生物合成己二酸的代谢工程研究进展

己二酸是一种重要的大宗化学品,主要用于合成尼龙和聚氨酯泡沫塑料,市场需求巨大,其高效生物合成至今还未实现。本文概括了酿酒酵母中构建和优化新的己二酸合成途径研究进展。首先,通过体内及体外活性测试,对催化每一步反应的酶进行筛选,构建初步的代谢途径。利用组学分析诊断和定位生物合成途径的瓶颈。对于途径中的限速酶采用蛋白质工程手段进行改造。其次,利用合成生物学和代谢工程手段优化代谢途径。优化手段具体包括:通过模块化优化,平衡各个基因之间的表达;利用蛋白支架,构建酶反应的流水线,减少中间产物的扩散,提高反应效率;通过RNA干扰技术抑制竞争代谢途径的流量,提高目标代谢途径的通量。最后,利用CRISPR/Cas9及全局转录机器工程(gTME)等最新技术进行基因组编辑、重排转录网络,最终获得己二酸的高产菌株及适用于高效生产其他芳香族化合物的底盘酵母菌株。     

酿酒酵母表达侧耳源单基因生物合成麦角硫因

运用生物信息学方法对侧耳属蘑菇（平菇、杏鲍菇和白灵菇）的麦角硫因合成酶基因Egt 1进行挖掘,对其蛋白结构和功能进行预测,并构建酿酒酵母表达载体,在酿酒酵母EC 1118中进行表达研究。结果表明：克隆得到侧耳属平菇、杏鲍菇和白灵菇麦角硫因合成基因PoEgt 1、PeEgt 1和PtEgt 1,三者核苷酸序列同源性为97.03%,氨基酸序列同源性为97.93%,联合菌体破碎法并通过体外酶促反应后检测到麦角硫因,产量为（2.5±0.08）mg/L,证明了该基因具有单基因合成麦角硫因的活性。     

酿酒酵母中β-葡聚糖生物合成的研究进展

本文详细地介绍了酵母β-葡聚糖（SCG）的生物合成的有关内容。酵母 β-葡聚糖是以尿嘧啶二磷酸葡萄糖（UDP-Glc）为前体物质经葡聚糖合成酶（GS）将葡萄糖转移到葡聚糖主链的非还原端而生成可溶性的SCG，在壳聚糖合成酶III（CSIII）作用下几丁质还原端以 β-1,4键/ β-1,2键的形式与 β-1，3葡聚糖链支链的非还原端共价连接，形成了不可溶的SCG。此外，还对葡聚糖合成酶（Gs）主要基因学的最新进展进行了阐述，其中FKSl和FKS2编码着β-1，6葡聚糖酶的基本组分蛋白，RH01基因则对β-1，3葡聚糖的合成进行调控；对β-1，6葡聚糖合成的基因尚不是很明确，目前已确定：KRE5、KRE6、CWH4p、ROT2和SKNl等基因对β-1，6葡聚糖的含量有着明显的影响。在此基础上，对有关酵母β-1，6葡聚糖的将来研究方向提出了自己的见解。     

番茄红素及其生物合成途径的研究

介绍番茄红素具有的理化特性和生理功能,植物中番茄红素的生物合成途径及其调控。由于番茄红素的特定分子结构,使其具有抗癌、保护皮肤、延缓衰老等功能特性,是对人类健康有益的食物成分。而随着番茄红素合成途径的阐明及对其合成调控的研究,运用基因工程手段调控番茄红素的生物合成已成为现实。     

过表达相关蛋白对酿酒酵母利用半乳糖的影响

以PGK1为启动子,以YEp352为载体,构建了SEC53和PGM2基因表达载体,获得酵母转化子AYSEC和AYPGM。通过对比重组菌株与亲本的发酵性能,研究了过表达SEC53、PGM2基因对酿酒酵母半乳糖代谢的影响。结果表明,过表达SEC53、PGM2基因可显著提高酿酒酵母利用半乳糖的速度;在半乳糖培养基中,重组菌株AYSEC和AYPGM的总发酵时间分别缩短了37.5%和25%;在混合糖培养基中,重组菌株在一定程度上降低了葡萄糖的阻遏效应,AYSEC和AYPGM总发酵时间分别缩短了28.6%和14.3%。     

