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。     

非常规酵母天然产物合成

以解脂耶氏酵母(Yarrowia lipolytica)、巴斯德毕赤酵母(Pichia pastoris)、马克斯克鲁维酵母(Kluyveromyces marxianus)、圆红冬孢酵母(Rhodosporidium toruloides)、多形汉逊酵母(Hansenula polymorpha)为代表的非常规酵母凭借较广的底物利用谱、较强的环境耐受性等优势,已成功实现多种天然产物的高效生产。随着合成生物学及基因编辑技术的发展,针对非常规酵母代谢工程改造的工具和策略也逐渐丰富。本文介绍了几类常见的非常规酵母的生理特性、工具开发及应用现状,并总结归纳了天然产物合成优化中常用的代谢工程策略;最后讨论了现阶段非常规酵母作为天然产物合成细胞工厂的优势和不足,并对后续研究和发展趋势进行了展望。     

多形汉逊酵母启动子的挖掘

多形汉逊酵母可以利用甲醇作为唯一的碳源和能量来源,是构建微生物细胞工厂的潜在宿主,在代谢工程改造和重组蛋白生产中引起了广泛关注.在合成生物学研究和代谢工程改造过程中,通常需要改变相关基因的转录水平来调节代谢通量,而这一过程需要借助不同种类、不同表达强度的启动子来实现.因此,对汉逊酵母糖酵解途径和活性氧(ROS)防御途径...     

历久弥新：进化中的代谢工程

20世纪90年代,Bailey及Stephanopoulos等提出了经典代谢工程的理念,旨在利用DNA重组技术对代谢网络进行改造,以达到细胞性能改善,目标产物增加的目的。自代谢工程诞生以来的30年,生命科学蓬勃发展,基因组学、系统生物学、合成生物学等新学科不断涌现,为代谢工程的发展注入了新的内涵与活力。经典代谢工程研究已进入到前所未有的系统代谢工程阶段。组学技术、基因组代谢模型、元件组装、回路设计、动态控制、基因组编辑等合成生物学工具与策略的应用,大大提升了复杂代谢的设计与合成能力;机器学习的介入以及进化工程与代谢工程的结合,为系统代谢工程的未来开辟了新的方向。文中对过去30年代谢工程的发展趋势作了梳理,介绍了代谢工程在发展中不断创新的理论与方法及其应用。     

嘌呤核苷及其衍生物的代谢工程

嘌呤核苷及其衍生物被广泛应用于食品和医药领域。利用诱变筛选技术可以获得嘌呤核苷类产品的工业生产菌株,但往往耗时,效率低,而且获得的某些高产菌株还存在不稳定的缺陷。菌株代谢调控与生理生化的研究为代谢工程优化嘌呤核苷类产品的合成提供了理论基础,利用代谢工程改造菌株合成嘌呤核苷也引起了研究人员的关注。系统地介绍了微生物嘌呤生物合成途径及其调控机制,综述了嘌呤核苷类产品及其衍生物的代谢工程研究进展,最后讨论了利用代谢工程改造菌株合成这些产品面临的问题及今后的研究方向。     

解脂耶氏酵母表达调控工具的开发及天然产物合成的研究进展

解脂耶氏酵母是一种重要的产油酵母,由于其能利用多种疏水性底物,具有良好的耐酸、耐盐等胁迫耐受性,具有高通量的三羧酸循环,可提供充足的乙酰辅酶A前体等特点,被认为是生产萜类、聚酮类和黄酮类等天然产物的理想宿主,在代谢工程领域有着广泛的应用。近年来,越来越多的基因编辑、表达和调控工具被逐渐开发,这促进了解脂耶氏酵母合成各种天然产物的研究。文中综述了近年来解脂耶氏酵母中基因表达和天然产物合成方面的研究进展,并探讨了在该酵母中异源合成天然产物所面临的挑战和可能的解决方案。     

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。     

合成生物制造

本文对2021年《生物工程学报》在合成生物制造领域发表的综述和论文进行了评述.重点讨论了大肠杆菌、枯草芽孢杆菌、谷氨酸棒杆菌、酿酒酵母、丝状真菌以及非模式细菌、非传统酵母等主要底盘细胞的设计改造,氨基酸、有机酸、维生素、高级醇、天然化合物(萜类、黄酮类、生物碱类)、抗生素类、酶与生物催化产品、生物聚合物等产品的生物制造...     

合成生物学开关在代谢工程中的应用

在代谢工程研究领域中合成生物学开关主要用于调控基因的表达。传统的代谢工程改造主要通过敲除和过表达来改变特定基因的表达量。但基因敲除通常会导致菌体生长的下降。因此,我们需要适时的关闭和激活特定基因的表达。合成生物学开关就是解决这一问题的关键工具。目前,在代谢工程中常用的合成生物学开关有光控开关、温度诱导开关、拨动开关和核糖开关。其中,拨动开关和核糖开关在动态调节基因表达上拥有更大优势。介绍了代谢工程中常用的几种合成生物学开关,以及它们在代谢工程的应用。     

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。     

The yeast Zygosaccharomyces bailii: a new host for heterologous protein production, secretion and for metabolic engineering applications

Molecular tools for the production of heterologous proteins and metabolic engineering applications of the non-conventional yeast Zygosaccharomyces bailii were developed. The combination of Z. bailii's resistance to relatively high temperature, osmotic pressure and low pH values, with a high specific growth rate renders this yeast potentially interesting for exploitation for biotechnological purposes as well as for the understanding of the biological phenomena and mechanisms underlying the respective resistances. Looking forward to these potential applications, here we present the tools required for the production and the secretion of different heterologous proteins, and one example of a metabolic engineering application of this non-conventional yeast, employing the newly developed molecular tools.     

