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

BACKGROUND: 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 (0.20mumol gDCW-1) showed different biocatalyst activity and efficiency in the hydroxylation of octane over a period of 120h. 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

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

Enhanced Synthesis of 2‑<i>O</i>‑α‑d‑Glucopyranosyl‑l‑ascorbic Acid from α‑Cyclodextrin by a Highly Disproportionating CGTase

2-<i>O</i>-α-d-Glucopyranosyl-l-ascorbic acid (AA-2G) is an industrially important derivative of vitamin C [l-ascorbic acid (l-AA)]. A useful synthetic route toward AA-2G is the selective glucosylation of l-AA by cyclodextrin glucanotransferase (CGTase). However, the cyclodextrin donor substrate is utilized rather inefficiently, because only one of its constituent glucosyl residues is coupled to the l-AA acceptor. A CGTase catalyzing disproportionation of the linear maltooligosaccharide chain formed in the initial coupling reaction might utilize a greater portion of the substrate for l-AA glucosylation and thus boost the AA-2G yield of cyclodextrin conversion. We present here a detailed characterization of the transfer reactions involved in the formation of AA-2G from α-cyclodextrin by a commercial CGTase preparation from <i>Thermoanaerobacter</i> sp. (Toruzyme 3.0L). We demonstrate that besides coupling, disproportionation constitutes a major route of glucosylation of l-AA by this enzyme. l-AA glucosides with oligoglucosyl chains between 1 and 12 units long were produced in the reaction. After chain-trimming hydrolysis with glucoamylase, however, AA-2G was recovered as the sole product of the enzymatic transglucosylation. The molar yield of AA-2G from cyclodextrin was 1.4, thus clearly exceeding the maximal yield of 1 for the coupling reaction. Using conditions optimized for transfer efficiency and productivity, we obtained AA-2G at the highest concentration (143 g/L, 424 mM) so far reported from an enzymatic glucosylation of l-AA. The synthetic yield was 30% based on l-AA (250 g/L, 1420 mM) offered in ≤4.6-fold molar excess over α-cyclodextrin

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