively. In conjunction with reduced acetic acid production, the GPD2-OP ald6Delta strain produced more glycerol and less ethanol than the wild-type. The growth rate and fermentation rate were similar for the modified and wild-type strains, although the fermentation rate for the GPD2 ald6Delta strain was slightly less than that of the other strains from 24h onwards. Analysis of the metabolome of the mutants revealed that genetic modification affected the production of some secondary metabolites of fermentation, including acids, esters, aldehydes and higher alcohols, many of which are flavour-active in wine. Modification of GPD2 and ALD6 expression represents an effective strategy to increase the glycerol and decrease the ethanol concentration during fermentation, and alters the chemical composition of the medium such that, potentially, novel flavour diversity is possible. The implications for the use of these modifications in commercial wine production require further investigation in wine yeast strains.     

Disruption of genes encoding pyruvate dehydrogenase kinases leads to retarded growth on acetate and ethanol in Saccharomyces cerevisiae

Two open reading frames, YIL042c (PKP1) and YGL059w, with 25% sequence similarity to human pyruvate dehydrogenase kinases, were shown to have protein kinase activity. Using GFP fusions, it was demonstrated that the proteins localize in discrete submitochondrial regions. Strains with a null mutation in these loci grew poorly on acetate and ethanol as carbon sources. Doubling times increased from ca. 4 h in the wild-type to > 6 h for the mutants. Growth rates of the mutants could be restored to wild-type levels by simultaneous disruption of the PDA1 gene, encoding the E1alpha subunit of the pyruvate dehydrogenase complex. This observation and the pyruvate dehydrogenase activities measured in the mutant strains and the wild-type grown on glucose or acetate suggest that the slow growth phenotype on C2 carbon sources is caused by a futile cycle in which phosphoenolpyruvate is converted back to acetyl coenzyme A.     

Isolation and characterization of Saccharomyces cerevisiae mutants with derepressed thiamine gene expression

Using a THI4-lacZ reporter gene, mutant strains have been isolated that display constitutive expression of thiamine genes in the presence of normally repressing levels of exogenous thiamine. In total, eight strains were isolated in which this derepressed expression on thiamine (Det(-)) phenotype was the result of single gene mutations. The Det(-) mutations of three of these strains were partially dominant in a heterozygous diploid configuration, whereas the other five were recessive. The partially dominant mutants DET1, DET12 and DET13, and the recessive mutant det2, all showed derepressed THI4-lacZ expression levels comparable to those of a fully induced normal strain. Use of other promoter-lacZ gene fusions revealed that these four mutants were pleiotropic; expression levels of all thiamine-regulated genes tested were also derepressed. Genetic analysis of the four mutants suggested that det2 and DET13 were allelic, whereas the others were at different loci; these four mutations therefore represent three different genes. None of the mutations were allelic with THI80, mutations of which have previously been shown to confer derepression on thiamine-regulated genes. Also, intracellular thiamine levels were close to normal and none of the four mutants excreted thiamine into the growth medium. All mutant strains were found to be prototrophic for thiamine and none of those tested were compromised for thiamine uptake. It is possible that some may be alleles of, or interact with, the activator gene THI3. Taken together, these results imply that DET1, det2, DET12 and DET13 represent new genes encoding negative regulators of thiamine-repressed genes.     

Quinoprotein alcohol dehydrogenase is involved in catabolic acetate production, while NAD-dependent alcohol dehydrogenase in ethanol assimilation in Acetobacter pasteurianus SKU1108

The relationship between quinoprotein alcohol dehydrogenase (ADH) and NAD-dependent ADH was studied by constructing quinoprotein ADH-deficient mutants. Quinoprotein ADH-deficient mutants were successfully constructed from Acetobacter pasteurianus SKU1108 by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis and also by adhA gene disruption with a kanamycin cassette. The NTG mutant exhibited a complete loss of its acetate-producing ability and acetic acid resistance, while the disruptant also exhibited a loss of its acetic acid resistance but retained a weak ADH activity. The immunoblot analysis of quinoprotein ADH indicated that there are no appreciable ADH subunits in the membranes of both mutant strains. The NTG mutant grew better than the wild-type strain in ethanol-containing medium, despite the absence of quinoprotein ADH. In the mutant, the activities of two NAD-dependent ADHs, present in a small amount in the wild-type strain, markedly increased in the cytoplasm when cultured in a medium containing ethanol, concomitant to the increase in the activities of the key enzymes in TCA and glyoxylate cycles. The disruptant showed a poorer growth than the wild-type strain, producing a lower amount of acetic acid in ethanol culture, and it induced one of the two NAD-dependent ADHs and some of the acetate-assimilating enzymes induced in the NTG mutant. This study clearly showed that quinoprotein ADH is extensively involved in acetic acid production, while NAD-dependent ADH only in ethanol assimilation through the TCA and glyoxylate cycles in acetic acid bacteria. The differences between the NTG mutant and the disruptant are also discussed.     

