海洋宏基因组学研究进展

宏基因组学作为研究微生物种群生态分布、群体遗传特征和基因相互作用的新兴学科,在未培养微生物资源的开发利用上取得了突破性进展,已成为海洋等极端环境中分离与鉴定新型工业酶制剂的有效工具。综述海洋宏基因组学研究进展,以及宏基因组学领域中如新一代测序技术等,以期为从海洋环境中开发具有工业潜力和应用价值的新型生物催化剂提供参考。     

利用元基因组学方法从瘤胃中筛选的功能酶及酶基因研究进展

瘤胃微生物群落是一个复杂、庞大的生物体系,其生物多样性极为丰富,蕴藏着巨大的基因和生态资源,是酶制剂开发的重要宝库。元基因组学方法避免了微生物培养条件的限制,通过直接对未培养微生物进行DNA提取、基因筛选与表达,从而不断扩大自然界中的基因数据库,为新型生物催化剂的开发及筛选提供了可能。对元基因组学技术及利用此技术从瘤胃中筛选的功能酶类及酶基因的研究进展进行了综述。     

宏基因组学技术在微生物功能酶开发中的应用

宏基因组学技术以特定环境样品中微生物复杂群落的基因组总和为研究对象,突破了传统微生物纯培养方法的局限,为不可培养微生物中丰富的基因资源的开发和利用提供了强有力的工具,已经取得了令人瞩目的研究进展。对宏基因组学技术及其在微生物功能酶新基因发现中的应用进行综述。     

新型微生物脂肪酶资源开发

微生物脂肪酶是一类重要的工业生物催化剂, 广泛应用于工农业生产的诸多领域。筛选、挖掘和开发出具有新型催化活性或高稳定性的微生物脂肪酶一直是脂肪酶研究的重点。本文从极端微生物、宏基因组技术、基因组数据库挖掘、定向进化技术、固定化技术和化学修饰技术等方面介绍了当前新型脂肪酶开发的途径和方法。     

耐有机溶剂微生物的耐受机制、改造策略和应用北大核心

耐有机溶剂微生物在生物燃料生产、全细胞催化、酶制剂的开发和生物修复等领域具有非常重要的作用。该文简要介绍了耐有机溶剂微生物及其耐受机制,并从基因过表达、基因敲除和全局转录机制三个方面总结了增强微生物有机溶剂耐受性的方法。通过对耐有机溶剂微生物应用的局限性进行分析,认为未来研究中应注重将微生物耐受表型和具体利用相结合,达到有机溶剂耐受和实际产出的最佳平衡状态,为耐有机溶剂工程菌的改造和选育提供参考。     

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。     

耐有机溶剂微生物的耐受机制、改造策略和应用

耐有机溶剂微生物在生物燃料生产、全细胞催化、酶制剂的开发和生物修复等领域具有非常重要的作用。该文简要介绍了耐有机溶剂微生物及其耐受机制,并从基因过表达、基因敲除和全局转录机制三个方面总结了增强微生物有机溶剂耐受性的方法。通过对耐有机溶剂微生物应用的局限性进行分析,认为未来研究中应注重将微生物耐受表型和具体利用相结合,达到有机溶剂耐受和实际产出的最佳平衡状态,为耐有机溶剂工程菌的改造和选育提供参考。     

宏基因组学技术在酶开发方面的应用

宏基因组技术是近些年发展起来的生物新技术。其针对环境样本中的全部微生物基因组DNA开展研究,可以覆盖过去无法研究的不可培养微生物,极大地拓宽了研究的广度,因而,一经提出就得到广泛应用,并取得丰富成果。本文主要针对宏基因组技术在酶开发方面的有关应用进行综述。     

宏基因组学结合合成生物学法挖掘新型生物催化剂的研究进展

生物催化剂是具有催化活性的细胞和酶的统称,因其高效和环境友好的特性被广泛应用于工业、农业、医药和能源等领域。传统的生物催化剂挖掘技术依赖于生物分离及体外培养,在一定程度上阻碍了生物催化剂的发展。宏基因组学挖掘新型生物催化剂通过直接从环境样品中提取全部微生物的DNA,构建宏基因组文库,再基于功能活性和序列分析筛选新型生物催化剂,避免了微生物的分离培养。合成生物学是利用工程学原理,通过元件的设计,组合和系统的构建对生物途径、有机体或生物系统进行重新设计。综述了宏基因组学方法挖掘新型生物催化剂的最新研究进展,并对利用合成生物学方法改进宏基因组筛选新型生物催化剂的效率进行了讨论。     

