Effect Of GndL And 2kgdH Double Knockout On Gluconic Acid Biosynthesis In Pseudomonas Plecoglossicida JUIM01

The aim of present study was to knock out the genes encoding gluconate dehydrogenase and 2-ketoglucose dehydrogenase and to verify their roles in 2-ketogluconic acid biosynthesis of Pseudomonas plecoglossicida.A gluconate-producting engineered strain was tried to construct using Pseudomonas plecoglossicida as chassis cells.GA could be extensively used in food,beverage,chemical,pharmaceutical and it was a kind of organic acid with a variety of physical and chemical properties,textile,leather,construction and other industries.This study was based on the domestic 2KGA industrial production strain Pseudomonas plecoglossicida JUIM01.Firstly,the full gene sequence of gad operon encoding GADH was cloned by LA-PCR and then its bioinformatics were predicted using some on-line tools;secondly,the structural genes gndS、gndL and gndC of gad operon knockout strains JUIM01ΔgndS、JUIM01ΔgndL and JUIM01ΔgndC were constructed respectively by methods of overlap-PCR、traceless knockout technology and homologous recombination;finally,2kgdH encoding 2KGDH was knocked out based on JUIM01ΔgndL and gndL 、 2kgdH double knockout strain JUIM01ΔgndLΔ2kgdH was constructed;then,the 2KGA fermentative characteristics of wild strain were compared with those of knockout strain and the 2KGA synthetic route of Pseudomonas plecoglossicida was determined.This study provided a theoretical basis for the construction of gluconic acid high-efficiency productive strain.The obtained conclusions were listed as follows:(1)A 4112 bp nucleotide sequence containing the entire gene of gad operon was firstly amplified from the strain P.plecoglossicida JUIM01 by LA-PCR.The gad operon consisted of three structural genes gndS、gndL、gndC of which the sequence length was 753 bp、1788 bp and 1314 bp respectively encoding the small subunit GndS、dehydrogenase subunit GndL and cytochrome c subunit GndC.The promoter site of the gad operon was located in 80 bp upstream of the structural gene gndS which was part of non-coding region.The-10 region was predicted to be 5′-GTCTAAGCT-3′ and the-35 region was predicted to be 5′-TTGTTC-3′.The terminator site was a GC-rich inverted repeat sequence located in its downstream non-coding region which could be capable of forming a hairpin structure(GGGCCGC-nt4-GCGGCCC).(2)The knockout strains JUIM01ΔgndS、JUIM01ΔgndL、JUIM01ΔgndC were constructed.The results of fermentation test showed that the deletion of gndS and gndC respectively had no effect on 2KGA and gluconic acid productivity,but the deletion of gndL could reduce 2KGA productivity by 15%.It suggested that there might be another 2KGA sythetic pathway in P.plecoglossicida.(3)The knockout strains JUIM01Δ2kgdH and JUIM01ΔgndLΔ2kgdH was constructed.The results of fermentation test showed that the deletion of 2kgdH could decrease the highest 2KGA yield by 5.7%;double deletion of gndL and 2kgdH could block 2KGA synthetic pathway and accumulate plentiful GA(57.14 g/L)in the fermentation broth.