甲醇酵母代谢工程研究进展

甲醇酵母由于独特优点被认为是绿色生物制造的潜在宿主。特别是其天然甲醇利用性能有望建立甲醇生物转化路线,拓展生物炼制底物,具有重要经济价值和环保意义。文中综述了代谢工程改造甲醇酵母合成蛋白质和化学品的最新研究进展,并比较了其与模式生物酿酒酵母作为细胞工厂的优缺点。随后,分析了甲醇酵母代谢工程改造面临的挑战,并展望了潜在解决方案。随着基因操作工具开发和细胞代谢阐释,甲醇酵母将在未来绿色生物制造发挥越来越重要的作用。     

Repair of UV damage in the fission yeast Schizosaccharomyces pombe

This review is concerned with repair and tolerance of UV damage in the fission yeast, Schizosaccharomyces pombe and with the differences between Sch. pombe and budding yeast, Saccharomyces cerevisiae in their response to UV irradiation. Sch. pombe is not as sensitive to ultra-violet radiation as Sac. cerevisiae nor are any of its mutants as sensitive as the most sensitive Sac. cerevisiae mutants. This can be explained in part by the fact that Sch. pombe, unlike budding yeast or mammalian cells, has an extra pathway (UVER) for excision of UV photoproducts in addition to nucleotide excision repair (NER). However, even in mutants lacking this additional pathway, there are significant differences between the two yeasts. Sch. pombe mutants that lack the alternative pathway are still more UV-resistant than wild-type Sac. cerevisiae; recombination mutants are significantly UV sensitive (unlike their Sac. cerevisiae equivalents); mutants lacking the second pathway are sensitized to UV by caffeine; and checkpoint mutants are relatively more sensitive than the budding yeast equivalents. In addition, Sch. pombe has no photolyase. Thus, the response to UV in the two yeasts has a number of significant differences, which are not accounted for entirely by the existence of two alternative excision repair pathways. The long G2 in Sch. pombe, its well-developed recombination pathways and efficient cell cycle checkpoints are all significant components in survival of UV damage.     

非常规酵母基因工程表达系统

非常规酵母系指除了酿酒酵母与粟裂殖酵母之外的酵母曹。非常规酵母可利用其自主复制序列构建载体,但整合载体是进行外源基因导入的主要方式。非常规酵母的转化有一定的宿主范围,可采用与酿酒酵母相同的方法,最常用的仍为化学法。高效表达元件可利用酿酒酵母的强启动子,也可以根据非常规酵母菌的代谢特点寻找强启动子．本文综述了近年来应用非常规酵母基因表达系统表达外源基因的一些实例。     

Regulation of bacterial photosynthesis genes by oxygen and light

When grown in the appropriate medium, several yeast species produce pectinases able to degrade pectic substances. It is mainly exocellular endopolygalacturonases that break pectins or pectate down by hydrolysis of alpha-1,4-glycosidic linkages in a random way. Biochemical characterisation of these enzymes has shown that they have an optimal pH in the acidic region and an optimal temperature between 40 and 55 degrees C. Their production by yeasts is a constitutive feature and is repressed by the glucose concentration and aeration. Pectic substances and their hydrolysis products are used as carbon sources by a limited number of yeasts and hence these enzymes must be involved in the colonisation of different parts of plants, including fruits. The first yeast pectic enzyme (encoded by the PSE3 gene) was cloned from Tichosporon penicillatum. Recently, a polygalacturonase-encoding gene from Saccharomyces cerevisiae has been cloned and overexpressed in several strains and the gene for an extracellular endopolygalacturonase from Kluyveromyces marxianus has also been described. Taking all the results together, the idea is now emerging that this type of yeast enzyme could offer an alternative to fungal enzymes for industrial applications.     

Application of yeasts in gene expression studies: a comparison of Saccharomyces cerevisiae,Hansenula polymorpha and Kluyveromyces lactis- a review

From the onset of gene technology yeasts have been among the most commonly used host cells for the production of heterologous proteins. At the beginning of this new development the attention in molecular biology and biotechnology focused on the use of the best characterized species, Saccharomyces cerevisiae, leading to an increasing number of production systems for recombinant compounds. In recent years alternative yeasts became accessible for the techniques of modern molecular genetics and, thereby, for potential applications in biotechnology. In this respect Kluyveromyces lactis, and the methylotrophs Hansenula polymorpha and Pichia pastoris have been proven to offer significant advantages over the traditional baker's yeast for the production of certain proteins. In the following article, the present status of the various yeast systems is discussed.     