优良本土戴尔有孢圆酵母的筛选及其在冰葡萄酒酿造中的应用

通过测定10株戴尔有孢圆酵母（Torulaspora delbrueckii）（编号为TD1～TD10）对葡萄糖、乙醇、酒石酸、SO2的耐受性能及其产β-葡萄糖苷酶性能,筛选发酵性能良好的戴尔有孢圆酵母,并将筛选菌株与酿酒酵母（Saccharomyces cerevisiae）ST混合发酵冰葡萄汁,最后对冰葡萄酒的基本理化指标进行测定。结果表明,除菌株TD2外,其他菌株均能够耐受葡萄糖500 g/L、乙醇体积分数4%、酒石酸16 g/L及SO2 350 mg/L,其中菌株TD6与TD9产β-葡萄糖苷酶活力较高,为优良本土戴尔有孢圆酵母。将菌株TD6、TD9分别与酿酒酵母ST混合发酵冰葡萄汁时均能在30 d内完成酒精发酵,且冰葡萄酒的基本理化指标均符合国标GB/T 25504—2010《冰葡萄酒》规定。此外,戴尔有孢圆酵母的接种对整个发酵过程的发酵速率与酿酒酵母的生长起到了一定的抑制作用,并且可以降低冰葡萄酒中乙酸和乙醇含量,提高总糖含量。     

Rag−mutations involved in glucose metabolism in yeast: Isolation and genetic characterization

Some natural isolates and many laboratory strains of the yeast Kluyveromyces lactis cannot grow on glucose when respiration is inhibited by antimycin A. The ability or inability to grow on glucose in the absence of mitochondrial respiration has been called Rag⁺ or Rag^? phenotype (resistance to antimycin on glucose, respectively). Rag^? strains, unable to grow on glucose in the presence of the respiratory drug, behave as if they were defective in fermentation. The Rag phenotype was first found to be determined by variant alleles of either of the two nuclear genes, RAG1 and RAG2, which code for a low-affinity glucose transport protein and for phosphoglucose isomerase, respectively. These findings suggested that the Rag^? phenotype can be used to obtain mutations of genes involved in glucose metabolism in K. lactis. We thus looked for other Rag^? mutants. Seventy-four mutants were isolated and genetically characterized. All of the mutations were nuclear recessive alleles, defining 11 new complementation groups, which we designate rag3 through rag13.     

Characterization of acetic acid bacteria in "traditional balsamic vinegar"

This study evaluated the glucose tolerance of acetic acid bacteria strains isolated from Traditional Balsamic Vinegar. The results showed that the greatest hurdle to acetic acid bacteria growth is the high sugar concentration, since the majority of the isolated strains are inhibited by 25% of glucose. Sugar tolerance is an important technological trait because Traditional Balsamic Vinegar is made with concentrated cooked must. On the contrary, ethanol concentration of the cooked and fermented must is less significant for acetic acid bacteria growth. A tentative identification of the isolated strains was done by 16S-23S-5S rDNA PCR/RFLP technique and the isolated strains were clustered: 32 strains belong to Gluconacetobacter xylinus group, two strains to Acetobacter pasteurianus group and one to Acetobacter aceti.     

Mutants of the methylotrophic yeast Pichia pinus defective in C2 metabolism

A collection of mutants of Pichia pinus which are unable to grow on ethanol but retain the ability to grow on glucose and methanol, was obtained. Genetic and biochemical analysis of these strains revealed mutations in seven nuclear genes affecting activities of isocitrate lyase (icl1), malate synthase (mls1), phosphoenolpyruvate carboxykinase (pck1), ‘malic’ enzyme (mdd1) and acetyl-CoA synthetase (acs1, acs2 and acs3). All mutations except acs1-acs3 have no effect on the activities of other enzymes involved in C₂ metabolism. Mutations acs1, acs2 and acs3 have a pleiotropic action, leading to partial reduction in activities of isocitrate lyase and malate synthase. Ethanol-induced repression of the synthesis of the methanol oxidative enzymes, alcohol oxidase and catalase, is not impaired in these seven mutant classes. On the other hand, C₂ compound-induced inactivation of alcohol oxidase and catalase is impaired in mutants acs1, acs2, acs3 and icl1. It was suggested that glyoxylate and acetate (or acetate precursors) act as low molecular weight effectors, ‘switching on’ inactivation and repression, respectively, of alcohol oxidase and catalase in the medium containing ethanol or acetate.     

Physiological profiles relevant for novel alcoholic beverage design among Dekkera bruxellensis strains from different provenances

A selection of D. bruxellensis strains from different geographical and beverage sources were tested for their potential to develop novel alcoholic beverages. Selected strains were initially clustered based on genetic similarities determined by PCR fingerprinting. Physiological profiles were subsequently determined during the fermentation experiments that were carried out in a defined synthetic medium supplemented with glucose and 4‐vinylphenol for 22 days, as static cultures under microaerobic conditions. There were significant differences (p ≤ 0.05) in ethanol, glycerol and acetic acid yields and in the growth rates between the strains. During prolonged fermentation, a reduction in ethanol and acetic acid was observed, ranging from 43 to 54% for ethanol and from 4 to 45% for acetic acid, which was strain or genetic group specific. Consumption of ethanol and acetic acid occurred during the stationary phase, suggesting that ethanol and acetic acid were utilized for processes other than growth and must have had an impact on the formation of the aromatic profile. The conversion of 4‐vinylphenol to 4‐ethylphenol was much more efficient and was completed within 4 days of fermentation. Although further investigation is needed, the results indicate a potential of this previously undesired microorganism to be useful for a wide range of applications. Copyright © 2016 The Institute of Brewing & Distilling     

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.     

