Ethanol fermentation by nystatin-resistant strains of Saccharomyces cerevisiae

Nystatin-resistant mutants of haploid and polyploid strains of Saccharomyces cerevisiae were isolated by plating on gradient plates with increasing nystatin concentrations (60–3000 U/ml). Some of the mutants were defective in ergosterol biosynthesis, and produced zymosterol and cholestatetraenol-like sterols. Those mutants which do not form ergosterol produce less ethanol than the parent strains. They also had lower viability during fermentation of glucose solutions (8–13% vs. 33–47%). This became more pronounced in fermentations of higher concentrations of glucose. A nystatin-resistant but ergosterol-forming mutant had a similar fermentation capacity to the parent strain.     

Effect of UV radiation on thermotolerance,ethanol tolerance and osmotolerance of Saccharomyces cerevisiae VS1 and VS3 strains

After a previous mass screening and enrichment programme for the isolation of thermotolerant yeasts, VS1, VS2, VS3 and VS4 strains isolated from soil samples, collected within the hot regions of Kothagudem Thermal Power Plant, AP, India, had a better thermotolerance, osmotolerance and ethanol tolerance than the other isolates. Among these isolates VS1 and VS3 were best performers. Efforts were made to further improve their osmotolerance, thermotolerance and ethanol tolerance by treating them with UV radiation. Mutants of VS1 and VS3 produced more biomass and ethanol than the parent strains at high temperature and glucose concentrations. The amount of biomass produced by VS1 and VS3 mutants was 0.25 and 0.20 g l(-1) more than the parent strains at 42 degrees C using 2% glucose. At high glucose concentrations VS1 and VS3 mutants produced biomass which was 0.70 and 0.30 g l(-1) at 30 degrees C and 0.10 and 0.20 g l(-1) at 40 degrees C more than the parent strains. The amount of ethanol produced by the mutants (VS1 and VS3) was 8.20 and 1.20 g l(-1) more than the parent strains at 42 degrees C using 150 g l(-1) glucose. More ethanol was produced by mutants (VS1 and VS3) than the parents at high glucose concentrations of 5.0 and 6.0 g l(-1) at 30 degrees C and 13.0 and 3.0 g l(-1) at 42 degrees C, respectively. These results indicated that UV mutagenesis can be used for improving thermotolerance, ethanol tolerance and osmotolerance in VS1 and VS3 yeast strains.     

Resistance of Escherichia coli to penicillins. IX. Genetics and physiology of class II ampicillin-resistant mutants that are galactose negative or sensitive to bacteriophage C21, or both

Ampicillin-resistant mutants of class II are determined by a doubling of chromosomally and episomally mediated ampicillin resistance on agar plates. Several mutants were isolated from a female as well as from an Hfr strain. The mutants differed from each other in various properties such as response to colicin E2 and sodium cholate, response to the phages T4 and C21, and fermentation of galactose. By conjugation and transduction experiments, it was shown that mutations in at least four loci gave the class II phenotype. The mutations were found to be in the galU gene, the ctr gene, and two new genes close to mtl denoted lpsA and lpsB. The carbohydrate compositions of the lipopolysaccharides of the mutants were investigated and found to be changed compared to the parent strains. GalU mutants lacked rhamnose and galactose and had 11% glucose compared to the parent strain. The lpsA mutant also lacked rhamnose and had only traces of galactose and 58% glucose, whereas the lpsB mutant contained 14% rhamnose, traces of galactose, and 81% glucose compared to the parent strain.     

Generation and characterisation of stable ethanol-tolerant mutants of Saccharomyces cerevisiae

