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

1511425 Cirino，帕特里克C. 拟议的研究旨在利用基因组工程，基因测序和遗传变异的高通量筛选的最新进展，以分离改良的转基因微生物。将确定基因组修饰的独特组合，使工程大肠杆菌能够更有效地将来自生物质的丰富和可再生的糖转化为前体代谢产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件。除了促进对可持续生物化学品生产的理解外，这些研究活动还将对代谢和微生物生理学产生新的见解，从而为许多生物学相关学科做出广泛贡献。研究计划还包括推广和教育活动。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者从大型组合基因组文库中找到菌株，这些菌株具有改进的化合物产量，而不与细胞生长相耦合。重点是一次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）的途径来增强生产。相同的重组菌株文库将与独特设计的遗传背景组合，以使得能够使用选择来鉴定遗传修饰的新组合，所述遗传修饰的新组合显示放松的基于氧化还原的调节和通过非发酵途径的增强的通量。 靶向产物分子直接与中枢代谢的关键分支点和与广泛生物化学物质相关的前体代谢物相关联。通过这项研究合成的各种各样的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。CBET部门的生物技术和生物化学工程项目的这一奖项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.