Gene Engineering And Directed Evolution Of Penicillin G Acylase

Industry of β-lactam antibiotics production is undergoing a huge reformation. Because the biocatalyzed processes are replacing the chemical synthesis processes. Penicillin G acylase (EC 3.5.1.11, PGA) now plays a central role in the production of β-lactam antibiotics. In view of drawbacks of the conventional chemical processes, such as the high toxicity of some reagents and the requirement for a lot of reaction steps, enzymes with higher synthesis capability facilitate synthetic applications in β-lactam antibiotics production. PGA that have been sequenced are those from the gram-negative bacteria family members Alcaligenes faecalis (Af), Escherichia coli (Ec), Kluyvera citrophila (Kc) and Providencia rettgeri (Pr) and from the gram-positive bacteria Bacillus megaterium (Bm) and Arthrobacter viscosus. Besides having similar substrate preferences, these enzymes are evolutionarily related. Their nucleotide sequences and amino acid sequences are mostly conserved.In chapter 1, Bacillus subtilis was utilized to express PGA from various sources. The pga gene from Alcaligenes faecalis (CICC AS1.767) was cloned (Genbank accession number is AF455356). The enzyme was extracelluarly expressed in Bacillus subtilis. In contrast to E.coli hosts where the enzymes were retained in the periplasm, B. subtilis cells secreted the recombinant enzyme into the medium. The expression yield of B. subtilis (pMAPGA) increased 109-fold higher than the native expression of A. faecalis CICC AS1.767. The enzyme was separated by (NH4)2SO4 fractionation and purified by DEAE-Sepharose CL-6B with a yield of 81%. The purified enzyme had a specific activity of 1.469U/mg. It was determined that the enzyme possessed a higher stability against organic solvent but a lower ratio of cephalexin synthesis to hydrolysis. Moreover, optimums of pH and temperature were explored. In chapter 2, the study focused on construction of some universal Bacillus subtilis vectors. The signal peptide sequence of BmPGA was modified according to analytic results of SignalP, the software for predicting signal peptide sequences. We studied promoters of HpaⅡand p43 and the signal peptides of BmPGAsp,AfPGAsp and SacB. Their effects on AfPGA expression were also elucidated.In chapter 3, Escherichia coli was utilized to express PGA from various sources. A gene encoding PGA was obtained from genomic DNA of Providenciarettgeri (ATCC29944) and its Genbank accession number was AF499615. PGA gene was expressed under the control of T7 promoter. The recombinant Escherichia coli gave a high level PGA expression, which increased 66-fold higher than the native expression of Providencia rettgeri (ATCC29944). Engineering organisms expressing AfPGA, EcPGA, KcPGA and PrPGA respectively, were subsequently constructed. Several factors were optimized for enzyme overproduction. In chapter 4, the 85.6kDa heterodimeric PGA from Bacillus megaterium was processed and functional displayed on the surface of phage fd. Phagemid pSurfscript was used to clone full-lenghth gene with amber stop codon of PGA from Bacillus megaterium. It was demonstrated that E.coli XL1-blue cells harboring pSurfpga could express PGA. Meanwhile, phages were rescued by M13KO7 helper phage particles. Results showed that the heterodimeric enzyme was expressed as a fusion protein that matures to an active biocatalyst connected to the coat protein of phage fd. The display of BmPGA not only offers the possibility of applying this technology for the selection of penicillin acylases with new side-chain specificities, but also facilitates the screen of mutant library constructed by using DNA shuffling technique. In chapter 5, Both BmPGA and PrPGA were mutated using DNA shuffling. It was demonstrated that the mutation rate of shuffling BmPGA β subunit was 1.07%. Because homologous sequences used in DNA family shuffling had been functional evolved during millions of years of natural selection, DNA family shuffling became a powerful tool for rapidly evolving en...