Special focus: Synthetic biology

Special focus: Synthetic biology What is synthetic biology? SynBERC – The Synthetic Biology Engineering Research Center Ars Synthetica iGEM – The International Genetically Engineered Machine competition Some synthetic biology companies Paper watch: Synthetic biology Building blocks for novel functions Knowledge-making distinctions in synthetic biology Scaffold design and manufacturing: From concept to clinic Peptidomimetics – a versatile route to biologically active compounds Metabolic engineering of E. coli E. coli needs safety valves Systems-level metabolic engineering Mammalian synthetic biology Chemical aspects of synthetic biology Synthesis of DNA fragments in yeast Synthetic biology and patentable subject matter Patenting artificial life? Metabolic effects of synthetic rewiring Engineering for biofuels Regulatory elements for synthetic biology Book highlight Systems Biology and Synthetic Biology     

Comparative genome analysis of a Saccharomyces cerevisiae wine strain

Many industrial strains of Saccharomyces cerevisiae have been selected primarily for their ability to convert sugars into ethanol efficiently despite exposure to a variety of stresses. To begin investigation of the genetic basis of phenotypic variation in industrial strains of S. cerevisiae, we have sequenced the genome of a wine yeast, AWRI1631, and have compared this sequence with both the laboratory strain S288c and the human pathogenic isolate YJM789. AWRI1631 was found to be substantially different from S288c and YJM789, especially at the level of single-nucleotide polymorphisms, which were present, on average, every 150 bp between all three strains. In addition, there were major differences in the arrangement and number of Ty elements between the strains, as well as several regions of DNA that were specific to AWRI1631 and that were predicted to encode proteins that are unique to this industrial strain.     

酿酒酵母染色体设计与合成研究进展

随着合成生物学的蓬勃发展,基因组学的研究正在由读取基因组信息拓展到以编写基因组信息为主的合成基因组学时代。2009年,由Jef D. Boeke教授提出的人工合成酵母基因组计划(Sc2.0)旨在合成世界上首个真核生物基因组。在美、中、英、法、澳大利亚、新加坡等多国科学家的努力下,目前已经完成1/3的酵母染色体的人工合成。本文从合成基因组学领域的发展历程出发,介绍了Sc2.0计划中酿酒酵母(Saccharomyces cerevisiae)染色体设计与合成的最新进展,包括酿酒酵母9号染色体右臂、3号染色体、2号染色体、5号染色体、6号染色体、10号染色体和12号染色体的设计与合成过程,阐述了其各自的合成策略以及生物学意义,以期为合成基因组学的深入开展提供借鉴与参考。     

Copper Tolerance and Biosorption of Saccharomyces cerevisiae during Alcoholic Fermentation

At high levels, copper in grape mash can inhibit yeast activity and cause stuck fermentations. Wine yeast has limited tolerance of copper and can reduce copper levels in wine during fermentation. This study aimed to understand copper tolerance of wine yeast and establish the mechanism by which yeast decreases copper in the must during fermentation. Three strains of Saccharomyces cerevisiae (lab selected strain BH8 and industrial strains AWRI R2 and Freddo) and a simple model fermentation system containing 0 to 1.50 mM Cu²⁺ were used. ICP-AES determined Cu ion concentration in the must decreasing differently by strains and initial copper levels during fermentation. Fermentation performance was heavily inhibited under copper stress, paralleled a decrease in viable cell numbers. Strain BH8 showed higher copper-tolerance than strain AWRI R2 and higher adsorption than Freddo. Yeast cell surface depression and intracellular structure deformation after copper treatment were observed by scanning electron microscopy and transmission electron microscopy; electronic differential system detected higher surface Cu and no intracellular Cu on 1.50 mM copper treated yeast cells. It is most probably that surface adsorption dominated the biosorption process of Cu²⁺ for strain BH8, with saturation being accomplished in 24 h. This study demonstrated that Saccharomyces cerevisiae strain BH8 has good tolerance and adsorption of Cu, and reduces Cu²⁺ concentrations during fermentation in simple model system mainly through surface adsorption. The results indicate that the strain selected from China’s stress-tolerant wine grape is copper tolerant and can reduce copper in must when fermenting in a copper rich simple model system, and provided information for studies on mechanisms of heavy metal stress.     