食品中主要真菌毒素生物合成途径研究进展

真菌毒素是真菌重要的次生代谢产物。食品和饲料等农产品在收获、储藏、加工过程中广泛受其污染,造成品质下降、经济损失巨大,并严重威胁人身健康。本文对食品中5种常见真菌毒素(黄曲霉毒素、赭曲霉毒素、伏马菌素、玉米赤霉烯酮和脱氧雪腐镰刀菌烯醇)的生物合成途径分别进行阐释。采用克隆基因的传统分子生物学方法和基因组测序的现代测序技术研究发现,真菌毒素的生物合成基因大多成簇存在,该簇中包含控制合成基因表达的调控基因,并受多种应对外部环境的调控基因所控制。因此本文还结合5种真菌毒素的生物合成基因簇及其基因功能进行综合解析。真菌毒素生物合成的研究将为食品和饲料等农产品的防控、预警以及脱毒提供理论基础和指导方向。     

谷氨酸发酵中由葡萄糖生物合成谷氨酸的代谢途径是什么？

以糖质原料发酵谷氨酸时,糖酵解经进EMP及HMP两条途径进行,生物素充足菌HMP所占的比例是38％,控制生物素亚适量的结果,发酵产酸期,EMP所占比例更大,HMP所占比例约为26％。生成丙酮酸后,一部分氧化脱羧生成乙酰CoA,一部分同定CO2生成草酰乙酸或苹果酸,草酰乙酸与乙酰CoA在柠檬酸合成酶的催化作用下,缩合成柠檬酸,继而生成异柠檬酸,再经氧化还原共轭的氨基化反应生成终产物谷氨酸。     

半乳糖发酵优良酿酒酵母单倍体的筛选

筛选半乳糖发酵优良酿酒酵母单倍体,对酿酒酵母AY5进行了单倍体的分离、鉴定,并以酿酒酵母AY5和克鲁维酵母匈3为对照,通过产气性能、耐酒精性能以及半乳糖发酵性能的分析,筛选出性能优良的单倍体菌株10株,其中4株a型、6株α型.筛选的优良单倍体菌株发酵半乳糖速度快,发酵液酒度高,还原糖含量低.     

微生物合成蒎烯的研究进展

蒎烯是自然界中广泛存在的单萜类化合物,在自然界中主要以α-蒎烯和β-蒎烯两种形式存在,被广泛应用于食品、能源、化工、军事、医药和材料等领域,具有重要的应用价值和发展前景。目前蒎烯的获得途径主要是通过从植物松节油中进行高效精馏分离提取,但是这种方式具有成本高、效率低、操作工艺复杂、污染环境等弊端。所以,随着微生物学代谢工程和发酵工程的发展,利用底盘生物构建蒎烯代谢通路来生产蒎烯已经成为一种重要趋势。该文主要总结了目前蒎烯在大肠杆菌和酿酒酵母中的合成途径以及提高蒎烯产量的策略,并且说明了提高宿主菌株对蒎烯的耐受性对蒎烯合成的影响,提出了蒎烯生物合成存在的问题以及解决思路,对未来蒎烯在生物中合成提供了参考和启示。     

Dfg5在酿酒酵母细胞壁合成中的功能分析

酿酒酵母中合成的糖基磷脂酰肌醇锚定蛋白(GPI-APs)一半停留在细胞膜上,另一半被转运至细胞壁的β-1,6-葡聚糖上形成GPI锚定细胞壁蛋白(CWPs)。有分析指出,Dfg5和Dcw1是定位于细胞膜的GPI锚定蛋白,可能以同工酶的形式参与GPI-APs转运。但这两个同源蛋白的分子作用机制尚不清楚。为探究Dfg5的作用机制,本研究构建了dfg5△PMET3DCW1条件突变菌株。研究发现在DCW1表达抑制型突变株中Cwp1在细胞膜上大量积累。对Dfg5中与甘露糖苷酶催化活性相关保守氨基酸位点分别定点突变,产生的突变蛋白Dfg5~(W71A)、Dfg5~(D122A)、Dfg5~(D123A)无法回补条件突变株的生长缺陷。然而截断Dfg5前导肽的分泌型Dfg5则可回补条件突变株的生长缺陷。上述结果可推测在GPI-APs锚定至细胞壁中,Dfg5至少具有糖苷酶,可将Cwps从细胞膜上的GPI锚中释放出来。     

Molecular cloning of the gamma-glutamylcysteine synthetase gene of Saccharomyces cerevisiae.

