Genetic Modification Of Cell Division Related Gene FtsZ1 Of Haloferax Mediterranei And Its Effect On PHA Production

Polyhydroxyalkanoates(PHAs)are a class of biodegradable plastic raw materials synthesized by microbial cells and are ideal substitutes for traditional plastics.Nowadays,the fermentation of polyhydroxyalkanoates by engineered microbial strains has become the main way of industrial-scale production of PHAs.PHAs synthesized by halophilic archaeal cells have better material properties.Previous studies have found that genetic modification of the cell division-related protein Fts Z in halophilic archaea leads to impaired division,and related research has been used to increase the production of PHAs in salt-tolerant bacteria.This article attempts to select the halophilic archaeon Haloferax mediterranei with the best PHA accumulation ability as the object,and achieve the goal of increasing PHA production by modifying its fts Z gene.The main research contents and results are as follows:1 、 Successful construction of a ftsZ1 gene promoter replacement strain of PHA-producing halophilic archaeon DF50.Because cell division protein is a lethal protein in microorganisms and cannot be knocked out by gene,we designed by genetic engineering technology,using Haloferax mediterranei DF50 as the research object,using the classic tryptophan-inducible expression regulatory promoter(ptna)in halophilic archaea to replace the promoter(pftsZ1)of the ftsZ1 gene.After the cloning,subcloning,transformation,homologous recombination vector transformation,double exchange of homologous arms,transposon screening and sequencing verification processes,we successfully constructed the Haloferax mediterranei engineering strain(Haloferax mediterranei DF50 ptna-ftsZ1),whose ftsZ1 gene expression level was regulated by tryptophan.The full length of the tryptophan-inducible expression promoter ptna includes 211 bp base pairs.2、Controlled expression of ftsZ1 gene in engineering strain and analysis of its growth and PHAs synthesis characteristics.(1).Using q PCR technology,it was found that ftsZ1 in DF50 ptna-ftsZ1 strain had a tryptophan concentration-dependent expression pattern,suggesting that the construction achieved the experimental purpose;further,under different concentrations of tryptophan induction,the cell morphology of DF50 ptna-ftsZ1 strain was observed by microscope,and compared with the wild-type strain DF50,the morphology of the replacement engineering strain showed irregular large cells and long rod-shaped cells,which again explained the successful replacement of ftsZ1 promoter from a macroscopic level.(2).Using high,medium and low concentrations of tryptophan to control the expression of ftsZ1,the results showed that under medium concentration of 0.25 m M induction,the growth of the strain was optimal,and the addition of tryptophan below or above this intermediate concentration would affect the growth of the strain,among which,the addition of too high concentration of tryptophan would significantly inhibit the growth of the strain.(3).Using gas chromatography method,PHAs production of DF50 ptna-ftsZ1 with different division characteristics induced by different concentrations of tryptophan was studied.It was found that within the set range of tryptophan concentration(0,0.25 m M,2.5 m M),the change of expression intensity of ftsZ1 gene did not cause significant change in PHA total production of engineering strain;it is worth noting that when ftsZ1 gene expression was induced by no addition of tryptophan(0 m M),the average content of 3-hydroxyvalerate(3HV),a key component of PHA in engineering strain,increased by 2.369% compared with wildtype strain,but there was no significant difference between them by statistical analysis.In summary,we ingeniously used a mature inducible promoter in halophilic archaea to replace the wild-type promoter of the lethal gene ftsZ1,and successfully achieved tryptophan concentration-mediated control of halophilic archaeal cell division.Under abnormal division state,this irregular enlargement or rod-shaped morphology of the cell may be beneficial to the production increase or material property optimization of DF50 ptna-ftsZ1 strain in PHA fermentation process.The above research proposed a possibility of changing PHA accumulation of archaeal cells by changing cell size,and also provided a more efficient engineering strain type for halophilic archaea to use a wide range of carbon sources to convert PHA.