“组学”技术在燃料乙醇生产用酿酒酵母菌株构建中的应用

酿酒酵母(Saccharomyces cerevisiae)是工业乙醇生产广泛使用的微生物,构建能同时发酵戊糖以及具有优良抑制物耐受能力的高产菌株,在燃料乙醇生产技术开发中具有十分重要的地位。基因组学、蛋白组学、转录组学、代谢组学和流量组学等"组学"技术可从基因型到表现型不同层次揭示生命活动规律,在燃料乙醇生产用酵母菌株的构建中具有广阔的应用前景,特别是目标基因的识别、代谢途径的优化、代谢机理的揭示等。运用"组学"技术可使菌株构建工作更具靶向性和明确性,更加易于获得目标性状,并大大缩短育种周期。综述了"组学"技术在燃料乙醇生产用酿酒酵母菌株构建中的应用及相关研究进展。     

酵母生物多样性开发及工业应用

酵母是一类包括酿酒酵母和非常规酵母在内的多种单细胞真菌的总称,其中酿酒酵母是应用较多的重要工业微生物,广泛应用于生物医药、食品、轻工和生物燃料生产等不同生物制造领域。近年来,研究者从不同生态环境中分离了大量的酵母菌株,鉴定了多个新种,也发现了抗逆性不同以及具有多种活性产物合成能力的菌株,证明天然酵母资源具有丰富的生物多样性和功能多样性。利用基因组挖掘以及转录组、蛋白组等多组学分析研究,可进一步开发利用酵母遗传多样性,获得酶和调节蛋白的基因以及启动子等遗传元件改造酵母菌株。除了利用酵母的天然遗传多样性,还可通过诱变、驯化、代谢工程改造及合成生物学等技术产生具有多种非天然多样性的菌株。此外,对天然遗传元件也可以进行突变和定向进化,所产生的新遗传元件可用于有效提升菌株的性能。开发利用酵母的生物多样性,对构建高效酵母细胞工厂,生产生物酶、疫苗以及多种活性天然产物等产品具有重要意义。文中对酵母生物多样性的研究现状进行综述,并对未来高效开发利用酵母菌株资源和遗传资源的研究进行了展望。文中所总结的研究方法和思路也可为研究其他工业微生物的多样性及进行高效菌株的选育提供参考。     

黑曲霉组学研究进展

黑曲霉作为重要的工业发酵菌株,被广泛用于多种有机酸和工业用酶的生产。随着组学技术的日益发展和成熟,黑曲霉的基因组、转录组、蛋白质组、代谢组等组学数据不断增长,宣告着黑曲霉生物过程研究大数据时代的到来。从单一组学的数据分析、多组学的比较到以基因组代谢网络模型为中心的多组学整合研究,人们对黑曲霉高效生产机制的理解不断深入和系统,这为通过遗传改造和过程调控对菌株的生产性能进行理性的全局优化提供了可能。本文回顾和总结了近年来黑曲霉的组学研究进展,并提出黑曲霉组学研究未来的发展方向。     

面向工业生物技术的系统生物学

工业生物技术是以微生物细胞工厂利用可再生的生物原料来生产能源、材料与化学品等的生物技术,在解决资源、能源与环境等问题方面起着越来越重要的作用。系统生物学是全面解析微生物细胞工厂及其发酵过程从"黑箱"到"白箱"的重要研究方法。系统生物学借助基因组、转录组、蛋白质组、代谢组以及代谢流组等多组学数据,可解析微生物细胞工厂在RNA、蛋白与代谢物等不同水平上的变化规律与调控机制。目前,系统生物学在微生物细胞工厂的设计创建与发酵工艺优化中起着越来越重要的指导作用,许多成功应用实例不断涌现,推动着工业生物技术的快速发展。文中重点综述基因组、转录组、蛋白质组、代谢组与代谢流组以及基因组规模的网络模型等各组学技术的最新发展及其在工业生物技术尤其是菌株改造与发酵优化中的应用,并就工业生物技术中系统生物学的未来发展方向进行展望。     

系统生物学和合成生物学研究在生物燃料生产菌株改造中的应用

基于生物质资源生产环境友好的生物燃料,对经济和社会的可持续发展具有重要意义,但其生产成本高的问题十分突出,而高效生产菌株的获得是解决这一问题的根本出路。以下综述了利用系统生物学研究所获得的信息进行菌种改造的过程,重点论述了生产菌株胁迫耐受性方面的研究进展,并讨论了系统生物学、合成生物学和代谢工程技术在改造生物燃料生产菌株中的应用,展望了合成生物学在构建高效生物能源生产菌株方面应用的前景。     

