TCA cycle-independent acetate metabolism via the glyoxylate cycle in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the accepted theory is that due to TCA cycle dysfunction, the Δcit1 mutant lacking the mitochondrial enzyme citrate synthase (Cit1) cannot grow on acetate, regardless of the presence of the peroxisomal isoenzyme (Cit2). In this study, we re-evaluated the roles of Cit1 and Cit2 in acetate utilization and examined the pathway of acetate metabolism by analysing mutants defective in TCA or glyoxylate cycle enzymes. Although Δcit1 cells showed significantly reduced growth on rich acetate medium (YPA), they exhibited growth similar to Δcit2 and the wild-type cells on minimal acetate medium (YNBA). Impaired acetate utilization by Δcit1Δcit2 cells on YNBA was restored by ectopic expression of either Cit2 or its cytoplasmically localized variants. Deletion of any of the genes for the enzymes solely involved in the TCA cycle (IDH1, KGD1 and LSC1), except for SDH1, caused little defect in acetate utilization on YNBA but resulted in significant growth impairment on YPA. In contrast, cells lacking any of the genes involved in the glyoxylate cycle (ACO1, FUM1, MLS1, ICL1 and MDH2) did not grow on either YNBA or YPA. Deletion of SFC1 encoding the succinate-fumarate carrier also caused similar growth defects on YNBA. Our results suggest that in S. cerevisiae the glyoxylate cycle functions as a competent metabolic pathway for acetate utilization on YNBA, while both the TCA and glyoxylate cycles are essential for growth on YPA.     

丙酮酸羧化酶对酿酒酵母积累琥珀酸的作用探究

为探究丙酮酸羧化酶及微量元素Ca~(2+)和生物素对酿酒酵母积累琥珀酸的作用,敲除了琥珀酸脱氢酶编码基因(SDH2)并过量表达来自米根霉的丙酮酸羧化酶基因(Ro PYC)。琥珀酸产量由(0. 011±0. 002) g/L提高至(0. 841±0. 020) g/L,较表达前提高了75. 45%。随后,外源添加不同浓度Ca~(2+)和生物素考察其对琥珀酸发酵的影响,结果发现,Ca~(2+)的添加促进了菌体的生长但不利于琥珀酸的积累,而外源添加浓度为16、32、64、96μg/L生物素时,琥珀酸产量分别提高75. 04%、84. 26%、69. 28%、66. 79%。其中生物素质量浓度为32μg/L时,琥珀酸产量达到最大为(0. 964±0. 02) g/L,且丙酮酸羧化酶(pyruvate carboxylase,PC)酶活提高14. 42%,说明PC酶活的提高促进了酿酒酵母中琥珀酸的积累。该研究为解决酿酒酵母琥珀酸合成过程中前体碳代谢流不足问题提供了新的解决思路。     

LEU2基因敲除对工业啤酒酵母高级醇生成量的影响

通过敲除啤酒酵母中β-丙基苹果酸脱氢酶基因(LEU2),研究该基因对工业啤酒酵母高级醇特别是异戊醇生成量的影响.通过醋酸锂一步转化法,将一段两端具有LEU2摹因序列同源区,中间为遗传霉素(G418)抗性基因的DNA片段转入酵母细胞中,与LEU2基因的ORF(open reading flames)进行同源重组,利用遗传霉素抗性进行筛选.将突变株与出发菌株进行发酵实验,测定其发酵性能和高级醇的生成量.筛选获得了LEU2摹因突变的工业啤酒酵母.在支链氨基酸含量较低的培养基中进行发酵测定,突变株发酵液中高级醇含量比出发菌株降低了9.97%,其中异戊醇的含量降低了11.82%,而酒精度、发酵速度等发酵性能没有明显变化.LEU2基因敲除可降低工业啤酒酵母在支链氨基酸含量较低的培养基中高级醇特别是异戊醇的生成量.     

