Metagenomics,biotechnology with non-culturable microbes

Metagenomics as a new field of research has been developed over the past decade to elucidate the genomes of the non-cultured microbes with the goal to better understand global microbial ecology on the one side, and on the other side it has been driven by the increasing biotechnological demands for novel enzymes and biomolecules. Since it is well accepted that the majority of all microbes has not yet been cultured, the not-yet-cultivated microbes represent a shear unlimited and intriguing resource for the development of novel genes, enzymes and chemical compounds for use in biotechnology. However, with respect to biotechnology, metagenomics faces now two major challenges. Firstly, it has to identify truly novel biocatalysts to fulfil the needs of industrial processes and green chemistry. Secondly, the already available genes and enzymes need to be implemented in production processes to further prove the value of metagenome-derived sequences.     

Improving the ‘tool box’ for robust industrial enzymes

The speed of sequencing of microbial genomes and metagenomes is providing an ever increasing resource for the identification of new robust biocatalysts with industrial applications for many different aspects of industrial biotechnology. Using ‘natures catalysts’ provides a sustainable approach to chemical synthesis of fine chemicals, general chemicals such as surfactants and new consumer-based materials such as biodegradable plastics. This provides a sustainable and ‘green chemistry’ route to chemical synthesis which generates no toxic waste and is environmentally friendly. In addition, enzymes can play important roles in other applications such as carbon dioxide capture, breakdown of food and other waste streams to provide a route to the concept of a ‘circular economy’ where nothing is wasted. The use of improved bioinformatic approaches and the development of new rapid enzyme activity screening methodology can provide an endless resource for new robust industrial biocatalysts.This mini-review will discuss several recent case studies where industrial enzymes of ‘high priority’ have been identified and characterised. It will highlight specific hydrolase enzymes and recent case studies which have been carried out within our group in Exeter.

     

Artificial enzymes with protein scaffolds: Structural design and modification

Recent development in biochemical experiment techniques and bioinformatics has enabled us to create a variety of artificial biocatalysts with protein scaffolds (namely ‘artificial enzymes’). The construction methods of these catalysts include genetic mutation, chemical modification using synthetic molecules and/or a combination of these methods. Designed evolution strategy based on the structural information of host proteins has become more and more popular as an effective approach to construct artificial protein-based biocatalysts with desired reactivities. From the viewpoint of application of artificial enzymes for organic synthesis, recently constructed artificial enzymes mediating oxidation, reduction and C–C bond formation/cleavage are introduced in this review article.     

Harnessing the power of enzymes for environmental stewardship

Enzymes are versatile catalysts with a growing number of applications in biotechnology. Their properties render them also attractive for waste/pollutant treatment processes and their use might be advantageous over conventional treatments. This review highlights enzymes that are suitable for waste treatment, with a focus on cell-free applications or processes with extracellular and immobilized enzymes. Biological wastes are treated with hydrolases, primarily to degrade biological polymers in a pre-treatment step. Oxidoreductases and lyases are used to biotransform specific pollutants of various nature. Examples from pulp and paper, textile, food and beverage as well as water and chemical industries illustrate the state of the art of enzymatic pollution treatment. Research directions in enzyme technology and their importance for future development in environmental biotechnology are elaborated. Beside biological and biochemical approaches, i.e. enzyme prospection and the design of enzymes, the review also covers efforts in adjacent research fields such as insolubilization of enzymes, reactor design and the use of additives. The effectiveness of enzymatic processes, especially when combined with established technologies, is evident. However, only a limited number of enzymatic field applications exist. Factors like cost and stability of biocatalysts need to be addressed and the collaboration and exchange between academia and industry should be further strengthened to achieve the goal of sustainability.     

The search for the ideal biocatalyst.

While the use of enzymes as biocatalysts to assist in the industrial manufacture of fine chemicals and pharmaceuticals has enormous potential, application is frequently limited by evolution-led catalyst traits. The advent of designer biocatalysts, produced by informed selection and mutation through recombinant DNA technology, enables production of process-compatible enzymes. However, to fully realize the potential of designer enzymes in industrial applications, it will be necessary to tailor catalyst properties so that they are optimal not only for a given reaction but also in the context of the industrial process in which the enzyme is applied.     

