Application of metagenomic techniques in mining enzymes from microbial communities for biofuel synthesis

Feedstock for biofuel synthesis is transitioning to lignocelluosic biomass to address criticism over competition between first generation biofuels and food production. As microbial catalysis is increasingly applied for the conversion of biomass to biofuels, increased import has been placed on the development of novel enzymes. With revolutionary advances in sequencer technology and metagenomic sequencing, mining enzymes from microbial communities for biofuel synthesis is becoming more and more practical. The present article highlights the latest research progress on the special characteristics of metagenomic sequencing, which has been a powerful tool for new enzyme discovery and gene functional analysis in the biomass energy field. Critical enzymes recently developed for the pretreatment and conversion of lignocellulosic materials are evaluated with respect to their activity and stability, with additional explorations into xylanase, laccase, amylase, chitinase, and lipolytic biocatalysts for other biomass feedstocks.     

土壤宏基因组学技术及其应用

传统的基于培养的研究方法只能反映土壤中少数(0.1%～10 %)微生物的信息,而大部分微生物目前还不能培养,因而这部分微生物资源尚难以被有效地开发利用.宏基因组学是分子生物学技术应用于环境微生物生态学研究而形成的一个新概念,主要技术包括土壤DNA的提取、文库的构建和目标基因克隆的筛选.它可为揭示微生物生态功能及其分子基础提供更全面的遗传信息,并已在微生物新功能基因筛选、活性物质开发和微生物多样性研究等方面取得了显著成果.本文对土壤宏基因组学技术的方法和应用作了详细介绍.     

Recent Advances in Function-based Metagenomic Screening

Metagenomes from uncultured microorganisms are rich resources for novel enzyme genes. The methods used to screen the metagenomic libraries fall into two categories, which are based on sequence or function of the enzymes. The sequence-based approaches rely on the known sequences of the target gene families. In contrast, the function-based approaches do not involve the incorporation of metagenomic sequencing data and, therefore, may lead to the discovery of novel gene sequences with desired functions. In this review, we discuss the function-based screening strategies that have been used in the identification of enzymes from metagenomes. Because of its simplicity, agar plate screening is most commonly used in the identification of novel enzymes with diverse functions. Other screening methods with higher sensitivity are also employed, such as microtiter plate screening. Furthermore, several ultra-high-throughput methods were developed to deal with large metagenomic libraries. Among these are the FACS-based screening, droplet-based screening, and the in vivo reporter-based screening methods. The application of these novel screening strategies has increased the chance for the discovery of novel enzyme genes.     

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。     

Synthetic biology approaches to improve biocatalyst identification in metagenomic library screening

There is a growing demand for enzymes with improved catalytic performance or tolerance to process‐specific parameters, and biotechnology plays a crucial role in the development of biocatalysts for use in industry, agriculture, medicine and energy generation. Metagenomics takes advantage of the wealth of genetic and biochemical diversity present in the genomes of microorganisms found in environmental samples, and provides a set of new technologies directed towards screening for new catalytic activities from environmental samples with potential biotechnology applications. However, biased and low level of expression of heterologous proteins in Escherichia coli together with the use of non‐optimal cloning vectors for the construction of metagenomic libraries generally results in an extremely low success rate for enzyme identification. The bottleneck arising from inefficient screening of enzymatic activities has been addressed from several perspectives; however, the limitations related to biased expression in heterologous hosts cannot be overcome by using a single approach, but rather requires the synergetic implementation of multiple methodologies. Here, we review some of the principal constraints regarding the discovery of new enzymes in metagenomic libraries and discuss how these might be resolved by using synthetic biology methods.     

Metagenomics for the development of new biocatalysts to advance lignocellulose saccharification for bioeconomic development

The world economy is moving toward the use of renewable and nonedible lignocellulosic biomasses as substitutes for fossil sources in order to decrease the environmental impact of manufacturing processes and overcome the conflict with food production. Enzymatic hydrolysis of the feedstock is a key technology for bio-based chemical production, and the identification of novel, less expensive and more efficient biocatalysts is one of the main challenges. As the genomic era has shown that only a few microorganisms can be cultured under standard laboratory conditions, the extraction and analysis of genetic material directly from environmental samples, termed metagenomics, is a promising way to overcome this bottleneck. Two screening methodologies can be used on metagenomic material: the function-driven approach of expression libraries and sequence-driven analysis based on gene homology. Both techniques have been shown to be useful for the discovery of novel biocatalysts for lignocellulose conversion, and they enabled identification of several (hemi)cellulases and accessory enzymes involved in (hemi)cellulose hydrolysis. This review summarizes the latest progress in metagenomics aimed at discovering new enzymes for lignocellulose saccharification.     

