UNS: Combining strain recombineering with molecular reporters and customized selections to overcome limitations in producing renewable hydrocarbons

1511425 Cirino, Patrick C.提出的研究旨在利用基因组工程、基因测序和高通量遗传变异筛选的最新进展来分离改良的转基因微生物。基因组修饰的独特组合将被确定，使工程大肠杆菌能够更有效地将来自生物质的丰富的可再生糖转化为前体代谢物和更高价值的化学产品，如生物燃料和用于生产天然产品的构建块。除了促进对可持续生化生产的理解外，这些研究活动还将对代谢和微生物生理学产生新的见解，从而对许多生物学相关学科做出广泛贡献。外联和教育活动也纳入了研究计划。克服许多限制微生物生化生产的协调代谢限制需要大量的遗传和上位性修饰。该研究计划旨在使用定制的内源性生物传感器或分子报告器，从大型组合基因组文库中发现菌株，这些菌株可以提高不与细胞生长耦合的化合物的产量。重点是同时筛选许多基因的表达水平文库，以通过消耗乙酰辅酶a和NADPH（甲羟戊酸合成/甲羟戊酸传感器），丙二酰辅酶a（三乙酸内酯合成/TAL传感器）和NADPH（木糖醇生产/ NADPH传感器）的途径增强生产。相同的重组菌株文库将与独特设计的遗传背景相结合，以便使用选择来识别新的遗传修饰组合，这些组合显示出轻松的基于氧化还原的调节和通过非发酵途径增强的通量。目标产物分子与中枢代谢的关键分支点和与广泛的生物化学相关的前体代谢物直接相关。通过这项研究合成的各种基因组文库将提供给科学界，用于选择/筛选其他感兴趣的生物合成途径。该奖项由CBET部门的生物技术和生化工程项目颁发，由分子和细胞生物学部门的系统和合成生物学项目共同资助。

1511425 Cirino, Patrick C. The proposed research aims to take advantage of recent advances in genome engineering, gene sequencing, and high-throughput screening of genetic variants to isolate improved, genetically modified microorganisms. Unique combinations of genome modifications will be identified that allow engineered Escherichia coli to more efficiently convert abundant and renewable sugars derived from biomass into precursor metabolites and higher value chemical products, such as biofuels and building blocks for producing natural products. In addition to advancing understanding in sustainable biochemicals production, these research activities will yield new insights into metabolism and microbial physiology, thereby contributing broadly to many biology-related disciplines. Outreach and educational activities are also incorporated into the research plan.Overcoming many coordinated metabolic constraints that limit microbial biochemical production requires a large number of genetic and epistatic modifications. This research plan aims to use customized, endogenous biosensors or molecular reporters to find strains from large combinatorial genome libraries with improved production of compounds not coupled to cell growth. The focus is on screening expression level libraries of many genes at once, to enhance production through pathways that consume acetyl-CoA and NADPH (mevalonate synthesis/mevalonate sensor), malonyl-CoA (triacetic acid lactone synthesis/TAL sensor), and NADPH (xylitol production/ NADPH sensor). The same recombineered strain libraries will be combined with uniquely designed genetic backgrounds to enable the use of selections to identify new combinations of genetic modifications that show relaxed redox-based regulation and enhanced flux through non-fermentative pathways. The targeted product molecules are directly linked to key branch points in central metabolism and precursor metabolites associated with a broad range of biochemicals. The wide variety of genomic libraries synthesized through this research will be made available to the scientific community, to be applied in selections/screens for other biosynthetic pathways of interest.This award by the Biotechnology and Biochemical Engineering Program of the CBET Division is co-funded by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biology.