The 4.4 kb SphI DNA fragment (GSH1) that complements the gamma-glutamylcysteine synthetase-deficient mutation (gsh1) of Saccharomyces cerevisiae YH1 was cloned into vector plasmid YEp24. Gene disruption of the cloned fragment confirmed that this segment was the same gene as gsh1. Mutant strain YH1 with this plasmid not only restored gamma-glutamylcysteine synthetase (GSH-I) activity but the glutathione content and the growth rate. DNA sequence analysis of the SphI fragment showed that the GSH1 structural gene contained 2034 bp and predicted a polypeptide of 678 amino acids. The deduced amino acid sequence had about a 45% homology to that of rat kidney GSH-I, but a very low homology (about 26%) to that of Escherichia coli GSH-I. Northern analysis showed that GSH1 had been transcribed into an approximately 2.7 kb mRNA fragment. Southern analysis showed that GSH1 mapped at chromosome X.     

Identification of a gene encoding a homocitrate synthase isoenzyme of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, most of the LYS structural genes have been identified except the genes encoding homocitrate synthase and α-aminoadipate aminotransferase. Expression of several LYS genes responds to an induction mechanism mediated by the product of LYS14 and an intermediate of the pathway, α-aminoadipate semialdehyde (αAASA) as an inducer. This activation is modulated by the presence of lysine in the growth medium leading to an apparent repression. Since the first enzyme of the pathway, homocitrate synthase, is feedback inhibited by lysine, it could be a major element in the control of αAASA supply. During the sequencing of chromosome IV of S. cerevisiae, the sequence of ORF D1298 showing a significant similarity with the nifV gene of Azotobacter vinelandii was reported. Disruption and overexpression of ORF D1298 demonstrate that this gene, named LYS20, encodes a homocitrate synthase. The disrupted segregants are able to grow on minimal medium and exhibit reduced but significant homocitrate synthase indicating that this activity is catalysed by at least two isoenzymes. We have also shown that the product of LYS20 is responsible for the greater part of the lysine production. The different isoforms are sensitive to inhibition by lysine but only the expression of LYS20 is strongly repressed by lysine. The N-terminal end of homocitrate synthase isoform coded by LYS20 contains no typical mitochondrial targeting sequence, suggesting that this enzyme is not located in the mitochondria.     

Para-aminobenzoate synthase gene of Saccharomyces cerevisiae encodes a bifunctional enzyme

The Saccharomyces cerevisiae gene for para-aminobenzoate (PABA) synthase has been identified based upon its ability to confer sulfonamide resistance when present on a multicopy episomal vector. The 3840 bp DNA sequence fragment reported here contains a 2199 bp open reading frame encoding a 733 amino acid protein with similarity to the two components of PABA synthase described for prokaryotes (Escherichia coli PabA and PabB), suggesting that PABA synthase is bifunctional in yeast. The cloned sequence was confirmed to be PABA synthase by gene disruption. Chromosome gel analysis places the gene for PABA synthase on chromosome XIV.     

Vitamin requirements and biosynthesis in Saccharomyces cerevisiae

Chemically defined media for yeast cultivation (CDMY) were developed to support fast growth, experimental reproducibility, and quantitative analysis of growth rates and biomass yields. In addition to mineral salts and a carbon substrate, popular CDMYs contain seven to nine B-group vitamins, which are either enzyme cofactors or precursors for their synthesis. Despite the widespread use of CDMY in fundamental and applied yeast research, the relation of their design and composition to the actual vitamin requirements of yeasts has not been subjected to critical review since their first development in the 1940s. Vitamins are formally defined as essential organic molecules that cannot be synthesized by an organism. In yeast physiology, use of the term “vitamin” is primarily based on essentiality for humans, but the genome of the Saccharomyces cerevisiae reference strain S288C harbours most of the structural genes required for synthesis of the vitamins included in popular CDMY. Here, we review the biochemistry and genetics of the biosynthesis of these compounds by S. cerevisiae and, based on a comparative genomics analysis, assess the diversity within the Saccharomyces genus with respect to vitamin prototrophy.     