土壤微生物中新型β-葡萄糖苷酶的挖掘与鉴定

【背景】通过培养微生物来获得新β-葡萄糖苷酶因只有少部分微生物可以被培养而受到限制,但宏基因组学技术可以挖掘非培养微生物来源的β-葡萄糖苷酶资源。【目的】利用宏基因组学技术挖掘土壤微生物来源的新型β-葡萄糖苷酶,并对其酶学性质进行初步分析。【方法】构建土壤微生物的宏基因组文库,利用七叶苷平板显色法对所构建的文库进行筛选获得阳性克隆,并对阳性克隆所含的β-葡萄糖苷酶基因进行异源表达和生物化学特性分析。【结果】通过筛选文库中的62万个克隆,获得一个具有β-葡萄糖苷酶活性的克隆,其插入片段中包含一个2 301 bp的ORF(YNBG3),蛋白同源性分析显示其属于β-葡萄糖苷酶第三家族。对YNBG3酶的生化分析确定其最佳反应温度为53°C,最适p H为5.2,有较好的热稳定性,对一定浓度范围内的DMSO、丙酮、乙醇等有机试剂有较好的耐受性,EDTA和尿素可增加该酶的活性。【结论】利用宏基因组学技术获得了一个有较好热稳定性及耐受一定浓度有机试剂和尿素的新β-葡萄糖苷酶。     

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.     

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.     

Modifying plants for biofuel and biomaterial production

The productivity of plants as biofuel or biomaterial crops is established by both the yield of plant biomass per unit area of land and the efficiency of conversion of the biomass to biofuel. Higher yielding biofuel crops with increased conversion efficiencies allow production on a smaller land footprint minimizing competition with agriculture for food production and biodiversity conservation. Plants have traditionally been domesticated for food, fibre and feed applications. However, utilization for biofuels may require the breeding of novel phenotypes, or new species entirely. Genomics approaches support genetic selection strategies to deliver significant genetic improvement of plants as sources of biomass for biofuel manufacture. Genetic modification of plants provides a further range of options for improving the composition of biomass and for plant modifications to assist the fabrication of biofuels. The relative carbohydrate and lignin content influences the deconstruction of plant cell walls to biofuels. Key options for facilitating the deconstruction leading to higher monomeric sugar release from plants include increasing cellulose content, reducing cellulose crystallinity, and/or altering the amount or composition of noncellulosic polysaccharides or lignin. Modification of chemical linkages within and between these biomass components may improve the ease of deconstruction. Expression of enzymes in the plant may provide a cost‐effective option for biochemical conversion to biofuel.     

Secretome of the Coprophilous Fungus Doratomyces stemonitis C8, Isolated from Koala Feces

Coprophilous fungi inhabit herbivore feces, secreting enzymes to degrade the most recalcitrant parts of plant biomass that have resisted the digestive process. Consequently, the secretomes of coprophilous fungi have high potential to contain novel and efficient plant cell wall degrading enzymes of biotechnological interest. We have used one-dimensional and two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization-time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), and quadrupole time-of-flight liquid chromatography-tandem mass spectrometry (Q-TOF LC-MS/MS) to identify proteins from the secretome of the coprophilous fungus Doratomyces stemonitis C8 (EU551185) isolated from koala feces. As the genome of D. stemonitis has not been sequenced, cross-species identification, de novo sequencing, and zymography formed an integral part of the analysis. A broad range of enzymes involved in the degradation of cellulose, hemicellulose, pectin, lignin, and protein were revealed, dominated by cellobiohydrolase of the glycosyl hydrolase family 7 and endo-1,4-β-xylanase of the glycosyl hydrolase family 10. A high degree of specialization for pectin degradation in the D. stemonitis C8 secretome distinguishes it from the secretomes of some other saprophytic fungi, such as the industrially exploited T. reesei. In the first proteomic analysis of the secretome of a coprophilous fungus reported to date, the identified enzymes provide valuable insight into how coprophilous fungi subsist on herbivore feces, and these findings hold potential for increasing the efficiency of plant biomass degradation in industrial processes such as biofuel production in the future.     

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.     

