Directed Evolution Of P450 And Its Application In Nylon 12 Monomer Biosynthesis

As an important engineering plastic,nylon 12(PA12)has many excellent properties such as low density,low water absorption,high temperature resistance,and corrosion resistance,which promote its applications in many fields such as automobiles,electronic appliances,pipeline cables,3D printing and household goods.However,the current industrial production of PA12 based on chemical synthesis has a number of issues,such as the use of toxic and harmful raw materials and difficult waste disposal causing environmental stress,complex process flow and difficult operation.As a more gentle and green alternative method,biosynthesis of PA 12 monomer from renewable feedstocks has attracted increasing attention.However,the yield is generally low,due to the presence of rate-limiting enzymes and the unsatisfying stability of the cell factory.This work aims to improve the biosynthetic efficiency of PA12 monomer using E.coli cell factories by means of enzyme directed evolution,multienzyme expression regulation,metabolic pathway optimization and tolerance engineering.First,as one of the important substrates for the biosynthesis of PA 12 monomer,the terminal hydroxylation efficiency of lauric acid(DDA)determines the yield of the target product.In order to improve the catalytic activity of the rate-limiting P450,a high-throughput screening method was established for its directed evolution.Based on the ABTS colorimetric method previously reported for screening P450 mutants with enhanced terminal hydroxylation activity of fatty acids,a modified ABTS 2.0 colorimetric method was developed to avoid false positives and improve the accuracy of detection.Application of this method for directed evolution of cyp153a-ncpG307A resulted in selection of mutant M3(cypl53a-ncpG307A/R14R/D629G)out of a total of 2000 clones,which increased the terminal hydroxylation activity towards DDA by 90.3%,leading to production of 2.78±0.08 g/L 12-hydroxylauric acid(ω-OHDDA)within 4 hours,with a conversion rate of 85.8%.Among the positive mutation sites,the Nterminal synonymous mutation R14R improved the soluble expression of P450,while the D629G mutation shortened the electron transfer distance between FMN and FAD.The resulting mutant also showed improved catalytic activity towards other similar substrates.Application of this mutant in the biosynthesis of PA12 monomer 12-aminododecanoic acid(co-AmDDA),together with replacement of the original alcohol dehydrogenase BsADH with the alcohol dehydrogenase AlkJ from P.putida GPOl,and balancing the expression of multiple enzymes using Ribosome-binding site(RBS)engineering to avoid the accumulation of intermediate product ω-OHDDA,constructed strain P1-1(E-M3-3/C-rbs3-alkJ),which produced 1.72±0.04 g/L of ω-AmDDA from DDA with a yield of 79.8%.Subsequently,by moderately up-regulating the hemB gene involved in heme synthesis,and knocking out the yfeX gene related to heme degradation,the heme synthesis in E.coli was improved,providing sufficient heme for the ω-hydroxylation activity of P450 and promoting the expression of active P450.The application of this strategy in biosynthesis of ω-AmDDA further promoted the production of ω-AmDDA to 2.02 ±0.03 g/L(93.6%yield).Finally,the P450 mutant and the heme synthesis engineering strategy were applied in de novo biosynthesis of ω-AmDDA,improving its production from glucose to 593.6 ± 32.8 mg/L within 20 h in shake flasks.In this study,the high throughput screening method established,the thermal mutation sites identified in the P450 reductase domain and the strategy used to enhance the supply of heme would provide helpful hints for engineering other P450-involved bioprocesses.Meanwhile,the engineered E.coli strains constructed to synthesize ω-AmDDA from DDA or glucose lay solid foundation for the bioproduction of PA12.