宏基因组学挖掘新型生物催化剂的研究进展

微生物蕴藏着大量具有工业应用潜力的生物催化剂。然而,传统培养方法只能从环境中获得不到1%的微生物。宏基因组学是通过提取某一特定环境中的所有微生物基因组DNA、构建基因组文库并对文库进行筛选,寻找和发现新的功能基因的一种方法。它绕过了微生物分离培养过程,成为研究环境样品中不可培养微生物的有力手段。因此,从宏基因组中挖掘新型生物催化剂一直倍受生物学家的关注。以下主要对宏基因组文库的样品来源、DNA提取方法、文库的构建和筛选策略的选择这4个方面的研究状况进行了综述,列举了近年来利用宏基因组技术所获得的新型生物催化剂,并对其今后的研究方向提出了展望。     

Characterization of two metagenome-derived esterases that reactivate chloramphenicol by counteracting chloramphenicol acetyltransferase

Function-driven metagenomic analysis is a powerful approach to screening for novel biocatalysts. In this study, we investigated lipolytic enzymes selected from an alluvial soil metagenomic library, and identified two novel esterases, EstDL26 and EstDL136. EstDL26 and EstDL136 reactivated chloramphenicol from its acetyl derivates by counteracting the chloramphenicol acetyltransferase (CAT) activity in Escherichia coli. These two enzymes showed only 27% identity in amino acid sequence to each other; however both preferentially hydrolyzed short-chain p-nitrophenyl esters (< or =C5) and showed mesophilic properties. In vitro, EstDL136 catalyzed the deacetylation of 1- and 3- acetyl and 1,3-diacetyl derivates; in contrast, EstDL26 was not capable of the deacetylation at C1, indicating a potential regioselectivity. EstDL26 and EstDL136 were similar to microbial hormone-sensitive lipase (HSL), and since chloramphenicol acetate esterase (CAE) activity was detected from two other soil esterases in the HSL family, this suggests a distribution of CAE among the soil microorganisms. The isolation and characterization of EstDL26 and EstDL136 in this study may be helpful in understanding the diversity of CAE enzymes and their potential role in releasing active chloramphenicol in the producing bacteria.     

Bacterial enzymes for lignin depolymerisation: new biocatalysts for generation of renewable chemicals from biomass

The conversion of polymeric lignin from plant biomass into renewable chemicals is an important unsolved problem in the biorefinery concept. This article summarises recent developments in the discovery of bacterial enzymes for lignin degradation, our current understanding of their molecular mechanism of action, and their use to convert lignin or lignocellulose into aromatic chemicals. The review also discusses the recent developments in screening of metagenomic libraries for new biocatalysts, and the use of protein engineering to enhance lignin degradation activity.     

Screening for novel lipolytic enzymes from uncultured soil microorganisms

The construction and screening of metagenomic libraries constitute a valuable resource for obtaining novel biocatalysts. In this work, we present the construction of a metagenomic library in Escherichia coli using fosmid and microbial DNA directly isolated from forest topsoil and screened for lipolytic enzymes. The library consisted of 33,700 clones with an average DNA insert size of 35 kb. Eight unique lipolytic active clones were obtained from the metagenomic library on the basis of tributyrin hydrolysis. Subsequently, secondary libraries in a high-copy-number plasmid were generated to select lipolytic subclones and to characterize the individual genes responsible for the lipolytic activity. DNA sequence analysis of six genes revealed that the enzymes encoded by the metagenomic genes for lipolytic activity were novel with 34–48% similarity to known enzymes. They had conserved sequences similar to those in the hormone-sensitive lipase family. Based on their deduced amino acid similarity, the six genes encoding lipolytic enzymes were further divided into three subgroups, the identities among which ranged from 33% to 45%. The six predicted gene products were successfully expressed in E. coli and secreted into the culture broth. Most of the secreted enzymes showed a catalytic activity for hydrolysis of p-nitrophenyl butyrate (C₄) but not p-nitrophenyl palmitate (C₁₆).     

宏基因组技术在新型生物燃料生产酶制剂开发中的应用

随着石化燃料的日益减少,以植物生物质为原料的可再生生物燃料成为石化燃料的理想替代品。然而微生物降解生物质效率低下,是生物燃料生产过程中一大难题,因此开发效率高、稳定性强的微生物酶制剂显得尤为重要。近年来,宏基因组技术的发展为生物燃料的生产提供了多种新型酶制剂。宏基因组技术是直接提取环境样品中的总DNA,通过构建文库,筛选目的基因或功能基因的方法,在用于燃料生产的新型酶制剂的开发中发挥着重要作用。本文概述了宏基因组技术的实施策略,总结了包括纤维素酶、蛋白酶、酯酶、脂肪酶等多种酶资源开发的最新研究进展,并综合和讨论了通过酶法将木质纤维素等生物材料有效转化为生物燃料的途径,为新酶的开发提供了新思路。     

Metagenomics: Concept,methodology, ecological inference and recent advances

Microorganisms constitute two third of the Earth's biological diversity. As many as 99% of the microorganisms present in certain environments cannot be cultured by standard techniques. Culture-independent methods are required to understand the genetic diversity, population structure and ecological roles of the majority of organisms. Metagenomics is the genomic analysis of microorganisms by direct extraction and cloning of DNA from their natural environment. Protocols have been developed to capture unexplored microbial diversity to overcome the existing barriers in estimation of diversity. New screening methods have been designed to select specific functional genes within metagenomic libraries to detect novel biocatalysts as well as bioactive molecules applicable to mankind. To study the complete gene or operon clusters, various vectors including cosmid, fosmid or bacterial artificial chromosomes are being developed. Bioinformatics tools and databases have added much to the study of microbial diversity. This review describes the various methodologies and tools developed to understand the biology of uncultured microbes including bacteria, archaea and viruses through metagenomic analysis.     