D-lactic acid production by metabolically engineered Saccharomyces cerevisiae

Poly D-lactic acid is an important polymer because it improves the thermostability of poly L-lactic acid by the stereo complex formation. We constructed a metabolically engineered Saccharomyces cerevisiae that produces D-lactic acid efficiently. In this recombinant, the coding region of pyruvate decarboxylase 1 (PDC1) was completely deleted, and two copies of the D-lactate dehydrogenase (D-LDH) gene from Leuconostoc mesenteroides subsp. mesenteroides strain NBRC3426 were introduced into the genome. The D-lactate production reached 61.5 g/l, the amount of glucose being transformed into D-lactic acid being 61.2% under neutralizing conditions. Additionally, the yield of free D-lactic acid was also shown to be 53.0% under non-neutralizing conditions. It was confirmed that D-lactic acid of extremely high optical purity of 99.9% or higher. Our finding obtained the possibility of a new approach for pure d-lactic acid production without a neutralizing process compared with other techniques involving lactic acid bacteria and transgenic Escherichia coli.     

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.     

酿酒酵母CHS3基因缺失对热耐受性的影响

研究真菌细胞壁几丁质合成酶CHS3基因在酵母热耐受性中的功能.采用长臂同源多聚酶链式反应(LFH-PCR)技术构建酵母CHS3基因缺失菌株:比较野生型与CHS3因缺失菌株的几丁质含量;梯度生长实验检测CHS3基因缺失菌株的热敏感性.结果显示,与野生型菌株相比,CHS3基因缺失菌株表现出热胁迫敏感性表型,此表型可被抗氧化剂N-乙酰半胱氨酸(NAC)弥补,表明该热敏感表型可能与热胁迫条件下活性氧积累引发的氧化损伤.     

酿酒酵母工程菌产青蒿酸的发酵动力学研究

以青蒿酸产量为考察指标,在50 L发酵罐中对酿酒酵母（Saccharomyces cerevisiae）工程菌1211发酵产青蒿酸的溶氧参数进行优化。在此基础上,根据Logistic方程及Luedeking-Piret方程构建酿酒酵母工程菌1211分批发酵产青蒿酸的动力学模型。结果表明,当溶氧参数为25%～30%时,青蒿酸产量最高,为（6 269.6±100.3）mg/L。青蒿酸合成与菌体生长呈现部分生长偶联型。通过Origin 9.0软件对动力学模型进行非线性拟合,发现S. cerevisiae工程菌1211的菌体繁殖生长、青蒿酸合成以及基质消耗动力学模型的拟合度R2分别达到了0.995 85、0.979 04和0.995 48,该动力学模型能够很好的描述S. cerevisiae工程菌1211分批发酵过程。该研究为青蒿酸的低成本发酵及工业化大规模发酵生产提供了理论基础。     

Effect of pre-fermentation on the production of cutinase by a recombinant Saccharomyces cerevisiae

The importance of controlling the expression of heterologous cutinase in a recombinant Saccharomyces cerevisiae SU50 strain was investigated. Maximum specific growth rate and the biomass yield increased 1.91 and 1.16 fold, respectively, when cutinase production was induced by galactose in a pre-fermentation step. However, only 19% of specific cell activity was obtained in comparison to other fermentations following a pre-fermentation step without induction of cutinase expression. Thus, the pre-fermentation step was performed using a selective medium not containing galactose, and the fermentation was performed with a cheaper and complex non-selective medium containing galactose. Under these conditions, and with the aim of maximising the specific cutinase activity, a pre-fermentation with low volume and high density of viable cells must be used. However, due to the low pre-fermentation volume, low yeast cell concentrations and low specific cell activities were obtained after 96 h of fermentation. Otherwise, when the aim was to maximise cutinase yield and productivity, a pre-fermentation volume of 10% (v/v) in relation to fermentation and in the exponential growth phase with a cell concentration between 1.1 and 1.8 g dcw/l should be used. A higher pre-fermentation volume, such as 20% (v/v), would still be economical in the case of a pre-fermentation with low cell density or low cell viability.     