Influence of choice of yeasts on volatile fermentation-derived compounds, colour and phenolics composition in Cabernet Sauvignon wine

Wine colour, phenolics and volatile fermentation-derived composition are the quintessential elements of a red wine. Many viticultural and winemaking factors contribute to wine aroma and colour with choice of yeast strain being a crucial factor. Besides the traditional Saccharomyces species S. cerevisiae, S. bayanus and several Saccharomyces interspecific hybrids are able to ferment grape juice to completion. This study examined the diversity in chemical composition, including phenolics and fermentation-derived volatile compounds, of an Australian Cabernet Sauvignon due to the use of different Saccharomyces strains. Eleven commercially available Saccharomyces strains were used in this study; S. cerevisiae (7), S. bayanus (2) and interspecific Saccharomyces hybrids (2). The eleven Cabernet Sauvignon wines varied greatly in their chemical composition. Nine yeast strains completed alcoholic fermentation in 19?days; S. bayanus AWRI 1375 in 26?days, and S. cerevisiae AWRI 1554 required 32?days. Ethanol concentrations varied in the final wines (12.7?C14.2?%). The two S. bayanus strains produced the most distinct wines, with the ability to metabolise malic acid, generate high glycerol concentrations and distinctive phenolic composition. Saccharomyces hybrid AWRI 1501 and S. cerevisiae AWRI 1554 and AWRI 1493 also generated distinctive wines. This work demonstrates that the style of a Cabernet Sauvignon can be clearly modulated by choice of commercially available wine yeast.     

BiotecVisions 2010, May–June

News: Ethanol biofuels from orange peels – Targeting leukaemia's gene addiction – Pea-derived solar cells – HIV is a kick in the head – Nano-scale DNA reader – Membrane in black – Cheese improves the immune response of elderly – Synthetic proteins built from standard parts – Therapeutic proteins produced in algae – Biosensor detects 100 mycoplasma cells – Protecting maggots against bacteria – Advanced biofuels from microbes – Fluorescent bacterial uptake – Two disparate stem cell states – Brachypodium genome sequenced Encyclopedia of Life Sciences: Nuclear transfer for cell lines WIREs Nanomedicine and Nanobiotechnology: Nanoparticle detection of respiratory infection Journal Highlights: Biocatalysis – Synthetic Biology In the news: Nanobiotech to detect cancer Most Read Industry News: Biomarker assays for personalized medicine – Bioplastic industry defies economic crisis – SDS-PAGE monitoring of mAB Awards: BTJ Editors elected members of the US National Academy of Engineering (NAE) Meeting highlight Writing tips: Figure preparation made simple – Some useful tutorials on the web Book Highlights: Molecular Biotechnology – Bacterial Signaling – Yeast Test your knowledge: Do you recognize this? WIREs Authors Spotlight: Nanotechnology and orthopedics     

合成芳香族化合物的酵母底盘改造策略*

芳香族化合物种类丰富,在多个行业具有广泛的用途,需求量大。通过构建微生物细胞工厂合成芳香族化合物具有独特的优势和工业化应用前景,其中酵母底盘因其清晰的遗传背景、完善的基因操作工具以及成熟的工业发酵体系等优势,常被用于构建细胞工厂。目前改造酵母底盘生产芳香族化合物的研究取得了一系列进展,并针对关键问题提出了一些可行的解决策略。针对酵母合成芳香族化合物的策略与挑战,从芳香族化合物合成路径改造、多样化碳源利用及转运系统改造、基因组多靶点改造、特殊酵母底盘及混菌系统构建、合成生物学高通量技术的应用这五个方面进行系统地梳理和阐述,为生产芳香族化合物的酵母底盘构建与改造提供思路。     

Engineering a microbial platform for de novo biosynthesis of diverse methylxanthines

Engineered microbial biosynthesis of plant natural products can support manufacturing of complex bioactive molecules and enable discovery of non-naturally occurring derivatives. Purine alkaloids, including caffeine (coffee), theophylline (antiasthma drug), theobromine (chocolate), and other methylxanthines, play a significant role in pharmacology and food chemistry. Here, we engineered the eukaryotic microbial host Saccharomyces cerevisiae for the de novo biosynthesis of methylxanthines. We constructed a xanthine-to-xanthosine conversion pathway in native yeast central metabolism to increase endogenous purine flux for the production of 7-methylxanthine, a key intermediate in caffeine biosynthesis. Yeast strains were further engineered to produce caffeine through expression of several enzymes from the coffee plant. By expressing combinations of different N-methyltransferases, we were able to demonstrate re-direction of flux to an alternate pathway and develop strains that support the production of diverse methylxanthines. We achieved production of 270 μg/L, 61 μg/L, and 3700 μg/L of caffeine, theophylline, and 3-methylxanthine, respectively, in 0.3-L bench-scale batch fermentations. The constructed strains provide an early platform for de novo production of methylxanthines and with further development will advance the discovery and synthesis of xanthine derivatives.     

qPCRTag Analysis - A High Throughput,Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII¹, and one synthetic chromosome arm, synIXR², have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.     

