高通量自动化微生物微液滴进化培养与筛选技术及其装备化

液滴微流控由于可以快速生成大量微液滴,并实现单个液滴独立的控制,每个液滴都可以作为独立的单元进行微生物培养,因此在微生物的高通量培养方面具有独特的应用优势。然而现有研究多停留在实验室搭建和使用阶段,存在操作要求高、影响因素多、缺乏自动化集成技术等关键问题,制约了液滴微流控技术在微生物研究中的应用。文中以解决液滴微流控技术用于微生物培养的装备化问题为目标,系统研究了微流控各单元模块的结构与功能,通过对液滴的发生、培养、检测、分割、融合、分选等多种操作的开发与集成,成功研制出了小型一体化、全自动高通量的微生物微液滴培养(Microbial Microdroplet Culture system,MMC)装备系统,可用于微生物的生长曲线测定、适应性进化、单因素多水平分析及代谢物检测等,为面向微生物菌种高效选育的进化培养和筛选提供了高通量仪器平台。     

Single-cell microliter-droplet screening system (MISS Cell): An integrated platform for automated high-throughput microbial monoclonal cultivation and picking

Solid plates have been used for microbial monoclonal isolation, cultivation, and colony picking since 1881. However, the process is labor- and resource-intensive for high-throughput requirements. Currently, several instruments have been integrated for automated and high-throughput picking, but complicated and expensive. To address these issues, we report a novel integrated platform, the single-cell microliter-droplet screening system (MISS Cell), for automated, high-throughput microbial monoclonal colony cultivation and picking. We verified the monoclonality of droplet cultures in the MISS Cell and characterized culture performance. Compared with solid plates, the MISS Cell generated a larger number of monoclonal colonies with higher initial growth rates using fewer resources. Finally, we established a workflow for automated high-throughput screening of Corynebacterium glutamicum using the MISS Cell and identified high glutamate-producing strains. The MISS Cell can serve as a universal platform to efficiently produce monoclonal colonies in high-throughput applications, overcoming the limitations of solid plates to promote rapid development in biotechnology.     

常压室温等离子体诱变育种与微生物液滴培养筛选技术应用进展

安全和高效的微生物突变及高通量筛选技术是微生物功能发掘、功能创制和生物产业技术创新的重要方向及重要支撑.有效的生物育种技术及高通量筛选技术成为该领域研究人员的关注点.其中,常压室温等离子体(atmospheric and room temperature plasma,ARTP)因具有活性粒子种类多、操作可控性强、基因...     

基于流式细胞仪高通量分选的深海微生物单细胞培养

【目的】建立适用于海洋微生物的流式细胞分选与高通量单细胞培养的方法,通过该方法从印度洋深海样品中分离微生物纯培养菌株。【方法】利用流式细胞仪单细胞分选功能,以前向角(FSC)和侧向角(SSC)散射光信号代替荧光信号作为分选逻辑,对深海水体和沉积物样品中微生物进行单细胞高通量分选和培养。【结果】确定了流式细胞分选的区域和条件,发现所建立方法适于分离海洋水体微生物,而不是沉积物微生物。从印度洋深海水体样品中获得61个潜在新菌株,分属于6个新属种,占分离菌株总数的26.29%,其16S rRNA基因序列与已培养的模式菌株相似性为89.79%–95.37%。【结论】本研究所建立的方法有助于提高发现海洋微生物新物种的效率,获得更多新的海洋微生物资源。     

基于工业环境扰动的微生物适应性进化

合成生物学技术的快速发展极大提升了微生物细胞工厂的构建能力,为化学品的绿色高效生产提供了重要策略。然而,微生物细胞难以耐受高强度工业环境、抗逆性差,成为了限制其生产性能的关键因素。适应性进化是一种人为施加定向选择压力,使微生物经过长期或短期驯化,获得适应特定环境的表型或生理性能的重要方法。近年来,随着微流控、生物传感器、组学分析等技术的发展,适应性进化为提升微生物细胞在工业环境下的生产性能奠定了基础。本文论述了适应性进化的关键技术及在提高微生物细胞工厂环境耐受性和生产效率方面的重要应用,并展望了适应性进化实现微生物细胞工厂在工业环境下高效运行的重要前景。     