酒精发酵过程中酿酒酵母耐受环境胁迫的研究进展

提高酿酒酵母细胞耐受环境胁迫（高渗透压、高浓度酒精和高温）的能力对酒精工业生产具有重要的意义。对提高酿酒酵母耐受性的研究方法和策略的发展历程进行了综述。基因组学、转录组学和蛋白质组学等现代组学技术在这一领域的研究获得了广泛的应用。这些技术将提供期待的信息,去理性改造并获得更加耐受胁迫的工业酵母菌株。     

典型产纤维小体梭菌的遗传改造及其在纤维素乙醇中的应用研究进展简

以解纤维梭菌（ Clostridium cellulolyticum）和热纤梭菌（ Clostridium thermocellum）为代表的产纤维小体梭菌可以直接完成从木质纤维素原料到乙醇的生物转化,是用于通过整合生物加工技术生产纤维素乙醇的优良候选菌株。然而,这些产纤维小体梭菌的纤维素降解效率及乙醇产量尚不能满足工业化生产的要求,其遗传改造技术的不成熟严重制约了通过定向代谢工程改造提高生产性能的进程。针对这些典型的产纤维小体菌株,各国科学家近年来在基于二类内含子的嗜中温及嗜高温遗传改造平台建立方面取得了较大突破,并通过靶向代谢工程改造,显著提高纤维素乙醇的产量。笔者对这些前期研究工作以及国内外相关研究成果进行系统的总结,并对构建的遗传改造工具的应用前景进行展望。     

基于基因组DNA诱变的遗传重组改造乙醇工业酵母的耐热性及发酵性能

酵母菌是乙醇发酵工业中非常重要的微生物细胞工厂,发酵过程中温度变化胁迫一直是影响生产效率的重要瓶颈之一,选育具有广泛温度适应性的酵母菌株对提高发酵性能和降低生产成本具有重要意义。通过化学诱变和基于基因组DNA诱变的遗传重组技术对乙醇工业酵母菌的温度适应性进行改造,获得耐热性能和发酵性能得到提高的重组酿酒酵母Saccharomyces cerevisiae T44-2。重组菌株T44-2的最高生长温度比原始菌株CE6提高了3 ℃,48 ℃和52 ℃热激处理1 h,重组菌株的细胞存活率分别是原始菌株的1.84     

海南热带雨林腐木上酵母菌物种多样性研究

用含木糖为唯一碳源(培养基X)和含葡萄糖及7.6%乙醇(培养基E)的两种富集培养基分别从采自海南热带雨林的56和57份腐木样品中分离到酵母菌67和75株.依据26S rDNA D1/D2区域序列分析并结合形态学特征对这些菌株进行了分类学研究,探讨了该地区腐木上的酵母菌物种多样性及其分布.从分离的142株酵母菌中鉴定出14个属63个种,其中疑似新种25个,占总种数的近40%,说明在热带雨林腐木中尚存在大量酵母菌新分类群有待被发现.从用培养基X和E分离的酵母菌中分别鉴定出7属37种和11属33种,优势属均为假丝酵母属Candida Berkhout和毕赤酵母属Pichia Hansen,但种类组成基本不同.用培养基x富集分离的菌株以Candida quercitrusa S.A.Meyer＆Phaff的地理分布最广,用培养基E富集分离的菌株以异常毕赤酵母Pichia anomala(Hansen)Kurtzman、酿酒酵母Saccharomyces cerevisiae Meyen ex Hansen和亚膜毕赤酵母Pichiasubpelliculosa Kurtzman分布最广泛.同一样品用两种不同富集培养基分离的菌株大多数属于不同的种,在对比的23份样品中,只从2份样品中分离到了同一个种的菌株.用培养基X和E分离的菌株分别属于可利用木糖和可耐受乙醇的酵母菌,用两种培养基同时分离到的菌株属于具备利用木糖和耐受高浓度乙醇两种能力的菌株.这些酵母菌在木质纤维素物质的生物乙醇转化技术中的应用价值值得进一步研究.     