Strategies for Enhancing the Effectiveness of Metagenomic-based Enzyme Discovery in Lignocellulolytic Microbial Communities

Producing cellulosic biofuels from plant material has recently emerged as a key US Department of Energy goal. For this technology to be commercially viable on a large scale, it is critical to make production cost efficient by streamlining both the deconstruction of lignocellulosic biomass and fuel production. Many natural ecosystems efficiently degrade lignocellulosic biomass and harbor enzymes that, when identified, could be used to increase the efficiency of commercial biomass deconstruction. However, ecosystems most likely to yield relevant enzymes, such as tropical rain forest soil in Puerto Rico, are often too complex for enzyme discovery using current metagenomic sequencing technologies. One potential strategy to overcome this problem is to selectively cultivate the microbial communities from these complex ecosystems on biomass under defined conditions, generating less complex biomass-degrading microbial populations. To test this premise, we cultivated microbes from Puerto Rican soil or green waste compost under precisely defined conditions in the presence dried ground switchgrass (Panicum virgatum L.) or lignin, respectively, as the sole carbon source. Phylogenetic profiling of the two feedstock-adapted communities using SSU rRNA gene amplicon pyrosequencing or phylogenetic microarray analysis revealed that the adapted communities were significantly simplified compared to the natural communities from which they were derived. Several members of the lignin-adapted and switchgrass-adapted consortia are related to organisms previously characterized as biomass degraders, while others were from less well-characterized phyla. The decrease in complexity of these communities make them good candidates for metagenomic sequencing and will likely enable the reconstruction of a greater number of full-length genes, leading to the discovery of novel lignocellulose-degrading enzymes adapted to feedstocks and conditions of interest.     

Enhanced cellulose degradation using cellulase-nanosphere complexes

Enzyme catalyzed conversion of plant biomass to sugars is an inherently inefficient process, and one of the major factors limiting economical biofuel production. This is due to the physical barrier presented by polymers in plant cell walls, including semi-crystalline cellulose, to soluble enzyme accessibility. In contrast to the enzymes currently used in industry, bacterial cellulosomes organize cellulases and other proteins in a scaffold structure, and are highly efficient in degrading cellulose. To mimic this clustered assembly of enzymes, we conjugated cellulase obtained from Trichoderma viride to polystyrene nanospheres (cellulase:NS) and tested the hydrolytic activity of this complex on cellulose substrates from purified and natural sources. Cellulase:NS and free cellulase were equally active on soluble carboxymethyl cellulose (CMC); however, the complexed enzyme displayed a higher affinity in its action on microcrystalline cellulose. Similarly, we found that the cellulase:NS complex was more efficient in degrading natural cellulose structures in the thickened walls of cultured wood cells. These results suggest that nanoparticle-bound enzymes can improve catalytic efficiency on physically intractable substrates. We discuss the potential for further enhancement of cellulose degradation by physically clustering combinations of different glycosyl hydrolase enzymes, and applications for using cellulase:NS complexes in biofuel production.     

Chloroplast molecular farming: efficient production of a thermostable xylanase by Nicotiana tabacum plants and long-term conservation of the recombinant enzyme

The high cost of recombinant enzymes for the production of biofuel from ligno-cellulosic biomass is a crucial factor affecting the economic sustainability of the process. The use of plants as biofactories for the production of the suitable recombinant enzymes might be an alternative to microbial fermentation. In the case of enzyme accumulation in chloroplasts, it is fundamental to focus on the issue of full photosynthetic efficiency of transplastomic plants in the field where they might be exposed to abiotic stress such as high light intensity and high temperature. Xylanases (EC 3.2.1.8), a group of enzymes that hydrolyse linear polysaccharides of beta-1,4-xylan into xylose, find an application in the biofuel industry favouring biomass saccharification along with other cell-wall degrading enzymes. In the present study, we analysed how a high level of accumulation of a thermostable xylanase in tobacco chloroplasts does not impact on photosynthetic performance of transplastomic plants grown outdoors. The recombinant enzyme was found to be stable during plant development, ex planta and after long-term storage.     

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.     

Plant-microbe interactions: novel applications for exploitation in multipurpose remediation technologies

Soil remediation that revitalizes degraded or contaminated land while simultaneously contributing to biomass biofuel production and carbon sequestration is an attractive strategy to meet the food and energy requirements of the burgeoning world population. As a result, plant-based remediation approaches have been gaining in popularity. The drawbacks of phytoremediation, particularly those associated with low productivity and limitations to the use of contaminant-containing biomass, could be addressed through novel biotechnological approaches that harness recent advances in our understanding of chemical interactions between plants and microorganisms in the rhizosphere and within plant tissues. This opinion article highlights three promising approaches that provide environmental and economic benefits of bioremediation: transgenics, low-input 'designer' plants and nanotechnology.