解脂耶氏酵母作为微生物细胞工厂的应用研究进展

解脂耶氏酵母（Yarrowia lipolytica）是一种重要的工业微生物菌种，被公认为食品级安全微生物。近年来，随着合成生物学和基因编辑技术的快速发展，科学家们利用合成生物学及基因编辑技术已经成功构建出了能够生产生物化学品、生物燃料、香料、药物、工业酶和药用蛋白等多种高附加值工业产品的解脂耶氏酵母细胞工厂，使得该酵母在食品、药品和生化能源等领域均具有巨大的应用潜力。本文将重点介绍解脂耶氏酵母表达系统、合成生物学元件和基因编辑方法的最新研究进展和应用情况，并对近年来以解脂耶氏酵母作为微生物细胞工厂生产高附加值产品的应用实例进行总结，希望为研究人员进一步利用解脂耶氏酵母进行底盘细胞设计、构建和优化相关合成途径并最终实现目的产物的高效合成提供有用的信息。     

微生物合成乳酸的细胞工厂构建研究进展

乳酸目前广泛应用于食品、医疗和化妆品行业,并因其作为可生物降解塑料聚乳酸的原料而备受关注。微生物发酵合成乳酸具有光学纯度高和底物成本低等优点,因此,研究人员利用合成生物学和代谢工程技术构建了新型微生物细胞工厂,以实现乳酸的高效合成。在此过程中,代谢通路的构建与优化、氧化还原平衡的调节、底物谱的拓宽以及细胞工厂鲁棒性的提高等方面是需要考虑的关键问题。文章详细介绍了微生物合成乳酸的细胞工厂构建研究进展,并对当前面临的挑战和未来的研究方向进行了探讨,旨在为工业微生物高效合成乳酸提供借鉴。     

微生物利用无机氮生产有机氮

在追求美好生活的新时代,全社会对食用蛋白的需求日益多元化、差异化和个性化。人口持续增长和生活水平的不断提升,迫切需要开发大规模、低成本、可持续、高质量的蛋白生产方式。随着合成生物学、代谢工程等新兴生物技术的发展,工程化改造微生物细胞,构建微生物细胞工厂,以可再生资源为原料,绿色高效合成氨基酸和微生物蛋白,为高品质蛋白可持续供给提供一条有前景的绿色发展路线。本文综述向自然界要铵,向微生物要氨基酸,向微生物要蛋白的研究进展,为将来更好地利用微生物细胞工厂生产氨基酸与蛋白质提供参考。     

谷氨酸棒杆菌细胞工厂构建与应用的研究进展

谷氨酸棒杆菌作为工业生产氨基酸的一种核心菌株,已被开发成为能利用可再生原料生产多种有价值的天然或非天然化学品的多功能微生物细胞工厂。该文针对近年来谷氨酸棒杆菌细胞工厂的构建新技术与应用领域,首先综述了构建谷氨酸棒杆菌细胞工厂的新型诱变、高通量筛选及基因编辑技术,其次介绍了工程改造后的微生物细胞工厂高效合成有机酸、生物燃料和萜类化合物的应用,最后列举了高效的微生物细胞工厂利用可再生原料生产高附加值产品的经典实例,以期为扩展谷氨酸棒杆菌产物谱和底物谱的研究提供参考。     

人溶菌酶基因在乳酸克鲁维酵母中的表达

人溶菌酶是一种天然广谱抑菌物质,在食品和医药工业有潜在应用前景。为获得高活性的人溶菌酶制剂,采用乳酸克鲁维酵母表达系统,对经密码子优化的人溶菌酶基因(h LYZ)进行分泌表达。将人工合成h LYZ插入到乳酸克鲁维酵母表达载体p KLAC1,构建重组载体p KLAC1-h LYZ,并用电脉冲法将SacⅡ线性化的重组质粒转化到乳酸克鲁维酵母GG799中。通过全细胞PCR鉴定,最后获得了一株多拷贝整合的基因工程菌h LYZ1。工程菌可以分泌表达分子量约14 ku的目的蛋白质,与预期大小相符。摇瓶发酵培养128 h,酶活最高达到1430 U/mL。抗菌活性检测结果显示,重组人溶菌酶对溶壁微球菌、大肠杆菌和枯草芽孢杆菌有较好的溶菌活性。本研究成功地在乳酸克鲁维酵母中表达了重组人溶菌酶,表达的蛋白具有较高的酶活性,试验结果为利用乳酸克鲁维酵母表达系统规模化生产重组人溶菌酶奠了基础。     