Impairment of Peroxisome Degradation in Pichia methanolica Mutants Defective in Acetyl-CoA Synthetase or Isocitrate Lyase

Single recessive mutations of the methylotrophic yeast Pichia methanolica acs1, acs2, acs3 and icl1 affecting acetyl-CoA synthetase and isocitrate lyase, and growth on ethanol as sole carbon and energy source, caused a defect in autophagic peroxisome degradation during exposure of methanol-grown cells to ethanol. As a control, a mutation in mdd1, which resulted in a defect of the ‘malic’ enzyme and also prevented ethanol utilization, did not prevent peroxisome degradation. Peroxisome degradation in glucose medium was unimpaired in all strains tested. Addition of ethanol to methanol-grown cells of acs1, acs2, acs3 and icl1 mutants led to an increase in average vacuole size. Thickening of peroxisomal membranes and tight contacts between groups of peroxisomes and vacuoles were rarely observed. These processes proceeded much more slowly than in wild-type or mdd1 mutant cells incubated under similar conditions. No peroxisomal remnants were observed inside vacuoles in the cells of acs1, acs2, acs3 and icl1 mutants after prolonged cultivation in ethanol medium. We hypothesize that the acs and icl mutants are defective in synthesis of the true effector—presumably glyoxylate—of peroxisome degradation in ethanol medium. Lack of the effector suspends peroxisome degradation at an early stage, namely signal transduction or peroxisome/vacuole recognition. Finally, these defects in peroxisome degradation resulted in mutant cells retaining high levels of alcohol oxidase which further led to increased levels of acetaldehyde accumulation upon incubation of mutant cells with ethanol. © 1997 by John Wiley & Sons, Ltd.     

酿酒酵母SOD1、SOD2基因缺失对胁迫耐受性的影响

以sod1Δ、sod2Δ、sod1Δsod2Δ酿酒酵母基因缺失菌株为遗传材料,采用休止细胞梯度生长法,分析SOD1和SOD2基因缺失对高温、乙醇毒性、高渗透压、高盐、乙酸毒性及营养饥饿胁迫条件耐受性的影响。结果显示,与野生型菌株相比,sod1Δ菌株对高温、高渗透压和乙酸胁迫的耐受性降低;sod2Δ菌株耐受性无明显变化;sod1Δsod2Δ双缺失菌株对高温、乙醇毒性、乙酸毒性、高渗透压和高盐的耐受性均下降,表明酵母超氧化物歧化酶基因与多种胁迫耐受性密切相关。     

巴氏醋杆菌Ap2012的生长及耐受性研究

考察巴氏醋杆菌Ap2012在不同碳源酵母膏平板的生长情况及其对乙醇、糖、酸、NaCl的耐受性。结果表明,在酵母膏平板上,Ap2012能利用葡萄糖、乙醇作为碳源生长并产酸,能较好地利用甘油、果糖、蔗糖、山梨糖,对甘露醇的利用较差。乙醇体积分数为12.4%时巴氏醋杆菌Ap2012的生长受明显抑制,达到15.8%时转酸能力受明显抑制。葡萄糖的质量浓度达到300 g/L,NaCl的质量浓度为15 g/L时,巴氏醋杆菌Ap2012的生长和转酸能力受到明显抑制。乙酸质量浓度达到42 g/L时,巴氏醋杆菌Ap2012的生长受到明显抑制。结果表明,巴氏醋杆菌Ap2012对高浓度糖及乙醇有较好的耐受性,对乙酸和NaCl的耐受性较差。     

诺丽自然发酵汁中醋酸菌的分离鉴定及发酵特性

为分析诺丽自然发酵汁中醋酸菌的多样性,丰富诺丽中醋酸菌种属信息,采用传统分离培养和16S rRNA基因序列分析相结合的方法,对诺丽自然发酵汁中的醋酸菌进行分离鉴定和发酵特性研究。结果表明,从不同发酵阶段诺丽自然发酵汁的67 个分离菌株中鉴定出了24 株醋酸菌,分别为Acetobacter fabarum（9 株）、Acetobacter syzygii（7 株）、Acetobacter pasteurianus（2 株）、Acetobacter tropicalis（1 株）、Acetobacter lambici（1 株）和Gluconobacter japonicus（4 株）,其中A. syzygii和A. fabarum为诺丽自然发酵过程中的优势菌种。在发酵性能方面,A. tropicalis N21性能最优,产酸量可达28.92 g/L,在40 ℃高温中仍能良好生长且产酸量为14.85 g/L,乙醇体积分数为7%时,产酸量略有下降,但依然可达23.60 g/L,其耐高温和耐乙醇能力均高于其他菌株。