形态工程在生物基化学品生产中的应用进展

合成生物学和代谢工程是构建微生物细胞工厂、实现化学品绿色生物制造的重要方法,目前主要集中在微生物代谢网络的改造及调控上,很少考虑到微生物细胞特性的影响。形态工程通过改造微生物细胞形态相关蛋白,有目的地对微生物细胞形态及分裂方式进行合理调控,从而优化微生物细胞的特性,是降低生物炼制成本的一种新兴生物工程技术。文中首先介绍了与微生物细胞形态相关的各类蛋白,并重点总结了形态工程在生物基化学品合成方面的应用进展,包括调控细胞体积以提高胞内产物积累量、改善细胞通透性以促进胞外产物分泌、实现高密度发酵以降低生产成本、控制产物水解程度以提高产品性能。最后,提出了形态工程面临的主要问题并展望了其未来的发展趋势。     

Inducible cell lysis systems in microbial production of bio-based chemicals

The release of products from microbial cells is an essential process for industrial scale production of bio-based chemicals. However, traditional methods of cell lysis, e.g., mechanical disruption, chemical solvent extraction, and immobilized enzyme degradation, account for a large share of the total production cost. Thus, an efficient cell lysis system is required to lower the cost. This review has focused on our current knowledge of two cell lysis systems, bacteriophage holin–endolysin system, and lipid enzyme hydrolysis system. These systems are controlled by conditionally inducible regulatory apparatus and applied in microbial production of fatty acids and polyhydroxyalkanoates. Moreover, toxin–antitoxin system is also suggested as alternative for its potential applications in cell lysis. Compared with traditional methods of cell disruption, the inducible cell lysis systems are more economically feasible and easier to control and show a promising perspective in industrial production of bio-based chemicals.     

Use of Surface Enhanced Blocking (SEB) Electrodes for Microbial Cell Lysis in Flow-Through Devices

By simultaneously subjecting microbial cells to high amplitude pulsed electric fields and flash heating of the cell suspension fluid, effective release of intracellular contents was achieved. The synergistic effect of the applied electric field and elevated temperature on cell lysis in a flow-through device was demonstrated for Gram-negative and Gram-positive bacteria, and Mycobacterium species. The resulting lysate is suitable for downstream nucleic acid amplification and detection without requiring further preparation. The lysis chamber employs surface enhanced blocking electrodes which possess an etched micro-structured surface and a thin layer of dielectric metal oxide which provides a large effective area and blocks transmission of electrical current. The surface enhanced blocking electrodes enable simultaneous suppression of the rapid onset of electric field screening in the bulk of the cell suspension medium and avoidance of undesired electrochemical processes at the electrode-electrolyte interface. In addition the blocking layer ensures the robustness of the cell lysis device in applications involving prolonged flow-through processing of the microbial cells.     

Maßgeschneiderte Mikroorganismen

Tailor‐made microorganisms Microbial diversity provides unlimited resources for the development of novel industrial processes and products. Since the beginning of the 20th century microorganisms have been successfully applied for the large scale production of bio‐based products. In recent years, modern methods of strain development and Synthetic Biology have enabled biotech engineers to design even more sophisticated and tailor‐made microorganisms. These microbes serve industrial processes for the production of bulk chemicals, enzymes, polymers, biofuels as well as plant‐derived ingredients such as Artemisinin in an ecologically and economically sustainable and attractive fashion. In the future, production of advanced biofuels, microbial fuel cells, CO₂ as feedstock and microbial cellulose are research topics as well as challenges of global importance. Continuous efforts in microbiology and biotechnology research will be pivotal for white biotechnology to gain more momentum in transforming the chemical industry towards a knowledge based bio‐economy.     

微生物胞外电子传递效率的合成生物学强化

电活性微生物(产电微生物和亲电微生物)通过与外界环境进行双向电子和能量传递来实现多种微生物电催化过程(包括微生物燃料电池、微生物电解电池、微生物电催化等),从而实现在环境、能源领域的广泛应用,并为开发有效且可持续性生产新能源或大宗精细化学品的工艺提供了新机会。但是,电活性微生物的胞外电子传递效率比较低,这已经成为限制微生物电催化系统在工业应用中的主要瓶颈。以下综述了近年来利用合成生物学改造电活性微生物的相关研究成果,阐明了合成生物学如何用于打破电活性微生物胞外电子传递途径低效率的瓶颈,从而实现电活性微生物与环境的高效电子传递和能量交换,推动电活性微生物电催化系统的实用化进程。     