裂殖酵母作为外源基因表达系统

虽然裂殖酵母与酿酒酵母同属于子囊真菌,但比其它的酵母相比,裂殖酵母与更高等的真核细胞有许多相似的性质,使得裂殖酵母在分子生物学研究中成为一种提供信息的、准确的真核实验模型.它在外源基因表达方面同样具有前景.主要介绍了裂殖酵母的优点,其表达载体的性质,以及外源蛋白表达的例子.     

Review: Ethanol production at elevated temperatures and alcohol concentrations: Part I – Yeasts in general

There are a number of process advantages which could be exploited through the use of thermophilic microorganisms for ethanol production. Energy savings through reduced cooling costs, higher saccharification and fermentation rates, continuous ethanol removal and reduced contamination have stimulated a search for routes to thermophilic or thermotolerant yeasts. These routes have included screening existing culture collections, temperature adaptation, mutagenesis and molecular techniques and finally isolating new strains. Varying success has been achieved, however, the most thermotolerant yeasts have come from fresh isolations from environments which experience high temperatures. Thermotolerant yeasts have been investigated for the following potential applications: simultaneous saccharification and fermentation of cellulose, where the high fermentation temperature allows more rapid and efficient enzymatic cellulose hydrolysis; whey fermentation, where high salt and low fermentable substrate concentrations make conditions difficult; and fermentation of D-xylose and cellobiose, which is essential for efficient conversion of woody biomass to ethanol. Ethanol and temperature tolerance are important characteristics for commercial yeast strains. Both characteristics are interactive and generally decrease with increasing temperature and ethanol concentration. Considerable research has been directed towards investigation of fatty acid composition changes in response to these stresses and the role of heat shock proteins in tolerance mechanisms. If thermotolerant yeasts are to be used in commercial processes, bioreactor configuration will play an important part in the design of production processes. Batch and fed-batch systems have been shown to be useful in some circumstances as have continuous flow systems, however, some of the newly isolated thermotolerant yeasts such as Kluyveromyces marxianus do not show the high growth rate under anaerobic conditions that is characteristic of Saccharomyces cerevisiae. Various immobilization techniques appear to offer a means of presenting and maintaining high biomass in anaerobic continuous flow reactors.     

Nonconventional yeasts: their genetics and biotechnological applications

To date, more than 500 species of yeasts have been described. Most of the genetic and biochemical studies have, however, been carried out with Saccharomyces cerevisiae. Although a considerable amount of knowledge has been accumulated on fundamental processes and biotechnological applications of this industrially important yeast, the large variety of other yeast genera and species may offer various advantages for experimental study as well as for product formation in biotechnology. The genetic investigation of these so-called unconventional yeasts is poorly developed and information about corresponding data is dispersed. It is the aim of this review to summarize and discuss the main results of genetic studies and biotechnological applications of unconventional yeasts and to serve as a guide for scientists who wish to enter this field or are interested in only some aspects of these yeasts.     

The role of acetaldehyde and glycerol in the adaptation to ethanol stress of Saccharomyces cerevisiae and other yeasts

Ethanol inhibition is a commonly encountered stress condition during typical yeast fermentations and often results in reduced fermentation rates and production yields. While past studies have shown that acetaldehyde addition has a significant ameliorating effect on the growth of ethanol-stressed Saccharomyces cerevisiae , this study investigated the potential ameliorating effect of acetaldehyde on a wide range of ethanol-stressed yeasts. Acetaldehyde does not appear to be a universal ameliorating agent for yeasts exposed to ethanol stress. It is also shown that as a result of an ethanol stress, most yeasts rapidly produce glycerol as an alternative means of NAD⁺ regeneration rather than having a specific requirement for glycerol. The results strongly suggest that both ethanol and acetaldehyde exposure have a direct effect on the cellular NAD⁺/NADH ratio, which can manifest itself as modulations in glycerol production.     

The eukaryote alternative: Advantages of using yeasts in place of bacteria in microbial biosensor development

The relationship between Man and yeast has been a successful and enduring one. The characteristics of yeast have made it an ideal tool in scientific research and as such, it has been used extensively. In this review some of the advantages, methods and applications of yeasts in the biosensor field are outlined. In doing so, we propose a eukaryotic alternative to the current battery of bacteria-based microbial biosensors.     

