代谢改造酿酒酵母合成萜类化合物的研究进展

萜类化合物是一类种类繁多、功能多样的化合物,部分具有抗癌、增强免疫力等作用,具有良好的生物活性,在食品、保健品以及医疗等领域应用广泛.近年来,随着对萜类化合物生物合成途径研究的深入,研究人员采用代谢工程手段构建了多种萜类产物的高产酿酒酵母工程菌株,部分已经达到或者接近工业化生产水平.因此,采用合成生物学相关技术手段合成...     

Introduction and expression of genes for metabolic engineering applications in Saccharomyces cerevisiae

Metabolic pathway engineering in the yeast Saccharomyces cerevisiae leads to improved production of a wide range of compounds, ranging from ethanol (from biomass) to natural products such as sesquiterpenes. The introduction of multienzyme pathways requires precise control over the level and timing of expression of the associated genes. Gene number and promoter strength/regulation are two critical control points, and multiple studies have focused on modulating these in yeast. This MiniReview focuses on methods for introducing genes and controlling their copy number and on the many promoters (both constitutive and inducible) that have been successfully employed. The advantages and disadvantages of the methods will be presented, and applications to pathway engineering will be highlighted.     

In silico aided metabolic engineering of Saccharomyces cerevisiae for improved bioethanol production

In silico genome-scale cell models are promising tools for accelerating the design of cells with improved and desired properties. We demonstrated this by using a genome-scale reconstructed metabolic network of Saccharomyces cerevisiae to score a number of strategies for metabolic engineering of the redox metabolism that will lead to decreased glycerol and increased ethanol yields on glucose under anaerobic conditions. The best-scored strategies were predicted to completely eliminate formation of glycerol and increase ethanol yield with 10%. We successfully pursued one of the best strategies by expressing a non-phosphorylating, NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase in S. cerevisiae. The resulting strain had a 40% lower glycerol yield on glucose while the ethanol yield increased with 3% without affecting the maximum specific growth rate. Similarly, expression of GAPN in a strain harbouring xylose reductase and xylitol dehydrogenase led to an improvement in ethanol yield by up to 25% on xylose/glucose mixtures.     

Production of natural products through metabolic engineering of Saccharomyces cerevisiae

1. Download : Download high-res image (77KB)
2. Download : Download full-size image

     

Ethylene production by metabolic engineering of the yeast Saccharomyces cerevisiae

The non-ethylene producing yeast, Saccharomyces cerevisiae, was transformed into an ethylene producer by introducing the ethylene forming enzyme from the plant pathogenic bacterium Pseudomonas syringae. Cultivation of the metabolically engineered strain was performed in well-controlled bioreactors as aerobic batch cultures with an on-line monitoring of ethylene production. The highest productivity was obtained during the respiro-fermentative growth on glucose but there was also a significant rate of formation during the subsequent phase of ethanol respiration. Furthermore, investigations were performed whether substitution of the original nitrogen source, NH(4)(+), for glutamate could improve productivity and yield of ethylene even more. The rationale being that one of the substrates for the enzyme is 2-oxoglutarate and this compound can be formed from glutamate in a single reaction. Indeed, there was a substantial improvement in the rate of production and the final yield of ethylene was almost three times higher when NH(4)(+) was replaced by glutamate.     

Impact of systems biology on metabolic engineering of Saccharomyces cerevisiae

Industrial biotechnology is a rapidly growing field. With the increasing shift towards a bio-based economy, there is rising demand for developing efficient cell factories that can produce fuels, chemicals, pharmaceuticals, materials, nutraceuticals, and even food ingredients. The yeast Saccharomyces cerevisiae is extremely well suited for this objective. As one of the most intensely studied eukaryotic model organisms, a rich density of knowledge detailing its genetics, biochemistry, physiology, and large-scale fermentation performance can be capitalized upon to enable a substantial increase in the industrial application of this yeast. Developments in genomics and high-throughput systems biology tools are enhancing one's ability to rapidly characterize cellular behaviour, which is valuable in the field of metabolic engineering where strain characterization is often the bottleneck in strain development programmes. Here, the impact of systems biology on metabolic engineering is reviewed and perspectives on the role of systems biology in the design of cell factories are given.     