代谢工程改造微生物合成单萜芳香产品的研究进展

单萜及其衍生物是重要的植物天然产物,且具有多种生物学功能。该类物质在多个领域中均表现出较高的开发利用价值,目前已被作为优质香精香料广泛应用于食品、饮料、化妆品和医药工业中,市场需求日益增长。从植物中提取这些单萜芳香产品存在着来源少、含量低和分离困难等缺点,很难满足市场需求。因此,开发生产单萜芳香产品可再生的微生物资源来补充甚至代替原有的植物资源就具有重要的理论意义和应用价值。近年来,研究人员利用代谢工程技术已经成功构建了合成单萜芳香产品的微生物细胞工厂,达到了利用微生物合成法生产该类工业产品的目的。本文主要从菌株改造、发酵优化及产物分离等角度总结了相关产物合成的代谢工程实例,并分析了目前利用代谢工程改造微生物合成单萜芳香产品所面临的瓶颈问题及其可能的解决方法,旨在为构建异源、廉价、高效生产单萜芳香产品的微生物细胞工厂并最终实现其绿色制造提供参考。     

渗透乳酸克鲁维酵母细胞方法与工艺的研究

研究了渗透乳酸克鲁维酵母细胞生产透性化细胞乳糖酶的方法,并确定了得到最大酶活的工艺条件。     

外源接菌对发酵辣椒微生物群落和挥发性风味化合物的影响

利用乳酸克鲁维酵母Km和植物乳杆菌QB3制备了多种发酵辣椒制品，理化指标分析发现QB3发酵辣椒消耗最多还原糖，导致发酵液的pH值下降最快，并且使短链脂肪酸和VA含量分别提高了62.5%和13.8%。发酵辣椒中微生物多样性和挥发性代谢组分析表明，接种QB3发酵具有生长优势并降低了微生物的α多样性；发酵辣椒中共检出73种挥发性化合物，46种挥发性化合物可能对风味具有贡献，相关性分析表明148种微生物可能参与71种化合物的合成，植物乳杆菌和乳酸克鲁维酵母可能参与26种挥发性化合物的合成，并且乳酸克鲁维酵母是风味物质形成的关键，而挥发性化合物的合成多数与植物乳杆菌呈负相关。该研究结果为通过接种乳酸菌和酵母菌发酵制备多种风味发酵辣椒制品提供理论依据和技术支撑，为发酵食品风味的多元化提供参考。     

米曲霉乳糖酶基因在乳酸克鲁维酵母中的表达

为获得高活性的乳糖酶制剂,根据米曲霉乳糖酶基因序列以及宿主细胞乳酸克鲁维酵母GG799密码子的偏爱性,对米曲霉乳糖酶基因进行优化。将已优化的米曲霉乳糖酶lacA基因片段插入到乳酸克鲁维酵母GG799表达载体pKLAC1中,构建重组载体p KLAC1-lacA,用电脉冲法将SacⅡ酶线性化的重组质粒转入乳酸克鲁维酵母GG799中。经过5 mmol/L酵母碳基础培养基(yeast carbon base,YCB)活性筛选,全细胞聚合酶链式反应(polymerase chain reaction,PCR)鉴定,最后获得了一株多拷贝整合的基因工程菌株lacA-1。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gelelectrophoresis,SDS-PAGE)和薄层层析分析(thin-layer chromatography,TLC),该重组菌株可胞外分泌乳糖酶,并具有生物活性。摇瓶发酵培养120 h,酶活力最高达到120.65 U/mL。本研究成功地将米曲霉乳糖酶基因在乳酸克鲁维酵母GG799中表达,获得的重组菌株lacA-1可胞外分泌的乳糖酶并有较高的乳糖酶活性,试验结果为利用乳酸克鲁维酵母GG799表达系统规模化生产乳糖酶奠定基础。     

