Rapid Construction Of Inducible Promoter Replacement System And Its Application In Zymomonas Mobilis

As a model ethanologen,Zymomonas mobilis has unique metabolic features and many ideal industrial characteristics.The genome sequence of the model strain ZM4 is only around2-Mb,making it an ideal microorganism as a synthetic industrial chassis.However,the limited information on gene function and their regulation especially those with hypothetical function impedes the development of synthetic chassis.The classical genetics approaches are usually to determine the effect of a target gene when it is knockout or overexpressed,which impedes the rapid characterization of candidate genes and their functions.The aim of this study is to develop an integrated inducible promoter replacement system to replace the native promoter of a target gene with an inducible promoter,which can help achieve the goal of determining the effect of gradient gene expression on phenotypic changes.The result can then be used to help decipher the function and necessity of genes.The Ptet promoter that has been proved to be regulated by the inducer tetracycline was selected to establish the system.In addition,the recombinant fragment construction method was improved resulting in an enhanced success rate by reducing the length of overlapping primers and optimizing the PCR reaction conditions.Using this system,we successfully obtained 78 mutant strains of Z.mobilis,and the growth of these strains and wild-type strain ZM4 was then examined under different induction conditions using both Bioscreen C and spot assay.Based on their growth performance,we artificially group these mutants into four types,which are termed as negative mutation death,mutation death,high expression inhibition and little/no effect.Two genes from a representative type were further investigated using bioinformatics tools to understand their functions,which are genes encoding pyruvate decarboxylase(pdc)and leucine regulatory protein(lrp).The inducible promoter replacement system established in this study thus provides a feasible approach to study gene function by obtaining the phenotypic changes associated with a gradient gene expression,which can be combined with systems biology datasets to reveal the potential function and regulation network of target genes.The information gathered through this effort can help identify essential genes and their functions to lay a foundation for future genome-minimized synthetic chassis development using Z.mobilis.