适应性实验室进化技术在微生物育种中的应用进展

适应性实验室进化(Adaptive laboratory evolution,ALE)技术已成为微生物学基础研究和工业微生物育种的强大工具,被广泛用来研究影响菌株表型、性能和稳定性的进化潜力以及快速获取含有有益突变的工业生产菌株。近年来,随着基因组测序技术的进步,关于微生物新陈代谢机理和动力学方面的研究变得更加广泛和深入,这也极大促进了适应性实验室进化技术的快速发展。文中主要介绍了长期、短期适应性实验室进化技术在微生物育种方面的应用实例,并总结归纳了该技术在快速高效构建优良菌株过程中的方式与作用。最后分析了目前ALE技术面临的瓶颈问题及其可能的解决方法,以期能够为该技术的未来发展提供有价值的参考依据。     

Improved bioconversion of lignocellulosic biomass by Saccharomyces cerevisiae engineered for tolerance to acetic acid

Lignocellulosic biomass has considerable potential for the production of fuels and chemicals as a promising alternative to conventional fossil fuels. However, the bioconversion of lignocellulosic biomass to desired products must be improved to reach economic viability. One of the main technical hurdles is the presence of inhibitors in biomass hydrolysates, which hampers the bioconversion efficiency by biorefinery microbial platforms such as Saccharomyces cerevisiae in terms of both production yields and rates. In particular, acetic acid, a major inhibitor derived from lignocellulosic biomass, severely restrains the performance of engineered xylose‐utilizing S. cerevisiae strains, resulting in decreased cell growth, xylose utilization rate, and product yield. In this study, the robustness of XUSE, one of the best xylose‐utilizing strains, was improved for the efficient conversion of lignocellulosic biomass into bioethanol under the inhibitory condition of acetic acid stress. Through adaptive laboratory evolution, we successfully developed the evolved strain XUSAE57, which efficiently converted xylose to ethanol with high yields of 0.43–0.50 g ethanol/g xylose even under 2–5 g/L of acetic stress. XUSAE57 not only achieved twofold higher ethanol yields but also improved the xylose utilization rate by more than twofold compared to those of XUSE in the presence of 4 g/L of acetic acid. During fermentation of lignocellulosic hydrolysate, XUSAE57 simultaneously converted glucose and xylose with the highest ethanol yield reported to date (0.49 g ethanol/g sugars). This study demonstrates that the bioconversion of lignocellulosic biomass by an engineered strain could be significantly improved through adaptive laboratory evolution for acetate tolerance, which could help realize the development of an economically feasible lignocellulosic biorefinery to produce fuels and chemicals.     

奶牛粪酸臭成分降解过程中的微生物群落变化

【目的】研究可降解成年泌乳奶牛粪中主要酸臭物的微生物群落的组成及动态变化。【方法】利用牛粪堆肥环境中的微生物进行了发酵优化、菌种驯化以及酸臭有机物降解规律的研究,结合r DNA高通量测序技术对有益微生物的组成及相对生物量进行了分析。【结果】实验发现,奶牛排泄物中的臭味来源主要为短链有机酸,堆肥自然环境中的微生物可以有效地对有机酸等污染物进行去除,经从低到高浓度的有机酸臭物(W/V,0.1%–0.2%)驯化发酵后,培养物中原核微生物以芽孢杆菌居多,而真核微生物主要由红曲霉及粉状毕赤酵母组成。【结论】进一步推测这几种微生物是耐受并降解有机酸臭物的优势微生物,可以应用于奶牛养殖过程中酸臭排泄物的生物控制。     

非天然氨基酸细胞工厂的构建与应用

非天然氨基酸在医药、农药、材料等领域得到广泛应用,其绿色、高效合成越来越受到关注.近年来,随着合成生物学的快速发展,微生物细胞工厂为非天然氨基酸的制造提供了重要手段.文中从合成途径的重构、关键酶的设计改造及与前体的协同调控、竞争性旁路途径的敲除、辅因子循环系统的构建等方面介绍了 一系列非天然氨基酸细胞工厂构建与应用的研...     