合成生物学技术在环境保护中的应用

合成生物学是一个基于生物学和工程学原理的科学领域,其目的是重新设计和重组微生物,以优化或创建具有增强功能的新生物系统。该领域利用分子工具、系统生物学和遗传框架的重编程,从而构建合成途径以获得具有替代功能的微生物。传统上,合成生物学方法通常旨在开发具有成本效益的微生物细胞工厂进而从可再生资源中生产化学物质。然而,近年来合成生物学技术开始在环境保护中发挥着更直接的作用。本综述介绍了基因工程中的合成生物学工具,讨论了基于基因工程的微生物修复策略,强调了合成生物学技术可以通过响应特定污染物进行生物修复来保护环境。其中,规律间隔成簇短回文重复序列(Clustered Regularly Interspersed Short Palindromic Repeats, CRISPR)技术在基因工程细菌和古细菌的生物修复中得到了广泛应用,生物修复领域也出现了很多新的先进技术,包括生物膜工程、人工微生物群落的构建、基因驱动、酶和蛋白质工程等。有了这些新的技术和工具,生物修复将成为当今最好和最有效的污染物去除方式之一。     

The environment,microbes and bioremediation: microbial activities modulated by the environment

Microorganisms in nature are largely responsible for the biodegradation and removal of toxic and non-toxic chemicals. Many organisms are also known to have specific ecological niches for proliferation and colonization. The nature of the environment dictates to a large extent the biodegradability of synthetic compounds by modulating the evolutionary processes in microorganisms for new degradative genes. Similarly, environmental factors often determine the extent of microbial gene expression by activating or repressing specific gene or sets of genes through a sensory signal transduction process. Understanding how the environment modulates microbial activity is critical for successful bioremediative applications.     

The microbial DNA cycle in soil.

Upon microbial cell death and lysis in soil, the free or naked DNA is exposed to the dynamic environment of the soil. The DNA can be enzymatically degraded by nucleases (DNases), bind to soil components, genetically transform competent bacterial cells and be a nutrient for other microorganisms. In this article we discuss the dual role of DNA as genetic material and as a nutrient source in the soil environment.     

Biomineralization of endolithic microbes in rocks from the McMurdo Dry Valleys of Antarctica: implications for microbial fossil formation and their detection

In some zones of Antarctica's cold and dry desert, the extinction of cryptoendolithic microorganisms leaves behind inorganic traces of microbial life. In this paper, we examine the transition from live microorganisms, through their decay, to microbial fossils using in situ microscopy (transmission electron microscopy, scanning electron microscopy in back-scattered electron mode) and microanalytical (energy dispersive X-ray spectroscopy) techniques. Our results demonstrate that, after their death, endolithic microorganisms inhabiting Commonwealth Glacier sandstone from the Antarctica McMurdo Dry Valleys become mineralized. In some cases, epicellular deposition of minerals and/or simply filling up of empty moulds by minerals leads to the formation of cell-shaped structures that may be considered biomarkers. The continuous deposition of allochthonous clay minerals and sulfate-rich salts fills the sandstone pores. This process can give rise to microbial fossils with distinguishable cell wall structures. Often, fossilized cell interiors were of a different chemical composition to the mineralized cell walls. We propose that the microbial fossil formation observed was induced by mineral precipitation resulting from inorganic processes occurring after the death of cryptoendolithic microorganisms. Nevertheless, it must have been the organic template that provoked the diffusion of mineral elements and gave rise to their characteristic distribution pattern inside the fossilized cells.     

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.     

Lignins and lignocellulosics: a better control of synthesis for new and improved uses

The composition and structure of lignified walls has a dramatic impact on the technological value of raw materials. The chemical flexibility of the secondary cell wall has been demonstrated and it is now possible to develop strategies to optimize its composition through genetic engineering. Thanks to functional genomics, new target genes of both plant and microbial origin are rapidly becoming available for this purpose and their use will open new avenues for producing tailor-made plant products with improved properties. Moreover, the major proportion of terrestrial plant biomass comprises lignified cell walls and this reservoir of carbon should be increasingly exploited for the production of chemicals and energy within the context of sustainable development. For example, the design of plants suitable for downstream conversion processes, such as the production of bioethanol, and the exploitation of microorganisms and microbial enzymes for biomass pretreatments or for the production of novel chemicals.     