Enantioselective biocatalysis optimized by directed evolution

Directed evolution methods are now widely used for the optimization of diverse enzyme properties, which include biotechnologically relevant characteristics like stability, regioselectivity and, in particular, enantioselectivity. In principle, three different approaches are followed to optimize enantioselective reactions: the development of whole-cell biocatalysts through the creation of designer organisms; the optimization of enzymes with existing enantioselectivity for process conditions; and the evolution of novel enantioselective biocatalysts starting from non-selective wild-type enzymes.     

An ultrahigh-throughput screening platform based on flow cytometric droplet sorting for mining novel enzymes from metagenomic libraries

Uncultivable microbial communities provide enormous reservoirs of enzymes, but their experimental identification by functional metagenomics is challenging, mainly due to the difficulty of screening enormous metagenomic libraries. Here, we propose a reliable and convenient ultrahigh-throughput screening platform based on flow cytometric droplet sorting (FCDS). The FCDS platform employs water-in-oil-in-water double emulsion droplets serving as single-cell enzymatic micro-reactors and a commercially available flow cytometer, and it can efficiently isolate novel biocatalysts from metagenomic libraries by processing single cells as many as 10⁸ per day. We demonstrated the power of this platform by screening a metagenomic library constructed from domestic running water samples. The FCDS assay screened 30 million micro-reactors in only 1 h, yielding a collection of esterase genes. Among these positive hits, Est WY was identified as a novel esterase with high catalytic efficiency and distinct evolutionary origin from other lipolytic enzymes. Our study manifests that the FCDS platform is a robust tool for functional metagenomics, with the potential to significantly improve the efficiency of exploring novel enzymes from nature.     

The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass

Lignocellulosic biomass is a renewable and abundant resource with great potential for bioconversion to value-added bioproducts. However, the biorefining process remains economically unfeasible due to a lack of biocatalysts that can overcome costly hurdles such as cooling from high temperature, pumping of oxygen/stirring, and, neutralization from acidic or basic pH. The extreme environmental resistance of bacteria permits screening and isolation of novel cellulases to help overcome these challenges. Rapid, efficient cellulase screening techniques, using cellulase assays and metagenomic libraries, are a must. Rare cellulases with activities on soluble and crystalline cellulose have been isolated from strains of Paenibacillus and Bacillus and shown to have high thermostability and/or activity over a wide pH spectrum. While novel cellulases from strains like Cellulomonas flavigena and Terendinibacter turnerae, produce multifunctional cellulases with broader substrate utilization. These enzymes offer a framework for enhancement of cellulases including: specific activity, thermalstability, or end-product inhibition. In addition, anaerobic bacteria like the clostridia offer potential due to species capable of producing compound multienzyme complexes called cellulosomes. Cellulosomes provide synergy and close proximity of enzymes to substrate, increasing activity towards crystalline cellulose. This has lead to the construction of designer cellulosomes enhanced for specific substrate activity. Furthermore, cellulosome-producing Clostridium thermocellum and its ability to ferment sugars to ethanol; its amenability to co-culture and, recent advances in genetic engineering, offer a promising future in biofuels. The exploitation of bacteria in the search for improved enzymes or strategies provides a means to upgrade feasibility for lignocellulosic biomass conversion, ultimately providing means to a ''greener'' technology.     

Screening for novel enzymes from metagenome and SIGEX,as a way to improve it

Metagenomics has been successfully applied to isolate novel biocatalysts from the uncultured microbiota in the environment. Two types of screening have been used to identify clones carrying desired traits from metagenomic libraries: function-based screening, and sequence-based screening. Both function- and sequence- based screening have individual advantages and disadvantages, and they have been applied successfully to discover biocatalysts from metagenome. However, both strategies are laborious and tedious because of the low frequency of screening hits. A recent paper introduced a high throughput screening strategy, termed substrate-induced gene-expression screening (SIGEX). SIGEX is designed to select the clones harboring catabolic genes induced by various substrates in concert with fluorescence activated cell sorting (FACS). This method was applied successfully to isolate aromatic hydrocarbon-induced genes from a metagenomic library. Although SIGEX has many limitations, it is expected to provide economic advantages, especially to industry.     

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.     

Recent advances in biocatalyst discovery,development and applications

Enzymes catalyze a wide range of biotransformations and have a great potential as environmentally friendly alternatives to classical chemical catalysts in various industrial applications. Recently, advanced techniques and strategies in enzyme discovery and engineering have led to the significant expansion of the quantity and functional diversity of biocatalysts, which has further allowed broader uses of biocatalysts in new processes, especially those traditionally enabled only by chemical catalysts. Here we highlight some of these recent advances with the focus on new approaches in biocatalyst discovery and development, and discuss new applications of selected biocatalysts including transaminases, cytochrome P450s, and Baeyer–Villiger monooxygenases.