体外多酶分子机器的现状和最新进展

体外多酶分子机器遵循所设计的多酶催化路径,将若干种纯化或部分纯化的酶元件进行合理的优化与适配,高效地在体外将特定的底物转化为目标化合物。体外多酶分子机器反应系统呈现元件化和模块化的特点,在设计、组装和调控方面具有较高的自由度。近年来,体外多酶分子机器在实现反应过程的精准调控和提高产品得率方面的优势逐渐体现,展示了其在生物制造领域重要的应用潜力。对体外多酶分子机器的相关研究已成为合成生物学的一个重要分支领域,日益受到广泛的关注。文中系统地综述了基于酶元件/模块的体外多酶分子机器的构建策略,以及改善该分子机器中酶元件/模块之间适配性的研究进展,并分析了该生物制造平台的发展前景与挑战。     

Non-Complexed Four Cascade Enzyme Mixture: Simple Purification and Synergetic Co-stabilization

Cell-free biosystems comprised of synthetic enzymatic pathways would be a promising biomanufacturing platform due to several advantages, such as high product yield, fast reaction rate, easy control and access, and so on. However, it was essential to produce (purified) enzymes at low costs and stabilize them for a long time so to decrease biocatalyst costs. We studied the stability of the four recombinant enzyme mixtures, all of which originated from thermophilic microorganisms: triosephosphate isomerase (TIM) from Thermus thermophiles, fructose bisphosphate aldolase (ALD) from Thermotoga maritima, fructose bisphosphatase (FBP) from T. maritima, and phosphoglucose isomerase (PGI) from Clostridium thermocellum. It was found that TIM and ALD were very stable at evaluated temperature so that they were purified by heat precipitation followed by gradient ammonia sulfate precipitation. In contrast, PGI was not stable enough for heat treatment. In addition, the stability of a low concentration PGI was enhanced by more than 25 times in the presence of 20 mg/L bovine serum albumin or the other three enzymes. At a practical enzyme loading of 1000 U/L for each enzyme, the half-life time of free PGI was prolong to 433 h in the presence of the other three enzymes, resulting in a great increase in the total turn-over number of PGI to 6.2×10⁹ mole of product per mole of enzyme. This study clearly suggested that the presence of other proteins had a strong synergetic effect on the stabilization of the thermolabile enzyme PGI due to in vitro macromolecular crowding effect. Also, this result could be used to explain why not all enzymes isolated from thermophilic microorganisms are stable in vitro because of a lack of the macromolecular crowding environment.     

工业应用导向的蛋白质结构与功能研究进展

在蛋白质工程、绿色生物制造以及合成生物学等研究领域中,对重要催化反应的重塑和合成路径的优化搭建,都依赖于对相关蛋白质结构与功能的深入了解。合成生物技术近年来的飞速发展对关键菌种及生物催化过程中的蛋白质的性能提出了更高要求,相关研究的关键是获得大批量、高纯度目的蛋白,并进行快速、准确的构效关系研究。中国科学院天津工业生物技术研究所建所10年来,在工业蛋白质领域进行了多年的积累,成功搭建成了蛋白质结构生物学平台;并在植物天然产物合成相关萜类合成酶、白色污染降解的聚对苯二甲酸乙二酯(polyethylene terephthalate, PET)塑料降解酶以及生物质转化利用相关酶等方面获得了一些进展,通过对这些蛋白进行结构和功能的研究,为许多研究工作提供了理论依据。蛋白质结构功能研究相关技术的不断发展,将加速合成生物学的学术和工业应用研究,推动我国生物制造领域的科技创新升级。     

甲醇生物转化合成化学品的研究进展

甲醇来源丰富、价格低廉,已成为生物制造行业极具吸引力的底物之一。构建微生物细胞工厂实现甲醇到增值化学品的生物转化,具有过程绿色、条件温和、产品体系多样等优势,不仅能拓展基于甲醇的产品链,还能缓解当前生物制造“与民争粮、与粮争地”的问题,是实现绿色生物制造的重要手段。因此,阐明不同天然甲基营养菌中涉及甲醇氧化、甲醛同化和异化途径对于后续基因工程改造工作至关重要,也更有利于构建新型非天然甲基营养菌。本文讨论了甲基营养菌中甲醇代谢途径的研究现状,并结合近年来天然和人工合成甲基营养菌在甲醇生物转化中的应用进展及面临的挑战。     