Effect of the nitrogen source on the fatty acid composition of Saccharomyces cerevisiae

The source and content of nitrogen in the medium are very important in the development of alcoholic fermentations since they both affect the growth of Saccharomyces cerevisiae. Furthermore, the composition of the growth medium and the environmental conditions are known to affect the cell membrane fatty acid composition. The aim of this work was to study how the nitrogen source affects the membrane fatty acid composition. A mixture of amino acids and ammonia delayed the yeast growth when a high content of yeast assimilable nitrogen was present in the media. Cells grown in the mixed nitrogen source had a lower content of total fatty acids with a higher unsaturation degree than cells grown on sole ammonia.     

Characterization of the recombinant succinic semi‐aldehyde dehydrogenase from Saccharomyces cerevisiae

The yeast succinic semi‐aldehyde dehydrogenase gene (SSADH; EC 1.2.1.16) was cloned and overexpressed in Escherichia coli. Based on SDS–PAGE, the molecular mass of the subunit was around 54 kDa, and the purified recombinant enzyme had a tetrameric molecular mass of ca. 200 kDa. The specific activity of the recombinant enzyme was 1.90 µm NADH formed/min/mg, and showed maximal activity at pH 8.4. The recombinant protein was highly specific for succinate semi‐aldehyde (K_m = 15.48 ± 0.14 µm ) and could use both NAD⁺ and NADP⁺ as co‐factors, with K_m values of 579.06 ± 30.1 µm and 1.017 ± 0.46 mm, respectively. Initial velocity studies showed that NADH was a competitive inhibitor with respect to NAD⁺ (K_i = 129.5 µm ) but a non‐competitive inhibitor with respect to succinate semi‐aldehyde. Adenine nucleotides of AMP, ADP and ATP inhibited yeast SSADH activity with K_i = 1.13–10.2 mm, and showed competitive inhibition with respect to NAD⁺ and mixed‐competitive, non‐competitive and non‐competitive inhibition, respectively, with respect to succinate semi‐aldehyde. The kinetic data suggest a 'ping‐pong' mechanism. Copyright © 2014 John Wiley & Sons, Ltd.     

MSN4基因的过表达对酿酒酵母耐受性的影响研究

以实验室现有菌种AY12a为出发菌株,URA3基因作为筛选标记,利用胞内重组,在MSN4基因的N端加上强启动子PGK1p以实现基因的过表达,最终通过多聚酶链式反应（PCR）验证,成功构建突变株AY12a-msn4。结果表明,该突变株具有一定的耐高温性能,在55 ℃条件下热击后仍能正常生长。同时将突变株AY12a-msn4与出发菌株AY12a进行玉米高温浓醪发酵,并测定发酵完成后的酒精度、残糖、48 h细胞存活率、CO2失重及发酵时间。结果表明,突变株AY12a-msn4发酵液酒精度提高3.85%,48 h细胞存活率上升,残糖含量下降14.5%。     

Saccharomy cescerevisiae氨基转移酶基因ARO8克隆及对3甲硫基丙醇合成的影响

酿酒酵母可通过艾希利（Ehrlich）途径将L-蛋氨酸转化为3-甲硫基丙醇等食品风味成分,本文研究了氨基转移酶及其基因在Ehrlich途径及3-甲硫基丙醇合成代谢的调控作用。从Saccharomyces cerevisiae S288C克隆氨基转移酶ARO8基因,并基于酿酒酵母-大肠杆菌穿梭型表达载体pYES-pgk,构建重组质粒表达载体pYES-pgk-ARO8,PEG/LiAc转化法将其导入S.cerevisiae S288C。结果表明,克隆的ARO8基因全长1503bp,与NCBI GenBank中酿酒酵母芳香族氨基转移酶Ⅰ编码基因ARO8的序列相似度为100%;构建的ARO8基因过表达工程菌S.cerevisiae AR8,其氨基转移酶活力为187U/mL,较野生型S288C菌株的酶活性提高1.63倍;工程菌AR8的摇瓶发酵3-甲硫基丙醇产量为0.76g/L,较野生型S288C提高28.8%。表明氨基转移酶Aro8p及其ARO8基因在S.cerevisiae 3-甲硫基丙醇合成代谢中发挥重要作用,增强其ARO8基因表达,有助于提高3-甲硫基丙醇的产量。      

