Metabolic Engineering of Saccharomyces cerevisiae

Comprehensive knowledge regarding Saccharomyces cerevisiae has accumulated over time, and today S. cerevisiae serves as a widley used biotechnological production organism as well as a eukaryotic model system. The high transformation efficiency, in addition to the availability of the complete yeast genome sequence, has facilitated genetic manipulation of this microorganism, and new approaches are constantly being taken to metabolicially engineer this organism in order to suit specific needs. In this paper, strategies and concepts for metabolic engineering are discussed and several examples based upon selected studies involving S. cerevisiae are reviewed. The many different studies of metabolic engineering using this organism illustrate all the categories of this multidisciplinary field: extension of substrate range, improvements of producitivity and yield, elimination of byproduct formation, improvement of process performance, improvements of cellular properties, and extension of product range including heterologous protein production.     

Opportunities for yeast metabolic engineering: Lessons from synthetic biology

Constant progress in genetic engineering has given rise to a number of promising areas of research that facilitated the expansion of industrial biotechnology. The field of metabolic engineering, which utilizes genetic tools to manipulate microbial metabolism to enhance the production of compounds of interest, has had a particularly strong impact by providing new platforms for chemical production. Recent developments in synthetic biology promise to expand the metabolic engineering toolbox further by creating novel biological components for pathway design. The present review addresses some of the recent advances in synthetic biology and how these have the potential to affect metabolic engineering in the yeast Saccharomyces cerevisiae. While S. cerevisiae for years has been a robust industrial organism and the target of multiple metabolic engineering trials, its potential for synthetic biology has remained relatively unexplored and further research in this field could strongly contribute to industrial biotechnology. This review also addresses are general considerations for pathway design, ranging from individual components to regulatory systems, overall pathway considerations and whole-organism engineering, with an emphasis on potential contributions of synthetic biology to these areas. Some examples of applications for yeast synthetic biology and metabolic engineering are also discussed.     

Advancing secondary metabolite biosynthesis in yeast with synthetic biology tools

Secondary metabolites are an important source of high-value chemicals, many of which exhibit important pharmacological properties. These valuable natural products are often difficult to synthesize chemically and are commonly isolated through inefficient extractions from natural biological sources. As such, they are increasingly targeted for production by biosynthesis from engineered microorganisms. The budding yeast species Saccharomyces cerevisiae has proven to be a powerful microorganism for heterologous expression of biosynthetic pathways. S. cerevisiae's usefulness as a host organism is owed in large part to the wealth of knowledge accumulated over more than a century of intense scientific study. Yet many challenges are currently faced in engineering yeast strains for the biosynthesis of complex secondary metabolite production. However, synthetic biology is advancing the development of new tools for constructing, controlling, and optimizing complex metabolic pathways in yeast. Here, we review how the coupling between yeast biology and synthetic biology is advancing the use of S. cerevisiae as a microbial host for the construction of secondary metabolic pathways.     

Oxidative versus reductive succinic acid production in the yeast Saccharomyces cerevisiae

Bio-based succinic acid is receiving increasing attention, as it could provide a cost-effective, ecologically sustainable alternative to the current petrochemical production process, thus promising a significantly higher market potential. The yeast Saccharomyces cerevisiae is a robust and well-established industrial production organism exhibiting an extraordinarily high acid- and osmotolerance. These features in conjunction with the sophisticated toolbox for genetic engineering make it particularly suitable for succinic acid production. The high tolerance towards acidity is a major advantage over previously established bacterial succinic acid production hosts, since it makes the use of neutralisation salts dispensable and thus enormously facilitates the downstream process. By constructing yeast strains capable of producing significant amounts of succinic acid, we have recently established S. cerevisiae as a promising host for succinic acid production. Our metabolic engineering strategy relied on the implementation of an oxidative production route using the glyoxylate cycle. We here discuss theoretical and practical aspects of oxidative and reductive succinic acid production routes in S. cerevisiae.     

Evolutionary engineering of Saccharomyces cerevisiae for improved industrially important properties

This article reviews evolutionary engineering of Saccharomyces cerevisiae. Following a brief introduction to the 'rational' metabolic engineering approach and its limitations such as extensive genetic and metabolic information requirement on the organism of interest, complexity of cellular physiological responses, and difficulties of cloning in industrial strains, evolutionary engineering is discussed as an alternative, inverse metabolic engineering strategy. Major evolutionary engineering applications with S. cerevisiae are then discussed in two general categories: (1) evolutionary engineering of substrate utilization and product formation and (2) evolutionary engineering of stress resistance. Recent developments in functional genomics methods allow rapid identification of the molecular basis of the desired phenotypes obtained by evolutionary engineering. To conclude, when used alone or in combination with rational metabolic engineering and/or computational methods to study and analyze processes of adaptive evolution, evolutionary engineering is a powerful strategy for improvement in industrially important, complex properties of S. cerevisiae.     