The industrial production of enzymes

The production of enzymes is a pursuit central to the modern biotechnology industry. Markets for traditional industrial enzymes continue to grow while the continued emphasis on biotechnological endeavours has generated demand for an ever increasing number of additional biocatalysts. The advent of genetic engineering has now facilitated the large-scale production of enzymes and other proteins which are produced naturally only in minute quantities. This development is particularly significant with regard to the production of enzymes and other proteins of therapeutic significance, which are now available in clinically useful quantities.

The level of downstream processing to which any enzyme is subjected is dependent upon its intended application. Industrial enzymes produced in bulk generally require little downstream processing, and hence are relatively crude preparations. Enzymes destined for therapeutic applications are subject to a far higher degree of downstream processing, often incorporating 3–4 chromatographic steps.

While enzymology is one of the longest established branches of the biochemical sciences, it continues to be an area of ongoing, active research. The continual discovery of new enzymes and a greater understanding of previously discovered enzymes and their functional significance suggests many novel applications for these catalytic activities. The intestinal production and utilization of enzymes will continue to be of central importance in the biotechnology industry.     

Cellulases and related enzymes in biotechnology

Basic and applied research on microbial cellulases, hemicellulases and pectinases has not only generated significant scientific knowledge but has also revealed their enormous potential in biotechnology. At present, cellulases and related enzymes are used in food, brewery and wine, animal feed, textile and laundry, pulp and paper industries, as well as in agriculture and for research purposes. Indeed, the demand for these enzymes is growing more rapidly than ever before, and this demand has become the driving force for research on cellulases and related enzymes. The present article is an overview of the biotechnological state-of-the-art for cellulases and related enzymes.     

Extremozyme

Extremozymes for biotechnological applications Industrial biotechnology is a fast growing and proliferating field of research. Biocatalysis gradually replaces chemical processes and is widely used in textile or food industry or in the sustainable production of fine chemicals. Although currently most of the enzymes in industry are of mesophilic origin, the focus is changing towards more robust biocatalysts from extremophilic organisms. Research on extremophiles will progressively supply novel extremozymes for biotechnological applications. In particular (hyper‐)thermophiles, acidophiles or salt‐tolerant microorganisms are a rich source of industrial applicable and robust extremozymes with optimal activity under harsh conditions.     

白色生物技术及其应用进展

“白色”生物技术也叫做工业生物技术,是利用某些微生物或酶进行物质转化,生产新产品或改进原有工业处理过程的技术。其产品可生物降解,生产过程能耗低,废弃物少。它是一门涉及生物学、微生物学、分子生物学、化学以及工程学等多学科的研究领域。综述了白色生物技术的产业优势及其涉及的研究领域,并从生物催化、生物材料、生物能源等方面概述了白色生物技术的应用进展。     

Potential biocatalysts originating from sea environments

This review is intended to give an account of the knowledge about known enzymes of marine origin described in literature thus stimulating future applications in biocatalysis that these biocatalysts can offer to a large spectra of end-users. The uniqueness of marine biocatalysts is not only based on habitat-related properties such as salt tolerance, hyperthermostability, barophilicity, cold adaptivity, etc. A marine enzyme in fact may carry more, e.g. novel chemical and stereochemical properties. This “chemical biodiversity” increases interest in this field; substrate specificity and affinity are evolved properties linked to the metabolic functions of the enzymes and to ecological asset related to the natural source and this is an important aspect in the bioprospecting for new biocatalysts. The importance of all examples reported should be sufficient to trigger the attention of the biocatalytically oriented scientific community towards marine environment as source of biocatalysts, and this could in turn enhance both new discovery and improvement of marine enzymes.     

