Toward protein engineering for phytoremediation: possibilities and challenges

The combination of rational protein engineering and directed evolution techniques allow for the redesign of enzymes with tailored properties for use in environmental remediation. This review summarizes current molecular methods for either altering or improving protein function and highlights examples of how these methods can address bioremediation problems. Although much of the protein engineering applied to environmental clean-up employs microbial systems, there is great potential for and significant challenges to translating these approaches to plant systems for phytoremediation purposes. Protein engineering technologies combined with genomic information and metabolic engineering strategies hold promise for the design of plants and microbes to remediate organic and inorganic pollutants.     

Enzymes from solvent-tolerant microbes: Useful biocatalysts for non-aqueous enzymology

Solvent-tolerant microbes are a newly emerging class that possesses the unique ability to thrive in the presence of organic solvents. Their enzymes adapted to mediate cellular and metabolic processes in a solvent-rich environment and are logically stable in the presence of organic solvents. Enzyme catalysis in non-aqueous/low-water media is finding increasing applications for the synthesis of industrially important products, namely peptides, esters, and other trans-esterification products. Solvent stability, however, remains a prerequisite for employing enzymes in non-aqueous systems. Enzymes, in general, get inactivated or give very low rates of reaction in non-aqueous media. Thus, early efforts, and even some recent ones, have aimed at stabilization of enzymes in organic media by immobilization, surface modifications, mutagenesis, and protein engineering. Enzymes from solvent-tolerant microbes appear to be the choicest source for studying solvent-stable enzymes because of their unique ability to survive in the presence of a range of organic solvents. These bacteria circumvent the solvent’s toxic effects by virtue of various adaptations, e.g. at the level of the cytoplasmic membrane, by degradation and transformation of solvents, and by active excretion of solvents. The recent screening of these exotic microbes has generated some naturally solvent-stable proteases, lipases, cholesterol oxidase, cholesterol esterase, cyclodextrin glucanotransferase, and other important enzymes. The unique properties of these novel biocatalysts have great potential for applications in non-aqueous enzymology for a range of industrial processes.     

Synthetic biosystems for the production of high-value plant metabolites

Plants display an immense diversity of specialized metabolites, many of which have been important to humanity as medicines, flavors, fragrances, pigments, insecticides and other fine chemicals. Apparently, much of the variation in plant specialized metabolism evolved through events of gene duplications followed by neo- or sub-functionalization. Most of the catalytic diversity of plant enzymes is unexplored since previous biochemical and genomics efforts have focused on a relatively small number of species. Interdisciplinary research in plant genomics, microbial engineering and synthetic biology provides an opportunity to accelerate the discovery of new enzymes. The massive identification, characterization and cataloguing of plant enzymes coupled with their deployment in metabolically optimized microbes provide a high-throughput functional genomics tool and a novel strain engineering pipeline.     

Molecular engineering of industrial enzymes: recent advances and future prospects

Many enzymes are efficiently produced by microbes. However, the use of natural enzymes as biocatalysts has limitations such as low catalytic efficiency, low activity, and low stability, especially under industrial conditions. Many protein engineering technologies have been developed to modify natural enzymes and eliminate these limitations. Commonly used protein engineering strategies include directed evolution, site-directed mutagenesis, truncation, and terminal fusion. This review summarizes recent advances in the molecular engineering of industrial enzymes and discusses future prospects in this field. We expect this review to increase interest in and advance the molecular engineering of industrial enzymes.     

Synthetic biology for manufacturing chemicals: constraints drive the use of non-conventional microbial platforms

Applied Microbiology and Biotechnology - Genetically modified microbes have had much industrial success producing protein-based products (such as antibodies and enzymes). However, engineering...     

Pathway engineering for functional isoprenoids

Pathway engineering is to engineer biosynthetic pathways for compounds of interests in heterologous organisms such as microbes and higher plants, which has also been one of the most important fields in metabolic engineering and synthetic biology. This review focuses on pathway engineering researches for the production of functional isoprenoids containing monoterpenes, sesquiterpenes, diterpenes, and triterpenes as well as carotenoids and for the elucidation of relevant biosynthesis genes and enzymes, which have been performed in the last two years. As microbial hosts, Escherichia coli and Saccharomyces cerevisiae have often been employed, since they, specifically the former, are fully amenable to genetic manipulations with extensive molecular resources. Various crops have also been used as the hosts for engineering pathways of functional isoprenoids of the plant origin, particularly carotenoids.     