Engineering of Saccharomyces cerevisiae for efficient anaerobic alcoholic fermentation of L-arabinose

For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as l-arabinose, is required. Wild-type strains of Saccharomyces cerevisiae, the organism used in industrial ethanol production, cannot ferment xylose and arabinose. Although metabolic and evolutionary engineering has enabled the efficient alcoholic fermentation of xylose under anaerobic conditions, the conversion of l-arabinose into ethanol by engineered S. cerevisiae strains has previously been demonstrated only under oxygen-limited conditions. This study reports the first case of fast and efficient anaerobic alcoholic fermentation of l-arabinose by an engineered S. cerevisiae strain. This fermentation was achieved by combining the expression of the structural genes for the l-arabinose utilization pathway of Lactobacillus plantarum, the overexpression of the S. cerevisiae genes encoding the enzymes of the nonoxidative pentose phosphate pathway, and extensive evolutionary engineering. The resulting S. cerevisiae strain exhibited high rates of arabinose consumption (0.70 g h(-1) g [dry weight](-1)) and ethanol production (0.29 g h(-1) g [dry weight](-1)) and a high ethanol yield (0.43 g g(-1)) during anaerobic growth on l-arabinose as the sole carbon source. In addition, efficient ethanol production from sugar mixtures containing glucose and arabinose, which is crucial for application in industrial ethanol production, was achieved.     

酿酒酵母代谢工程技术

自20世纪90年代初期诞生以来,代谢工程历经了30年的快速发展。作为代谢工程的首选底盘细胞之一,酿酒酵母细胞工厂已被广泛应用于大量大宗化学品和新型高附加值生物活性物质的生物制造,在能源、医药和环境等领域取得了巨大的突破。近年来,合成生物学、生物信息学以及机器学习等相关技术也极大地促进了代谢工程的技术发展和应用。文中回顾了近30年来酿酒酵母代谢工程重要的技术发展,首先总结了经典代谢工程的常用方法和策略,以及在此基础上发展而来的系统代谢工程和合成生物学驱动的代谢工程技术。最后结合最新技术发展趋势,展望了未来酿酒酵母代谢工程发展的新方向。     

酿酒酵母戊糖转运蛋白及C6/C5共代谢菌株的研究进展

充分利用木质纤维素中的糖分是提高以此类生物质为原料生产二代燃料乙醇经济盈利性的基本要求,也是实现其他生物基化学品规模化生产的基础。传统的乙醇生产微生物酿酒酵母Saccharomyces cerevisiae具有独特的生产性能及内在优势,是备受关注的底盘细胞,但其不能有效地利用戊糖。利用代谢工程、合成生物学策略,对二代燃料乙醇生产专用酿酒酵母的精准构制持续研究了30余年,已明显改善了其对木糖/葡萄糖的乙醇共发酵能力。近年来关注点集中在早期忽略的限速步骤即糖转运环节的研究上,以期实现不同糖分各行其道、高效专一性转运蛋白各行其责的二代燃料乙醇生产特种酿酒酵母所需的糖转运理想状态。文中主要综述了酿酒酵母戊糖转运蛋白的研究进展,及酿酒酵母的木糖和L-阿拉伯糖代谢工程的研究现状。     

酿酒酵母转化木糖生产化学品的研究进展

木糖的有效利用是木质纤维素生产生物燃料或化学品经济性转化的基础.30年来,通过理性代谢改造和适应性进化等工程策略,显著提高了传统乙醇发酵微生物——酿酒酵母Saccharomyces cerevisiae的木糖代谢能力.因此,近年来在酿酒酵母中利用木糖生产化学品的研究逐步展开.研究发现,酿酒酵母分别以木糖和葡萄糖为碳源时...     