生物催化剂发现与改造的研究进展

生物催化是指将酶或生物有机体用于有用的化学转化的过程,在人们对传统化学催化的环境影响抱有忧虑的情况下,生物催化提供了一种有吸引力的选择。在过去的几十年里,对生物催化剂的研究每出现一次大的进步,生物催化的发展就会出现一次高潮。因此,生物催化剂的发现与改造已成为当今研究的热点。宏基因组文库技术的出现克服了许多微生物不可培养的障碍,人们能够从自然资源中获得丰富的潜在的生物催化剂。而基于理性设计的分子改造技术的发展,可以使得人们对潜在的生物催化剂进行快速而有效的改造以满足工业化生产的需求。随着生物催化剂发现与改造的手段不断进步,更多的优良生物催化剂得到了广泛的应用,生物催化在工业生产中也得到了更深入的应用。结合作者的研究工作,总结了生物催化剂发现与改良的一些研究进展,以为获得更多优良的、能够实现工业应用的生物催化剂奠定理论基础。     

Microfluidic tools toward industrial biotechnology

Microfluidics is a technology that operates with small amounts of fluids and makes possible the investigation of cells, enzymes, and biomolecules and encapsulation of biocatalysts in a greater variety of conditions than permitted using conventional methods. This review discusses technological possibilities that can be applied in the field of industrial biotechnology, presenting the principal definitions and fundamental aspects of microfluidic parameters to better understand advanced approaches. Specifically, concentration gradient generators, droplet‐based microfluidics, and microbioreactors are explored as useful tools that can contribute to industrial biotechnology. These tools present potential applications, inclusive as commercial platforms to optimizing in bioprocesses development as screening cells, encapsulating biocatalysts, and determining critical kinetic parameters. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1372–1389, 2016     

极端微生物：一种新型的酶资源

极端微生物具有自身独特的特点和代谢产物 ,在食品工业、化工、药用工业和环境生物技术领域都有潜在的应用。一些酶已经得到纯化 ,其基因在宿主中已成功克隆。主要介绍和讨论极端微生物的类型、基因组及极端酶类的生产、分离与应用。     

Microbial formation of esters

Small aliphatic esters are important natural flavor and fragrance compounds and have numerous uses as solvents and as chemical intermediates. Besides the chemical or lipase-catalyzed formation of esters from alcohols and organic acids, small volatile esters are made by several biochemical routes in microbes. This short review will cover the biosynthesis of esters from acyl-CoA and alcohol condensation, from oxidation of hemiacetals formed from aldehydes and alcohols, and from the insertion of oxygen adjacent to the carbonyl group in a straight chain or cyclic ketone by Baeyer–Villiger monooxygenases. The physiological role of the ester-forming reactions can allow degradation of ketones for use as a carbon source and may play a role in detoxification of aldehydes or recycling cofactors. The enzymes catalyzing each of these processes have been isolated and characterized, and a number of genes encoding the proteins from various microbes have been cloned and functionally expressed. The use of these ester-forming organisms or recombinant organisms expressing the appropriate genes as biocatalysts in biotechnology to make specific esters and chiral lactones has been studied in recent years.     

Biocatalysis for synthesis of pharmaceuticals

Chirality is a key factor in the safety and efficacy of many drug products and thus the production of single enantiomers of drug intermediates and drugs has become important and state of the art in the pharmaceutical industry. There has been an increasing awareness of the enormous potential of microorganisms and enzymes (biocatalysts) for the transformation of synthetic chemicals with high chemo-, regio- and enatioselectivities providing products in high yields and purity. In this article, biocatalytic processes are described for the synthesis of key chiral intermediates for development pharmaceuticals.     

