BRIGE: A combinatorial engineering approach for identifying determinants of complex phenotypes

1032487KaoPROJECT SUMMARY智能优点利用微生物系统生产有价值的化合物和燃料提供了一条潜在的更绿色和可持续的化学品生产路线。然而，微生物生物燃料和生物可再生能源生产中的挑战之一是微生物宿主对原料和产品中有毒化合物的低耐受性。不幸的是，目前，由于缺乏对相关分子机制的了解，提高微生物健壮性的工程受到限制。我们的长期目标是在全球范围内确定微生物适应微生物系统代谢工程原料和产品中存在的生长抑制剂的机制。短链乳杆菌是乳酸菌中的一员，最近被鉴定出对纤维素生物质原料中存在的几种生长抑制剂和第二代生物燃料丁醇表现出耐受性。然而，短乳杆菌缺乏许多生物合成途径，营养需求高，可能不是理想的生产平台。这项研究的重点是从短乳杆菌中鉴定S基因和/或一组基因(S)，当这些基因异源表达时，将使大肠杆菌对丁醇耐受，而大肠杆菌是生物技术中不挑剔的主力。具体地说，该研究计划建议建立表达L.brevis基因的大肠杆菌单一整合体的文库。在大肠杆菌单整合子之间的基因组改组将被用来建立表达两个基因组座位上的短乳杆菌基因组合的大肠杆菌双整合子文库。将开发一系列浓缩策略，从文库中筛选含有短链球菌基因的整合子，这些基因赋予大肠杆菌对丁醇的耐受性。拟议工作的结果将被用作后续提案的初步结果，以进一步开发和扩大这一组合工程方法在微生物生产宿主中理想的复杂表型的应用。教育推广计划旨在扩大妇女和少数群体在科学和工程领域的参与和留住他们。首先，我们希望通过在实验室建立暑期实习计划来鼓励女性和/或少数族裔高中生在大学攻读工程学学位；这将通过与当地高中理科教师的合作来实现。第二，我们将通过为理工科本科生提供研究机会和导师，解决女性和少数族裔在获得工程学科高级学位方面的低留存率和比例下降的问题。此外，还将开设代谢工程本科生/研究生课程，以激发人们对攻读工程学高级学位的兴奋和兴趣；这将通过将工程学与科学相结合来解决当前的社会挑战来实现。首先，短乳杆菌对大肠杆菌丁醇耐受性的基因将被识别，并用于进一步改造更强大的生物丁醇产生菌(例如，大肠杆菌和乙酰丁酸杆菌)。第二，开发的组合工程方法将广泛适用于各种理想的复杂表型。第三，教育推广计划将激发妇女和少数民族对工程领域的兴趣并增加他们的参与度。成功地设计出对可持续原料中存在的产品和抑制剂具有高度耐受性的微生物宿主，将显著提高工业生物技术的经济可行性。因此，拟议的活动将对开始国际和平协会的独立事业和对社会产生重大好处。

1032487KaoPROJECT SUMMARYIntellectual merit The use of microbial systems for the production of valued compounds and fuels provide a potentially greener and sustainable route for chemical production. However, one of the challenges in microbial biofuel and biorenewables production is the low tolerance of the microbial hosts to toxic compounds in the feedstock and products. Unfortunately, the engineering of microorganisms for increased robustness is currently restricted by the lack of knowledge of the molecular mechanisms involved. Our long-term goal is to globally identify the mechanisms involved in microbial adaptation to growth inhibitors present in the feedstock and products for the metabolic engineering of microbial systems. A member of the lactic acid bacteria, Lactobacillus brevis, was recently identified to exhibit tolerance to several growth inhibitors present in cellulosic biomass feedstock and to the second-generation biofuel, butanol. However, L. brevis lacks many biosynthetic pathways and has high nutritional requirements, and thus may not be an ideal production platform. The proposed research focuses on identifying gene(s) and/or sets of genes(s) from L. brevis, when heterologously expressed, will confer butanol tolerance to Escherichia coli, the non-fastidious workhorse of biotechnology. Specifically, the research plan proposes to generate libraries of E. coli single integrants that express L. brevis genes. Genome shuffling between the E. coli single integrants will be used to generate libraries of E. coli double integrants expressing combinations of L. brevis genes at two genomic loci. Serial enrichment strategies will be developed to select for integrants containing L. brevis genes that confer butanol tolerance to E. coli from the libraries. The results from the proposed work will be used as preliminary results for subsequent proposals to further develop and expand the application of this combinatorial engineering approach for desirable complex phenotypes in microbial production hosts. The educational outreach plan aims to broaden the participation and enhance the retention of women and minorities in science and engineering fields. First, we hope to encourage female and/or minority high school students to pursue engineering degrees in college by establishing a summer internship program in the lab; this will be accomplished through the collaboration with local high school science teachers. Second, we will tackle the problem of low retention and decrease in proportion of women and minorities in achieving advanced degrees in engineering disciplines by providing research opportunities and mentorships to science and engineering undergraduates. In addition, an undergraduate/graduate course in metabolic engineering will be established to generate excitement and interest in pursuing advanced degrees in engineering; this will be accomplished by focusing on the integration of engineering and science to solve current societal challenges.Broader impact The broader impact of the proposed work is three fold. First, L. brevis genes that confer butanol tolerance to E. coli will be identified and used to further engineer more robust biobutanol producers (e.g. E. coli, and C. acetobutylicum). Two, the combinatorial engineering approach developed will be broadly applicable to a variety of desirable complex phenotypes. Third, the educational outreach plans will stimulate interest and increase participation of females and minorities in engineering fields. The successful engineering of microbial hosts with high tolerance to products and inhibitors present in sustainable feedstock will significantly improve the economical viability of industrial biotechnology. Thus, the proposed activities will have significant benefit on the initiation of the PI's independent career and on society.