Enhancing sesquiterpene production in Saccharomyces cerevisiae through in silico driven metabolic engineering

A genome-scale metabolic model was used to identify new target genes for enhanced biosynthesis of sesquiterpenes in the yeast Saccharomyces cerevisiae. The effect of gene deletions on the flux distributions in the metabolic model of S. cerevisiae was assessed using OptGene as the modeling framework and minimization of metabolic adjustments (MOMA) as objective function.Deletion of NADPH-dependent glutamate dehydrogenase encoded by GDH1 was identified as the best target gene for the improvement of sesquiterpene biosynthesis in yeast. Deletion of this gene enhances the available NADPH in the cytosol for other NADPH requiring enzymes, including HMG-CoA reductase. However, since disruption of GDH1 impairs the ammonia utilization, simultaneous over-expression of the NADH-dependent glutamate dehydrogenase encoded by GDH2 was also considered in this study.Deletion of GDH1 led to an approximately 85% increase in the final cubebol titer. However, deletion of this gene also caused a significant decrease in the maximum specific growth rate. Over-expression of GDH2 did not show a further effect on the final cubebol titer but this alteration significantly improved the growth rate compared to the GDH1 deleted strain.     

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。     

Stepwise increase of resveratrol biosynthesis in yeast Saccharomyces cerevisiae by metabolic engineering

Resveratrol is a unique, natural polyphenolic compound with diverse health benefits. In the present study, we attempted to improve resveratrol biosynthesis in yeast by different methods of metabolic engineering. We first mutated and then re-synthesized tyrosine ammonia lyase (TAL) by replacing the bacteria codons with yeast-preferred codons, which increased translation and improved p-coumaric acid and resveratrol biosynthesis drastically. We then demonstrated that low-affinity, high-capacity bacterial araE transporter could enhance resveratrol accumulation, without transporting resveratrol directly. Yeast cells carrying the araE gene produced up to 2.44-fold higher resveratrol than control cells. For commercial applications, resveratrol biosynthesis was detected in sucrose medium and fresh grape juice using our engineered yeast cells. In collaboration with the Chaumette Winery of Missouri, we were able to produce resveratrol-containing white wines, with levels comparable to the resveratrol levels found in most red wines.     

Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle

When xylose metabolism in yeasts proceeds exclusively via NADPH-specific xylose reductase and NAD-specific xylitol dehydrogenase, anaerobic conversion of the pentose to ethanol is intrinsically impossible. When xylose reductase has a dual specificity for both NADPH and NADH, anaerobic alcoholic fermentation is feasible but requires the formation of large amounts of polyols (e.g., xylitol) to maintain a closed redox balance. As a result, the ethanol yield on xylose will be sub-optimal. This paper demonstrates that anaerobic conversion of xylose to ethanol, without substantial by-product formation, is possible in Saccharomyces cerevisiae when a heterologous xylose isomerase (EC 5.3.1.5) is functionally expressed. Transformants expressing the XylA gene from the anaerobic fungus Piromyces sp. E2 (ATCC 76762) grew in synthetic medium in shake-flask cultures on xylose with a specific growth rate of 0.005 h(-1). After prolonged cultivation on xylose, a mutant strain was obtained that grew aerobically and anaerobically on xylose, at specific growth rates of 0.18 and 0.03 h(-1), respectively. The anaerobic ethanol yield was 0.42 g ethanol x g xylose(-1) and also by-product formation was comparable to that of glucose-grown anaerobic cultures. These results illustrate that only minimal genetic engineering is required to recruit a functional xylose metabolic pathway in Saccharomyces cerevisiae. Activities and/or regulatory properties of native S. cerevisiae gene products can subsequently be optimised via evolutionary engineering. These results provide a gateway towards commercially viable ethanol production from xylose with S. cerevisiae.     

Chromosome-shuffling technique for selected chromosomal segments in Saccharomyces cerevisiae

We describe a novel chromosome engineering technique for shuffling selected regions of chromosomes from two strains in Saccharomyces cerevisiae: The technique starts with the construction of MAT a and MATα strains in which a particular chromosome is split at exactly the same site in both strains such that the split chromosomes generated are marked with different markers. The two strains are then crossed, and the resultant diploid is cultivated in nutrient medium to induce loss of the split chromosome originating from either of the strains. We predicted that some of these clones that are hemizygous for the split chromosome would spontaneously restore a homozygous configuration of the split chromosome during cultivation. We verified this prediction by tetrad analysis and quantitative Southern hybridization analysis, indicating that it is possible to create diploid hybrids in which a selected region of a chromosome from one strain is replaced by the corresponding chromosomal region from another strain. We also found that some chromosomal segments maintain a hemizygous state. This novel technique, which we call ‘chromosome shuffling’, could provide a new tool to analyze phenotypic alterations caused by the replacement or hemizygosity of a selected chromosomal region in not only laboratory but also industrial strains of S. cerevisiae.     

