The Genomic Analysis And Promoter Screening Of Gluconobacter Thailandicus

Gluconobacter thailandicus,also known as Gluconobacter frateurii,is a relatively new species in the Gluconobacter genus.G.thailandicus contains a large number of dehydrogenase systems,such as glucose dehydrogenase,sorbitol dehydrogenase,glycerol dehydrogenase,these etc.,which often use pyrroloquinoline quinone（PQQ）as a prosthetic group to catalyze the formation of the corresponding substrates to form important industrial products such as L-sorbitose,dihydroxyacetone,2-keto-L-gulonic acid.The transcription of coding genes of these enzyme systems is regulated by promoters.Selecting promoters with strong activity provides powerful regulatory elements for the later metabolic engineering transformation of these dehydrogenase systems.Thus this study aims to obtain the gene structure and basic metabolic pathway of G.thailandicus through in-depth analysis of the genome of G.thailandicus,to obtain strong promoters by genomic key enzyme gene analysis,phylogenetic tree analysis and transcriptome gene expression level analysis,to increase the production of 1,3-dihydroxyacetone（DHA）and to shorten the time of glycerol fermentation by overexpressing sld AB gene,to obtain powerful promoter regulatory elements.The specific research results are as follows:Firstly,the complete genome structure of G.thailandicus HD924 was obtained by genome sequencing,and the genome chromosome and plasmid map were assembled.The metabolic pathway of G.thailandicus HD924 was obtained by annotation of KEGG,GO and COG protein databases.In the genome annotation and predicted metabolic pathways,it was proved that there were a large number of dehydrogenase lines in G.thailandicus HD924,some of which had PQQ as a prothetic group,some of which had NAD⁺as a coenzyme,and some had both enzyme systems.In addition,it was confirmed that the TCA cycle pathway of G.thailandicus HD924 was not complete.Secondly,through genome annotation,promoter sequences encoding eight key enzymes,GDHA,GDHB,ADHA,ADHB,ALDHA,ALDHB,MDH and pqq-SDH,were finally screened:P_gdha,P_gdhb,P_adha,P_adhb,P_aldha,P_aldhb,P_mdh,P_pqq-sdh.By constructing a phylogenetic tree of 91 genomes,the phylogenetic evolutionary distance close species G.oxydans WSH-003 was obtained.The phylogenetic tree of 91 genomes was constructed to obtain the species G.oxydans WSH-003,which is phylogenetically close to G.thailandicus HD924.According to the results of the study on the promoter of G.oxydans WSH-003,the genes dnak and flb with the same function as the strong promoter gene of G.oxydans WSH-003 were selected in the genome of G.thailandicus HD924,and the promoters P_dnak and P_flb were obtained.Through RNA transcriptome sequencing of G.thailandicus HD924 in four stages of glycerol fermentation,the study selected the gene prediction promoters that were stably expressed and relatively high in expression throughout the fermentation stage,and the upstream promoters P_cpb,P_csp and P_uosof three functional genes,cpb,csp and uos were finally screened out.Thirdly,with P_tuf as the strong promoter control,the gene transcription level,promoter fluorescence intensity and the growth density of G.thailandicus HD924 cells were determined to evaluate the activity intensity of the above 13 promoters,and P_gdhb was finally selected as the strongest promoter.The fluorescence of G.thailandicus HD924 p BBR-P_gdhb-egfp was the brightest when blue light irradiated the middle and late fermentation cells of all promoter recombinant bacteria.The relative transcription level of egfp in P_gdhb was about 10.2 times that of the strong promoter P_tuf（control）,and the ratio of fluorescence intensity to cell density in P_gdhb was about 17 times that of the strong promoter P_tuf（control）.At last,quantitative PCR was used to detect sld AB gene overexpression in recombinant strain G.thailandicus HD924 p BBR-Pgdhb-sld AB,and high performance liquid chromatography（HPLC）was used to determine glycerol and DHA content in glycerol fermentation process.The results of fluorescence quantitative PCR showed that the relative expression level of sld AB gene in recombinant G.thailandicus HD924 p BBR-Pgdhb-sld AB was 5.8 times and 3.3 times higher than that in wild bacteria and control bacteria with strong promoter P_tuf.The results of HPLC showed that the wild strain entered the stable growth stage after 36 h fermentation,the conversion rate of glycerol was 36.0%,and the yield of DHA was 21.4 g/L.The control strain G.thailandicus HD924 p BBR-P_tuf-sld AB was cultured for 28 h,the conversion rate of glycerol was 46.8%,and the yield of DHA was 38.9 g/L.G.thailandicus HD924 p BBR-Pgdhb-sld AB was cultured for 20 h,the conversion rate of glycerol was 73.7%,and the yield of DHA was 70.5 g/L.Compared with the wild bacteria,G.thailandicus HD924 p BBR-Pgdhb-sld AB increased DHA production by 2.3 times and reduced glycerol conversion time by 44.4%.The results proved that the P_gdhb promoter could be used as an element in the regulation of sld AB gene in the metabolic engineering of converting glycerol to DHA in strain HD924 of G.thailandicus and increase the production of DHA...