Optimization Of Bacillus Licheniformis Industrial Enzyme Expression System

Enzymes are the core components of modern bio-manufacturing industry and play a pivotal and irreplaceable role in national economic and social development.The prerequisite for realizing the industrial application of enzyme preparations is obtaining enzyme molecules with industrial application value and the creation,optimization and application of an industrial-grade expression system suitable for high-efficiency expression of enzyme preparations.In the previous research,we have successfully created a world-leading Bacillus licheniformis industrial expression system,which has been successfully applied to the efficient preparation and industrial production of various major industrial enzyme preparations in our country.On the basis of existing research and industrial practice,the expression vectors and new technologies for the construction of high-yielding strains were further enriched in this study,and the following main research results were obtained:（1）Construction and functional evaluation of new expression vectors of B.licheniformis.Based on the expression vector p HY-WZX,eight new expression vectors were constructed and obtained by combining/replacing the promoters:p_{amy L},p_{amy E},p_cry3A and signal peptides:S_{amy L},S_chi,S_{amy E},among which 4 vectors（p HY-318,p HY-318b,p HY-319,p HY-319b）contain dual promoter combinations.By expressing thermophilicα-amylase（Amy S）,mesophilicα-amylase（Amy Q）and pullulanase（Pul A）,the eight new vectors can effectively mediate their secretion and expression,while there were differences in the expression efficiency.Among them,p HY-318 could mediate the best expression of Amy S and Amy Q（the enzyme activities of shake flask fermentation reached 6971 U/m L and 460 U/m L,respectively）,while Pul A was mediated by p HY-113 to achieve the highest level of expression.Similar to the results of previous studies,this study also found that the recombinant plasmids were unstable when expressingα-amylase and pullulanase from episomal plasmids,that is,the recombinant plasmids were lost to different degrees in the process of fermentation and culture of transformants and was related to the type of enzymes expressed.Based on the expression vector p HY-113,the structure of the ribosome binding site（RBS）in the5’-UTR of p_{amy E} and its nearby sequences were fine-tuned to obtain the expression vector p HD₁-113,whose RBS sequence was in the open region of an independent loop,when this vector mediates the expression of amy S and amy Q,the expression level was significantly reduced,and the expression level of the enzyme could not be increased as expected.（2）The TA system of B.licheniformis was analyzed and rearranged,and it was applied to the construction of high-yielding strains.Through bioinformatics analysis,it was confirmed that B.licheniformis contains a typical TA system,encoded by the operon ydc DE,which encodes a specific m RNA endonuclease and its specific inhibitor.The endo AI was cloned into the non-functional region of the expression vector p HY-318 and its transcription was restricted to be controlled by the temperature-inducible promoterλp _L p _R to obtain the expression vector p HA-318;the gene encoding the target enzyme was further cloned and transferred into enzyme expresses host B.licheniformis D402,and simultaneously uses the homologous recombination method to delete the endo AI in the host cell genome.When the recombinant strain A318amy S obtained by this method was cultivated at the normal fermentation and growth temperature of 37-42°C,the enzyme production level of shake flask fermentation reached 5930 U/m L,which was 30.9%higher than that of the control strain 318amy S.After the curing of the recombinant plasmid,the host cell apoptosis because the specific inhibitor of endonuclease cannot be obtained,thereby effectively ensuring the dominant position of the enzyme-producing recombinant strain in the fermentation culture population.（3）On the basis of newly obtained helper vector,the enzyme-encoding gene could integrated and amplified in the host genome mediated by integrated expression vector,a high-yielding enzyme-producing strain could be quickly constructed.The m RNA endonuclease coding gene maz F from Escherichia coli under the control of a lactose-inducible promoter was cloned into p T2^ts and the kanamycin resistance marker was replaced by a tetracycline resistance marker to obtain a new helper vector p UB-Maz F with"suicide"ability;The rep F from p T2^ts was disrupted and an expression cassette from p HY-318 was inserted into the multiple colning sites to obtaining an integrated expression vector p UB’-EX1 that cannot independently replicate autonomously in B.licheniformis.The recombinant plasmid p UB’-amy S was further constructed based on p UB’-EX1 and transformed into B.licheniformis D402（p UB-Maz F）to obtain a transformant BL-amy S,which was then incubated at 42°C with 1 mmol/L IPTG The high-yielding strains were cultured in liquid medium with500μg/m L kanamycin and screened on plates,and a group of high-yielding enzyme-producing strains were facilly obtained.Among them,the recombinant strain BLi S-002could stably produce enzymes in a 50 L fermenter,and the highest enzyme production level reached 50753 U/m L.（5）The ammonia-inducible promoters in B.licheniformis were systematically analyzed and functionally identified,and the value of such promoters in the novel application of enzyme expression was further explored.The transcriptome of B.licheniformis was studied and analyzed,and six ammonia-inducible promoter candidates,p_{cop A},p_{sac A},p_ald,p_{pdb X},p_plpand p_dfp,were selected.Molecular cloning and fermentation experiments were applied to confirm the strength of the selected ammonia promoter candidates,and the strength of the ammonia promoters to activate the transcription of amy L gene from B.licheniformis was p_plp>p_{sac A}>p_{pdb X}>p_{cop A}>p_ald.The ammonia-inducible promoter p_plp was used to mediate the expression of amy L,and ammonia was applied to adjust the p H of fermentation broth,the enzyme production level increased by 23%compared with the control fermentation process.