Gene Cloning,Expression And Directed Evolution Of Isopenicillin N Acyltransferase From Penicillium Chrysogenum

Penicillin is a kind of important antibiotic for low mammalian toxicity which widely used in clinic. Penicillium chrysogenum is an industrial production strain of penicillin. It is an important research project for penicillin industrial production to obtain higher yield through strain improvement. Modern molecular biology techniques, especially the progress of metabolic pathway engineering, allowed us to leading-in the foreign gene to transform the metabolic pathway of Penicillium chrysogenum. That will breakthrough in production bottlenecks and even creat the new product. The synthetic pathway of penicillinG in Penicillium chrysogenum starts withα-amino adipic acid and cysteine and valine. PenicillinG was generated by the catalysis of ACV synthetase, iso-penicillin synthase, Isopenicillin N Acyltransferase and phenylacetyl CoA ligase, and phenylacetic acid as the side chain precursor. In which two key enzymes are important to the type of side chain of penicillin. One is PCL which catalyzes phenyl acetic acid and CoA to generate phenylacetyl CoA, the other is IAT which catalyzes phenyl acetic acid and 6APA to generate penicillinG.In this study, the penDE gene was cloned from Penicillium chrysogenum and expressed in E.coli BL21（DE3）. The main results were as follow:（1） The degree of the reaction was indicated using acid-base indicator according to the changes in pH of the enzyme system. The microtiter plate was used to establish the screening model. The avantage of this model is small amount of sample, detection sensitivity and its lowest concentration was 1mmol/L. This method can be used to dectect the enzyme activity and screening the mutant of enzyme by directed evolution.（2） In this study, RT-PCR was used to clone penDE from Penicillium chrysogenum. The result showed that the cloned gene was 1074 bp by sequencing, of which the amino acid sequence was 100% homology with IAT from a number of Penicillium chrysogenum （NCBI rReference sequence numbers were: XM₀02569066.1,AM920436.1,EF601124.1,DQ192518.1 and M31454.1） whereas 83% homology with IAT from Aspergillus （NCBI reference sequence numbers were: XM₀02380606.1 and XM₀01825397.1）.（3） The prokaryotic expression vector pET-penDE was constructed. The optimized Induction expression condition in E.coli was established for increasing the solubility of IAT from inactive 40kDa inclusion protein. The results showed that more soluble IAT was induced at 16℃after the addition of 10mmol/L lactose at OD600≈1.0.（4） By studying the substrate specificity of IAT, it is showed that the natural enzyme substrate was phenylacetic acid, and not thiophene malonic and hydroxyphenylglycine.（5） By the random strategy, enzyme Trp120Arg which has weakly catalytic activity for thiophene malonic acid was obtained.