氨基酸生物传感器的开发及应用研究进展

氨基酸是蛋白质的基本组成单元,对人和动物的营养健康十分重要,广泛应用于饲料、食品、医药和日化等领域。目前,氨基酸主要通过微生物发酵可再生原料生产,氨基酸产业是我国生物制造的重要支柱产业之一。氨基酸菌株主要通过随机诱变和代谢工程改造结合筛选获得。菌株生产水平进一步提高的核心限制之一是缺乏高效、快速和准确的筛选方法,因此,发展氨基酸菌株的高通量筛选方法对关键功能元件挖掘及高产菌株的创制筛选至关重要。本文综述了氨基酸生物传感器的设计,及其在功能元件、高产菌株的高通量进化筛选和代谢途径动态调控中的应用研究进展,讨论了现有氨基酸生物传感器存在的问题和性能提升改造策略,并展望了开发氨基酸衍生物生物传感器的重要性。     

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Consolidated bioprocessing (CBP) by using microbial consortium was considered as a promising approach to achieve direct biofuel production from lignocellulose. In this study, the interaction mechanism of microbial consortium consisting of Thermoanaerobacterium thermosaccharolyticum M5 and Clostridium acetobutylicum NJ4 was analyzed, which could achieve efficient butanol production from xylan through CBP. Strain M5 possesses efficient xylan degradation capability, as 19.73 g/L of xylose was accumulated within 50 hr. The efficient xylose utilization capability of partner strain NJ4 could relieve the substrate inhibition to hydrolytic enzymes of xylanase and xylosidase secreted by strain M5. In addition, the earlier solventogenesis of strain NJ4 was observed due to the existence of butyrate generated by strain M5. The mutual interaction of these two strains finally gave 13.28 g/L of butanol from 70 g/L of xylan after process optimization, representing a relatively high butanol production from hemicellulose. Moreover, 7.61 g/L of butanol was generated from untreated corncob via CBP. This successfully constructed microbial consortium exhibits efficient cooperation performance on butanol production from lignocellulose, which could provide a platform for the emerging butanol production from lignocellulose.     

Starter culture of <Emphasis Type="Italic">Pseudomonas veronii</Emphasis> strain B for aerobic granulation

Aggregation of bacterial cells is used in formation of microbial granules. Aerobically grown microbial granules can be used as the bio-agents in the treatment of wastewater. However, there are problems with start up of microbial granulation and biosafety of this process. Aim of this research was selection and testing of safe microbial strain with high cell aggregation ability to shorten period of microbial granules formation. Five bacterial strains with cell aggregation index higher than 50% have been isolated from the granules. Strain of Pseudomonas veronii species was considered as most probably safe starter culture for granulation because other strains belonged to the species known as human pathogens. The microbial granules were formed after 3 days of cultivation in case when P. veronii strain B was applied to start-up aerobic granulation process using model wastewater. The granules were produced from activated sludge after 9 days of cultivation. Microbial aggregates produced from starter culture of P. veronii strain B were more compact (sludge volume index was 70 ml/g) than those produced from activated sludge (sludge volume index was 106 ml/g). It is a first proof that application of selected safe starter pure culture with high cell aggregation ability can accelerate and enhance formation of microbial granules.     