工业酵母菌株转化体系的建立

本文通过敏感度实验测定了工业酵母菌株SK—1、SPSC—1、NAN—27在不同的培养基上对G418及Cu^2 的敏感浓度,确定了3株工业菌株转化子的筛选方法。针对它们各自的生理生化、遗传特性,用改进的醋酸锂完整细胞转化和原生质体转化两种方法,分别建立了3株菌的转化系统,初步解决了工业酵母菌株转化的问题,并为下一步工业酵母菌株的代谢工程改造奠定了基础。     

<Emphasis Type="Italic">Phaffia rhodozyma</Emphasis>: colorful odyssey

Phaffia rhodozyma was isolated by Herman Phaff in the 1960s, during his pioneering studies of yeast ecology. Initially, the yeast was isolated from limited geographical regions, but isolates were subsequently obtained from Russia, Chile, Finland, and the United States. The biological diversity of the yeast is more extensive than originally envisioned by Phaff and his collaborators, and at least two species appear to exist, including the anamorph Phaffia rhodozyma and the teleomorph Xanthophyllomyces dendrorhous. The yeast has attracted considerable biotechnological interest because of its ability to synthesize the economically important carotenoid astaxanthin (3,3-dihydroxy-, -carotene-4,4-dione) as its major pigment. This property has stimulated research on the biology of the yeast as well as development of the yeast as an industrial microorganism for astaxanthin production by fermentation. Our laboratory has isolated several mutants of the yeast affected in carotenogenesis, giving colonies a vivid array of pigmentation. We have found that nutritional and environmental conditions regulate astaxanthin biosynthesis in the yeast, and have demonstrated that astaxanthin protects P. rhodozyma from damage by reactive oxygen species. We proposed in the 1970s that P. rhodozyma could serve as an economically important pigment source in animal diets including salmonids, lobsters, and the egg yolks of chickens and quail, in order to impart characteristic and desirable colors. Although P. rhodozyma/Xanthomyces dendrorhous has been studied by various researchers for nearly 30 years, it still attracts interest from yeast biologists and biotechnologists. There is a bright and colorful outlook for P. rhodozyma/X. dendrorhous from fundamental and applied research perspectives.     

Properties of mitochondria from cells of the "fermentative" variant of Endomyces magnusii]

The properties of mitochondria from the cells of the "fermentative" variant of End. magnusii were studied. The induced fermentative transformation was brought about by a non-balanced vitamin cultivation. It was shown that the "fermentative" variant of End. magnusii represents an interesting model, in which the energy required for the cell functioning is provided for by a high fermentative activity and a normally functioning respiratory chain. The "fermentative" variant mitochondria were tightly coupled and possessed theoretical efficiency during oxidation of NAD-dependent substrates, which suggested the existence of all the three sites of energy coupling and phosphorylation at the substrate level. A specificity of energy regulation of the End. magnusii "fermentative" variant mitochondria, e. g. tight coupling during oxidation of succinate and lack of tight coupling during oxidation of exogenous NADH, is discussed. The tight coupling during succinate oxidation is confirmed by the observation of reverse electron transfer. Thus, the energy-dependent reduction of NAD during succinate oxidation has been firstly demonstrated for the mitochondria of yeast grown on a fermentable substrate.     

Production of ethanol by alginate-entrapped Saccharomyces cerevisiae strain "14-12"

Cells of S. cerevisiae strain "14-12" of different ages were immobilized in sodium alginate and used for conversion of glucose to ethanol. Immobilized cells of 48 hr old were the most potential. Employment of high counts of alginate-entrapped cells shortened the period required for production of the maximal alcohol yield. However, the percentage surviving cells decreased with increasing initial cell counts. Maximal accumulation of ethanol (4.18 g/100 ml) was obtained after 4 days of static fermentation with 1.8 X 10(8) immobilized yeast cells. The residual viable cell count was found to represent 3-fold the surviving percentage in a control experiment using an inoculum of the free yeast cells. Immobilized yeast cells could convert about 85% of the available sugars to ethanol over 28 days of the repeated-batch fermentation. The immobilized cells retained 50% of their viability for 16 days. After 48 days of repeated fermentation only 6% of the yeast cells were viable, and on the 52nd day no viable cells could be detected.     