酿酒酵母异源合成柠檬烯的研究进展

植物萜类化合物是以异戊二烯为结构单位的一大类植物天然的次生代谢产物。柠檬烯属于单萜类化合物,具有抑菌、增香、抗癌、止咳、平喘等多种功能,因此在食品、药品、化妆品、医疗等领域具有广泛的应用前景。目前柠檬烯的生产主要是从植物中提取,受到季节性原材料、产物分离纯化复杂、产率低等因素的限制,而化学合成又存在能耗高、污染严重等缺点。随着合成生物学技术的兴起,诞生了以微生物生物合成法生产柠檬烯的新方法,该方法具有能耗低、绿色环保、可持续等优势。然而微生物法合成柠檬烯也存在低产量、低效率等问题,这就限制了其商业化,因此构建高效的异源合成柠檬烯微生物细胞工厂,实现微生物发酵法替换传统的植物提取法,具有重要的经济与社会效益。本文主要回顾了近几年利用代谢工程改造酿酒酵母异源合成柠檬烯取得的成就,阐述了以酿酒酵母作为底盘微生物,利用代谢工程和合成生物学的手段构建高产柠檬烯的合成策略,还讨论了如何减轻柠檬烯对宿主细胞的毒性和提高宿主对柠檬烯的耐受性。     

The renaissance of yeasts as microbial factories in the modern age of biomanufacturing

Yeasts are essential for many processes, including the production of some of our most beloved foods and beverages. Less is known to the public about the far-reaching impacts of yeasts on other products such as biofuels, pharmaceuticals, and industrial products. By leveraging and combining newly discovered yeast genetic diversity with now affordable and efficient genetic engineering and synthetic biology tools, academic and industrial yeast labs have designed yeast cell factories for a wide range of novel applications such as the production of medicines, components of human breast milk, heme for meat substitutes, bioplastics, and other biomaterials. This review covers the newest technologies developed for yeast research including synthetic biology and their use in the engineering of yeast cell factories for emerging applications.     

Uses of biotechnology and technology transfer to keep food safe

The era of biology is composed of 1) the definition of molecular laws of biology, 2) the exponential expansion of the data base, and 3) the establishment of the first generation molecular and cellular tool kit; this era is driving the development and commercialization of biotechnological products and processes for agriculture and the food system. These products and processes should have a major impact in maintaining and improving food safety. Several meeting and organizational initiatives on biotechnology and food safety are summarized. Possible roles of biotechnology in areas of food safety involve microbial contaminants, nutritional quality, natural antimetabolites, allergens, toxicants, and synthetic chemical residues. Biotechnology will have an impact on all these areas through both improved ability to measure as well as to modify microbes, animals, and plants used as food. Diagnostics for microbial contaminants and biobased alternatives to synthetic chemicals are most advanced. However, all these biotechnological products and processes for food safety are in very early stages of development and commercialization.     

Microencapsulation of microbial cells

Microencapsulation involves coating or entrapping of a core material with a polymeric material to generate microspheres in the size range of 1–1000 μm. This versatile technology has been used to encapsulate a wide array of products such as pharmaceuticals, flavors, volatile oils, plant extracts, enzymes and others. In the recent decades, this technology has also been applied to the area of microbial cell immobilization owing to its numerous advantages over other cell immobilization techniques such as higher cell loading capacity, enhanced cell survival and increased production rate of the desired microbial products. The confinement of microbial cells within a semipermeable polymeric matrix enables the physical isolation of cells from the external environment while maintaining a hospitable internal micro-environment. It has found application in various biotechnological processes such as probiotic encapsulation in food industries, in biotransformation and fermentation processes producing antibiotics, organic acids, enzymes, and alcohols as well as environmental decontamination such as waste water treatment. The judicious selection of materials and methods for the production of microspheres is critical for ensuring minimum damage to the viability of the encapsulated microbial cells. The conventional methods used for microencapsulation of microbial cells are reviewed along with the recent advances in the respective methods. The effect of microencapsulation on the microbial cells, the stability of the microspheres as well as the techniques for enumeration of the encapsulated cells are also discussed, followed by a summary of recent applications of microencapsulation in different biotechnological processes.     

微生物脂肪酶的应用

微生物脂肪酶主要来源于真菌和细菌 ,它们不但能催化脂水解反应而且能催化长链脂肪酸甘油酯的合成反应 ,并且具有高底物专一性、区域选择性和对映选择性 ,被广泛应用于油脂、食品、医药、化妆品、洗涤剂、香料和其它有机合成领域。本文综述了脂肪酶在油脂、食品等诸多工业领域的应用。     

Cell factory applications of the yeast Kluyveromyces marxianus for the biotechnological production of natural flavour and fragrance molecules