Recent progress in consolidated bioprocessing

Consolidated bioprocessing, or CBP, the conversion of lignocellulose into desired products in one step without added enzymes, has been a subject of increased research effort in recent years. In this review, the economic motivation for CBP is addressed, advances and remaining obstacles for CBP organism development are reviewed, and we comment briefly on fundamental aspects. For CBP organism development beginning with microbes that have native ability to utilize insoluble components of cellulosic biomass, key recent advances include the development of genetic systems for several cellulolytic bacteria, engineering a thermophilic bacterium to produce ethanol at commercially attractive yields and titers, and engineering a cellulolytic microbe to produce butanol. For CBP organism development, beginning with microbes that do not have this ability and thus requiring heterologous expression of a saccharolytic enzyme system, high-yield conversion of model cellulosic substrates and heterologous expression of CBH1 and CBH2 in yeast at levels believed to be sufficient for an industrial process have recently been demonstrated. For both strategies, increased emphasis on realizing high performance under industrial conditions is needed. Continued exploration of the underlying fundamentals of microbial cellulose utilization is likely to be useful in order to guide the choice and development of CBP systems.     

Production of squalene by microbes: an update

Squalene, a naturally occurring linear triterpene formed via MVA or MEP biosynthetic pathway, is widely distributed in microorganisms, plants and animals. At present, squalene is used extensively in the food, cosmetic and medicine industries because of its antioxidant, antistatic and anti-carcinogenic properties. Increased consumer demand has led to the development of microbial bioprocesses for the commercial production of squalene, in addition to the traditional methods of isolating squalene from the liver oils of deep-sea sharks and plant seed oils. As knowledge of the biosynthetic enzymes and of regulatory mechanisms modulating squalene production increases, opportunities arise for the genetic engineering of squalene production in hosts. In this review, we present the various strategies used up to date to improve and/or engineer squalene production in microbes and analyze yields.     

Current prospects for the production of coenzyme Q10 in microbes

Coenzyme Q or ubiquinone (UQ) is a naturally occurring coenzyme formed from the conjugation of a benzoquinone ring and an isoprenoid chain of varying length. UQ-10, the main UQ species produced by humans, provides therapeutic benefits in certain human diseases, such as cardiomyopathy, when administered orally. Increased consumer demand has led to the development of bioprocesses for the commercial production of UQ-10. Up to now, these processes have relied on microbes that produce high levels of UQ-10 naturally. However, as knowledge of the biosynthetic enzymes and of regulatory mechanisms modulating UQ production increases, opportunities arise for the genetic engineering of UQ-10 production in hosts, such as Escherichia coli, that are better suited for commercial fermentation. We present the various strategies used up to now to improve and/or engineer UQ-10 production in microbes and analyze yields obtained in light of the current knowledge on the biosynthesis of this molecule.     

可用于二氧化碳捕获过程的微生物碳酸酐酶的挖掘与改造

二氧化碳排放量的急剧上升引起全球温室效应加剧。碳酸酐酶是地球上反应速率最快的几种酶之一,可以大幅提高CO_2捕获和生物矿化的效率,从而降低大气中CO_2的排放量。但捕获过程在高温条件,而CO_2生物矿化形成CaCO_3的过程则需要碱性条件。因此,迫切需要筛选出既嗜热又耐碱的碳酸酐酶以用于CO_2捕获,极端微生物是这类酶的重要来源之一。文中系统、深入地介绍了目前从极端微生物或利用蛋白质工程技术获取嗜热、耐碱的碳酸酐酶的最新研究进展,同时简要介绍了一些新型固定化碳酸酐酶的方法。最后指出当前研究的重点应致力于拓宽寻找碳酸酐酶的范围,改良蛋白质工程改造技术,研发高效廉价、易于放大的固定化方法,为减轻温室效应、延缓全球变暖这一迫切需要解决的问题提供新思路。     

Structure-driven protein engineering for production of valuable natural products

Proteins are the most frequently used biocatalysts, and their structures determine their functions. Modifying the functions of proteins on the basis of their structures lies at the heart of protein engineering, opening a new horizon for metabolic engineering by efficiently generating stable enzymes. Many attempts at classical metabolic engineering have focused on improving specific metabolic fluxes and producing more valuable natural products by increasing gene expression levels and enzyme concentrations. However, most naturally occurring enzymes show limitations, and such limitations have hindered practical applications. Here we review recent advances in protein engineering in synthetic biology, chemoenzymatic synthesis, and plant metabolic engineering and describe opportunities for designing and constructing novel enzymes or proteins with desirable properties to obtain more active natural products.     

