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

BRIGE：一种用于识别复杂表型决定因素的组合工程方法

• 批准号: 1032487
• 负责人: Katy Kao
• 金额: $ 17.5万
• 依托单位: Texas A&M Engineering Experiment Station
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1032487&HistoricalAwards=false
• 关键词: BRIGE; combinatorial; engineering; approach; identifying

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.

利用微生物系统生产有价值的化合物和燃料为化工生产提供了一条潜在的更环保和可持续的途径。然而，微生物生物燃料和生物可再生能源生产面临的挑战之一是微生物宿主对原料和产品中的有毒化合物的耐受性较低。不幸的是，目前由于缺乏对相关分子机制的了解，增加稳健性的微生物工程受到限制。我们的长期目标是在全球范围内确定微生物对存在于微生物系统代谢工程的原料和产品中的生长抑制剂的适应机制。乳酸菌的一种成员，短乳杆菌，最近被发现对纤维素生物质原料中的几种生长抑制剂和第二代生物燃料丁醇具有耐受性。然而，短乳杆菌缺乏许多生物合成途径，且营养需求高，因此可能不是理想的生产平台。拟议的研究重点是鉴定来自短链乳杆菌的基因和/或基因组，当异源表达时，将赋予大肠杆菌丁醇耐受性，而大肠杆菌是生物技术中不挑剔的工作马。具体而言，本研究计划拟建立表达短乳杆菌基因的大肠杆菌单整合物文库。大肠杆菌单整合子之间的基因组洗牌将用于生成表达短乳杆菌基因在两个基因组位点组合的大肠杆菌双整合子文库。将开发系列富集策略，从文库中选择含有短链乳杆菌基因的整合物，使其对大肠杆菌具有丁醇耐受性。这项工作的结果将作为后续建议的初步结果，以进一步发展和扩大这种组合工程方法在微生物生产宿主中所需复杂表型的应用。教育推广计划的目的是扩大妇女和少数民族在科学和工程领域的参与并加强其保留。首先，我们希望通过在实验室建立暑期实习项目，鼓励女性和/或少数族裔高中生在大学攻读工程学位；这将通过与当地高中科学教师的合作来完成。其次，我们将通过为理工科本科生提供研究机会和指导，解决在工程学科获得高级学位的女性和少数族裔的保留率低和比例下降的问题。此外，将设立代谢工程的本科/研究生课程，以激发追求工程高级学位的激情和兴趣；这将通过专注于工程和科学的整合来解决当前的社会挑战。更广泛的影响拟议工作的更广泛影响有三个方面。首先，将鉴定赋予大肠杆菌丁醇耐受性的短乳杆菌基因，并将其用于进一步设计更健壮的生物丁醇生产者（例如大肠杆菌和C. acetobutylicum）。第二，所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女性和少数族裔对工程领域的兴趣和参与。对可持续原料中存在的产品和抑制剂具有高耐受性的微生物宿主的成功工程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PI的独立职业生涯的开始和社会有重大的好处。