生物丁醇代谢工程的研究进展

丁醇作为一种重要的大宗化学品具有广泛的用途,同时又是一种潜在的生物燃料。随着能源与环境危机的日益加重,利用可再生原料通过微生物法生产丁醇受到全世界的普遍关注。代谢工程为定向改造微生物生产丁醇提供了有力的工具。通过改造经典的丙酮丁醇发酵和定向改造模式微生物生产丁醇是生物丁醇研究的两个重要方向。笔者从代谢工程改造的角度评述近5年来生物丁醇研究的进展,同时讨论了生物丁醇研究中需要着力解决的问题。     

制造生物燃料丁醇的新技术

木质茎、稻草、农业残余物、玉米纤维和外皮都含有大量的纤维素和部分木质素,这些木质纤维材料都可用于制造生物丁醇。丁醇被认为是一种优于乙醇的生物燃料,因为它的腐蚀性更小,热量值更高。如同乙醇一样,丁醇也可添加到汽油中。     

丁醇基因在大肠杆菌中表达的现状与展望

随着化石能源过度开采带来的能源短缺与环境恶化,丁醇凭借着其优越的理化性质成为了最具潜力的绿色燃料之一。近几年微生物在生物能源生产研究中受到广泛关注,主要集中在梭菌丁醇合成途径的异源表达。目前利用大肠杆菌产丁醇的产量已经接近产丁醇的天然菌株的产量。然而,大肠杆菌产丁醇仍存在很多限制性因素。主要从乙酰辅酶A依赖途径评述大肠杆菌生产丁醇的限制因素,并讨论提高丁醇产量需要解决的问题。     

丙酮丁醇梭菌XY16丁醇发酵特性

以诱变选育的1株突变菌株丙酮丁醇梭菌XY16为对象,对影响该菌发酵特性的相关因素(N源、生长因子、热激)进行研究。结果显示:无机N源乙酸铵比其他N源更有利于丙酮丁醇的发酵,玉米浆或玉米蛋白可以直接替代生长因子进行丙酮丁醇发酵,热激可以提高总溶剂产量,最高可以达到21.28 g/L。该菌还可以同时利用葡萄糖和木糖,当葡萄糖利用完后,木糖才能被有效利用。     

生物丁醇：新生代生物能源

随着石油资源的大量消耗及其价格的不断攀升,世界上许多化学公司已经开始进行重大的战略转向,用生物资源代替石油资源,用生物技术路线代替化学技术路线进行生物燃料及化学品的生产。一种新生代生物能源——生物丁醇正在进入人们的视野。     

丙酮丁醇梭菌丁醇耐受性

丁醇在发酵培养基中的积累所产生的毒性问题是限制丁醇产量的重要因素,然而对于Clostridium acetobutylicum是如何适应丁醇胁迫,进而调节菌体生长和代谢的,目前尚缺乏系统研究,不能全面揭示C.acetobutylicum的丁醇耐受性机制.对丙酮丁醇梭菌丁醇耐受性有关的研究成果进行了综述,旨在深入理解菌株丁醇耐受性发生改变的相关分子基础.希望为进行微生物丁醇耐受性分子机制的改造、提高菌株的丁醇耐受性提供新的研究思路.     

生物丁醇研发及相关专利分析

目前,全球对于石油、天然气和煤炭等不可再生能源的需求日益增加,但资源储量却在逐渐枯竭。因此,开发包括生物质燃料在内的新能源,就成为关注的重点。在生物燃料的产品中,生物丁醇比起目前市场上常见的生物乙醇、生物柴油等,有着无可比拟的优势：首先,丁醇的能量密度高,     