酶标仪分光光度法在微生物耐性研究上的应用

微生物在环境中的耐性是该微生物在相应环境发挥作用的重要基础。为了获得一种用于微生物耐性分析的快速简便方法,传统平板分离法和酶标仪分光光度法被用于评估其在细菌和链霉菌紫外耐受水平检测中的差异,并分析了酶标仪分光光度法在微生物其他耐性水平检测中的适用性。结果显示,两种检测方法均能体现细菌和链霉菌对紫外线的耐受水平,前者经过稀释、涂布、培养、菌落计数,获得的是菌株在紫外线照射后的存活浓度,而后者经过接种96孔培养板、培养、吸光度检测,获得的是菌株经紫外线照射后的生长曲线,并从生长曲线获知菌株的生长速率、增殖能力等信息。此外,酶标仪分光光度法同样适用于细菌对pH和盐的耐受水平分析,对于链霉菌耐受性分析有一定的适用性。酶标仪法除了能获得与平板分离法相似的耐性水平检测外,还能获得菌株在不同耐性水平上的增殖潜力,且在操作上比平板分离法省时、省力,可用于微生物耐性分析、高通量筛选等研究工作。     

Biodiversity of rhizobia isolated from a wide range of forest legumes in Brazil

Tropical forests have a high diversity of plant species; are they associated with a correspondingly rich microbial flora? We addressed this question by examining the symbiotic rhizobium bacteria that nodulate a diverse pool of forest legume species in Brazil. The 44 strains studied had been isolated from 29 legume tree species representing 13 tribes including all three subfamilies of the Leguminosae, and were chosen to represent major groups from a larger sample that had previously been characterized by SDS–PAGE of total proteins. Partial 16S rRNA gene sequence was determined, corresponding to positions 44–303 in the Escherichia coli sequence. Fifteen sequences were found, including six novel ones. However, all but one of them could be assigned to a genus because they grouped closely with sequences from previously described rhizobial species. Fast-growing strains had sequences similar to Rhizobium spp., Sinorhizobium spp. or Mesorhizobium spp., while the slow-growing strains had sequences similar to Bradyrhizobium spp. One strain with an intermediate growth rate had a unique sequence which indicated that the strain might belong to the genus Azorhizobium. Although the strains showed a variety of sequences, it was surprising that these strains isolated from taxonomically very diverse host plants in previously unexplored environments were mostly very similar to strains described previously, largely from agricultural systems.     

饲用微生物的分离鉴定及其对杏鲍菇菌糠发酵的效果

【目的】菌糠的营养素含量齐全,但纤维素含量过高是阻碍其饲料化利用的主要因素。故本研究筛选适合于发酵杏鲍菇菌糠的微生物菌株,以改善其饲用品质。【方法】首先,本研究采用纤维素-刚果红、苯胺蓝和MRS-Ca (De Man, Rogosa, Sharpe-Ca)筛选培养基,结合纤维素、木质素酶活力及抑菌活性的测定,从EM (effective microorganisms)原液发酵的杏鲍菇菌糠中分离筛选具有较强纤维素、木质素降解能力及抑菌能力的细菌/真菌。通过细菌16S rRNA和真菌18S rDNA基因序列分析确定菌株所属种属。其次,将筛选出的菌株菌液等体积混合制成复合菌剂用于固态发酵杏鲍菇菌糠。测定不同发酵时长菌糠营养成分含量以确定最佳发酵时间,并与相同工艺条件下EM原液发酵的杏鲍菇菌糠进行饲用品质比较。【结果】筛选并鉴定得到纤维素酶活性较高的特基拉芽孢杆菌(Bacillus tequilensis)菌株P11、发酵毕赤酵母(Pichia fermentans)菌株R8和马克斯克鲁维应变酵母(Kluyveromyces marxianus)菌株MU5;木质素酶活性较高的解淀粉芽孢杆菌(Bacillus amyloliquefaciens subsp.plantarum)菌株MU7;抑菌活性较高的类肠膜魏斯氏菌(Weissella paramesenteroides)菌株R4和乳酸片球菌(Pediococcus acidilactici)菌株R9。使用以上菌株复合发酵杏鲍菇菌糠7 d后,各项指标达到稳定。与EM原液发酵的杏鲍菇菌糠相比,复合菌剂发酵杏鲍菇菌糠的NDF和ADF分别显著降低了19.6%和21.44%(P0.05);CP (crude protein)、CA (crude ash)和EE (ether extract)含量分别显著提高了10.44%、5.26%和123.53%(P0.05)。【结论】本研究筛选得到的芽孢杆菌、酵母菌和乳酸菌优势菌株复合后用于发酵杏鲍菇菌糠可以很好地改善其饲用品质,效果优于生产中常用市售EM原液。     

