Metabolic Engineering Modification Of Gellan Gum Production Strain And Its Gel Production Performance

Gellan gum produced by fermentation of Sphingomonas elodea JLJ is a macromolecular linear anionic extracellular capsular polysaccharide.It has good heat resistance,acid and alkali resistance,non-toxic and harmless properties,and is widely used in various fields.In the production process of gellan gum fermentation,with the accumulation of gellan gum,the fermentation liquid will become more viscous.Therefore,the contradiction between supply and demand of dissolved oxygen in the fermentation process has been a serious problem to be solved.This study intends to use the CRISPR/Cas9 technology to knock the Vitreoscilla hemoglobin gene(vgb)gene into the Sphingomonas elodea cells to modify its metabolic network,thereby improving the ability of the strain to take up and utilize oxygen,and to solve the oxygen in the process of gellan gum fermentation.The contradiction between supply and demand,effectively improve the production of gellan gum;the fermentation medium and fermentation conditions of the recombinant strain were modified,and finally the yield of gellan gum was significantly improved.The main findings are:(1)The acetyltransferase gene(nat)fragment was amplified by PCR,and the nat gene was identified in the strain JLJ genome,which was selected as the target site.(2)The strain JLJ was modified by CRISPR/Cas9 technology,the Vitreoscilla hemoglobin gene(vgb)was knocked into the genome,and the nat gene was knocked out to obtain the recombinant strain JLJ-vgb.PCR,SDS-PAGE and CO difference spectroscopy indicated that the vgb gene was successfully knocked into the strain JLJ genome and successfully expressed active hemoglobin(VHb).(3)The optimum fermentation medium for recombinant strain JLJ-vgb was determined by single factor test and response surface optimization test: soluble starch 7%,soybean cake powder 0.55%,KH2PO4 0.05%,K2HPO4 0.05%,MgSO4 0.06%,pH7.0;The optimum speed is 200 r/min.Under this condition,the amount of gel produced by the recombinant strain JLJ-vgb was 41.62±1.72 g/L,which was 127.32% higher than that of the starting strain.