Metabolic Engineering Of Ectoine Producing Pathway And Optimization Of Fermentation Process In Halomonas Hydrothermalis Y2

Ectoine is a type of compatible solutes that are mostly produced in halophilic bacteria and play a role in balancing intraosmotic pressure in cells.Due to its anti-stress protection effect,it has received high attention in practical applications such as,cosmetics industry,enzyme preparations and pharmaceutical industries.The Halomonas hydrothermalis Y2 studied in this paper was isolated from pulpmill wastewater,which can occupy the survival advantage under the environment of high salt and strong alkali in the wastewater,this mechanism of salt-tolerance makes the strain have the potential to use seawater to achieve industrial production without sterilization and continuous fermentation.In this study,based on genome analyses of the H.hydrothermalis Y2,some gene clusters for ectoine synthesis and metabolism were found in the genome,and the pathway for the synthesis and degradation of ectoine in H.hydrothermalis Y2 was predicted.The ectoine synthesis pathway was further verified by the mutant constration of ectA.The mutant showed a loss of ability to synthesis ectoine and decreased tolerance for high salt,indicating that ectoine played an important role in H.hydrothermalis Y2 high salt tolerance.Seeds and fermentation medium was optimized based on NaCl,Glucose,Monosodium glutamate(MSG)and pH in MG medium.Finally,4%NaCl,1%Glucose(Glc),1%MSG was identified as the best combination for seed optimization.Salt concentration and pH are the two main factors affecting the production of ectoine during the fermentation medium optimization process.The results show that H.hydrothermalis Y2 is dependent on high salt and high pH during the production of ectoine,10%NaCl and pH 9 are the optimal conditions for the production of ectoine by fermentation.In order to increase the yield of ectoine synthesis in H.hydrothermalis Y2,by reviewing the literature and combining the genome sequencing results of the H.hydrothermalis Y2,the genes related to the ectoine metabolic pathway were analyzed and the ectoine hydrolase gene doeA and hydroxylase gene ectD was knocked out separately.After knocking out ectD alone,its ectoine production increased by 16%.On the contrast,the double mutant that lacking ectD and doeA has a yield increased of 63.8%,indicating that Doe metabolic pathway is the main route for ectoine degradation in H.hydrothermalis Y2.In addition,in the double mutant H.hydrothermalis Y2/?ectD/?doeA,a decrease in the production of ectoine can still be detected in the late fermentation period.Therefore,it is speculated that there may be other unknown pathways for the degradation of ectoine in H.hydrothermalis Y2.Based on the previous research of the research group,single and double mutant of NhaD2 and Mrp type Na+/H+ antiporters were constructed separately to investigate their effects on the strain salt tolerance.The results showed that under high pH fermentation conditions,the salt dependence of ectoine produced by the strain was not reduced after knocking out nhaD2;and after the mrp was knocked out,the optimal salt concentration of ectoine production is reduced by 20 g/L and its ectoine yield is equivalent to H.hydrothermalis Y2/?ectD/?doeA;after both nhaD2 and mrp were deleted,the optimal salt concentration for the ectoine production by fermentation is reduced while its yield is reduced.This further proves that at pH 9,the mrp-type Na+/H+antiporter plays a major role in the salt tolerance of the strain.Therefore,H.hydrothermalis Y2/?ectD/?doeA/?mrp was identified as a strain with high production of ectoine in a reduced salinity medium.In addition,in this study,the batch fermentation capacity of H.hydrothermalis Y2,H.hydrothermalis Y2/?ectD/?doeA,H.hydrothermalis Y2/?ectD/?doeA/?mrp to product ectoine was compared in 1-L fermentor.The highest ectoine yields of different mutants were 5.5 g/L,7.2 g/L and 7.2 g/L,respectively.The secondary growth of the bacteria can be observed during the batch fermentation process.The mrp triple mutant has the fastest growth rate in the early stage of fermentation due to the relatively low salt concentration required for fermentation,and Glc and MSG are also rapidly consumed,which indicated that the lower The NaCl is beneficial to the growth of the strain,and it will not affect the production of ectoine in the H.hydrothermalis Y2/?ectD/?doeA/?mrp.Compared with the wild strain Y2,the double mutant and the mrp triple mutant in the late fermentation period caused the reduction of the ectoine degradation rate due to the blocking of the ectoine metabolic pathway.In order to further increase the ectoine production of H.hydrothermalis Y2/?ectD/?doeA/?mrp,the mutant was cultured in a 1-L fermentor containing 0.5 L MMG medium,and batch feeding during the fermentation.The initial NaCl concentration of the medium was 20 g/L,which was increased to 60 g/L after 18 h of fermentation,the MSG concentration was maintained at about 20 g/L,and the dissolved oxygen in the medium was adjusted by the speed and aeration to be higher than 30%.During the fed-batch fermentation,no secondary growth of the cells was observed.In the early stage of fermentation,the bacteria grew rapidly and reached the maximum biomass at 18 h.The maximum tire of 10.5 g/L was achieved in 48 h.During the entire fermentation process,a total of 50 g of MSG was consumed and a yield of 0.21 g g-1 monosodium glutamate.The decrease of intracellular ectoine was detected in the late stage of fermentation,and combined with factors such as the decline in biomass at this time,it is presumed that the ectoine is released into the culture medium from the cell.