Hepatic stellate cells (vitamin A-storing cells) change their cytoskeleton structure by extracellular matrix components through a signal transduction system

 When cultured on a polystyrene surface or aminoalkylsilane-coated cover glasses, rat and human hepatic stellate cells exhibit a flattened, fibroblast-like shape with well-developed stress fibers. However, culturing the cells on type I collagen gel results in the elongation of long, multipolar cellular processes, whereas cells cultured on Matrigel maintain their round shapes. Dual fluorescence staining of microtubules and fibrillar actin indicated that the processes extend together with collagen fibers and contained microtubules as the core, whereas the periphery contained fibrillar actin. Immunofluorescence staining of vinculin showed that the focal adhesions were distributed mainly in lamellipodia when cultured on aminoalkylsilane-coated cover glasses, whereas in the cells cultured on type I collagen gel they were localized to the tips of the processes and along their bottom surface contacting collagen fibers. Wortmannin, as well as staurosporin and herbimycin A, inhibited the elongation process and induced the retraction of elongated processes. The wortmannin treatment also resulted in an alteration in focal adhesion distribution from the processes to cell bodies. These results indicate that the cell surface integrin binding to interstitial collagen fibers induces the elongation of processes through signaling events and the subsequent cytoskeleton assembly in hepatic stellate cells. Accepted: 12 February 1998     

Extraction of high molecular weight DNA from microbial mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes. The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 μg of HMW DNA (35-50 kb) per gram of mat sample, with an A(260/280) ratio of 1.6. Furthermore, amplification of 16S rRNA genes suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.     

Impact of pesticides on soil microbiological parameters and possible bioremediation strategies

Intensive agriculture is spectacularly successful since last couple of decades due to the inputs viz; fertilizers and pesticides along with high yielding varieties. The mandate for agriculture development was to feed and adequate nutrition supply to the expanding population by side the agriculture would be entering to into new area of commercial and export orientation. The attention of public health and proper utilization natural resources are also the main issues related with agriculture development. Concern for pesticide contamination in the environment in the current context of pesticide use has assumed great importance [1]. The fate of the pesticides in the soil environment in respect of pest control efficacy, non-target organism exposure and offsite mobility has been given due consideration [2]. Kinetics and pathways of degradation depend on abiotic and biotic factors [6], which are specific to a particular pesticide and therefore find preference. Adverse effect of pesticidal chemicals on soil microorganisms [3], may affect soil fertility [4] becomes a foreign chemicals major issue. Soil microorganisms show an early warning about soil disturbances by foreign chemicals than any other parameters. But the fate and behavior of these chemicals in soil ecosystem is very important since they are degraded by various factors and have the potential to be in the soil, water etc. So it is indispensable to monitor the persistence, degradation of pesticides in soil and is also necessary to study the effect of pesticide on the soil quality or soil health by in depth studies on soil microbial activity. The removal of metabolites or degraded products should be removed from soil and it has now a day’s primary concern to the environmentalist. Toxicity or the contamination of pesticides can be reduced by the bioremediation process which involves the uses of microbes or plants. Either they degrade or use the pesticides by various co metabolic processes.     

Bioflavoring and beer refermentation

Various techniques are used to adjust the flavors of foods and beverages to new market demands. Although synthetic flavoring chemicals are still widely used, flavors produced by biological methods (bioflavors) are now more and more requested by consumers, increasingly concerned with health and environmental problems caused by synthetic chemicals. Bioflavors can be extracted from plants or produced with plant cell cultures, microorganisms or isolated enzymes. This Mini-Review paper gives an overview of different systems for the microbial production of natural flavors, either de novo, or starting with selected flavor precursor molecules. Emphasis is put on the bioflavoring of beer and the possibilities offered by beer refermentation processes. The use of flavor precursors in combination with non-conventional or genetically modified yeasts for the production of new products is discussed.     

An improved protocol and a new grinding device for extraction of genomic DNA from microorganisms by a two-step extraction procedure

Current protocols to extract genomic DNA from microorganisms are still laborious, tedious and costly, especially for the species with thick cell walls. In order to improve the effectiveness of extracting DNA from microbial samples, a novel protocol, defined as two-step extraction method, along with an improved tissue-grinding device, was developed. The protocol included two steps, disruption of microbial cells or spores by grinding the sample together with silica sand in a new device and extraction of DNA with an effective buffer containing cell lysis chemicals. The device was prepared by using a commercial electric mini-grinder, adapted with a grinding stone, and a sample cup processed by lathing from a polytetrafluoroethylene rod. We tested the method with vegetative cells of four microbial species and two microbial spores that have thick cell walls and are therefore hard to process; these included Escherichia coli JM109, Bacillus subtilis WB600, Sacchromyces cerevisiae INVSc1, Trichoderma viride AS3.3711, and the spores of S. cerevisiae and T. viride, respectively, representing Gram-positive bacteria, Gram-negative bacteria, yeast, filamentous fungi. We found that this new method and device extracted usable quantities of genomic DNA from the samples. The DNA fragments that were extracted exceeded 23 kb. The target sequences up to about 5 kb were successfully and exclusively amplified by PCR using extracted DNA as the template. In addition, the DNA extraction was finalized within 1.5 h. Thus, we conclude that this two-step extraction method is an effective and improved protocol for extraction of genomic DNA from microbial samples.     

