Comparative Genomics Analysis Of High Ectoine Production Mechanism And Fermentation Process Optimization Of Ultraviolet Mutant Strain G₈-52

Objective The whole genomes of wild strain Halomona campaniensis sp.XH26and UV mutant strain G₈-52 were sequenced using a third-generation high-throughput Pac Bio Sequel sequencing platform to screen functional genes affecting ectoine production by comparative genomics.Meanwhile,the fermentation conditions were optimized and a large biodigester was used for supplemental batch fermentation with the aim of enhancing ectoine production.Methods The whole genome sequencing and bioinformatics analysis were performed by Pac Bio Sequel sequencing platform using UV mutant strain G8-52 and wild strain XH26 as the target of the study.Comparative genomics was used to analyze the functional genes affecting ectoine yield from genomic characterization and screening of differential functional genes.Based on Box-Benhnken central combinatorial experimental design principles,a three-factor,three-level response surface analysis was used to model the microbial fermenter（5.0 L）batch fermentation and explore the conditions of ectoine industrial production.Results The whole genome sequencing results of mutant strain G₈-52 showed that the whole genome size was 4,098,386 bp,3,945 predicted coding genes,total coding gene length was 3,670,458 bp,GC content of coding genes was 53.19%,and total coding gene length to genome size was 89.56%.The number of genes annotated based on COG database was 3,260（82.64%）,2,301（58.33%）from KEGG database and 2,632（66.72%）from GO database,for a total of 3,675（93.16%）annotated genes.After comparative genomic analysis,four single base mutation sites（SNP）,ten insertion fragments（Insertion）,and ten deletion fragments（Deletion）were found in the mutant strain G₈-52.Among them,the mutated genes gab D and dav T were involved in the tricarboxylic acid cycle and indirectly in the synthesis process of ectoine,which was tentatively speculated to possibly lead to the increased production of ectoine.Compared with wild strain XH26,the ectoine synthesis of mutant strain G₈-52 was increased from 0.45 g/L to 1.50 g/L.Batch fermentation was carried out using a 5.0 L biodigester,and the optimal fermentation conditions were optimized by response surface methodology for salt concentration of 1.5 mol/L,p H 8.0,fermentation temperature of 35°C,and supplemental batch fermentation,which increased the ectoine yield to 3.26 g/L.Conclusion Comparative genomic analysis revealed a total of 24 mutations in the mutant strain,among which the differential genes dav T and gab D enhanced the TCA cycle,while the upstream TCA（succinic acid,ferredoxin and oxaloacetate）of ectoine biosynthesis was closely related,and preliminary analysis of the genes dav T and gab D may indirectly lead to the ectoine accumulation outbreak.In addition,the fermentation parameters were controlled by the combination of DO value and p H,resulting in ectoine synthesis and yield of 3.26 g/L and 1.10 g/（L·d）,respectively,and controlling the whole fermentation time to 72 h,showing the advantage of cost reduction.