Adaptation of the yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> to heat shock

The adaptive response of the yeast Yarrowia lipolytica to heat shock has been studied. Experiments showed that, after 10 min of incubation at 45°C, the survival rate of Yarrowia lipolytica cells was less than 0.1%. Stationary-phase yeast cells were found to be more thermotolerant than exponential-phase cells. A 60-min preincubation of cells at 37°C or pretreatment with low concentrations of H₂O₂ (0.5 mM) or menadione (0.05 mM) made them more tolerant to heat and to oxidative stress (120 mM hydrogen peroxide). The pH dependence of yeast thermotolerance has also been studied. The adaptation of yeast cells to heat shock and oxidative stress was found to be associated with a decrease in the intracellular level of cAMP and an increase in the activity of antioxidant enzymes (catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase).     

Two-dimensional protein map of an "ale"-brewing yeast strain: proteome dynamics during fermentation

The first protein map of an ale-fermenting yeast is presented in this paper: 205 spots corresponding to 133 different proteins were identified. Comparison of the proteome of this ale strain with a lager brewing yeast and the Saccharomyces cerevisiae strain S288c confirmed that this ale strain is much closer to S288c than the lager strain at the proteome level. The dynamics of the ale-brewing yeast proteome during production-scale fermentation was analysed at the beginning and end of the first and the third usage of the yeast (called generation in the brewing industry). During the first generation, most changes were related to the switch from aerobic propagation to anaerobic fermentation. Fewer changes were observed during the third generation but certain stress-response proteins such as Hsp26p, Ssa4p and Pnc1p exhibited constitutive expression in subsequent generations. The ale brewing yeast strain appears to be quite well adapted to fermentation conditions and stresses.     

Yeast "N"-hybrid systems for protein-protein and drug-protein interaction discovery

The majority of small molecule drugs act on protein targets to exert a therapeutic function. It has become apparent in recent years that many small molecule drugs act on more than one particular target and consequently, approaches which profile drugs to uncover their target binding spectrum have become increasingly important. Classical yeast two-hybrid systems have mainly been used to discover and characterize protein-protein interactions, but recent modifications and improvements have opened up new routes towards screening for small molecule-protein interactions. Such yeast "n"-hybrid systems hold great promise for the development of drugs which interfere with protein-protein interactions and for the discovery of drug-target interactions. In this review, we discuss several yeast two-hybrid based approaches with applications in drug discovery and describe a protocol for yeast three-hybrid screening of small molecules to identify their direct targets.     

A widely distributed "CAT" family of repetitive DNA sequences

The yeast genome contains a family of repetitive sequences consisting primarily of a tandemly arranged trinucleotide, CAT, or a closely related CGT sequence. To characterize similar sequences in divergent organisms, a previously isolated "CAT" sequence was used to isolate homologous genomic clones from a human cell line, an insect and a higher plant. Sequence analyses show that comparable repetitive sequences are widely distributed and may be present in all eukaryotic genomes. In situ hybridization analyses indicate that in yeast, the CAT elements are dispersed among all the chromosomes, and a more detailed analysis in Drosophila indicates that at least one of these sequences maps on the X chromosome between known genetic loci which are actively expressed. Repeated searches of yeast cDNA libraries indicate that these CAT clusters are not expressed but substantial effects on the expression of a cloned gene strongly suggest that they play an important role in gene regulation.     

Fed-batch fermentation of indole-3-acetic acid production in stirred tank fermenter by red yeast <Emphasis Type="Italic">Rhodosporidium paludigenum</Emphasis>

Indole-3-acetic acid (IAA) is a significant secondary metabolite that is the most important auxin of plant hormones. Production of IAA is considered to be a key trait to support plant growth. The improvement of IAA production by a basidiomycetous red yeast Rhodosporidium paludigenum DMKU-RP301 was investigated. Batch and fed-batch fermentation of R. paludigenum DMKU-RP301 were conducted in a 2 L stirred tank fermenter. Using batch fermentation, it was found that when cultivated at an agitation speed of 200 rpm and a 3 L/min aeration rate, this yeast produced IAA at its maximum level of 1,627.1 mg/L (9.7 mg/L/h). In fed-batch fermentation, a higher level of maximum IAA production than that found in batch fermentation was observed, i.e. 2,743.9 mg/L (25.4 mg/L/h). It is therefore suggested that fed-batch fermentation improves the efficiency of IAA production in terms of product concentration and IAA productivity. Moreover, yeast carotenoid production was also investigated using R. paludigenum DMKU-RP301, and found a maximum carotenoid production of 3.05 mg/L.