Electron transfer mechanisms between microorganisms and electrodes in bioelectrochemical systems

Microbes have been shown to naturally form veritable electric grids in which different species acting as electron donors and others acting as electron acceptors cooperate. The uptake of electrons from cells adjacent to them is a mechanism used by microorganisms to gain energy for cell growth and maintenance. The external discharge of electrons in lieu of a terminal electron acceptor, and the reduction of external substrates to uphold certain metabolic processes, also plays a significant role in a variety of microbial environments. These vital microbial respiration events, viz. extracellular electron transfer to and from microorganisms, have attracted widespread attention in recent decades and have led to the development of fascinating research concerning microbial electrochemical sensors and bioelectrochemical systems for environmental and bioproduction applications involving different fuels and chemicals. In such systems, microorganisms use mainly either (1) indirect routes involving use of small redox-active organic molecules referred to as redox mediators, secreted by cells or added exogenously, (2) primary metabolites or other intermediates, or (3) direct modes involving physical contact in which naturally occurring outer-membrane c-type cytochromes shuttle electrons for the reduction or oxidation of electrodes. Electron transfer mechanisms play a role in maximizing the performance of microbe?Celectrode interaction-based systems and help very much in providing an understanding of how such systems operate. This review summarizes the mechanisms of electron transfer between bacteria and electrodes, at both the anode and the cathode, in bioelectrochemical systems. The use over the years of various electrochemical approaches and techniques, cyclic voltammetry in particular, for obtaining a better understanding of the microbial electrocatalysis and the electron transfer mechanisms involved is also described and exemplified.     

Effects of encapsulation of microorganisms on product formation during microbial fermentations

This paper reviews the latest developments in microbial products by encapsulated microorganisms in a liquid core surrounded by natural or synthetic membranes. Cells can be encapsulated in one or several steps using liquid droplet formation, pregel dissolving, coacervation, and interfacial polymerization. The use of encapsulated yeast and bacteria for fermentative production of ethanol, lactic acid, biogas, l-phenylacetylcarbinol, 1,3-propanediol, and riboflavin has been investigated. Encapsulated cells have furthermore been used for the biocatalytic conversion of chemicals. Fermentation, using encapsulated cells, offers various advantages compared to traditional cultivations, e.g., higher cell density, faster fermentation, improved tolerance of the cells to toxic media and high temperatures, and selective exclusion of toxic hydrophobic substances. However, mass transfer through the capsule membrane as well as the robustness of the capsules still challenge the utilization of encapsulated cells. The history and the current state of applying microbial encapsulation for production processes, along with the benefits and drawbacks concerning productivity and general physiology of the encapsulated cells, are discussed.     

On the future fermentation

Microbial fermentations produce chemicals, materials, biofuels, foods and medicines for many years. The processes are less competitive compared to chemical industries. To increase its competitiveness, technologies must be developed to address the following issues including fresh water shortage, heavy energy consumption, microbial contaminations, complexity of sterile operations, poor oxygen utilization in the cultures, food-related ingredients as substrates, low substrate to product conversion efficiency, difficult cells and broth separation, large amount of wastewater, discontinuous processes, heavy labour involvements and expensive bioreactors. Future industrial fermentations should be more effective with the above issues reasonably addressed. Recently, extremophilic bacteria have well addressed the above issues for future fermentation.     

提高微生物可培养性的方法和措施

目前自然界中只有极少部分微生物能够得到培养,严重阻碍了对微生物生命活动规律的研究和微生物资源的开发。改进传统培养方法,采用新型培养技术,提高微生物可培养性,大量培养自然界中存在的微生物,从而更全面、准确地了解微生物细胞的生命规律、获悉微生物群落中各种微生物之间的动态相互作用和相互协调的规律,对环境微生物工艺进行准确地设计、精细地调控和高效地利用。简要介绍了微生物不可培养的原因,系统总结了有关提高微生物可培养性方法的最新研究进展,提出研究中存在的问题,并阐述了模拟自然环境条件、强调微生物相互关系是提高微生物可培养性的关键。