Cloning and characterization of a panel of constitutive promoters for applications in pathway engineering in Saccharomyces cerevisiae

Saccharomyces cerevisiae is an important platform organism for synthesis of chemicals and fuels. However, the promoters used in most pathway engineering studies in S. cerevisiae have not been characterized and compared in parallel under multiple conditions that are routinely operated in laboratory and the number of known promoters is rather limited for the construction of large biochemical pathways. Here a total of 14 constitutive promoters from S. cerevisiae were cloned and characterized using a green fluorescent protein (GFP) as a reporter in a 2 μ vector pRS426, under varying glucose and oxygen concentrations. The strengths of these promoters varied no more than sixfold in the mean fluorescence intensity of GFP, with promoter TEF1p being the strongest and promoter PGI1p the weakest. As an example of application for these promoters in metabolic engineering, the genes involved in xylan degradation and zeaxanthin biosynthesis were subsequently cloned under the control of promoters with medium to high strength and assembled into a single pathway. The corresponding construct was transformed to a S. cerevisiae strain integrated with a D-xylose utilizing pathway. The resulting strain produced zeaxanthin with a titer of 0.74 ± 0.02 mg/L directly from birchwood xylan.     

Engineering Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: reflections and perspectives

Conversion of the abundant lignocellulosic biomass into ethanol is an environmentally sustainable solution to the energy crisis. Fermentation of lignocellulosic hydrolysates by Saccharomyces cerevisiae is not cost-effective yet as substantial amounts of xylose in the hydrolysates cannot be utilized by native S. cerevisiae strains. Extensive studies including both metabolic and evolutionary engineering have been carried out to develop an efficient xylose-fermenting S. cerevisiae strain, yet the ethanol yield and productivity from xylose fermentation of the best one are still far below expectation. This review compares the engineering approaches and resulted anaerobic xylose fermentation performance of recently reported xylose-utilizing S. cerevisiae strains, with the aim to understand the intrinsic reason for their low xylose fermentation capabilities. These comparative analyses revealed that some of the current engineering targets and the so-called "hot issues" might be overrated. Our opinions on the underrated parts and future efforts in this field are also presented. Overall, this review serves as a comprehensive reference to understanding xylose fermentation by S. cerevisiae.     

Metabolic engineering of recombinant protein secretion by Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a widely used cell factory for the production of fuels and chemicals, and it is also provides a platform for the production of many heterologous proteins of medical or industrial interest. Therefore, many studies have focused on metabolic engineering S. cerevisiae to improve the recombinant protein production, and with the development of systems biology, it is interesting to see how this approach can be applied both to gain further insight into protein production and secretion and to further engineer the cell for improved production of valuable proteins. In this review, the protein post-translational modification such as folding, trafficking, and secretion, steps that are traditionally studied in isolation will here be described in the context of the whole system of protein secretion. Furthermore, examples of engineering secretion pathways, high-throughput screening and systems biology applications of studying protein production and secretion are also given to show how the protein production can be improved by different approaches. The objective of the review is to describe individual biological processes in the context of the larger, complex protein synthesis network.     

Metabolic engineering of Saccharomyces cerevisiae for production of carboxylic acids: current status and challenges

To meet the demands of future generations for chemicals and energy and to reduce the environmental footprint of the chemical industry, alternatives for petrochemistry are required. Microbial conversion of renewable feedstocks has a huge potential for cleaner, sustainable industrial production of fuels and chemicals. Microbial production of organic acids is a promising approach for production of chemical building blocks that can replace their petrochemically derived equivalents. Although Saccharomyces cerevisiae does not naturally produce organic acids in large quantities, its robustness, pH tolerance, simple nutrient requirements and long history as an industrial workhorse make it an excellent candidate biocatalyst for such processes. Genetic engineering, along with evolution and selection, has been successfully used to divert carbon from ethanol, the natural endproduct of S. cerevisiae , to pyruvate. Further engineering, which included expression of heterologous enzymes and transporters, yielded strains capable of producing lactate and malate from pyruvate. Besides these metabolic engineering strategies, this review discusses the impact of transport and energetics as well as the tolerance towards these organic acids. In addition to recent progress in engineering S. cerevisiae for organic acid production, the key limitations and challenges are discussed in the context of sustainable industrial production of organic acids from renewable feedstocks.     

Genetic mapping of quantitative phenotypic traits in Saccharomyces cerevisiae

Saccharomyces cerevisiae has become a favorite production organism in industrial biotechnology presenting new challenges to yeast engineers in terms of introducing advantageous traits such as stress tolerances. Exploring subspecies diversity of S. cerevisiae has identified strains that bear industrially relevant phenotypic traits. Provided that the genetic basis of such phenotypic traits can be identified inverse engineering allows the targeted modification of production strains. Most phenotypic traits of interest in S. cerevisiae strains are quantitative, meaning that they are controlled by multiple genetic loci referred to as quantitative trait loci (QTL). A straightforward approach to identify the genetic basis of quantitative traits is QTL mapping which aims at the allocation of the genetic determinants to regions in the genome. The application of high-density oligonucleotide arrays and whole-genome re-sequencing to detect genetic variations between strains has facilitated the detection of large numbers of molecular markers thus allowing high-resolution QTL mapping over the entire genome. This review focuses on the basic principle and state of the art of QTL mapping in S. cerevisiae. Furthermore we discuss several approaches developed during the last decade that allow down-scaling of the regions identified by QTL mapping to the gene level. We also emphasize the particular challenges of QTL mapping in nonlaboratory strains of S. cerevisiae.     