Metabolic engineering for the utilization of carbohydrate portions of lignocellulosic biomass

The petrochemical industry has grown to meet the need for massive production of energy and commodities along with an explosive population growth; however, serious side effects such as greenhouse gas emissions and global warming have negatively impacted the environment. Lignocellulosic biomass with myriad quantities on Earth is an attractive resource for the production of carbon-neutral fuels and chemicals through environmentally friendly processes of microbial fermentation. This review discusses metabolic engineering efforts to achieve economically feasible industrial production of fuels and chemicals from microbial cell factories using the carbohydrate portion of lignocellulosic biomass as substrates. The combined knowledge of systems biology and metabolic engineering has been applied to construct robust platform microorganisms with maximum conversion of monomeric sugars, such as glucose and xylose, derived from lignocellulosic biomass. By comprehensively revisiting carbon conversion pathways, we provide a rationale for engineering strategies, as well as their features, feasibility, and recent representative studies. In addition, we briefly discuss how tools in systems biology can be applied in the field of metabolic engineering to accelerate the development of microbial cell factories that convert lignocellulosic biomass into carbon-neutral fuels and chemicals with economic feasibility.     

A systems-level approach for metabolic engineering of yeast cell factories

The generation of novel yeast cell factories for production of high-value industrial biotechnological products relies on three metabolic engineering principles: design, construction, and analysis. In the last two decades, strong efforts have been put on developing faster and more efficient strategies and/or technologies for each one of these principles. For design and construction, three major strategies are described in this review: (1) rational metabolic engineering; (2) inverse metabolic engineering; and (3) evolutionary strategies. Independent of the selected strategy, the process of designing yeast strains involves five decision points: (1) choice of product, (2) choice of chassis, (3) identification of target genes, (4) regulating the expression level of target genes, and (5) network balancing of the target genes. At the construction level, several molecular biology tools have been developed through the concept of synthetic biology and applied for the generation of novel, engineered yeast strains. For comprehensive and quantitative analysis of constructed strains, systems biology tools are commonly used and using a multi-omics approach. Key information about the biological system can be revealed, for example, identification of genetic regulatory mechanisms and competitive pathways, thereby assisting the in silico design of metabolic engineering strategies for improving strain performance. Examples on how systems and synthetic biology brought yeast metabolic engineering closer to industrial biotechnology are described in this review, and these examples should demonstrate the potential of a systems-level approach for fast and efficient generation of yeast cell factories.     

Application of the Cre-loxP system for multiple gene disruption in the yeast Kluyveromyces marxianus

The yeast Kluyveromyces marxianus presents several interesting features that make this species a promising industrial yeast for the production of several compounds. In order to take full advantage of this yeast and its particular properties, proper tools for gene disruption and metabolic engineering are needed. The Cre-loxP system is a very versatile tool that allows for gene marker rescue, resulting in mutant strains free of exogenous selective markers, which is a very important aspect for industrial application. As the Cre-loxP system works in some non-conventional yeasts, namely Kluyveromyces lactis, we wished to know whether it also works in K. marxianus. Here, we report the validation of this system in K. marxianus CBS 6556, by disrupting two copies of the LAC4 gene, which encodes a beta-galactosidase activity.     

植物源二萜类化合物微生物合成研究进展

植物源二萜类天然产物结构复杂且功能多样,具有抗癌、抗炎和抗菌等多种药理活性,在药品、化妆品和食品添加剂等方面广泛应用。近年来,基于植物源二萜类化合物(diterpenoids)生物合成途径中功能基因的逐步揭示和合成生物技术的发展,科研人员采用代谢工程技术构建了多种二萜类化合物的微生物细胞工厂,且多个化合物达到克级产量。本文对植物源二萜类化合物微生物细胞工厂的构建情况进行综述,介绍并探讨植物源二萜类化合物微生物合成的研究进展和改造策略,为高产二萜类化合物细胞工厂构建和工业化生产提供参考。     

Lactobacilli and pediococci as versatile cell factories – Evaluation of strain properties and genetic tools

This review discusses opportunities and bottlenecks for cell factory development of Lactic Acid Bacteria (LAB), with an emphasis on lactobacilli and pediococci, their metabolism and genetic tools. In order to enable economically feasible bio-based production of chemicals and fuels in a biorefinery, the choice of product, substrate and production organism is important. Currently, the most frequently used production hosts include Escherichia coli and Saccharomyces cerevisiae, but promising examples are available of alternative hosts such as LAB. Particularly lactobacilli and pediococci can offer benefits such as thermotolerance, an extended substrate range and increased tolerance to stresses such as low pH or high alcohol concentrations. This review will evaluate the properties and metabolism of these organisms, and provide an overview of their current biotechnological applications and metabolic engineering. We substantiate the review by including experimental results from screening various lactobacilli and pediococci for transformability, growth temperature range and ability to grow under biotechnologically relevant stress conditions. Since availability of efficient genetic engineering tools is a crucial prerequisite for industrial strain development, genetic tool development is extensively discussed. A range of genetic tools exist for Lactococcus lactis, but for other species of LAB like lactobacilli and pediococci such tools are less well developed. Whereas lactobacilli and pediococci have a long history of use in food and beverage fermentation, their use as platform organisms for production purposes is rather new. By harnessing their properties such as thermotolerance and stress resistance, and by using emerging high-throughput genetic tools, these organisms are very promising as versatile cell factories for biorefinery applications.     

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。