Saccharomyces spp. are widely used for ethanologenic fermentations, however yeast metabolic rate and viability decrease as ethanol accumulates during fermentation, compromising ethanol yield. Improving ethanol tolerance in yeast should, therefore, reduce the impact of ethanol toxicity on fermentation performance. The purpose of the current work was to generate and characterise ethanol-tolerant yeast mutants by subjecting mutagenised and non-mutagenised populations of Saccharomyces cerevisiae W303-1A to adaptive evolution using ethanol stress as a selection pressure. Mutants CM1 (chemically mutagenised) and SM1 (spontaneous) had increased acclimation and growth rates when cultivated in sub-lethal ethanol concentrations, and their survivability in lethal ethanol concentrations was considerably improved compared with the parent strain. The mutants utilised glucose at a higher rate than the parent in the presence of ethanol and an initial glucose concentration of 20 g l⁻¹. At a glucose concentration of 100 g l⁻¹, SM1 had the highest glucose utilisation rate in the presence or absence of ethanol. The mutants produced substantially more glycerol than the parent and, although acetate was only detectable in ethanol-stressed cultures, both mutants produced more acetate than the parent. It is suggested that the increased ethanol tolerance of the mutants is due to their elevated glycerol production rates and the potential of this to increase the ratio of oxidised and reduced forms of nicotinamide adenine dinucleotide (NAD⁺/NADH) in an ethanol-compromised cell, stimulating glycolytic activity.     

Biochemical Properties of Haploid and Diploid Strains of Penicillium chrysogenum

An intensive parasexual genetics program in which industrial strains of Penicillium chrysogenum were used culminated in the isolation of a number of heterozygous diploid strains. The diploid clones were selected from heterokaryons formed from matings between mutant strains having complementary biochemical and conidial color markers. Several diploid cultures were compared with their haploid wild-type parents and other distantly related production strains on the basis of a variety of cultural and physiological criteria. The diploid strains characteristically produced conidia of larger volume and higher deoxyribonucleic acid content. Some were vigorous with respect to growth rate and onset and degree of conidiation. One diploid strain (WC-9) had a 46% greater oxygen uptake rate and oxidized glucose at a 57% greater rate than its haploid parent (M-2). It also produced 33% higher concentrations of β-galactosidase, 66% more alkaline protease, and 53% more glucose oxidase than the M-2 haploid parent. The selection of rare stable diploid mold cultures through the use of parasexual genetics offers a unique approach to the direct selection of mutants with potential for increased enzyme formation.     

降低光滑球拟酵母电子传递链活性加速丙酮酸合成

光滑球拟酵母CCTCCM2 0 2 0 19经溴化乙锭诱变 ,挑选假阳性呼吸缺陷型菌株共 4 0株。对其中 7株丙酮酸产量提高的突变株进行发酵性底物 (葡萄糖 )和非发酵性底物 (甘油、乙酸 )的利用能力测试 ,鉴定得到 3株呼吸缺陷型突变株RD 16、RD 17和RD 18。相对于出发菌株 ,呼吸缺陷型突变株生长速率下降 ,最终菌体浓度降低 2 1%～2 9% ,胞内ATP含量下降 15 %～ 2 1% ,但单位细胞耗葡萄糖能力和单位细胞产丙酮酸能力分别提高了 2 0 7%～30 7%和 30 7%～ 5 5 5 %。进一步研究发现 ,呼吸缺陷型突变株线粒体复合体Ⅰ、Ⅰ Ⅲ、Ⅱ Ⅲ和Ⅳ的活性分别下降了 34%～ 4 1%、38 6 %～ 5 2 6 %、2 1%～ 2 5 %、15 0 %～ 6 30 % ,表明线粒体电子传递链氧化NADH的功能受到抑制。为使酵解产生的NADH正常氧化 ,在RD 18菌株的对数生长期流加 2 1mmol L外源电子受体乙醛。发现细胞合成丙酮酸能力提高 2 1 6 % ,且葡萄糖消耗速度明显加快 ,发酵周期缩短 14h。结果表明适当削弱能量代谢能够提高真核微生物中心代谢途径的速度     

Selection and study of potent lactose-fermenting yeasts

Whey-fermenting Kluyveromyces cultures were revealed among 105 yeast strains assimilating lactose. Eighteen most potent strains isolated from milk products fermented galactose, sucrose, and raffinose, in addition to lactose. Many yeast strains fermented inulin. Most strains were resistant to cycloheximide and grew in medium containing glucose, NaCl, and ethanol at concentrations of up to 50, 11-12, and 10-12%, respectively (4 degrees C). Three strains had mycocinogenic activity. After fermentation of whey with selected yeast strains at 30 degrees C for 2-3 days, ethanol concentration was 4-5%.     