Bioreactor systems for in vitro production of foreign proteins using plant cell cultures

Plant cells have been demonstrated to be an attractive heterologous expression host (using whole plants and in vitro plant cell cultures) for foreign protein production in the past 20years. In recent years in vitro liquid cultures of plant cells in a fully contained bioreactor have become promising alternatives to traditional microbial fermentation and mammalian cell cultures as a foreign protein expression platform, due to the unique features of plant cells as a production host including product safety, cost-effective biomanufacturing, and the capacity for complex protein post-translational modifications. Heterologous proteins such as therapeutics, antibodies, vaccines and enzymes for pharmaceutical and industrial applications have been successfully expressed in plant cell culture-based bioreactor systems including suspended dedifferentiated plant cells, moss, and hairy roots, etc. In this article, the current status and emerging trends of plant cell culture for in vitro production of foreign proteins will be discussed with emphasis on the technological progress that has been made in plant cell culture bioreactor systems.     

Immobilized enzymes: crystals or carriers?

The advantages of immobilized over soluble enzymes arise from their enhanced stability and ease of separation from the reaction media, leading to significant savings in enzyme consumption. Immobilization methods range from binding to prefabricated carrier materials to packaging in enzyme crystals or powders. During their use, mass-transfer effects can produce substrate or pH gradients, which reduce the reaction rates and product yields. The costs of immobilized enzymes must be minimized in order to increase their competitiveness for technical applications.     

透明质酸的生物合成及其代谢工程的研究进展

透明质酸(hyaluronic acid,HA)是广泛存在于生物体内的功能性糖胺高分子聚合物,在日化、医疗和食品领域应用前景广阔.随着基因工程与代谢工程等合成生物学技术的发展,人们对HA的生物合成过程和机理解析越发深入的同时,也伴随一些新的挑战来临.该综述从分子生物学角度总结了HA的关键合成酶基因及合成途径,对不同来源...     

Metabolic engineering in dark fermentative hydrogen production; theory and practice

Dark fermentation is an attractive option for hydrogen production since it could use already existing reactor technology and readily available substrates without requiring a direct input of solar energy. However, a number of improvements are required before the rates and yields of such a process approach those required for a practical process. Among the options for achieving the required advances, metabolic engineering offers some powerful tools for remodeling microbes to increase product production rates and molar yields. Here we review the current metabolic engineering tool box that is available, discuss the current status of engineering efforts as applied to dark hydrogen production, and suggest areas for future improvements.     

Production of biocommodities and bioelectricity by cell‐free synthetic enzymatic pathway biotransformations: Challenges and opportunities

Cell‐free synthetic (enzymatic) pathway biotransformation (SyPaB) is the assembly of a number of purified enzymes (usually more than 10) and coenzymes for the production of desired products through complicated biochemical reaction networks that a single enzyme cannot do. Cell‐free SyPaB, as compared to microbial fermentation, has several distinctive advantages, such as high product yield, great engineering flexibility, high product titer, and fast reaction rate. Biocommodities (e.g., ethanol, hydrogen, and butanol) are low‐value products where costs of feedstock carbohydrates often account for ～30–70% of the prices of the products. Therefore, yield of biocommodities is the most important cost factor, and the lowest yields of profitable biofuels are estimated to be ca. 70% of the theoretical yields of sugar‐to‐biofuels based on sugar prices of ca. US$ 0.18 per kg. The opinion that SyPaB is too costly for producing low‐value biocommodities are mainly attributed to the lack of stable standardized building blocks (e.g., enzymes or their complexes), costly labile coenzymes, and replenishment of enzymes and coenzymes. In this perspective, I propose design principles for SyPaB, present several SyPaB examples for generating hydrogen, alcohols, and electricity, and analyze the advantages and limitations of SyPaB. The economical analyses clearly suggest that developments in stable enzymes or their complexes as standardized parts, efficient coenzyme recycling, and use of low‐cost and more stable biomimetic coenzyme analogs, would result in much lower production costs than do microbial fermentations because the stabilized enzymes have more than 3 orders of magnitude higher weight‐based total turn‐over numbers than microbial biocatalysts, although extra costs for enzyme purification and stabilization are spent. Biotechnol. Bioeng. 2010. 105: 663–677. © 2009 Wiley Periodicals, Inc.     

