Molecular Modification And Expression Optimization Of Pyrococcus Yayanosii CH1 L-asparaginase

L-asparaginase?EC 3.5.1.1?,which catalyses the conversion of L-asparagine to L-aspartate and ammonia,has high application value in medicine and food industry.In clinical applications,L-asparaginase is used to treat lymphoblastic leukaemia,as it removes the L-asparaginate required for the survival of lymphoblastic leukaemia cells.In the food industry,L-asparaginase is used to reduce carcinogenic acrylamide by removing the precursor L-asparagine.However,the poor thermostability,low specific activity and yield restricted the application of L-asparaginase in medicine and food industry.Therefore,it is of great significance to construct L-asparaginase with better thermal stability,higher enzyme activity and high-yield fermentation.In this study,the enzymatic characteristics of thermophilic bacteria Pyrococcus yayanosii CH1 L-asparaginase were characterized,and the key functional domains responsible for thermal stability were identified;Mutant strains of L-asparaginase with higher enzyme activity and thermal stability were obtained by high throughput screening;High-yield fermentation of L-asparaginase was realized through screening of expression elements and optimization of fermentation process,.The main results are listed as follows:?1?Bacillus subtilis 168 was used as the host strain to express non-thermophilic bacteria?Escherichia coli and Bacillus subtilis B11-06?L-asparaginase and thermophilic bacteria?P.yayanosii CH1,Thermococcus gammatolerans EJ3,Pyrococcus furiosus DSM 3638?L-asparaginase,respectively.In shaking flask fermentation,P.yayanosii CH1 L-asparaginase?PYA?had highest activity yield(89.03U?mL^-1),showing potential for large-scale fermentation.The optimum temperature and pH of P.yayanosii CH1 L-asparaginase were 95°C and 8.0,respectively.The L-asparaginase had high specific activity(1483.81 U?mg^-1),and a great thermostability and alkaline pH stability.The half-life of the L-asparaginase at 85°C(T_?1/2,85°C?)was105 min,and it was only a slight decrease in the activity of up to 90%after storage at37°C for one month;After incubation at pH=10 for 30 hours,the retained activity of L-asparaginase was more than 80%.?2?Homologs and residues conservatism of 1,000 thermophilic and non-thermophilic L-asparaginase were analyzed by bioinformatics.It was found that there were“specific residues”near the highly conserved regions of thermophilic and non-thermophilic L-asparaginase.These residues were highly conserved in thermophilic and non-thermophilic L-asparaginase,respectively,but they were different each other.These“special residues”were hypothesized as the key amino acid residues responsible for the high thermal stability of thermophilic asparaginases.Verification with interchanging these residues and structure domains between thermophilic L-asparaginase and non-thermophilic L-asparaginase,the key residues?residues D51 and K298 of PYA,corresponding residues G57 and L305 of E.coli L-asparaginase?for thethermostability were identified,and the change of the loop rigidity,surface charge near the activity sides,and the enzyme C terminal tightness were key for L-asparaginase thermostability.Based on these,we mutated the key residues of E.coli L-asparaginase structure domains,and improved the thermal stability at 37°C and specific activity to1.61 and 2.74 times of wild-type,respectively.?3?The high throughput screening method for screening thermophilic L-asparaginase with high thermal stability and specific activity was established by simultaneous pyrolysis cells with enzyme activity determination.Two rounds of error-prone PCR?epPCR?were used to construct the mutagenesis library of PYA gene.880positive mutants were identified from 6,000 mutants by high throughput screening.It was found that the mutants containing residual S17G,A90S,R156S and K272A accounted for 31%,28%,39%and 25%of all positive mutants respectively,which were much higher than other mutants.The mutation of these sites may lead to the improvement of activity or thermostability.The specific activity of single point mutations PYA-S17G,PYA-R156S and PYA-K272A were increased to 1.72,1.62 and1.27 times of PYA,respectively.The T_?1/2,85°C?of PYA-A90S was 45 min longer than that of PYA.Further,the combinatorial mutant PYA-FH showed specific activity which2.1 times of PYA,and T_?1/2,85°C?was 40 min longer than that of PYA.?4?Through screening promoters,the transcription level of PYA gene with Bacillus subtilis 168 was increased to 8.2 times,but the activity of PYA was only increased to1.32 times.The mismatch between the transcription level and the activity yield may be due to the lower translation level than that of the original bacteria.Hence,three hundred RBS sequences with a gradient of translation initiation rates?2,000 to 5,687,190 au?were designed through RBS Calculator?https://www.denovodna.com/software/?.With the most suitable RBS,L-asparaginase production was increased to 2.09 times than that of original strain.Meanwhile,through signal peptide prediction and protein N terminal sequencing et,the secretion of P.yayanosii L-asparaginase in B.subtilis was identified to depend on non-classical protein secretion pathway.By signal peptide screening,the Tat-pathway signal peptide SP_phoD was found to be beneficial for the L-asparaginase secretion in B.subtilis.With SP_phoD and molecular chaperone PrsA co-expression,the secretion of the L-asparaginase was 72.11%higher than that of the original one.And then,through fermentation medium and fermentation process optimization,the active production of PYA was reached 6324.23 U?mL^-1,which was highest yield ever reported.