The influence of microbial physiology on biocatalyst activity and efficiency in the terminal hydroxylation of n-octane using Escherichia coli expressing the alkane hydroxylase, CYP153A6

Biocatalyst improvement through molecular and recombinant means should be complemented with efficient process design to facilitate process feasibility and improve process economics. This study focused on understanding the bioprocess limitations to identify factors that impact the expression of the terminal hydroxylase CYP153A6 and also influence the biocatalytic transformation of n–octane to 1-octanol using resting whole cells of recombinant E. coli expressing the CYP153A6 operon which includes the ferredoxin (Fdx) and the ferredoxin reductase (FdR). Results: Specific hydroxylation activity decreased with increasing protein expression showing that the concentration of active biocatalyst is not the sole determinant of optimum process efficiency. Process physiological conditions including the medium composition, temperature, glucose metabolism and product toxicity were investigated. A fed-batch system with intermittent glucose feeding was necessary to ease overflow metabolism and improve process efficiency while the introduction of a product sink (BEHP) was required to alleviate octanol toxicity. Resting cells cultivated on complex LB and glucose-based defined medium with similar CYP level (0.20 μmol gDCW -1) showed different biocatalyst activity and efficiency in the hydroxylation of octane over a period of 120 h. This was influenced by differing glucose uptake rate which is directly coupled to cofactor regeneration and cell energy in whole cell biocatalysis. The maximum activity and biocatalyst efficiency achieved presents a significant improvement in the use of CYP153A6 for alkane activation. This biocatalyst system shows potential to improve productivity if substrate transfer limitation across the cell membrane and enzyme stability can be addressed especially at higher temperature. Conclusion: This study emphasises that the overall process efficiency is primarily dependent on the interaction between the whole cell biocatalyst and bioprocess conditions

Improving the production of a thermostable amidase through optimising IPTG induction in a highly dense culture of recombinant Escherichia coli.

The production of a novel thermostable amidase (EC 3.5.1.4) from Geobacillus pallidus RAPc8 using recombinant Escherichia coli BL21 (DE3) was investigated. Volumetric and specific enzyme activities were investigated in relation to inducer concentration in a batch process using a defined medium with glucose as the carbon source. While IPTG is routinely used to induce expression of genes under the control of lac promoter, the impact of high biomass concentration on IPTG induction has not been reported rigorously. In this study, biomass production was unaffected by IPTG concentration across the range 0–1000 M. Induction of recombinant protein expression by 400 M IPTG at late lag phase of growth (3rd hour) inhibited cell growth while induction at early exponential phase of growth (5th hour) gave a 3 fold increase in volumetric amidase activity compared to induction at mid exponential phase (8th hour). Protein production increased by a factor of two with IPTG addition, independent of IPTG concentration in the range of 40–1000 M. Amidase activity, measured on a volumetric basis and relative to protein and biomass concentrations, increased with increasing IPTG concentration up to 400 M. While inducer concentrations are typically reported on a volumetric basis, their mode of action is consistent with a biomass dependence. Analysis of the data across a range of biomass concentration confirmed that induction was a function of inducer concentration per unit biomass. The amidase enzyme was predominantly soluble and cytoplasmic with less than 3% retained within the cell debris

Whole-cell hydroxylation of n-octane by Escherichia coli strains expressing the CYP153A6 operon

CYP153A6 is a well-studied terminal alkane hydroxylase which has previously been expressed in Pseudomonas putida and Escherichia coli by using the pCom8 plasmid. In this study, CYP153A6 was successfully expressed in E. coli BL21(DE3) by cloning the complete operon from Mycobacterium sp. HXN-1500, also encoding the ferredoxin reductase and ferredoxin, into pET28b(+). LB medium with IPTG as well as auto-induction medium was used to express the proteins under the T7 promoter. A maximum concentration of 1.85 μM of active CYP153A6 was obtained when using auto-induction medium, while with IPTG induction of LB cultures, the P450 concentration peaked at 0.6–0.8 μM. Since more biomass was produced in auto-induction medium, the specific P450 content was often almost the same, 0.5–1.0 μmol P450 gDCW−1 , for both methods. Analytical scale whole-cell biotransformations of n-octane were conducted with resting cells, and it was found that high P450 content in biomass did not necessarily result in high octanol production. Whole cells from LB cultures induced with IPTG gave higher specific and volumetric octanol formation rates than biomass from auto-induction medium. A maximum of 8.7 g octanol LBRM−1 was obtained within 24 h (0.34 g LBRM−1 h−1 ) with IPTG-induced cells containing only 0.20 μmol P450 gDCW−1 , when glucose (22 g LBRM−1 ) was added for cofactor regeneration