Effect of cell permeability and dehydrogenase expression on octane activation by CYP153A6-based whole cell Escherichia coli catalysts

Abstract

BackgroundThe regeneration of cofactors and the supply of alkane substrate are key considerations for the biocatalytic activation of hydrocarbons by cytochrome P450s. This study focused on the biotransformation of n-octane to 1-octanol using resting Escherichia coli cells expressing the CYP153A6 operon, which includes the electron transport proteins ferredoxin and ferredoxin reductase. Glycerol dehydrogenase was co-expressed with the CYP153A6 operon to investigate the effects of boosting cofactor regeneration. In order to overcome the alkane supply bottleneck, various chemical and physical approaches to membrane permeabilisation were tested in strains with or without additional dehydrogenase expression.ResultsDehydrogenase co-expression in whole cells did not improve product formation and reduced the stability of the system at high cell densities. Chemical permeabilisation resulted in initial hydroxylation rates that were up to two times higher than the whole cell system, but severely impacted biocatalyst stability. Mechanical cell breakage led to improved enzyme stability, but additional dehydrogenase expression was necessary to improve product formation. The best-performing system (in terms of final titres) consisted of mechanically ruptured cells expressing additional dehydrogenase. This system had an initial activity of 1.67 ± 0.12 U/gDCW (32% improvement on whole cells) and attained a product concentration of 34.8 ± 1.6 mM after 24 h (22% improvement on whole cells). Furthermore, the system was able to maintain activity when biotransformation was extended to 72 h, resulting in a final product titre of 60.9 ± 1.1 mM.ConclusionsThis study suggests that CYP153A6 in whole cells is limited by coupling efficiencies rather than cofactor supply. However, the most significant limitation in the current system is hydrocarbon transport, with substrate import being the main determinant of hydroxylation rates, and product export playing a key role in system stability.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0763-0) contains supplementary material, which is available to authorized users.

Deconstruction of the CYP153A6 Alkane Hydroxylase System: Limitations and Optimization of In Vitro Alkane Hydroxylation

“…In this respect the stability of CYP153A6 is remarkable, with 50% of the initial CYP still active after 48 h according to CO-difference spectra. White et al had similarly noticed that even in cell-free reactions at least 75% of CYP153A6 was still intact after 24 h [18]. This again indicates that instability of other enzymes be considered.…”

“…Unfortunately, these whole-cell systems also suffer from several limitations [17], including substrate uptake, product toxicity and inefficient cofactor regeneration. Substrate uptake can be improved through permeabilization of the whole cells or co-expression of transport proteins such as AlkL [13,18]. Product toxicity was confirmed through the addition of 1-octanol to whole-cell biotransformations using CYP153A6, requiring in situ product removal through a second organic phase such as bis(2-ethylhexyl) phthalate (BEHP) to alleviate the inhibition [19].…”

“…Co-expression of additional enzymes such as glycerol dehydrogenase (GlyDH) with the P450 enzymes in E. coli for adequate cofactor regeneration have also been investigated. Despite improved cofactor regeneration, this additional metabolic burden often is at the cost of P450 concentration [18].…”

“…White et al (2017) had found that an E. coli strain over expressing an additional E. coli glycerol dehydrogenase together with the CYP153A6 operon yielded excellent results when the cells were partially broken [18]. With this system, activity could be maintained for 72 h and a final product titer of 61 mM was achieved.…”

“…The glucose dehydrogenase (GDH) from Bacillus megaterium was kindly provided by Dr. Dirk Holtmann (Dechema, Germany) and sub-cloned into pET28b(+) using NdeI and XhoI. The glycerol dehydrogenase (GlyDH) from E. coli, previously cloned in pCDFDUET [18], were sub-cloned into pET28b(+) via NdeI and AvrII.…”