Effect of low-temperature fermentation on yeast nitrogen metabolism

The aim of this study was to analyse the influence of low-temperature wine fermentation on nitrogen consumption and nitrogen regulation. Synthetic grape must was fermented at 25 and 13°C. Low-temperature decreased both the fermentation and the growth rates. Yeast cells growing at low-temperature consumed less nitrogen than at 25°C. Specifically, cells at 13°C consumed less ammonium and glutamine, and more tryptophan. Low-temperature seemed to relax the nitrogen catabolite repression (NCR) as deduced from the gene expression of ammonium and amino acid permeases (MEP2 and GAP1) and the uptake of some amino acids subjected to NCR (i.e. arginine and glutamine). Low-temperature influences the quantity and the quality of yeast nitrogen requirements. Nitrogen-deficient grape musts and low temperature are two of the main prevalent causes of sluggish fermentations and, therefore, the effects of both growth conditions on yeast metabolism are of considerable interest for wine making.     

Modulation of volatile sulfur compounds by wine yeast

Sulfur compounds in wine can be a ‘double-edged sword’. On the one hand, certain sulfur-containing volatile compounds such as hydrogen sulfide, imparting a rotten egg-like aroma, can have a negative impact on the perceived quality of the wine, and on the other hand, some sulfur compounds such as 3-mercaptohexanol, imparting fruitiness, can have a positive impact on wine flavor and aroma. Furthermore, these compounds can become less or more attractive or repulsive depending on their absolute and relative concentrations. This presents an interesting challenge to the winemaker to modulate the concentrations of these quality-determining compounds in wine in accordance with consumer preferences. The wine yeast Saccharomyces cerevisiae plays a central role in the production of volatile sulfur compounds. Through the sulfate reduction sequence pathway, the HS^- is formed, which can lead to the formation of hydrogen sulfide and various mercaptan compounds. Therefore, limiting the formation of the HS^- ion is an important target in metabolic engineering of wine yeast. The wine yeast is also responsible for the transformation of non-volatile sulfur precursors, present in the grape, to volatile, flavor-active thiol compounds. In particular, 4-mercapto-4-methylpentan-2-one, 3-mercaptohexanol, and 3-mercaptohexyl acetate are the most important volatile thiols adding fruitiness to wine. This paper briefly reviews the metabolic processes involved in the production of important volatile sulfur compounds and the latest strategies in the pursuit of developing wine yeast strains as tools to adjust wine aroma to market specifications.     

Genotype-dependent sulphite tolerance of Australian Dekkera (Brettanomyces) bruxellensis wine isolates

Aims: The aim of this study was to determine sulphite tolerance for a large number of Dekkera bruxellensis isolates and evaluate the relationship between this phenotype and previously assigned genotype markers. Methods and Results: A published microplate‐based method for evaluation of yeast growth in the presence of sulphite was benchmarked against culturability following sulphite treatment, for the D. bruxellensis type strain (CBS 74) and a reference wine isolate (AWRI 1499). This method was used to estimate maximal sulphite tolerance for 41 D. bruxellensis isolates, which was found to vary over a fivefold range. Significant differences in sulphite tolerance were observed when isolates were grouped according to previously assigned genotypes and ribotypes. Conclusions: Variable sulphite tolerance for the wine spoilage yeast D. bruxellensis can be linked to genotype markers. Significance and Impact of the Study: Strategies to minimize risk of wine spoilage by D. bruxellensis must take into account at least a threefold range in effective sulphite concentration that is dependent upon the genotype group(s) present. The isolates characterized in this study will be a useful resource for establishing the mechanisms conferring sulphite tolerance for this industrially important yeast species.     

Single-particle tracking of quantum dot-conjugated prion proteins inside yeast cells

Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way toward the individual tracking of proteins of interest inside living yeast cells.     

酵母基因组大尺度遗传操纵工具研究进展

基因组大尺度遗传操纵是指对基因组大片段DNA的敲除、整合、易位等遗传改造。相较于小规模基因编辑,基因组大尺度遗传操纵可实现更多遗传信息的同步改造,对于探究多基因相互作用等复杂机制的理解有重要意义。同时,基因组大尺度遗传操纵技术可对基因组开展更大规模的设计重构,甚至创建全新的基因组,在复杂功能重塑方面具有重要创新潜力。酵母是一种重要的真核模式生物,因其安全性和易于操作而被广泛应用。本文系统总结了酵母基因组大尺度遗传操纵的工具包,包括重组酶介导的大尺度操纵、核酸酶介导的大尺度操纵、从头合成大片段DNA以及其他大尺度操纵工具,介绍了它们的基本工作原理与典型应用案例。最后,对大尺度遗传操纵面临的挑战和发展进行了展望。     