Screening for novel biocatalysts

This review attempts to demonstrate the importance of goal-orientated screening for new biocatalysts. Examples of enzymes and microorganisms that have been developed and that have acquired commercial applications are described so as to illustrate the technological potential of biocatalysts. A survey of screening techniques and recently reported examples of screening from food, chemical, pharmaceutical and waste disposal applications etc. are also presented to demonstrate the feasibility of this approach for generating new biocatalysts. An appreciation of some of the difficulties involved, the achievements of Japanese researchers and some examples of the cornucopia of largely unrecognized and potentially valuable microbial activities are also given. An increased effort in screening would have the following benefits: an increased range of biocatalysts with different enzyme activities would be available and more biocatalysts with improved characteristics, suitable for use under industrial conditions, such as resistance to elevated temperatures, extremes of pH and organic solvents would be discovered. Secondly the manpower and other resources required to carry out screening programmes would be reduced, for instance by developing automated techniques. Thirdly, screening procedures would be made much more accessible to non-specialists. Fourthly, improved efforts and expertise in screening would supplement other emerging techniques such as protein engineering. The development of selective, non-random, goal-orientated screening techniques, methods of evaluating biocatalyst performance under operational conditions, and an approach that is more orientated towards commercially desirable goals are essential if these objectives are to be achieved. Screening of naturally occurring microorganisms still appears to be the best way to obtain new strains and/or enzymes for commercial applications. However, two major problems appear to exist. Firstly in identifying applications that are technically feasible and that have sufficient commercial potential to justify the research and development required to generate a new commercially viable biocatalyst and secondly the relatively small number of scientists outside Japan with skill and experience in screening for biocatalysts.     

The Impact of Non-cultivated Biodiversity on Enzyme Discovery and Evolution

The search for novel enzymes with biotechnological potential in the fine chemical, food and feed, detergent and cosmetics industries is driven by the need to improve existing processes and applications, to design novel processes for innovative products or intermediates or to avoid intellectual property related operative restrictions. Strategies for obtaining novel biocatalysts will be based on screening natural biodiversity or a combination of nature derived scaffolds and optimization by directed evolution technology. Considering the enormous potential of in vitro mutational and recombinatorial strategies to alter genes and improve enzyme properties, we propose that it might be advantageous to select improved molecular starting points before embarking on the arduous walk through sequence space towards optimized performance.     

Yeast cell-surface display—applications of molecular display

In a cell-surface engineering system established using the yeast Saccharomyces cerevisiae, novel, so-called arming yeasts are constructed that are armed with biocatalysts in the form of enzymes, functional proteins, antibodies, and combinatorial protein libraries. Among the many advantages of the system, in which proteins are genetically displayed on the cell surface, are easy reproduction of the displayed biocatalysts and easy separation of product from catalyst. As proteins and peptides of various kinds can be displayed on the yeast cell surface, the system is expected to allow the preparation of tailor-made functional proteins. With its ability to express many of the functional proteins necessary for post-translational modification and in a range of different sizes, the yeast-based molecular display system appears uniquely useful among the various display systems so far developed. Capable of conferring novel additional abilities upon living cells, cell-surface engineering heralds a new era of combinatorial bioengineering in the field of biotechnology. This mini-review describes molecular display using yeast and its various applications.     

Extremophiles as a source of novel enzymes for industrial application

Extremophilic microorganisms are adapted to survive in ecological niches such as at high temperatures, extremes of pH, high salt concentrations and high pressure. These microorganisms produce unique biocatalysts that function under extreme conditions comparable to those prevailing in various industrial processes. Some of the enzymes from extremophiles have already been purified and their genes successfully cloned in mesophilic hosts. In this review we will briefly discuss the biotechnological significance of extreme thermophilic (optimal growth 70–80 °C) and hyperthermophilic (optimal growth 85–100 °C) archaea and bacteria. In particular, we will focus on selected extracellular-polymer-degrading enzymes, such as amylases, pullulanases, cyclodextrin glycosyltransferases, cellulases, xylanases, chitinases, proteinases and other enzymes such as esterases, glucose isomerases, alcohol dehydrogenases and DNA-modifying enzymes with potential use in food, chemical and pharmaceutical industries and in environmental biotechnology. Received: 14 August 1998 / Received revision: 17 February 1999 / Accepted: 19 February 1999     

Engineering of molecular and cellular biocatalysts: selected contributions by James E. Bailey

James (Jay) E. Bailey was a pioneer in biotechnology and biochemical engineering. During his 30 years in academia he made seminal contributions to many fields of chemical engineering science, including catalysis and reaction engineering, bioprocess engineering, mathematical modeling of cellular processes, recombinant DNA technology, enzyme engineering, and metabolic engineering. This article celebrates some of his contributions to the engineering of molecular and cellular biocatalysts, and identifies the influence he had on current and future research in biotechnology.