Building a genome engineering toolbox in nonmodel prokaryotic microbes

The realization of a sustainable bioeconomy requires our ability to understand and engineer complex design principles for the development of platform organisms capable of efficient conversion of cheap and sustainable feedstocks (e.g., sunlight, CO₂, and nonfood biomass) into biofuels and bioproducts at sufficient titers and costs. For model microbes, such as Escherichia coli, advances in DNA reading and writing technologies are driving the adoption of new paradigms for engineering biological systems. Unfortunately, microbes with properties of interest for the utilization of cheap and renewable feedstocks, such as photosynthesis, autotrophic growth, and cellulose degradation, have very few, if any, genetic tools for metabolic engineering. Therefore, it is important to develop “design rules” for building a genetic toolbox for novel microbes. Here, we present an overview of our current understanding of these rules for the genetic manipulation of prokaryotic microbes and the available genetic tools to expand our ability to genetically engineer nonmodel systems.     

Engineering the robustness of industrial microbes through synthetic biology

Microbial fermentations and bioconversions play a central role in the production of pharmaceuticals, enzymes and chemicals. To meet the demands of industrial production, it is desirable that microbes maintain a maximized carbon flux towards target metabolites regardless of fluctuations in intracellular or extracellular environments. This requires cellular systems that maintain functional stability and dynamic homeostasis in a given physiological state, or manipulate transitions between different physiological states. Stable maintenance or smooth transition can be achieved through engineering of dynamic controllability, modular and hierarchical organization, or functional redundancy, three key features of biological robustness in a cellular system. This review summarizes how synthetic biology can be used to improve the robustness of industrial microbes.     

Biomedical Applications of Polyhydroxyalkanoates

Polyhydroxyalkanoates (PHA) are produced by a large number of microbes under stress conditions such as high carbon (C) availability and limitations of nutrients such as nitrogen, potassium, phosphorus, magnesium, and oxygen. Here, microbes store C as granules of PHAs—energy reservoir. PHAs have properties, which are quite similar to those of synthetic plastics. The unique properties, which make them desirable materials for biomedical applications is their biodegradability, biocompatibility, and non-toxicity. PHAs have been found suitable for various medical applications: biocontrol agents, drug carriers, biodegradable implants, tissue engineering, memory enhancers, and anticancer agents.     

Microbial amylolytic enzymes

Starch-degrading, amylolytic enzymes are widely distributed among microbes. Several activities are required to hydrolyze starch to its glucose units. These enzymes include alpha-amylase, beta-amylase, glucoamylase, alpha-glucosidase, pullulan-degrading enzymes, exoacting enzymes yielding alpha-type endproducts, and cyclodextrin glycosyltransferase. Properties of these enzymes vary and are somewhat linked to the environmental circumstances of the producing organisms. Features of the enzymes, their action patterns, physicochemical properties, occurrence, genetics, and results obtained from cloning of the genes are described. Among all the amylolytic enzymes, the genetics of alpha-amylase in Bacillus subtilis are best known. Alpha-Amylase production in B. subtilis is regulated by several genetic elements, many of which have synergistic effects. Genes encoding enzymes from all the amylolytic enzyme groups dealt with here have been cloned, and the sequences have been found to contain some highly conserved regions thought to be essential for their action and/or structure. Glucoamylase appears usually in several forms, which seem to be the results of a variety of mechanisms, including heterogeneous glycosylation, limited proteolysis, multiple modes of mRNA splicing, and the presence of several structural genes.     

Integrating enzyme immobilization and protein engineering: An alternative path for the development of novel and improved industrial biocatalysts

Enzyme immobilization often achieves reusable biocatalysts with improved operational stability and solvent resistance. However, these modifications are generally associated with a decrease in activity or detrimental modifications in catalytic properties. On the other hand, protein engineering aims to generate enzymes with increased performance at specific conditions by means of genetic manipulation, directed evolution and rational design. However, the achieved biocatalysts are generally generated as soluble enzymes, ?thus not reusable- and their performance under real operational conditions is uncertain.Combined protein engineering and enzyme immobilization approaches have been employed as parallel or consecutive strategies for improving an enzyme of interest. Recent reports show efforts on simultaneously improving both enzymatic and immobilization components through genetic modification of enzymes and optimizing binding chemistry for site-specific and oriented immobilization. Nonetheless, enzyme engineering and immobilization are usually performed as separate workflows to achieve improved biocatalysts.In this review, we summarize and discuss recent research aiming to integrate enzyme immobilization and protein engineering and propose strategies to further converge protein engineering and enzyme immobilization efforts into a novel “immobilized biocatalyst engineering” research field. We believe that through the integration of both enzyme engineering and enzyme immobilization strategies, novel biocatalysts can be obtained, not only as the sum of independently improved intrinsic and operational properties of enzymes, but ultimately tailored specifically for increased performance as immobilized biocatalysts, potentially paving the way for a qualitative jump in the development of efficient, stable biocatalysts with greater real-world potential in challenging bioprocess applications.     