低双乙酰酿酒酵母的DNA标记研究

对15株酿酒酵母菌株的外观发酵度、双乙酰生成和还原能力进行了比较,通过随机扩增多态性DNA(RAPD)和HSP150编码基因的长度多态性对不同酿酒酵母菌株的基因组DNA分子差异进行了分析。结果显示,菌株YZU-APK和SS-05的外观发酵度分别为77.2%和76.8%,双乙酰峰值分别为0.26mg/L和0.25mg/L,并在发酵第8d均降至0.09 mg/L。在46条随机引物中筛选出的2条引物P07和P42能够扩增出具有较好多态性的RAPD指纹图谱,可以将15个酵母菌株分成9个遗传型;对细胞壁热休克蛋白HSP150编码基因的PCR扩增,并根据扩增条带长度的不同可以将15株酵母分成6个遗传型。综合2种分析结果,并对低双乙酰酿酒酵母菌株YZU-APK和SS-05进行DNA分子标记,确定了其基因型分别是(P07:1,P42:1,hsp150:1)和(P07:1,P42:5,hsp150:2)。     

A Review of Phenotypes in Saccharomyces cerevisiae

A summary of previously defined phenotypes in the yeast Saccharomyces cerevisae is presented. The purpose of this review is to provide a compendium of phenotypes that can be readily screened to identify pleiotropic phenotypes associated with primary or suppressor mutations. Many of these phenotypes provide a convenient alternative to the primary phenotype for following a gene, or as a marker for cloning a gene by genetic complementation. In many cases a particular phenotype or set of phenotypes can suggest a function for the product of the mutated gene. © 1997 John Wiley & Sons, Ltd.     

酿酒酵母产乙醇脱氢酶发酵条件的研究

选择了一株酿酒酵母菌(Saccharomyces cerevisiae)As2.399,通过单因素和正交实验得到酿酒酵母产乙醇脱氢酶(Alcohol Dehydrogenase,ADH)的最优发酵条件。结果表明:产ADH的最优培养条件是温度为25℃、pH为5、通气量为250mL、接种量为7%,液体发酵培养中pH对ADH活性的影响最大。      

Cloning and disruption of a gene required for growth on acetate but not on ethanol: the acetyl-coenzyme A synthetase gene of Saccharomyces cerevisiae.

A DNA fragment of Saccharomyces cerevisiae with high homology to the acetyl-coenzyme A (acetyl-CoA) synthetase genes of Aspergillus nidulans and Neurospora crassa has been cloned, sequenced and mapped to chromosome I. It contains an open reading frame of 2139 nucleotides, encoding a predicted gene product of 79.2 kDa. In contrast to its ascomycete homologs, there are no introns in the coding sequence. The first ATG codon of the open reading frame is in an unusual context for a translational start site, while the next ATG, 24 codons downstream, is in a more conventional context. Possible implications of two alternative translational start sites for the cellular localization of the enzyme are discussed. A stable mutant of this gene, obtained by the gene disruption technique, had the same low basal activity of acetyl-CoA synthetase as wild-type cells when grown on glucose but completely lacked the strong increase in activity upon entering the stationary phase, providing direct proof that the gene encodes an inducible acetyl-CoA synthetase (ACS1) of yeast. As expected, the mutant was unable to grow on acetate as sole carbon source. Nevertheless, it showed normal induction of isocitrate lyase on acetate media, indicating that activity of acetyl-CoA synthetase is dispensable for induction of the glyoxylate cycle in S. cerevisiae. Surprisingly, disruption of the ACS1 gene did not affect growth on media containing ethanol as the sole carbon source, demonstrating that there are alternative pathways leading to acetyl-CoA under these conditions.