酿酒酵母人造纤维小体的研究进展简

纤维素乙醇的统合生物加工过程（consolidated bioprocessing,CBP）是将（半）纤维素酶生产、纤维素水解和乙醇发酵过程组合,通过一种微生物完成的生物加工过程。 CBP有利于降低生物转化过程的成本,受到研究者的普遍关注。酿酒酵母（ Saccharomyces cerevisiae）作为传统的乙醇生产菌株,是极具潜力的CBP底盘细胞。纤维小体是某些厌氧微生物细胞表面由纤维素酶系与支架蛋白组成的大分子复合物,它能高效降解木质纤维,在酿酒酵母表面展示纤维小体已成为构建CBP细胞的研究热点。笔者综述了人造纤维小体在酿酒酵母细胞表面展示组装的研究进展,重点阐述了纤维小体各元件的设计和改造,并针对酿酒酵母分泌途径的改造,提出提高人造纤维小体分泌组装的可能性策略。     

Diversion of flux toward sesquiterpene production in Saccharomyces cerevisiae by fusion of host and heterologous enzymes

The ability to transfer metabolic pathways from the natural producer organisms to the well-characterized cell factory Saccharomyces cerevisiae is well documented. However, as many secondary metabolites are produced by collaborating enzymes assembled in complexes, metabolite production in yeast may be limited by the inability of the heterologous enzymes to collaborate with the native yeast enzymes. This may cause loss of intermediates by diffusion or degradation or due to conversion of the intermediate through competitive pathways. To bypass this problem, we have pursued a strategy in which key enzymes in the pathway are expressed as a physical fusion. As a model system, we have constructed several fusion protein variants in which farnesyl diphosphate synthase (FPPS) of yeast has been coupled to patchoulol synthase (PTS) of plant origin (Pogostemon cablin). Expression of the fusion proteins in S. cerevisiae increased the production of patchoulol, the main sesquiterpene produced by PTS, up to 2-fold. Moreover, we have demonstrated that the fusion strategy can be used in combination with traditional metabolic engineering to further increase the production of patchoulol. This simple test case of synthetic biology demonstrates that engineering the spatial organization of metabolic enzymes around a branch point has great potential for diverting flux toward a desired product.     

酿酒酵母木糖发酵酒精途径工程的研究进展

途径工程(Pathway engineering),被称为第三代基因工程,改变代谢流向,开辟新的代谢途径是途径工程的主要目的。利用途径工程理念,对酿酒酵母（Saccharomyces cerevisiae）代谢途径进行理性设计,以拓展这一传统酒精生产菌的底物范围,使其充分利用可再生纤维质水解物中的各种糖分,是酿酒酵母酒精途径工程的研究热点之一。这里介绍了近年来酿酒酵母以木糖为底物的酒精途径工程的研究进展。     

Metabolic engineering of Saccharomyces cerevisiae for production of novel lipid compounds

The yeast Saccharomyces cerevisiae has been modified successfully for production of numerous metabolites and therapeutic proteins through metabolic engineering, but has not been utilized to date for the production of lipid-derived compounds. We developed a lipid metabolic engineering strategy in S. cerevisiae based upon culturing techniques that are typically employed for studies of peroxisomal biogenesis; cells were grown in media containing fatty acids as a sole carbon source, which promotes peroxisomal proliferation and induction of enzymes associated with fatty acid beta-oxidation. Our results indicate that growth of yeast on fatty acids such as oleate results in extensive uptake of these fatty acids from the media and a subsequent increase in total cellular lipid content from 2% to 15% dry cell weight. We also show that co-expression of plant fatty acid desaturases 2 and 3 ( FAD2 and FAD3), using a fatty acid-inducible peroxisomal gene promoter, coupled the processes of fatty acid uptake with the induction of a new metabolic pathway leading from oleic acid (18:1) to linolenic acid (18:3). Finally, we show that cultivation of yeast cells in the presence of triacylglycerols and exogenously supplied lipase promotes extensive incorporation of triglyceride fatty acids into yeast cells. Collectively, these results provide a framework for bioconversion of low-cost oils into value-added lipid products.