丙酮丁醇梭菌发酵菊芋汁生产丁醇

对丙酮丁醇梭菌Clostridium acetobutylicum L7发酵菊芋汁酸水解液生产丁醇进行了初步研究。实验结果表明,以该水解液为底物生产丁醇,不需要添加氮源和生长因子。当水解液初始糖浓度为48.36 g/L时,其发酵性能与以果糖为碳源的对照组基本相同,发酵终点丁醇浓度为8.67 g/L,丁醇、丙酮和乙醇的比例为0.58∶0.36∶0.06,但与以葡萄糖为碳源的对照组相比,发酵时间明显延长,表明该菌株葡萄糖转运能力强于果糖。当水解液初始糖浓度提高到62.87 g/L时,发酵终点残糖浓度从3.09 g/L增加到3.26 g/L,但丁醇浓度却提高到11.21 g/L,丁醇、丙酮和乙醇的比例相应为0.64∶0.29∶0.05,表明适量糖过剩有助于C.acetobutylicum L7胞内代谢从丙酮合成向丁醇合成途径调节;继续提高水解液初始糖浓度,发酵终点残糖浓度迅速升高,丁醇生产的技术经济指标受到明显影响。     

纤维燃料丁醇研究进展

随着能源危机与粮食安全问题的日趋加重,以纤维质为原料生产石油替代燃料已成为生物质能研究的重点。分析了丁醇作为燃料的优点,归纳了丁醇发酵微生物的种类与研究现状,综述了纤维原料生产燃料丁醇的研究进展,最后对我国纤维燃料丁醇的产业化优势和前景进行了分析与展望。     

利用核糖体工程选育丙酮丁醇菌提高丁醇产量

利用核糖体工程技术对丙酮丁醇梭菌Clostridium acetobutylicum L7进行诱变筛选,以获得丁醇高产菌株。使用链霉素诱变C.acetobutylicum L7并结合设计的平板转接逐次提高链霉素浓度的筛选路线,获得丁醇产量较高的菌株S3。结果表明,S3丁醇产量为(12.48±0.03)g/L,乙醇产量为(1.70±0.07)g/L,相对于原始菌分别提高了11.2%及50%;丁醇/葡萄糖转化率由原始菌的0.19提高到0.22,丁醇生产率达到0.24 g/(L.h),相比提高30.5%;耐受丁醇浓度由原始菌的12 g/L提高到14 g/L;发酵液粘度下降到4 mPa/s,同比降低了60%,利于后续分离工作的进行,降低发酵成本。进一步研究工作表明,S3菌株遗传稳定性良好。因此,核糖体工程技术是一种选育丁醇高产菌株的有效方法。     

Environmental impacts and limitations of third‐generation biobutanol: Life cycle assessment of n‐butanol produced by genetically engineered cyanobacteria

Photosynthetic cyanobacteria have attracted interest as production organisms for third‐generation biofuels, where sunlight and CO₂ are used by microbes directly to synthesize fuel molecules. A particularly suitable biofuel is n‐butanol, and there have been several laboratory reports of genetically engineered photosynthetic cyanobacteria capable of synthesizing and secreting n‐butanol. This work evaluates the environmental impacts and cumulative energy demand (CED) of cyanobacteria‐produced n‐butanol through a cradle‐to‐grave consequential life cycle assessment (LCA). A hypothetical production plant in northern Sweden (area 1 ha, producing 5–85 m³ n‐butanol per year) was considered, and a range of cultivation formats and cellular productivity scenarios assessed. Depending on the scenario, greenhouse gas emissions (GHGe) ranged from 16.9 to 58.6 gCO₂eq/MJ_BuOH and the CED from 3.8 to 13 MJ/MJ_BuOH. Only with the assumption of a nearby paper mill to supply waste sources for heat and CO₂ was the sustainability requirement of at least 60% GHGe savings compared to fossil fuels reached, though placement in northern Sweden reduced energy needed for reactor cooling. A high CED in all scenarios shows that significant metabolic engineering is necessary, such as a carbon partitioning of >90% to n‐butanol, as well as improved light utilization, to begin to displace fossil fuels or even first‐ and second‐generation bioethanol.     