High throughput automated microbial bioreactor system used for clone selection and rapid scale‐down process optimization

High throughput automated fermentation systems have become a useful tool in early bioprocess development. In this study, we investigated a 24 x 15 mL single use microbioreactor system, ambr 15f, designed for microbial culture. We compared the fed‐batch growth and production capabilities of this system for two Escherichia coli strains, BL21 (DE3) and MC4100, and two industrially relevant molecules, hGH and scFv. In addition, different carbon sources were tested using bolus, linear or exponential feeding strategies, showing the capacity of the ambr 15f system to handle automated feeding. We used power per unit volume (P/V) as a scale criterion to compare the ambr 15f with 1 L stirred bioreactors which were previously scaled‐up to 20 L with a different biological system, thus showing a potential 1,300 fold scale comparability in terms of both growth and product yield. By exposing the cells grown in the ambr 15f system to a level of shear expected in an industrial centrifuge, we determined that the cells are as robust as those from a bench scale bioreactor. These results provide evidence that the ambr 15f system is an efficient high throughput microbial system that can be used for strain and molecule selection as well as rapid scale‐up. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:58–68, 2018     

Adaptive Evolution in Producing Microtiter Cultivations Generates Genetically Stable Escherichia coli Production Hosts for Continuous Bioprocessing

The production of recombinant proteins usually reduces cell fitness and the growth rate of producing cells. The growth disadvantage favors faster-growing non-producer mutants. Therefore, continuous bioprocessing is hardly feasible in Escherichia coli due to the high escape rate. The stability of E. coli expression systems under long-term production conditions and how metabolic load triggered by recombinant gene expression influences the characteristics of mutations are investigated. Iterated fed-batch-like microbioreactor cultivations are conducted under production conditions. The easy-to-produce green fluorescent protein (GFP) and a challenging antigen-binding fragment (Fab) are used as model proteins, and BL21(DE3) and BL21^Q strains as expression hosts. In comparative whole-genome sequencing analyses, mutations that allowed cells to grow unhindered despite recombinant protein production are identified. A T7 RNA polymerase expression system is only conditionally suitable for long-term cultivation under production conditions. Mutations leading to non-producers occur in either the T7 RNA polymerase gene or the T7 promoter. The host RNA polymerase-based BL21^Q expression system remains stable in the production of GFP in long-term cultivations. For the production of Fab, mutations in lacI of the BL21^Q derivatives have positive effects on long-term stability. The results indicate that adaptive evolution carried out with genome-integrated E. coli expression systems in microtiter cultivations under industrial-relevant production conditions is an efficient strain development tool for production hosts.     

Cultivating the uncultured: limits, advances and future challenges

Since the invention of the Petri dish, there have been continuous efforts to improve efficiency in microbial cultivation. These efforts were devoted to the attainment for diverse growth conditions, simulation of in situ conditions and achievement of high-throughput rates. As a result, prokaryotes catalysing novel redox reactions as well as representatives of abundant, but not-yet cultured taxa, were isolated. Significant insights into microbial physiology have been made by studying the small number of prokaryotes already cultured. However, despite these numerous breakthroughs, microbial cultivation is still a low-throughput process. The main hindrance to cultivation is likely due to the prevailing lack of knowledge on targeted species. In this review, we focus on the limiting factors surrounding cultivation. We discuss several cultivation obstacles, including the loss of microbial cell–cell communication following species isolation. Future research directions, including the refinement of culture media, strategies based on cell–cell communication and high-throughput innovations, are reviewed. We further propose that a combination of these approaches is urgently required to promote cultivation of uncultured species, thereby dawning a new era in the field.