Mechanism and yields in enzyme catalysed equilibrium and kinetically controlled synthesis of β-lactam antibiotics,peptides and other condensation products

Hydrolases can be used to catalyse the synthesis of condensation products such as β-lactam antibiotics, peptides, oligosaccharides and glycerides. In biotechnological processes, synthesis to achieve maximum yields may be carried out either as an equilibrium controlled or kinetically controlled reaction. Only in the later case is the yield of condensation product influenced by the properties of the enzyme that act as a transferase in this reaction. With the same amount of enzyme the maximum yield is also obtained much more rapidly than in the equilibrium controlled process. Hydrolases with high ratios of transferase to hydrolase activity are favourable for use. Recent results on the mechanisms of enzyme catalysed condensations allow a rational analysis of how yield controlling factors (pH, temperature, ionic strength, enzyme and substrate properties) may be changed to obtain optimum yields. This can be used to evaluate whether these biotechnological processes can compete with the chemical methods currently used for the synthesis of these products. It can also be used to plan rational protein engineering of the enzymes that in kinetically controlled synthesis of the condensation products may give yields that can compete favourably with the existing chemical processes to produce these compounds.     

Non-sterile fermentations for the economical biochemical conversion of renewable feedstocks

Heavy reliance on petroleum-based products drives continuous exploitation of fossil fuels, and results in serious environmental and climate problems. To address such an issue, there is a shift from petroleum sources to renewable ones. Biochemical conversion via fermentation is a primary platform for converting renewable sources to biofuels and bulk chemicals. In order to provide cost-competitive alternatives, it is imperative to develop efficient, cost-saving, and robust fermentation processes. Non-sterile fermentation offers several benefits compared to sterile fermentation, including elimination of sterility, reduced maintenance requirements, relatively simple bioreactor design, and simplified operation. Thus, cost effectiveness of non-sterile fermentation makes it a practical platform for low cost, large volume production of biofuels and bulk chemicals. Many approaches have been developed to conduct non-sterile fermentation without sacrificing the yields and productivities of fermentation products. This review focuses on the strategies for conducting non-sterile fermentation. The challenges facing non-sterile fermentation are also discussed.     

系统生物科学与工程的高科技产业化

系统生物科学最早开创于贝塔郎菲的理论生物学和一般系统论。系统生物科学或生物系统科学研究分子、细胞、器官和群体各层次的生物系统,尤其是心智和遗传的信息系统。系统生物工程(曾邦哲1994年)采用实验分析和系统逻辑的双重方法论,探索改造和仿造生物系统的工程技术,包括:1)生化工程、生态工程,2)生物反应器、遗传工程,3)高分子传感器、仿生工程,4)生物遗传计算、智能工程等领域,将促进系统医学、转基因生物反应器、生物计算机、智能机器人和中医药现代化的发展。现代城市生态、工业网络的系统藕合、循环、再生的无污染、无烟化工业生态工程将带来自然、工业、人文之间的和谐。     

