Promoter Optimization And Low-Salt Fermentation For Ectoine Production In Halomonas Hydrothermalis Y2

To cope with pressures of high salinity,osmotic pressures and extreme temperatures,ectoine is widely synthesized by bacteria,fungi and some archaea.It has excellent anti-adversity protection properties,and therefore has a wide range of application potential in the development of biotechnology,skin care and medicine.Halomonas hydrothermalis Y2 investigated in this study is a strain isolated from papermaking waste liquid and has a survival advantage in the environment of high salinity and strong alkalinity in the waste liquid.Based on the analysis of the transcriptome data of H.hydrothermalis Y2 at three different pHs(pH6,pH8,pH 10)in the early stage of the laboratory,15 possible strong constitutive promoters were found,and the promoters were determined by the promoter probe vector Vigor,a strong constitutive promoter P2155 was found.In order to apply P2155 to the synthesis of ectoine,a 40 bp core region of P2155 was determined by truncation experiments.5'RACE analyzes the transcription initiation site of ectABC in the ectoine synthesis pathway,and the promoter types were determined by analyzing the sequences of-10 and-35 regions.Salt sensitivity experiment confirms that the promoter of ectA in H.hydrothermalis Y2 is a salt-sensitive promoter,while the promoter of ectB is salt sensitive.And the promoter of ectA in Halomonas salinas DSM5928T is not salt sensitive.To alleviate the pressure from fermenters,downstream processing equipment and wastewater treatment,and to reduce the salt dependence in the process of ectoine synthesis,the ectA promoter was replaced with the promoter P2155 in the outer membrane porin of H.hydrothermalis Y2.Shaking flask experiments under different salt concentrations revealed that replacing the promoter reduced the salt-dependence of ectoine synthesis.The strains of promoter replacement had significantly enhanced extracellular ectoine synthesis ability,especially under low salt conditions.After72 h fed-batch fermentation in a 1 L fermentor containging 80 g L'1 NaCl,the ectoine yield of pp/Y2/?ectD/?doeA was 13.1 g L-1,with an increase of 13%.Similarly,in this study,the ectA pro-promoter of the ectoine-secreting strain H.salinas DSM5928T was used to replace the ectA pro-promoter in H.hydrothermalis Y2,and similar results were obtained.In order to reduce the degradation of ectoine in the later stage of fermentation,the pathway of ectoine absorption was analyzed,and the substrate binding protein TeaA was knocked out to disable the pathway.After the knockout of strain Y2/?ectD/?doeA/?mrp/?teaA,the batch fermentation yield of ectoine was 4.1 g L-1,the fed batch fermentation yield was 14.4 g L-1,and the optimal salt concentration was 60 g L-1.Ectoine is a protective agent and stabilizer,and its synthesis pathway exists only in some moderately halophilic bacteria.But in these strains,the high yield of ectoine is often dependent on the high salt environment.Therefore,this paper attempted to introduce the ectoine synthesis pathway into Corynebacterium glutamate Lys to construct the heterologous production strain of ectoine.The lysine producing strain of C.glutamicum Lys with high lysine yield was selected as the starting strain,which was weakly competitive lysine branch,and the lysine exogenous gene on the genome was knocked out to improve the production of ectoine.On this basis,ectABC gene cluster from H.hydrothermalis Y2 was introduced into C.glutamate to construct ECT-1 strain.Finally,3.7 g L-1 ectoine was synthesized by shaking flask fermentation.