Isolation and characterisation ofCandida sp. mutants enriched in S-adenosylmethionine (SAM)

The ability to accumulate S-adenosylmethionine (SAM) of 572 yeast strains isolated from environmental sources were surveyed. An S-adenosylmethionine enriching strain S42-12, identified asCandida sp., was chose to develop a SAM-accumulating mutant successfully. The final SAM-accumulating mutant strain YQ-5 was isolated by UV radiation or by NTG treatment using ethionine selection and nystatin selection method. The mutant strain YQ-5 accumulated 112.1 mg per gram biomass, was 3.14-fold higher than the original strain S42-12. When cultivated in the optimal medium with a favourable fermentation conditions, SAM content of the mutant strain reached at 1740 mg L^?1. Trend of SAM and ergosterol contents and methionine adenosyltransferase activity of SAM-accumulating mutants during fermentation were analysed. The results suggested that one of the reasons why the mutants accumulated SAM in significantly high amounts may be the lower consumption of SAM for ergosterol biosynthesis, other than improvement of methionine adenosyltransferase activity.     

Sterol level in Saccharomyces cerevisiae mutants with altered ergosterol biosynthesis

The sterol content in Saccharomyces cerevisiae mutants defective in the synthesis of cyclic ergosterol precursors has been studied. It was found that strains with mutational blocks involving the stages of zymosterol side chain methylation at C24 and delta 8----delta 7 isomerization accumulated twice more sterols as compared to parent strains. Regulation of the ergosterol biosynthesis is discussed.     

影响酵母细胞麦角固醇的发酵控制参数的分析

研究了麦角固醇发酵过程中的参数。比较了溶氧、OUR、Ph和残糖浓度与酿酒酵母的生物量和麦角固醇含量的关系 ,并研究了这些参数之间的内在联系。研究表明 ,溶氧能比较准确地反映酵母的生长状况 ,是发酵过程中的一个较好的控制参数。将溶氧控制在 12 %左右能有效提高单位体积发酵液中的麦角固醇的产量。     

Cloning and characterization of a gene complementing the mutation of an ethanol-sensitive mutant of sake yeast

Ethanol-sensitive mutants (esl to es10) were isolated from sake yeast, Saccharomyces cerevisiae SY-32. These mutants were unable to grow at 7% ethanol at which the wild type strain SY-32 does grow. The mutants had a variety of fermentation rates and viabilities in the presence of ethanol. The gene ERG6, complementing the ethanol-sensitive mutation of es5, was cloned from an SY-32 gene library. ERG6 encodes S-adenosylmethionine: delta 24-sterol-C-methyltransferase (EC 2.1.1.41) in the ergosterol synthetic pathway. Mutant es5 had a reduced ability to synthesize ergosterol. An erg6 disruptant was also ethanol-sensitive. These results suggested that ERG6 plays an important role in the ethanol tolerance of S. cerevisiae.     

Use of catabolite repression mutants for fermentation of sugar mixtures to ethanol

Use of agricultural biomass, other than corn-starch, to produce fuel ethanol requires a microorganism that can ferment the mixture of sugars derived from hemicellulose. Escherichia coli metabolizes a wide range of substrates and has been engineered to produce ethanol in high yield from sugar mixtures. E. coli metabolizes glucose in preference to other sugars and, as a result, utilization of the pentoses in hemicellulose-derived sugar mixtures is delayed and may be incomplete. Residual sugar lowers the ethanol yield and is problematic for downstream processing of fermentation products. Therefore, a catabolite repression mutant that simultaneously utilizes glucose and pentoses would be useful for fermentation of complex substrate mixtures. We constructed ethanologenic E. coli strains with a glucose phosphotransferase (ptsG) mutation and used the mutants to ferment glucose, arabinose, and xylose, singly and in mixtures, to ethanol. Yields were 87-94% of theoretical for both the wild type and mutants, but the mutants had an altered pattern of mixed sugar utilization. Phosphotransferase mutants metabolized the pentoses simultaneously with glucose, rather than sequentially. Based upon fermentations of sugar mixtures, a catabolite-repression mutant of ethanologenic E. coli is expected to provide more efficient fermentation of hemicellulose hydrolysates by allowing direct utilization of pentoses.     