Microbial formation, biotechnological production and applications of 1,2-propanediol

This short review covers metabolic pathways, genetics and metabolic engineering of 1,2-propanediol formation in microbes. 1,2-Propanediol production by bacteria and yeasts has been known for many years and two general pathways are recognized. One involves the metabolism of deoxyhexoses, where lactaldehyde is formed during the glycolytic reactions and is then reduced to 1,2-propanediol. The second pathway derives from the formation of methylglyoxal from dihydroxyacetonephosphate and its subsequent reduction to 1,2-propanediol. The enzymes involved in the reduction of methylglyoxal can generate isomers of lactaldehyde or acetol, which can be further reduced by specific reductases, giving chiral 1,2-propanediol as the product. The stereospecificity of the enzymes catalyzing the two reduction steps is important in deriving a complete pathway. Through genetic engineering, appropriate combinations of enzymes have been brought together in Escherichia coli and yeast to generate 1,2-propanediol from glucose. The optimization of these strains may yield microbial processes for the production of this widely used chemical. Received: 25 May 2000 / Received revision: 24 July 2000 / Accepted: 25 July 2000     

Acidophilic bacteria and archaea: acid stable biocatalysts and their potential applications

Acidophiles are ecologically and economically important group of microorganisms, which thrive in acidic natural (solfataric fields, sulfuric pools) as well as artificial man-made (areas associated with human activities such as mining of coal and metal ores) environments. They possess networked cellular adaptations to regulate pH inside the cell. Several extracellular enzymes from acidophiles are known to be functional at much lower pH than the cytoplasmic pH. Enzymes like amylases, proteases, ligases, cellulases, xylanases, α-glucosidases, endoglucanases, and esterases stable at low pH are known from various acidophilic microbes. The possibility of improving them by genetic engineering and directed evolution will further boost their industrial applications. Besides biocatalysts, other biomolecules such as plasmids, rusticynin, and maltose-binding protein have also been reported from acidophiles. Some strategies for circumventing the problems encountered in expressing genes encoding proteins from extreme acidophiles have been suggested. The investigations on the analysis of crystal structures of some acidophilic proteins have thrown light on their acid stability. Attempts are being made to use thermoacidophilic microbes for biofuel production from lignocellulosic biomass. The enzymes from acidophiles are mainly used in polymer degradation.     

平台化学品短链支链脂肪酸和短链支链醇的微生物代谢工程

短链支链脂肪酸和短链支链醇均为重要的平台化学品,是合成多种高附加值产品的前体物质,市场需求巨大。目前两者的生产主要是利用基于石化原料的化学合成法。化学合成法存在着严重依赖化石燃料、反应效率低以及极易造成环境污染等缺点。微生物代谢工程的快速发展为这些平台化学品的生产提供了一条极具潜力的生物合成路线。利用微生物代谢工程技术构建生产这些平台化学品的微生物细胞工厂具有绿色清洁、可持续发展和经济效益好等独特优势。本文系统综述了近年来微生物代谢工程技术在短链支链脂肪酸和短链支链醇合成方面的研究进展,包括所涉及的宿主菌株、关键酶、代谢途径及其改造等,并探讨了未来的发展前景。     

Structurally diverse dehydroshikimate dehydratase variants participate in microbial quinate catabolism

Quinate and shikimate can be degraded by a number of microbes. Dehydroshikimate dehydratases (DSDs) play a central role in this process, catalyzing the conversion of 3‐dehydroshikimate to protocatechuate, a common intermediate of aromatic degradation pathways. DSDs have applications in metabolic engineering for the production of valuable protocatechuate‐derived molecules. Although a number of Gram‐negative bacteria are known to catabolize quinate and shikimate, only limited information exists on the quinate/shikimate catabolic enzymes found in these organisms. Here, we have functionally and structurally characterized a putative DSD designated QuiC1, which is present in some pseudomonads. The QuiC1 protein is not related by sequence with previously identified DSDs from the Gram‐negative genus, Acinetobacter, but instead shows limited sequence identity in its N‐terminal half with fungal DSDs. Analysis of a Pseudomonas aeruginosa quiC1 gene knock‐out demonstrates that it is important for growth on either quinate or shikimate. The structure of a QuiC1 enzyme from P. putida reveals that the protein is a fusion of two distinct modules: an N‐terminal sugar phosphate isomerase‐like domain associated with DSD activity and a novel C‐terminal hydroxyphenylpyruvate dioxygenase‐like domain. The results of this study highlight the considerable diversity of enzymes that participate in quinate/shikimate catabolism in different microbes.