Process limitations of a whole-cell P450 catalyzed reaction using a CYP153A-CPR fusion construct expressed in Escherichia coli

Abstract

Cytochrome P450s are interesting biocatalysts due to their ability to hydroxylate non-activated hydrocarbons in a selective manner. However, to date only a few P450-catalyzed processes have been implemented in industry due to the difficulty of developing economically feasible processes. In this study, we have used the CYP153A heme domain from Marinobacter aquaeolei fused to the reductase domain of CYP102A1 from Bacillus megaterium (BM3) expressed in Escherichia coli. This self-sufficient protein chimera CYP153A-CPRBM3 G307A mutant is able to selectively hydroxylate medium and long chain length fatty acids at the terminal position. ω-Hydroxylated fatty acids can be used in the field of high-end polymers and in the cosmetic and fragrance industry. Here, we have identified the limitations for implementation of a whole-cell P450-catalyzed reaction by characterizing the chosen biocatalyst as well as the reaction system. Despite a well-studied whole-cell P450 catalyst, low activity and poor stability of the artificial fusion construct are the main identified limitations to reach sufficient biocatalyst yield (mass of product/mass of biocatalyst) and space-time yield (volumetric productivity) essential for an economically feasible process. Substrate and product inhibition are also challenges that need to be addressed, and the application of solid substrate is shown to be a promising option to improve the process.

Biosynthesis of the Nylon 12 Monomer, ω‐Aminododecanoic Acid with Novel CYP153A, AlkJ, and ω‐TA Enzymes

“…On the contrary, higher concentrations of glucose beyond certain limit can increase the generation of reactive oxygen species (ROS). ROS are involved in the oxidative modifications of proteins and alteration in enzyme stability . Generation of ROS and the consequent instability of the MprCYP153A can be one of the possible reasons for the lower productivity at higher glucose concentrations beyond 1%.…”

“…This finding can be explained in terms of the initial formation of ω‐OHDDA, which was then converted to ω‐ODDA and ω‐AmDDA due to the activity of AlkJ and M. loti ω‐TA. At 6 h, ω‐AmDDA may degrade, and α,ω‐DCA accumulates in the reaction due to over‐oxidation of the ω‐OHDDA . However, the precise reasons for the degradation of ω‐AmDDA over time remain elusive.…”

Semirational Protein Engineering of CYP153AM.aq.‐CPRBM3 for Efficient Terminal Hydroxylation of Short‐ to Long‐Chain Fatty Acids

“…Further evaluations of the best hits demonstrated an increased resistance to greater concentrations of C 12:0 and of ω‐OHC 12:0 and an improved activity confirmed through in vivo tests. The next step towards larger‐scale implementation for the production of hydroxylated compounds would require taking the optimized conditions previously reported for the successful accumulation of ω‐OHC 12:0 and the best variants described in this study …”

“…The G307A variant, termed CPR2 mut , showed >95 % regioselectivity for the terminal hydroxylation of fatty acids, together with increased activity and enhanced coupling efficiency relative to the wild‐type enzyme . However, recent studies have described challenges relating to substrate and product inhibition by dodecanoic acid (C 12:0 ) and the corresponding ω‐hydroxylated product (ω‐OHC 12:0 ) as well as low activity of the fused biocatalyst . In view of the identified limitations associated with the system, the purpose of this study was to engineer a suitable catalyst with higher activity for the terminal hydroxylation of fatty acids and thus to improve the whole‐cell production of ω‐OHC 12:0 prior to industrial implementation …”

Maximizing the stability of metabolic engineering‐derived whole‐cell biocatalysts

“…Therefore, the amount of the catalytically active protein(s) and thus the specific activity of non-growing biocatalysts often is highest at the beginning of the bioconversion. Under nitrogen starvation, the specific activity of non-growing E. coli harboring inherently unstable oxygenases declined rather quickly as a consequence of protein instability/degradation [140,142,165,177]. In contrast, the redox-state (intracellular NADH/NAD + ratio) and cell integrity of nitrogenstarved resting cells appeared to be rather constant in the presence of glucose [63,165].…”