Solid-state (solid-substrate) food/beverage fermentations involving fungi

“Solid-substrate” fermentation developed in the Orient is a very useful fermentation method. It is presently used to produce a variety of foods, beverages and related products. Solid-substrate fermentation products utilizing fungi including soy sauce, miso and tempe, ontjom, sake, and bread have been produced for centuries at the home and village level. They are examples of economical methods of preserving and improving the flavor, texture and nutritive values of cereal/legume substrates. “Solid-substrate” fermentation is also applied to animal products such as milk to produce Roquefort and Camembert cheeses which diversify the food flavors available to man “Solid-substrate”fermentation has certain advantages. The substrate is concentrated; the product can be extracted with relatively small quantities of solvent; the product can be easily dehydrated; moisture level can be controlled favoring the desired organisms; enzyme concentration is generally higher than is submerged fermentation; product concentration is generally higher than in submerged cultures; it is the only technique that yields true mushroom fruiting bodies and it can be used not only for production of crude enzyme concentrates (koji) but also for raising the protein content of high starch substrates. It also can be used to increase the content of vitamins at low cost. Disadvantages of “solid-substrates”from the modern industrial processing view point are the greater difficulty of handling solid substrate and the greater difficulty of controlling the fermentation parameters, temperature, pH and oxygen, and rate of microbial growth compared with liquid submerged fermentations.     

Artificially designed routes for the conversion of starch to value-added mannosyl compounds through coupling in vitro and in vivo metabolic engineering strategies

Starch/cellulose has become the major feedstock for manufacturing biofuels and biochemicals because of their abundance and sustainability. In this study, we presented an artificially designed “starch-mannose-fermentation” biotransformation process through coupling the advantages of in vivo and in vitro metabolic engineering strategies together. Starch was initially converted into mannose via an in vitro metabolic engineering biosystem, and then mannose was fermented by engineered microorganisms for biomanufacturing valuable mannosyl compounds. The in vitro metabolic engineering biosystem based on phosphorylation/dephosphorylation reactions was thermodynamically favorable and the conversion rate reached 81%. The mannose production using whole-cell biocatalysts reached 75.4 g/L in a 30-L reactor, indicating the potential industrial application. Furthermore, the produced mannose in the reactor was directly served as feedstock for the fermentation process to bottom-up produced 19.2 g/L mannosyl-oligosaccharides (MOS) and 7.2 g/L mannosylglycerate (MG) using recombinant Corynebacterium glutamicum strains. Notably, such a mannose fermentation process facilitated the synthesis of MOS, which has not been achieved under glucose fermentation and improved MG production by 2.6-fold than that using the same C-mole of glucose. This approach also allowed access to produce other kinds of mannosyl derivatives from starch.     

代谢工程改造解脂耶氏酵母合成植物萜类化合物的研究进展

萜类化合物是一类广泛存在于植物中的天然产物,其在食品、药品和化工等多个领域中均有广泛的用途,市场潜力巨大。因此,开发生产萜类化合物等植物天然产物可再生的微生物资源来补充甚至代替原有稀少和珍贵的植物资源,具有重要的理论意义和潜在的应用价值。解脂耶氏酵母是目前使用最广泛的非常规酵母底盘细胞之一。近年来,利用代谢工程及合成生物学技术在解脂耶氏酵母底盘细胞中重构与优化萜类化合物的合成途径以实现目标代谢产物的高效合成,已经成为一项研究热点。本文系统总结了有关利用解脂耶氏酵母作为底盘细胞异源生产植物萜类化合物的具体实例和最新进展,包括所涉及的宿主菌株、关键酶、代谢途径及改造策略等,并在最后对该领域的未来发展方向进行了展望。     

Biosynthesis of lactate-containing polyesters by metabolically engineered bacteria

Due to increasing concerns about environmental problems, climate change and limited fossil resources, bio-based production of chemicals and polymers is gaining attention as one of the solutions to these problems. Polyhydroxyalkanoates (PHAs) are polyesters that can be produced by microbial fermentation. PHAs are synthesized using monomer precursors provided from diverse metabolic pathways and are accumulated as distinct granules inside the cells. On the other hand, most so-called bio-based polymers including polybutylene succinate, polytrimethylene terephthalate, and polylactic acid (PLA) are synthesized by a chemical process using monomers produced by fermentation. PLA, an attractive biomass-derived plastic, is currently synthesized by heavy metal-catalyzed ring opening polymerization of L-lactide that is made from fermentation-derived L-lactic acid. Recently, a complete biological process for the production of PLA and PLA copolymers from renewable resources has been developed by direct fermentation of recombinant bacteria employing PHA biosynthetic pathways coupled with a novel metabolic pathway. This could be accomplished by establishing a pathway for generating lactyl-CoA and engineering PHA synthase to accept lactyl-CoA as a substrate combined with systems metabolic engineering. In this article, we review recent advances in the production of lactate-containing homo- and co-polyesters. Challenges remaining to efficiently produce PLA and its copolymers and strategies to overcome these challenges through metabolic engineering combined with enzyme engineering are discussed.     