Kluyveromyces marxianus is emerging as a new platform organism for the production of flavour and fragrance (F&F) compounds. This food‐grade yeast has advantageous traits, such as thermotolerance and rapid growth, that make it attractive for cell factory applications. The major impediment to its development has been limited fundamental knowledge of its genetics and physiology, but this is rapidly changing. K. marxianus produces a wide array of volatile molecules and contributes to the flavour of a range of different fermented beverages. Advantage is now being taken of this to develop strains for the production of metabolites such as 2‐phenylethanol and ethyl acetate. Strains that were selected from initial screens were used to optimize processes for production of these F&F molecules. Most developments have focused on optimizing growth conditions and the fermentation process, including product removal, with future advancement likely to involve development of new strains through the application of evolutionary or rational engineering strategies. This is being facilitated by new genomic and molecular tools. Furthermore, synthetic biology offers a route to introduce new biosynthetic pathways into this yeast for F&F production. Consumer demand for biologically‐synthesized molecules for use in foods and other products creates an opportunity to exploit the unique potential of K. marxianus for this cell factory application. Copyright © 2014 John Wiley & Sons, Ltd.     

Xylitol: A Review on Bioproduction,Application, Health Benefits,and Related Safety Issues

Xylitol is a pentahydroxy sugar-alcohol which exists in a very low quantity in fruits and vegetables (plums, strawberries, cauliflower, and pumpkin). On commercial scale, xylitol can be produced by chemical and biotechnological processes. Chemical production is costly and extensive in purification steps. However, biotechnological method utilizes agricultural and forestry wastes which offer the possibilities of economic production of xylitol by reducing required energy. The precursor xylose is produced from agricultural biomass by chemical and enzymatic hydrolysis and can be converted to xylitol primarily by yeast strain. Hydrolysis under acidic condition is the more commonly used practice influenced by various process parameters. Various fermentation process inhibitors are produced during chemical hydrolysis that reduce xylitol production, a detoxification step is, therefore, necessary. Biotechnological xylitol production is an integral process of microbial species belonging to Candida genus which is influenced by various process parameters such as pH, temperature, time, nitrogen source, and yeast extract level. Xylitol has application and potential for food and pharmaceutical industries. It is a functional sweetener as it has prebiotic effects which can reduce blood glucose, triglyceride, and cholesterol level. This review describes recent research developments related to bioproduction of xylitol from agricultural wastes, application, health, and safety issues.     

Vanillin biotechnology: the perspectives and future

The biotechnological production of fragrances is a recent trend that has expanded rapidly in the last two decades. Vanillin is the second most popular flavoring agent after saffron and is extensively used in various applications, e.g., as a food additive in food and beverages and as a masking agent in various pharmaceutical formulations. It is also considered a valuable product for other applications, such as metal plating and the production of other flavoring agents, herbicides, ripening agents, antifoaming agents, and personal and home-use products (such as in deodorants, air fresheners, and floor-polishing agents). In general, three types of vanillin, namely natural, biotechnological, and chemical/synthetic, are available on the market. However, only natural and nature-identical (biotechnologically produced from ferulic acid only) vanillins are considered as food-grade additives by most food-safety control authorities worldwide. In the present review, we summarize recent trends in fermentation technology for vanillin production and discuss the importance of the choice of raw materials for the economically viable production of vanillin. We also describe the key enzymes used in the biotechnological production of vanillin as well as their underlying genes. Research to advance our understanding of the molecular regulation of different pathways involved in vanillin production from ferulic acid is still ongoing. The enhanced knowledge is expected to offer new opportunities for the application of metabolic engineering to optimize the production of nature-identical vanillin. © 2018 Society of Chemical Industry     

微生物利用一碳底物生产单细胞蛋白研究进展

世界人口的持续增长导致肉类、乳制品等高蛋白食品的需求大幅增加，给我国食品蛋白的供应带来了较大挑战。微生物能够利用二氧化碳、甲烷、甲醇等一碳化合物生产高质量的单细胞蛋白，这种新型蛋白可应用于食品工业。建立微生物蛋白绿色生物制造的食品蛋白生产体系，对保障国家食物蛋白供给安全十分重要。此外，微生物转化一碳化合物制备单细胞蛋白的过程还可以减少碳排放、缓解温室效应，实现可持续发展。本文主要总结微生物单细胞蛋白在食品工业中的应用；论述近年来微生物利用一碳化合物高效生产单细胞蛋白的研究进展；阐述天然一碳利用微生物的代谢网络机制以及改造前景；展望了利用合成生物学改造微生物从一碳底物生产单细胞蛋白的前景，旨在为微生物单细胞蛋白的商业化生产提供思路。