Breeding of high ergosterol-producing yeast strains

High ergosterol-producing yeast strains YEH-28 and YEH-56 were constructed by hybridization of two haploids with opposite mating types from different species. The fermentation conditions of hybrid strain YEH-56 were studied. The highest level of ergosterol was obtained in 30 h at 28°C and 200 rpm, when 60 ml of culture in 250-ml shake flasks was grown in fermentation medium that consisted of (per liter): 100 g glucose; 10 g peptone and 10 g yeast extract. The initial pH was adjusted to 6.0. The optimal inoculum volume was 10% (v/v). Under optimal conditions, the yield of ergosterol of YEH-28 strain is 1.96 and 1.56 times that of the parental strains YE39 and YE244, whereas that of YEH-56 is 1.98 and 1.57 times that of the parental strains YE39 and YE244, respectively. Analysis of genetic stability showed that hybrid strains YEH-28 and YEH-56 are stable genetically. Journal of Industrial Microbiology & Biotechnology (2000) 25, 39–44. Received 13 November 1999/ Accepted in revised form 09 March 2000     

Stress tolerance in doughs of Saccharomyces cerevisiae trehalase mutants derived from commercial Baker's yeast.

Accumulation of trehalose is widely believed to be a critical determinant in improving the stress tolerance of the yeast Saccharomyces cerevisiae, which is commonly used in commercial bread dough. To retain the accumulation of trehalose in yeast cells, we constructed, for the first time, diploid homozygous neutral trehalase mutants (Deltanth1), acid trehalase mutants (Deltaath1), and double mutants (Deltanth1 ath1) by using commercial baker's yeast strains as the parent strains and the gene disruption method. During fermentation in a liquid fermentation medium, degradation of intracellular trehalose was inhibited with all of the trehalase mutants. The gassing power of frozen doughs made with these mutants was greater than the gassing power of doughs made with the parent strains. The Deltanth1 and Deltaath1 strains also exhibited higher levels of tolerance of dry conditions than the parent strains exhibited; however, the Deltanth1 ath1 strain exhibited lower tolerance of dry conditions than the parent strain exhibited. The improved freeze tolerance exhibited by all of the trehalase mutants may make these strains useful in frozen dough.     

Regulation by heme of sterol uptake in Saccharomyces cerevisiae

The leaky heme mutants G204, G216, and G214 are shown to accumulate exogenous sterols. Unlike hem mutants which have complete blocks in the heme pathway, these strains do not require ergosterol, methionine, or unsaturated fatty acids for growth. The addition of aminolevulinic acid to the growth medium inhibited sterol uptake in G204 96% but had only a slight effect on sterol uptake by strains G214 and G216. Sterol uptake in all three strains was inhibited 83-94% when cells were grown in the presence of hematin. Sterol analysis of these strains grown in the presence and absence of either aminolevulinic acid or hematin revealed that saturation of the cell membrane with ergosterol was not responsible for the dramatic decrease in sterol uptake. These results suggest that sterol uptake by yeast cells is controlled by heme, and explain the non-viability of yeast strains that are heme competent and auxotrophic for sterols.     

Improved strains of recombinant Escherichia coli for ethanol production from sugar mixtures

Hemicellulose hydrolysates of agricultural residues often contain mixtures of hexose and pentose sugars. Ethanologenic Escherichia coli that have been previously investigated preferentially ferment hexose sugars. In some cases, xylose fermentation was slow or incomplete. The purpose of this study was to develop improved ethanologenic E. coli strains for the fermentation of pentoses in sugar mixtures. Using fosfomycin as a selective agent, glucose-negative mutants of E. coli KO11 (containing chromosomally integrated genes encoding the ethanol pathway from Zymomonas mobilis) were isolated that were unable to ferment sugars transported by the phosphoenolpyruvate-dependent phosphotransferase system. These strains (SL31 and SL142) retained the ability to ferment sugars with independent transport systems such as arabinose and xylose and were used to ferment pentose sugars to ethanol selectively in the presence of high concentrations of glucose. Additional fosfomycin-resistant mutants were isolated that were superior to strain KO11 for ethanol production from hexose and pentose sugars. These hyperproductive strains (SL28 and SL40) retained the ability to metabolize all sugars tested, completed fermentations more rapidly, and achieved higher ethanol yields than the parent. Both SL28 and SL40 produced 60 gl^–1 ethanol from 120 gl^–1 xylose in 60 h, 20% more ethanol than KO11 under identical conditions. Further studies illustrated the feasibility of sequential fermentation. A mixture of hexose and pentose sugars was fermented with near theoretical yield by SL40 in the first step followed by a second fermentation in which yeast and glucose were added. Such a two-step approach can combine the attributes of ethanologenic E. coli for pentoses with the high ethanol tolerance of conventional yeasts in a single vessel.     