Tailoring cyanobacterial cell factory for improved industrial properties

Photosynthetic biomanufacturing provides a promising solution for sustainable production of biofuels and biochemicals. Cyanobacteria are among the most promising microbial platforms for the construction of photosynthetic cell factories. Metabolic engineering of cyanobacteria has enabled effective photosynthetic synthesis of diverse natural or non-natural metabolites, while commercialization of photosynthetic biomanufacturing is usually restricted by process and economic feasibilities. In actual outdoor conditions, active cell growth and product synthesis is restricted to narrow light exposure windows of the day-night cycles and is threatened by diverse physical, chemical, and biological environmental stresses. For biomass harvesting and bioproduct recovery, energy and cost consuming processing and equipment is required, which further decreases the economic and environmental competitiveness of the entire process. To facilitate scaled photosynthetic biomanufacturing, lots of efforts have been made to engineer cyanobacterial cell properties required by robust & continual cultivation and convenient & efficient recovery. In this review, we specifically summarized recently reported engineering strategies on optimizing industrial properties of cyanobacterial cells. Through systematically re-editing the metabolism, morphology, mutualism interaction of cyanobacterial chassis cells, the adaptabilities and compatibilities of the cyanobacterial cell factories to the industrial process could be significantly improved. Cell growth and product synthesis of the tailored cyanobacterial cells could be expanded and maintained at night and in stressful environments, while convenient biomass harvesting could also be expected. For developing more feasible cyanobacterial photosynthetic biomanufacturing in large scale, we here propose the importance of tailoring industrial properties of cyanobacteria and outline the directions that should be exploited in the future.     

大数据-模型混合驱动下生物过程优化与放大的新机遇与挑战

当前,生物制造技术和产业是世界关注的热点。然而,生物过程优化与放大过程中普遍面临以下几个难题,包括:过程检测手段缺乏,难以满足关键指标参数的监控;细胞代谢认知匮乏,无法理性实现过程最优化调控;反应器环境差异大,导致逐级放大效率低下。文中针对以上亟待解决的关键问题,通过案例分析介绍发酵过程实时检测-动态调控-理性放大全链条关键技术创新。在未来,生物过程设计将以集成细胞生理学(时空多尺度细胞代谢模型)和流体动力学(CFD模型)的全生命周期模型为指导,推进计算机辅助设计与开发,加速生物过程实现大规模智能化生产,开启绿色生物制造新时代。     

Comparison of fluidized bed and ultrasonic cell-retention systems for high cell density mammalian cell culture

Economically viable biopharmaceutical production is to a high degree dependent on high product yields and stable fermentation systems that are easy to handle. In the current study we have compared two different fermentation systems for the production of recombinant protein from CHO cells. Both systems are fully scaleable and can be used for industrial high cell density bioprocesses. As a model cell line we have used a recombinant CHO cell line producing the enzyme arylsulfatase B (ASB). CHO cells were cultivated as adherent cell culture attached on Cytoline macroporous microcarrier (Amersham Biosciences, Sweden) using a Cytopilot Mini fluidized bed bioreactor (FBR, Vogelbusch-Amersham Biosciences, Austria) and as suspension culture using a stirred tank bioreactor equipped with a BioSep ultrasonic resonator based cell separation device (Applikon, The Netherlands). Both systems are equally well-suited for stable, long-term high cell density perfusion cell culture and provide industrial scalability and high yields. For products such as the recombinant ASB, high perfusion rates and therefore short product bioreactor residence times may be of additional benefit.