The yeast expression system for recombinant glycosyltransferases

Glycosyltransferases are increasingly being used for in vitro synthesis of oligosaccharides. Since these enzymes are difficult to purify from natural sources, expression systems for soluble forms of the recombinant enzymes have been developed. This review focuses on the current state of development of yeast expression systems. Two yeast species have mainly been used, i.e. Saccharomyces cerevisiae and Pichia pastoris. Safety and ease of fermentation are well recognized for S. cerevisiae as a biotechnological expression system; however, even soluble forms of recombinant glycosyltransferases are not secreted. In some cases, hyperglycosylation may occur. P. pastoris, by contrast, secrete soluble orthoglycosylated forms to the supernatant where they can be recovered in a highly purified form. The review also covers some basic features of yeast fermentation and describes in some detail those glycosyltransferases that have successfully been expressed in yeasts. These include beta1,4galactosyltransferase, alpha2,6sialyltransferase, alpha2,3sialyltransferase, alpha1,3fucosyltransferase III and VI and alpha1,2mannosyltransferase. Current efforts in introducing glycosylation systems of higher eukaryotes into yeasts are briefly addressed.     

Recent developments in yeast cell surface display toward extended applications in biotechnology

Yeasts are promising hosts for industrial bio-refinery applications. In yeast cell surface displays, functional proteins, such as cellulases or lipases, are genetically fused to an anchor protein and expressed on the cell surface. Saccharomyces cerevisiae is the most commonly used yeast for cell surface display. Engineered yeasts have been utilized for a variety of applications, such as bioethanol production, chemicals synthesis, adsorption of environmental pollutants, and protein evolution. Here, we summarize recent developments in yeast cell surface display techniques for bio-refinery applications, including methods using hosts such as Pichia pastoris, Yarrowia lipolytica, and S. cerevisiae, focusing on the characteristics of anchor proteins and applications.     

Conversion of pentoses by yeasts

The utilization and conversion of D-xylose, D-xylulose, L-arabinose, and xylitol by yeast strains have been investigated with the following results: (1) The majority of yeasts tested utilize D-xylose and produce polyols, ethanol, and organic acids. The type and amount of products formed varies with the yeast strains used. The most commonly detected product is xylitol. (2)The majority of yeasts tested utilize D-xylulose aerobically and fermentatively to produce ethanol, xylitol, D-arabitol, and organic acids. The type and amount of products varies depending upon the yeast strains used. (3) Xylitol is a poor carbon and energy source for most yeasts tested. Some yeast strains produce small amounts of ethanol from xylitol. (4) Most yeast strains utilize L-arabinose, and L-arabitol is the common product. Small amounts of ethanol are also produced by some yeast strains. (5) Of the four substrates examined, D-xylulose was the perferred substrate, followed by D-xylose, L-arabinose, and xylitol. (6) Mutant yeast strains that exhibit different metabolic product patterns can be induced and isolated from Candida sp. Saccharomyces cerevisiae, and other yeasts. These mutant strains can be used for ethanol production from D-xylose as well as for the study of metabolic regulation of pentose utilization in yeasts.     

Overexpression of csc1-1. A plausible strategy to obtain wine yeast strains undergoing accelerated autolysis

The potential of several alternative genetic engineering based strategies in order to accelerate Saccharomyces cerevisiae autolysis for wine production has been studied. Both constitutively autophagic and defective in autophagy strains have been studied. Although both alternatives lead to impaired survival under starvation conditions, only constitutively autophagic strains, carrying a multicopy plasmid with the csc1-1 allele under the control of the TDH3 promoter, undergo accelerated autolysis in the experimental conditions tested. Fermentation performance is impaired in the autolytic strains, but industrial strains carrying the above-mentioned construction are still able to complete second fermentation of a model base wine. We suggest the construction of industrial yeasts showing a constitutive autophagic phenotype as a way to obtain second fermentation yeast strains undergoing accelerated autolysis.     

Phenotypic expression of Kluyveromyces lactis killer toxin against Saccharomyces spp.

The secretion of killer toxins by some strains of yeasts is a phenomenon of significant industrial importance. The activity of a recently discovered Kluyveromyces lactis killer strain against a sensitive Saccharomyces cerevisiae strain was determined on peptone-yeast extract-nutrient agar plates containing as the carbon source glucose, fructose, galactose, maltose, or glycerol at pH 4.5 or 6.5. Enhanced activity (50 to 90% increase) was found at pH 6.5, particularly on the plates containing galactose, maltose, or glycerol, although production of the toxin in liquid medium was not significantly different with either glucose or galactose as the carbon source. Results indicated that the action of the K. lactis toxin was not mediated by catabolite repression in the sensitive strain. Sensitivities of different haploid and polyploid Saccharomyces yeasts to the two different killer yeasts S. cerevisiae (RNA-plasmid-coded toxin) and K. lactis (DNA-plasmid-coded toxin) were tested. Three industrial polyploid yeasts sensitive to the S. cerevisiae killer yeast were resistant to the K. lactis killer yeast. The S. cerevisiae killer strain itself, however, was sensitive to the K. lactis killer yeast.     