基于一体化生物加工过程的木质纤维素合成生物丁醇的研究进展

一体化生物加工过程(Consolidated bioprocessing,CBP)是在一个生物反应器中完成水解酶生产、酶解、微生物发酵等多步生物过程的工艺。因其过程步骤简单、成本低,被认为是生产二代生物燃料最具发展前景的工艺。然而,由于木质纤维素降解与丁醇合成路径的复杂性,鲜有天然微生物可以直接利用木质纤维素合成丁醇。随着合成生物学技术的发展,在纤维素降解梭菌中引入丁醇合成途径,可以使单菌利用木质纤维素直接合成丁醇。但是该策略存在菌株代谢负荷重、丁醇产量低等问题。而混菌策略可以通过不同菌株的劳动分工,使单菌代谢负担得到缓解,因此进一步提高了丁醇合成效率。文中从单菌策略和混菌策略分析了近年来一体化生物加工过程利用木质纤维素合成丁醇的相关研究进展,为生物丁醇以及其他生物燃料的一体化生物加工过程研究提供借鉴。     

Energy crops: current status and future prospects

Energy crops currently contribute a relatively small proportion to the total energy produced from biomass each year, but the proportion is set to grow over the next few decades. This paper reviews the current status of energy crops and their conversion technologies, assesses their potential to contribute to global energy demand and climate mitigation over the next few decades, and examines the future prospects. Previous estimates have suggested a technical potential for energy crops of～400 EJ yr^?1 by 2050. In a new analysis based on energy crop areas for each of the IPCC SRES scenarios in 2025 (as projected by the IMAGE 2.2 integrated assessment model), more conservative dry matter and energy yield estimates and an assessment of the impact on non‐CO₂ greenhouse gases were used to estimate the realistically achievable potential for energy crops by 2025 to be between 2 and 22 EJ yr^?1, which will offset～100–2070 Mt CO₂‐eq. yr^?1. These results suggest that additional production of energy crops alone is not sufficient to reduce emissions to meet a 550 μmol mol^?1 atmospheric CO₂ stabilization trajectory, but is sufficient to form an important component in a portfolio of climate mitigation measures, as well as to provide a significant sustainable energy resource to displace fossil fuel resources. Realizing the potential of energy crops will necessitate optimizing the dry matter and energy yield of these crops per area of land through the latest biotechnological routes, with or without the need for genetic modification. In future, the co‐benefits of bioenergy production will need to be optimized and methods will need to be developed to extract and refine high‐value products from the feedstock before it is used for energy production.     

Biotechnological production of muconic acid: current status and future prospects

Muconic acid (MA), a high value-added bio-product with reactive dicarboxylic groups and conjugated double bonds, has garnered increasing interest owing to its potential applications in the manufacture of new functional resins, bio-plastics, food additives, agrochemicals, and pharmaceuticals. At the very least, MA can be used to produce commercially important bulk chemicals such as adipic acid, terephthalic acid and trimellitic acid. Recently, great progress has been made in the development of biotechnological routes for MA production. This present review provides a comprehensive and systematic overview of recent advances and challenges in biotechnological production of MA. Various biological methods are summarized and compared, and their constraints and possible solutions are also described. Finally, the future prospects are discussed with respect to the current state, challenges, and trends in this field, and the guidelines to develop high-performance microbial cell factories are also proposed for the MA production by systems metabolic engineering.     

代谢工程在D-核糖生产中研究现状及应用前景

简要介绍了代谢工程的进展情况,并较为详细地从宿主的选择、加速限速反应、改变代谢流和生产过程的优化等方面论述了代谢工程在D-核糖基因工程菌构建方面的应用及其应用前景。     

Current status of,and prospects for,immobilized biocatalyst technology in China

Summary Studies on immobilized enzymes and whole cells have been made since 1970 in China. A new method for immobilization was developed by using the bifunctional reagent, -sulphatoethyl-sulphonylaniline to activate polysaccharides. More than ten immobilized biocatalysts such as polynucleotide phosphorylase and penicillin acylase, have been used in the pharmaceutical industry, the production of high value chemicals, and in pollutant treatment. The prospects and scientific constraints in this field are discussed in this paper.