Yeast identification in grape juice concentrates from Argentina

Aims: The purpose of this study was to identify yeast species present in spoiled and unspoiled grape juice concentrates from Argentine industries. Methods and Results: Osmophilic and osmotolerant yeasts were isolated from spoiled – visually effervescent – and unspoiled – without any visible damage – grape juice concentrates by the spread‐plate technique in two culture media. Yeast identification was done by classical and molecular methods. Zygosaccharomyces rouxii was the only species isolated from spoiled grape juice concentrates. In unspoiled samples, five different species were identified: Z. rouxii was isolated at a higher frequency, followed in decreasing order by Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia anomala and Kluyveromyces delphensis. Conclusions: Yeasts isolated from grape juice concentrates were characterized by a limited taxonomic diversity, where Z. rouxii was the main species isolated. Significance and Impact of the Study: Grape production in Argentina is mainly devoted to the industry where wine and grape juice concentrates represent major types of commercial products. Little information on common yeast contaminants is available for grape juice concentrates. This study constitutes the first report of osmophilic yeast species present in spoiled and unspoiled grape juice concentrates elaborated in Argentina.     

非常规酵母的分子遗传学及合成生物学研究进展

先进的合成生物学技术与传统的分子遗传学技术的结合更有助于实现酵母底盘细胞的快速改造和优化。酵母合成生物学研究最早开始于常规酵母——酿酒酵母(Saccharomyces cerevisiae),近些年来又迅速扩展至一些非常规酵母,包括巴斯德毕赤酵母(Pichiapastoris)、解脂耶氏酵母(Yarrowialipolytica)、乳酸克鲁维酵母(Kluyveromyces lactis)和多形汉逊酵母(Hansenula polymorpha)等。借助合成生物学技术与工具,目前科学家们已经成功开发出了能够高效生产生物材料、生物燃料、生物基化学品、蛋白质制剂、食品添加剂和药物等工业产品的重组非常规酵母工程菌株。本文系统总结了合成生物学工具(主要是基因组编辑工具)、合成生物学组件(主要是启动子和终止子)和相关分子遗传学方法在上述非常规酵母系统(底盘细胞)中的最新研究进展和应用情况,并讨论了其他合成生物学技术在这些非常规酵母表达系统中的潜在适用性和应用前景。这为研究人员利用合成生物学方法在这一新型非模式微生物底盘细胞中设计和构建各种高附加值工业产品的异源合成模块并最终实现目标化合物的高效生物合成提供了科学的理论指导。     

Biotech networks and awards

Meeting preview: Costa Brava in October – Protein Design and Evolution for Biocatalysis James Briscoe awarded 2008 EMBO Gold Medal Chemical biology: Anti-Alzheimer's agent RTS Life Science wins 2008 North West “innovation in engineering” award Synthetic Biology: Funders move to address social and ethical challenges Novartis to accelerate TB drug development Project highlight: Metagenomic sequencing Biotech round the world: Focus on Uganda     

Evaluation of Non-Saccharomyces Yeasts for the Reduction of Alcohol Content in Wine

Over recent decades, the average ethanol concentration of wine has increased, largely due to consumer preference for wine styles associated with increased grape maturity; sugar content increases with grape maturity, and this translates into increased alcohol content in wine. However, high ethanol content impacts wine sensory properties, reducing the perceived complexity of flavors and aromas. In addition, for health and economic reasons, the wine sector is actively seeking technologies to facilitate the production of wines with lower ethanol content. Nonconventional yeast species, in particular, non-Saccharomyces yeasts, have shown potential for producing wines with lower alcohol content. These yeast species, which are largely associated with grapes preharvest, are present in the early stages of fermentation but, in general, are not capable of completing alcoholic fermentation. We have evaluated 50 different non-Saccharomyces isolates belonging to 24 different genera for their capacity to produce wine with a lower ethanol concentration when used in sequential inoculation regimes with a Saccharomyces cerevisiae wine strain. A sequential inoculation of Metschnikowia pulcherrima AWRI1149 followed by an S. cerevisiae wine strain was best able to produce wine with an ethanol concentration lower than that achieved with the single-inoculum, wine yeast control. Sequential fermentations utilizing AWRI1149 produced wines with 0.9% (vol/vol) and 1.6% (vol/vol) (corresponding to 7.1 g/liter and 12.6 g/liter, respectively) lower ethanol concentrations in Chardonnay and Shiraz wines, respectively. In Chardonnay wine, the total concentration of esters and higher alcohols was higher for wines generated from sequential inoculations, whereas the total concentration of volatile acids was significantly lower. In sequentially inoculated Shiraz wines, the total concentration of higher alcohols was higher and the total concentration of volatile acids was reduced compared with those in control S. cerevisiae wines, whereas the total concentrations of esters were not significantly different.