Transportome-wide engineering of Saccharomyces cerevisiae

Synthetic biology enables the production of small molecules by recombinant microbes for pharma, food, and materials applications. The secretion of products reduces the cost of separation and purification, but it is challenging to engineer due to the limited understanding of the transporter proteins' functions. Here we describe a method for genome-wide transporter disruption that, in combination with a metabolite biosensor, enables the identification of transporters impacting the production of a given target metabolite in yeast Saccharomyces cerevisiae. We applied the method to study the transport of xenobiotic compounds, cis,cis-muconic acid (CCM), protocatechuic acid (PCA), and betaxanthins. We found 22 transporters that influenced the production of CCM or PCA. The transporter of the 12-spanner drug:H(+) antiporter (DHA1) family Tpo2p was further confirmed to import CCM and PCA in Xenopus expression assays. We also identified three transporter proteins (Qdr1p, Qdr2p, and Apl1p) involved in betaxanthins transport. In summary, the described method enables high-throughput transporter identification for small molecules in cell factories.     

The role of metabolic engineering in the improvement of Saccharomyces cerevisiae: utilization of industrial media

Metabolic engineering has become a very important approach to strain improvement in parallel with classical strain development. Although Saccharomyces cerevisiae has been domesticated for ethanol and bread production, there are still some fundamental problems associated with its industrial use. The industrially used carbon sources often consist of a sugar mixture, and due to glucose repression these sugars are utilized sequentially, resulting in prolonged production time. In this article we discuss the application of metabolic engineering for construction of glucose-derepressed strains and specify advantages as well as difficulties associated with this approach.     

Optimization of ethanol production in Saccharomyces cerevisiae by metabolic engineering of the ammonium assimilation

Ethanol is still one of the most important products originating from the biotechnological industry with respect to both value and amount. In addition to ethanol, a number of byproducts are formed during an anaerobic fermentation of Saccharomyces cerevisiae. One of the most important of these compounds, glycerol, is produced by yeast to reoxidize NADH, formed in synthesis of biomass and secondary fermentation products, to NAD+. The purpose of this study was to evaluate whether a reduced formation of surplus NADH and an increased consumption of ATP in biosynthesis would result in a decreased glycerol yield and an increased ethanol yield in anaerobic cultivations of S. cerevisiae. A yeast strain was constructed in which GLN1, encoding glutamine synthetase, and GLT1, encoding glutamate synthase, were overexpressed, and GDH1, encoding the NADPH-dependent glutamate dehydrogenase, was deleted. Hereby the normal NADPH-consuming synthesis of glutamate from ammonium and 2-oxoglutarate was substituted by a new pathway in which ATP and NADH were consumed. The resulting strain TN19 (gdh1-A1 PGK1p-GLT1 PGK1p-GLN1) had a 10% higher ethanol yield and a 38% lower glycerol yield compared to the wild type in anaerobic batch fermentations. The maximum specific growth rate of strain TN19 was slightly lower than the wild-type value, but earlier results suggest that this can be circumvented by increasing the specific activities of Gln1p and Glt1p even more. Thus, the results verify the proposed concept of increasing the ethanol yield in S. cerevisiae by metabolic engineering of pathways involved in biomass synthesis.     

代谢工程改造酿酒酵母合成肌醇

【目的】肌醇别名环己六醇,是一种具有生物活性的糖醇,在医药、食品和饲料等领域具有重要的应用价值。为获得生产肌醇的微生物细胞工厂,通过代谢工程改造,构建生产肌醇的酿酒酵母工程菌株。【方法】对酿酒酵母肌醇合成途径的正负调控同时改造,过表达肌醇-3-磷酸合成酶基因ino1,敲除肌醇生物合成的转录抑制子基因opi1和抗性基因kan MX,获得重组菌。利用气相色谱法检测重组菌发酵液中肌醇含量。【结果】构建了生物安全性的产肌醇基因工程菌株,摇瓶培养产量为1.021 g/L。【结论】通过过表达ino1和敲除opi1来改造酿酒酵母,能够有效提高重组菌的肌醇产量,为下一步的微生物发酵法产肌醇的工业应用奠定基础。