β-Cryptoxanthin Production in Escherichia coli by Optimization of the Cytochrome P450 CYP97H1 Activity

Abstract

Cytochromes P450, forming a superfamily of monooxygenases containing heme as a cofactor, show great versatility in substrate specificity. Metabolic engineering can take advantage of this feature to unlock novel metabolic pathways. However, the cytochromes P450 often show difficulty being expressed in a heterologous chassis. As a case study in the prokaryotic host Escherichia coli, the heterologous synthesis of β-cryptoxanthin was addressed. This carotenoid intermediate is difficult to produce, as its synthesis requires a monoterminal hydroxylation of β-carotene whereas most of the classic carotene hydroxylases are dihydroxylases. This study was focused on the optimization of the in vivo activity of CYP97H1, an original P450 β-carotene monohydroxylase. Engineering the N-terminal part of CYP97H1, identifying the matching redox partners, defining the optimal cellular background and adjusting the culture and induction conditions improved the production by 400 times compared to that of the initial strain, representing 2.7 mg/L β-cryptoxanthin and 20% of the total carotenoids produced.

Constructing Protein-Scaffolded Multienzyme Assembly Enhances the Coupling Efficiency of the P450 System for Efficient Daidzein Biosynthesis from (2S)-Naringenin

“…It has been reported that CPR derived from different origins can impact the efficiency of the coupled redox reaction involving cytochrome P450. , To enhance the activity of CYP82D26, we coexpressed CPR genes from Catharanthus roseus (Cr_CPR), Oryza sativa (Os_CPR), and Lotus japonicas (Lj_CPR) in E. coli BL21­(DE3) along with CYP82D26, resulting in strains S2–S4.…”

“…It has been reported that CPR derived from different origins can impact the efficiency of the coupled redox reaction involving cytochrome P450. 37,38 To enhance the activity of CYP82D26, we coexpressed CPR genes from Catharanthus roseus (Cr_CPR), 5 Oryza sativa (Os_CPR), 5 and Lotus japonicas (Lj_CPR) 37 in E. coli BL21(DE3) along with CYP82D26, resulting in strains S2−S4. Based on the results shown in Figure 1C, coexpression of CrCPR and CYP82D26 resulted in the highest genistein titer of 19.8 mg/L (strain S2) compared to other strains (Figure 1C).…”

Bioflavour 2022 - Biotechnology of Flavours, Fragrances, and Functional Ingredients

“…Altogether, a 400-fold improvement in cell-based β-cryptoxanthin production was achieved, resulting in 407 mg of β-cryptoxanthin per kilogram of wet E. coli cells. 15 ■ SUMMARY AND OUTLOOK Bioflavour 2022 not only provided cutting-edge science but also gave sufficient time for socializing and networking. This was especially true for the afternoon excursion to the Eberbach monastery and the conference dinner on the river Rhine.…”

“…Additional improvements could be made by using the optimal redox partner and by optimization of culture and induction parameters. Altogether, a 400-fold improvement in cell-based β-cryptoxanthin production was achieved, resulting in 407 mg of β-cryptoxanthin per kilogram of wet E. coli cells …”

Systems Metabolic Engineering for Efficient Violaxanthin Production in Yeast

“…Cataldo et al also reported coexpression of RFNR1 and FD3 from Arabidopsis thaliana increased the violaxanthin content by 47% . Similarly, Lautier et al improved β-cryptoxanthin production by 6-fold via coexpressing the ferredoxin FER1 and ferredoxin reductase from Spinacia oleracea . Moreover, the cofactor pairs NADP + /NADPH, FMN/FMNH 2 , and FAD/FADH 2 , as important components for electron transfer, are probably essential for enhancing violaxanthin biosynthesis efficiency.…”