Engineering Of P450 And Its Application In Steroid Biosynthesis

Cortisol is an important glucocorticoid in humans,which plays a vital role in the physiological adaptation of stress,energy mobilization and regulation of immune responses.It is also used as an intermediate in the synthesis of other glucocorticoids.The chemical synthesis of cortisol involves many steps leading to low yield,so the current industrial production mainly adopts semi-synthetic methods,in which 11β-OH is introduced to 11-deoxycortisol to produce cortisol,and this microbial transformation is mainly carried out with fungal cultures.However,the selectivity of the fungal catalytic process is poor and often accompanied by the production of hydroxylation byproducts in other locations.Besides,the culture and transformation times of fungi are generally long.Therefore,the development of alternative biocatalysts with higher selectivity and efficiency is of great industrial value.In this work,the human-derived mitochondrial 11β-hydroxylase P450 CYP11B1 was used as the research object,and a biocatalytic process based on Escherichia coli was developed for efficient production of cortisol,via condition optimization for expression of recombinant proteins and whole-cell reactions,P450 redox partner engineering and protein engineering.In order to solve the problems of low expression and poor stability encountered in the heterologous expression of eukaryotic P450 in prokaryotes,the expression vector and polycistronic order of CYP11B1 and its homologous redox companion bovine AdR and bovine Adx in E.coli were first optimized.Under the conditions of optimal expression vector and expression order,the relative expression intensity of the three proteins was coordinated by screening for the optimal RBS combination.Subsequently,the cell culture conditions were optimized,including culture temperature,induction conditions,and media composition.In the whole cell reaction system,the effects of different cell membrane co-penetrants and substrate co-solvents on the catalytic activity of cells were investigated,and the substrate concentration of the reaction system was increased under the application of the preferred co-solvent.Through the optimization of expression and reaction conditions,the spatiotemporal yield of cortisol was successfully increased from 343.6 mg·L-1·d-1 to 1470.0 mg·L-1·d-1.Subsequently,the redox partner of CYP11B1 was engineered to obtain the optimal redox chaperone,in order to increase the electron transfer rate and thus improve the whole-cell catalytic efficiency.First,the reductase domain of natural self-sufficient enzymes P450BM3 and P450RhF were fused with CYP11B1 to form artificial selfsufficient P450s,but the fusion enzymes led to a complete loss of CYP11B1 activity.Subsequently,four groups of redox partners from different sources were selected for co-expression with CYP11B1,and cross-combinations of different ferroxedoxin and ferroxedoxin reductases were constructed,showing significant differences in the effect on the catalytic activity of whole cells.Finally,two different molecular scaffolds,CipB and PCNAs,were introduced to form efficient electron channels through self-assembly function,but they did not show a positive effect on improving catalytic activity.Although the attempt at redox partner engineering did not directly improve the catalytic activity of whole cells,it demonstrated the potential of redox chaperone replacement to improve the catalytic efficiency of P450 enzymes.Finally,CYP11B1 was modified by protein engineering.Based on the understanding of the molecular structure of CYP11B1,some residues constituting the substrate channel were selected,and alanine scanning was used to find the key sites affecting the entry and exit of the substrate.Rational design was conducted for the residues in the substrate binding pocket,in order to shorten the distance between C11 of the substrate and the heme-Fe.However,the mutants generated did not show a beneficial effect on activity improvement,and some mutations even caused severe loss of activity.Subsequently,the solubility and stability of CYP11B1 were improved by applying computational design tools,and multiple mutants showed increase in catalytic activity.The three-point mutant M3(S169V/H354D/L463F)increased the spatiotemporal yield of cortisol to 2756.3 mg·L-1·d-1,which is 3.3 times of the highest reported cortisol yield obtained by this type of biotransformation.This work significantly improved the biocatalytic efficiency of cortisol production from 11-deoxycortisol dependent on CYP11B1,showing the potential of microbial cortisol production.Meanwhile,the process optimization,redox partner engineering and enzyme engineering strategies might provide reference for the P450-catalyzed bioproduction of other steroids.