(29p) A Cotransformation Strategy for Combinatorial Engineering Paralogous Genes in Clostridium Acetobutylicum

Paralogous genes involved in metabolic and regulatory pathway, widely exist in microorganisms, which can increase the robustness of biological systems. It is challenging to explore the phenotypes of paralogous genes with all permutations. How to construct a mutant library with all permutations is necessary but a tough work. Here, we present a multi-plasmid cotransformation strategy that efficiently achieves all combinatorial inactivation of several paralogous genes in one round. Taking five paralogous histidine kinase genes of Clostridium acetobutylicum as a demonstration, a library containing all the possible histidine kinase-inactivated combinations of 22 survival mutants was constructed after systematically optimizing several factors. The cotransformation strategy required 11 days in one round compared to conventional method with successive rounds. Based on mutant library, we elucidated the regulatory function of histidine kinases on cell survival, metabolism and development. Moreover, the mutant library facilitated the screening of hyper butanol-producing strains. Six mutants showed the enhancement in butanol biosynthesis with excellent stress tolerance, one of which could produce 54.2% more butanol than wild type. In view of the pleiotropic function of paralogous family genes, combinatorial engineering multiple genes by cotransformation strategy provides a novel and effective approach to investigate functional prioritization and interaction in a time-efficient manner, which may be applied to other organisms.