Construction of phosphatidylethanolamine-less strain of Saccharomyces cerevisiae. Effect on amino acid transport

A triple yeast mutant was constructed which lacks BST1, the gene for sphingosine lyase, besides the phosphatidylserine decarboxylases PSD1 and PSD2. In this yeast mutant, which can only be grown in the presence of exogenous ethanolamine, phosphatidylethanolamine can be depleted to very low levels. Under those conditions, respiration as well as glucose and 3-O-methylglucose uptake proceed unaffected. Plasma membrane ATPase is as active in these cells as that of control cells grown in the presence of ethanolamine. Drastically decreased, however, are H+/amino acid symporters. The activities of arginine (Can1p), proline (Put4p) and general amino acid permease (Gap1p) are decreased more than 20-fold. Amino acid transport in yeast is dependent on coupling to the proton motive force. It can be envisaged that phosphatidylethanolamine might play a role in this process or in the early steps of the secretion pathway common for all amino acid permeases or, eventually, it could affect the transport proteins directly at the plasma membrane Transformation of the triple mutant with a CEN plasmid harbouring BST1 wild-type gene totally reversed its phenotype to that observed in the double mutant.     

Mapping of the trifunctional fatty acid synthetase gene FAS2 on chromosome XVI of Saccharomyces cerevisiae

The trifunctional FAS2 gene encoding subunit alpha of the Saccharomyces cerevisiae fatty acid synthetase complex was mapped on the left arm of chromosome XVI 24 centimorgans proximal to GAL4 and 39 centimorgans distal to PEP4 relative to the centromere. Mapping was achieved by three independent methods: meiotic co-segregation of FAS2 and ARO7 in recombination-deficient spo11-mutants: tetrad analysis of crosses between FAS2, GAL4 and PEP4; and Southern hybridization of purified FAS2 DNA with individual yeast chromosomes separated by pulsed-field gel electrophoresis.     

柠檬酸钠提高酿酒酵母谷氨酸合成效率

为增加酵母抽提物鲜度以更好地满足其在食品领域的需求,在10 L发酵罐水平研究了柠檬酸钠-糖蜜补料策略对酿酒酵母J-5生长及胞内谷氨酸合成的影响。糖蜜流加补料发酵中存在显著的溢流代谢,乙醇浓度(10 h)高达22 g/L,干重和胞内谷氨酸分别达到36.36 g/L(21 h)和2.28%(质量分数)(18 h)。而3～14 h采用50 mL/h的初始速度流加糖蜜(体积1.8 L,含72 g柠檬酸钠)和每2 h流速增加50 mL的策略,能有效地减少碳溢流代谢,乙醇质量浓度(10 h)仅为对照的54.5%。同时,显著提高了胞内谷氨酸合成效率,其峰值含量达到了3.7%(质量分数)(11 h),比优化前提高了62.3%。该文确定了柠檬酸钠浓度/峰值菌体干重为0.238±0.007的优化参数,为工业化放大实验提供了参数依据。     

Recovery of gene function by gene duplication in Saccharomyces cerevisiae

A prototroph revertant (Rev9) selected from an ATCase^? mutant of the URA2 gene containing three nonsense mutations was shown to contain two ATCase coding sequences. We cloned both ATCase coding areas to show that the duplicated locus (dl9) was the only functional one. Its size corresponded roughly to the second half of the URA2 wild-type gene. Sequence analysis of the 5′ end of dl9 indicated that this duplicated sequence was inserted within the intergenic region close to the MRS3 gene and was transcribed from an unknown promoter divergently from the MRS3 gene. The event leading to the revertant strain Rev9 included a rearrangement that increased the size of chromosome X by about 60 kb. In agreement with such a rearrangement, recombination was undetectable in the vicinity of the locus dl9. Genetic mapping confirms that the MRS3 gene is 2 cM distal to the URA2 gene on the right arm of chromosome X.