Genome shuffling enhanced L-lactic acid production by improving glucose tolerance of Lactobacillus rhamnosus

Genome shuffling is a powerful strategy for rapid engineering of microbial strains for desirable industrial phenotypes. Here we applied the genome shuffling to improve the glucose tolerance of Lactobacillus rhamnosus ATCC 11443 while simultaneously enhancing the L-lactic acid production. The starting population was generated by ultraviolet irradiation and nitrosoguanidine mutagenesis and then subjected for the recursive protoplast fusion. The positive colonies from library created by fusing the inactivated protoplasts were more likely to be screened on plates containing different concentrations of high glucose and 2% CaCO(3). Characterization of all mutants and wild-type strain in the shake flask indicated the compatibility of two optimal phenotypes of glucose tolerance and lactic acid enhancement. The lactic acid production, cell growth and glucose consumption of the best performing strain from the second round genome shuffled populations were 71.4%, 44.9% and 62.2% higher than those of the wild type at the initial glucose concentration of 150 g/l in the 16l bioreactor. Furthermore, the higher lactic acid concentrations were obtained when the initial glucose concentrations increased to 160 and 200 g/l in batch fermentation.     

酿酒酵母呼吸缺陷型和野生型酒精发酵特性的比较分析*

比较了酒精发酵生产菌株IFFI1300及其呼吸缺陷型突变株在酒精产量、发酵动力学、耐酒精能力及与酒精发酵相关的乙醇脱氢酶活性等方面的特性。结果表明：1)发酵终期的酒精产量,45株呼吸缺陷型的平均值与野生型没有显著性差异;但部分缺陷型的酒精产量高于野生型。2)酒精发酵动力学结果显示,呼吸缺陷型酒精产生速度略高于野生型。3)单位重量干菌体的乙醇脱氢酶活性,呼吸缺陷型高于野生型。以上结果提示：呼吸缺陷型用于酒精发酵以提高酒精产量和缩短发酵周期是有潜力的。4)单位体积发酵液的乙醇脱氢酶活性则野生型高于呼吸缺陷型,主要原因在于呼吸缺陷型的生物量明显低于野生型。5)呼吸缺陷型菌株之间的耐酒精能力差别很小,耐酒精能力的高低与酒精产量的高低没有明显的正相关性。一般的,酒精产量高的菌株耐酒精能力较强。在实验结果的基础上,对呼吸缺陷型用于酒精发酵的优越性和可行性进行了讨论。     

Inverse Diauxy in the Yeast Hansenula anomala: Mutants Derepressed for Malic Acid Utilization in the Presence of Glucose

Utilization of l-malic acid by yeast strain Hansenula anomala IGC 4380 is subject to glucose repression. Derepressed mutants were obtained with UV light by use of the nonmetabolizable glucose analog 2-deoxyglucose as a selective agent. Three mutant strains degraded l-malic acid in the presence of up to 30% (wt/vol) glucose and are of potential interest for the biological deacidification of grape must. The mutant strains, as compared with the parent strain, displayed inverse diauxy in glucose-malate medium, glucose being metabolized only after malate consumption had been completed.     

Genetic improvement of Escherichia coli for ethanol production: chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II

Zymomonas mobilis genes for pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) were integrated into the Escherichia coli chromosome within or near the pyruvate formate-lyase gene (pfl). Integration improved the stability of the Z. mobilis genes in E. coli, but further selection was required to increase expression. Spontaneous mutants were selected for resistance to high level of chloramphenicol that also expressed high levels of the Z. mobilis genes. Analogous mutants were selected for increased expression of alcohol dehydrogenase on aldehyde indicator plates. These mutants were functionally equivalent to the previous plasmid-based strains for the fermentation of xylose and glucose to ethanol. Ethanol concentrations of 54.4 and 41.6 g/liter were obtained from 10% glucose and 8% xylose, respectively. The efficiency of conversion exceeded theoretical limits (0.51 g of ethanol/g of sugar) on the basis of added sugars because of the additional production of ethanol from the catabolism of complex nutrients. Further mutations were introduced to inactivate succinate production (frd) and to block homologous recombination (recA).