Efficient Whole-cell P450 Biocatalyst Using Engineered Escherichia Coli With Fine-tuned Heme Biosynthesis

Cytochrome P450 enzymes（P450s,CYP）belong to a superfamily of hemoproteins.Due to the diversity of substrate structure and the catalytic reaction types of P450s,they can specifically catalyze functional group reactions（e.g.,hydroxylation,epoxidation,etc.）in the post-modification stage of natural products biosynthesis,broadening the structural complexity and biological functional diversity of natural products.Thus,P450s can be the powerful tools for the biosynthesis and modification of natural products.However,there are still some limitations to using P450s for the efficient biosynthesis of natural products in microbial hosts.One limitation is that the heterologous expression of P450s is often inefficient in microbial hosts.Another limitation is that the insufficient supply of intracellular heme is a bottleneck in preparing highly active P450s in microbial hosts.Another limitation is that the biosynthesis of intracellular heme and the expression of P450s are not balanced.In addition,the complex enzyme isolation and purification processes for P450s and coenzymes are required,increasing the cost of catalytic reactions.Moreover,the requirements of costly cofactors limit the catalytic efficiency of P450s.To solve these problems,strain E.coli was used as the host and the expression of P450genes was optimized in this study.The heme biosynthesis was improved by the metabolic engineering of the host’s endogenous heme biosynthetic pathway and the heme level is fine-tuned by the combined use of mutated heme-sensitive biosensor and small regulatory RNA systems.Based on the engineered E.coli with fine-tuned heme biosynthesis,the efficient whole-cell P450 biocatalysts for the synthesis of natural products were constructed by using the intracellular cofactors combined with redox partner engineering and enzymatic engineering.The main research contents and results of this study are as follows.（1）Construction of the expression system for P450s and the heme transport system.Take the self-sufficient P450 BM3_A82F/A328F(BM3_mut)and the three-component P450sca-2G52S/T85F/F89I/T119S/P159A/V194N/D269E/T323A/N363Y/E370V（sca-2mut）as representative P450s,three host strains,E.coli BL21（DE3）,C41（DE3）,and C43（DE3）,and plasmids of different copy numbers,a high-copy number plasmid pRSFDuet-1 and a medium-copy number plasmid p ETDuet-1,were used to construct the suitable expression system for P450s.The E.coli C41（DE3）strain and pRSFDuet-1 plasmid were selected as the best expression system of P450s by comparison.The highest activity of self-sufficient P450 BM3_mut（29.4±1.5 U/g DCW）,using 7-ethoxycoumarin as a substate,was obtained in the C41-pRSF-BM3_mutstrain.The highest activity of three-component P450 sca-2_mut（5.8±0.5 U/g DCW）,using mevastatin as a substate,was obtained in the C41-pRSF-sca-2_mut strain.In the optimized expression system,the gene encoding the heme receptor ChuA in the outer membrane from E.coli Nissle 1917 was selected to construct the heme importers using the plasmid pCDFDuet-1and 2.5 nM hemin could be transported into the intracellular.The chuA gene was expressed an inducible T7 promoter using the plasmid pCDFDuet-1,increasing the whole-cell activity of self-sufficient P450 BM3_mut,three-component P450 sca-2_mut by 16.1%（34.1 U/g DCW）and29.4%（7.5 U/g DCW）,respectively.（2）Enhancing intracellular heme supply by the engineering of heme synthetic pathway.The intracellular heme supply was improved by enhancing the endogenous heme biosynthetic pathway.By comparing the effect of episomal expression and integrated expression of the key enzymes on P450 activities,the genes encoding the key enzymes（glutamyl-tRNA reductase,glutamate-1-semialdehyde 2,1-aminomutase,porphobilinogen synthase,porphobilinogen deaminase,uroporphyrinogen Ⅲ synthase,and ferrochelatase）in the heme biosynthetic pathway were integrated expression and the rate-limiting enzymes（porphobilinogen synthase,porphobilinogen deaminase,and uroporphyrinogen Ⅲ synthase）were assembled by the DNA-guided scaffolds.The intracellular concentration of heme in the engineering strains（0.22～0.43 mg/L）increased 22-fold to 43-fold compared to the C41（DE3）strain（0.01 mg/L）.The engineering strain HEME-S13 was used to preparing the whole-cell biocatalyst,increasing the whole-cell activity of self-sufficient P450 BM3_mut,three-component P450sca-2_mutby 2-fold（87.6 U/g DCW）and 2.8-fold（21.9 U/g DCW）,respectively,compared to the C41（DE3）strain.（3）Fine-tuning heme synthesis to balance the expression of P450s.The regulator HrtR and its binding site HrtO_Lfrom Lactococcus lactis was used to design an E.coli heme biosensor.The mutant HrtR_H149K,which sensitivity lower than that of wild-type HrtR,was obtained through the saturation mutagenesis of His-149.The fine-tuned heme system was designed comminated the use of the mutated heme biosensor and s RNA regulatory systems.The intracellular concentration of heme in HEME-R11 strain could be better controlled（0.06～0.12 mg/L）based on the heme fine-tuned system.The engineering strain HEME-R11was used to prepare the whole-cell P450 biocatalyst,increasing the whole-cell activity of self-sufficient P450 BM3_mut,three-component P450 sca-2_mutby 3.3-fold（125.9 U/g DCW）and 6.9-fold（45.4 U/g DCW）,respectively,compared to the C41（DE3）strain.The expression levels of P450 BM3_mut were similar in the C41（DE3）strain and HEME-R11 strain.The ratio of holo-BM3_mut(mol heme/mol BM3_mut)increased from 17.7%in the C41-pRSF-BM3_mutstrain to 71.5%in the HEME-R11-BM3_mut strain.The expression level of three-component P450 sca-2_mutin the HEME-R11 strain could be increased 79.7%compared to the C41（DE3）strain.In addition,the ratio of holo-sca-2_mut(mol heme/mol sca-2_mut)increased from 12.9%in the C41-pRSF-BM3_mut strain to 61.8%in the HEME-R11-BM3_mut strain.The whole-cell conversion efficiency（68.4%）towards mevastatin in HEME-R11-sca-2_mut was 1.3-fold higher than the previously reported conversion efficiency in E.coli（53.9%）.（4）Synthesis of hydroxylated flavones natural products using efficient whole-cell P450s biocatalysts.Whole-cell P450s biocatalysts were prepared using the HEME-R11 strain to synthesize the natural products.Compared with the original strain C41（DE3）,the catalytic efficiencies of coumarin and resveratrol were increased by 290%and 80%for the synthesis of3-hydroxycoumarin and piceatannol,respectively.The three-component P450 sca-2_mutwas found the outstanding 3’-hydroxylation activity towards flavonoids.The whole-cell activity of sca-2_mut was enhanced by using a novel combination of flavodoxin Fld and flavodoxin reductase Fpr from E.coli.Subsequently,the whole-cell activity of sca-2_mutR88A/S96A was improved through the enzymatic engineering and the optimization of whole-cell biocatalytic conditions.Finally,eriodictyol,dihydroquercetin,quercetin,luteolin,and7,3’,4’-trihydroxyisoflavone,as examples of flavanone,flavanonol,flavonol,flavone,and isoflavone,were produced by whole-cell biocatalysis using naringenin,dihydrokaempferol,kaempferol,apigenin,and daidzein as the substrates,with the conversion yield of 80.6%,66.3%,5.7%,31.8%,and 75.1%,respectively.