---

Resumen Desde 1970 se estan llevando a cabo en China estudios sobre enzimas inmovilizados y células enteras. Un nuevo método para la inmovilización fue diseñado utilizando el reactivo bifuncional -sulfatoetilsulfonilanilina para activar polisacaridos. Màs de 10 biocatalizadores inmovilizados, tales como polinucleotidofosforilasa y penicillin-acilasa, se han utilizado en la industria farmaceútica, en la producción de compuetos químicos de alto coste y en el tratamiento de polucionantes. En este trabajo se discuten las perspectivas y las dificultades científicas en este campo.

---

Résumé Des études sur les enzymes fixés et les cellules immobilisées ont été entreprises en Chine à partir de 1970. II a été mis au point une nouvelle méthode d'immobilisation, utilisant pour l'activation des polysaccharides un réactif bifonctionnel, la -sulphato-éthyl-sulphonyl-aniline. Plus de 10 biocatalyseurs immobilisés, dont la polynucléotide-phosphorylase et la pénicilline-acylase, ont été employés dans l'industrie pharmaceutique, pour la production de composés chimiques à haute valeur commerciale et pour la dépollution. Les perspectives et les contraintes scientifiques dans le domaine considéré sont discutées.

---

---

Paper presented at the VII International Conference on the Global Impacts of Applied Microbiology, Helsinki, 12–16 August 1985.     

Selective separation of biobutanol from acetone-butanol-ethanol fermentation broth by means of sorption methodology based on a novel macroporous resin

The traditional distillation method for recovery of butanol from fermentation broth is an energy-intensive process. Separation of butanol based on adsorption methodology has advantages in terms of biocompatibility and stability, as well as economy, and therefore gains much attention. However, the application of the commercial adsorbents in the integrated acetone-butanol-ethanol (ABE) fermentation process is restricted due to the low recovery (less than 85%) and the weak capability of enrichment in the eluent (3-4 times). In this study, we investigated the sorption properties of butanol onto three kinds of adsorbents with different polarities developed in our laboratory, that is, XD-41, H-511, and KA-I resin. The sorption behaviors of single component and ABE ternary mixtures presented in the fermentation broths on KA-I resin were investigated. KA-I resin had higher affinity for butanol than for acetone, ethanol, glucose, acetic acid, and butyric acid. Multicomponent ABE sorption on KA-I resin was modeled using a single site extended Langmuir isotherm model. In a desorption study, all the adsorbed components were desorbed in one bed volume of methanol, and the recovery of butanol from KA-I resin was 99.7%. The concentration of butanol in the eluent was increased by a factor of 6.13. In addition, KA-I resin was successfully regenerated by two bed volumes of water. Because of its quick sorption, high sorption capacity, low cost, and ease of desorption and regeneration, KA-I resin exhibits good potential for compatibility with future ABE fermentation coupled with in situ recovery product removal techniques.     

Biotransformation of lignin: Mechanisms,applications and future work

As one of the most abundant polymers in biosphere, lignin has attracted extensive attention as a kind of promising feedstock for biofuel and bio-based products. However, the utilization of lignin presents various challenges in that its complex composition and structure and high resistance to degradation. Lignin conversion through biological platform harnesses the catalytic power of microorganisms to decompose complex lignin molecules and obtain value-added products through biosynthesis. Given the heterogeneity of lignin, various microbial metabolic pathways are involved in lignin bioconversion processes, which has been characterized in extensive research work. With different types of lignin substrates (e.g., model compounds, technical lignin, and lignocellulosic biomass), several bacterial and fungal species have been proved to own lignin-degrading abilities and accumulate microbial products (e.g., lipid and polyhydroxyalkanoates), while the lignin conversion efficiencies are still relatively low. Genetic and metabolic strategies have been developed to enhance lignin biodegradation by reprogramming microbial metabolism, and diverse products, such as vanillin and dicarboxylic acids were also produced from lignin. This article aims at presenting a comprehensive review on lignin bioconversion including lignin degradation mechanisms, metabolic pathways, and applications for the production of value-added bioproducts. Advanced techniques on genetic and metabolic engineering are also covered in the recent development of biological platforms for lignin utilization. To conclude this article, the existing challenges for efficient lignin bioprocessing are analyzed and possible directions for future work are proposed.