Construction of a flocculent Saccharomyces cerevisiae strain secreting high levels of Aspergillus niger β-galactosidase

A flocculent Saccharomyces cerevisiae strain secreting Aspergillus niger beta-galactosidase activity was constructed by transforming S. cerevisiae NCYC869-A3 strain with plasmid pVK1.1 harboring the A. niger beta-galactosidase gene, lacA, under the control of the ADH1 promoter and terminator. Compared to other recombinant S. cerevisiae strains, this recombinant yeast has higher levels of extracellular beta-galactosidase activity. In shake-flask cultures, the beta-galactosidase activity detected in the supernatant was 20 times higher than that obtained with previously constructed strains (Domingues et al. 2000a). In bioreactor culture, with cheese-whey permeate as substrate, a yield of 878.0 nkat/gsubstrate was obtained. The recombinant strain is an attractive alternative to other fungal beta-galactosidase production systems as the enzyme is produced in a rather pure form. Moreover, the use of flocculating yeast cells allows for enzyme production with high productivity in continuous fermentation systems with facilitated downstream processing.     

Phenotypic expression of Kluyveromyces lactis killer toxin against Saccharomyces spp

The secretion of killer toxins by some strains of yeasts is a phenomenon of significant industrial importance. The activity of a recently discovered Kluyveromyces lactis killer strain against a sensitive Saccharomyces cerevisiae strain was determined on peptone-yeast extract-nutrient agar plates containing as the carbon source glucose, fructose, galactose, maltose, or glycerol at pH 4.5 or 6.5. Enhanced activity (50 to 90% increase) was found at pH 6.5, particularly on the plates containing galactose, maltose, or glycerol, although production of the toxin in liquid medium was not significantly different with either glucose or galactose as the carbon source. Results indicated that the action of the K. lactis toxin was not mediated by catabolite repression in the sensitive strain. Sensitivities of different haploid and polyploid Saccharomyces yeasts to the two different killer yeasts S. cerevisiae (RNA-plasmid-coded toxin) and K. lactis (DNA-plasmid-coded toxin) were tested. Three industrial polyploid yeasts sensitive to the S. cerevisiae killer yeast were resistant to the K. lactis killer yeast. The S. cerevisiae killer strain itself, however, was sensitive to the K. lactis killer yeast.     

Genetic analyses of yeast protein serine/threonine phosphatases

Abstract Protein phosphorylation is an important regulatory phenomenon in yeasts just as in other eukaryotic cells and controls a wide variety of cellular processes. The importance of protein phosphatases as well as protein kinases as key elements in such control is becoming increasingly clear. Over the past four years since the first yeast protein phosphatase gene was isolated, many more such genes have been described and the number of genes encoding protein phosphatase catalytic subunits in Saccharomyces cerevisiae has comfortably entered double figures. Given the genetic approaches available, yeasts offer powerful systems for addressing the cellular roles of these enzymes. This review summarises the results of genetic studies aimed at determining the functions of protein serine/threoninc phosphatases in yeast.     

Engineering yeasts for raw starch conversion

Next to cellulose, starch is the most abundant hexose polymer in plants, an import food and feed source and a preferred substrate for the production of many industrial products. Efficient starch hydrolysis requires the activities of both α-1,4 and α-1,6-debranching hydrolases, such as endo-amylases, exo-amylases, debranching enzymes, and transferases. Although amylases are widely distributed in nature, only about 10?% of amylolytic enzymes are able to hydrolyse raw or unmodified starch, with a combination of α-amylases and glucoamylases as minimum requirement for the complete hydrolysis of raw starch. The cost-effective conversion of raw starch for the production of biofuels and other important by-products requires the expression of starch-hydrolysing enzymes in a fermenting yeast strain to achieve liquefaction, hydrolysis, and fermentation (Consolidated Bioprocessing, CBP) by a single organism. The status of engineering amylolytic activities into Saccharomyces cerevisiae as fermentative host is highlighted and progress as well as challenges towards a true CBP organism for raw starch is discussed. Conversion of raw starch by yeast secreting or displaying α-amylases and glucoamylases on their surface has been demonstrated, although not at high starch loading or conversion rates that will be economically viable on industrial scale. Once efficient conversion of raw starch can be demonstrated at commercial level, engineering of yeast to utilize alternative substrates and produce alternative chemicals as part of a sustainable biorefinery can be pursued to ensure the rightful place of starch converting yeasts in the envisaged bio-economy of the future.