Walking a Fine Line with Sucrose Phosphorylase: Efficient Single-Step Biocatalytic Production of l-Ascorbic Acid 2-Glucoside from Sucrose.

The 2-O-α-d-glucoside of l-ascorbic acid (AA-2G) is a highly stabilized form of vitamin C, with important industrial applications in cosmetics, food, and pharmaceuticals. AA-2G is currently produced through biocatalytic glucosylation of l-ascorbic acid from starch-derived oligosaccharides. Sucrose would be an ideal substrate for AA-2G synthesis, but it lacks a suitable transglycosidase. We show here that in a narrow pH window (pH 4.8-6.0, with sharp optimum at pH 5.2), sucrose phosphorylases catalyzed the 2-O-α-glucosylation of l-ascorbic acid from sucrose with high efficiency and perfect site-selectivity. Optimized synthesis with the enzyme from Bifidobacterium longum at 40 °C gave a concentrated product (155 g L-1 ; 460 mm), from which pure AA-2G was readily recovered in ∼50 % overall yield, thus providing the basis for advanced production. The peculiar pH dependence is suggested to arise from a "reverse-protonation" mechanism in which the catalytic base Glu232 on the glucosyl-enzyme intermediate must be protonated for attack on the anomeric carbon from the 2-hydroxyl of the ionized l-ascorbate substrate.

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 (0.20 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.

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 g (DCW)⁻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 L (BRM)⁻¹ was obtained within 24 h (0.34 g L (BRM)⁻¹ h⁻¹) with IPTG-induced cells containing only 0.20 µmol P450 g (DCW)⁻¹, when glucose (22 g L (BRM)⁻¹) was added for cofactor regeneration.

Identification and characterization of 4-hexylbenzoic acid and 4-nonyloxybenzoic acid as substrates of CYP102A1.

CYP102A1 is an efficient medium- to long-chain fatty acid hydroxylase that is able to accept a wide range of non-natural substrates which bear no resemblance to the natural ones. 4-Hexylbenzoic acid (HBA) and 4-nonyloxybenzoic acid (NOBA) were identified as CYP102A1 substrates via screening studies using the BD Oxygen Biosensor System. Spectroscopic binding studies showed that these two substrates bind in the active site of CYP102A1 with K(d) values of 2.6 ± 0.1 µM for HBA and 1.9 ± 0.2 µM for NOBA. NADPH consumption rates in the presence of HBA and NOBA were 45 ± 1 min(-1) and 61 ± 1 min(-1), respectively. The coupling efficiency for NADPH was 57% for NOBA, while it was 77% for HBA. During whole-cell biotransformations, HBA was converted into ω-1- and ω-2-hydroxyhexylbenzoic acid, whereas NOBA was oxidized to ω-2-hydroxynonyloxybenzoic acid and ω-2,ω-4-dihydroxynonyloxybenzoic acid. HBA was used as a fatty acid mimic to compare whole-cell biotransformations with cell-free extracts. Whole-cell biotransformations carried out in a biphasic system resulted in 86% conversion of 5 mM HBA, producing 3.8 mM ω-2- and 0.5 mM ω-1-hydroxyhexylbenzoic acid in 4 h with a turnover number of 4.1 min(-1), whereas 100% conversion of 5 mM HBA was obtained in 1 h with crude cell extracts and a cofactor regeneration system, giving a turnover number of 10.5 min(-1).

Screening for cytochrome P450 expression in Pichia pastoris whole cells by P450-carbon monoxide complex determination.

Cytochrome P450 (CYP) enzymes are useful biocatalysts for the pharmaceutical and biotechnological industries. A high-throughput method for quantification of CYP expression in yeast is needed in order to fully exploit the yeast expression system. Carbon monoxide (CO) difference spectra of whole cells have been successfully used for the quantification of heterologous CYP expressed in Escherichia coli in the 96-well format; however, very few researchers have shown whole-cell CO difference spectra with yeast cells using 1-cm path length. Spectral interference from the native hemoproteins often obscures the P450 peak, challenging functional CYP quantification in whole yeast cells. For the first time, we describe the high-throughput determination of CO difference spectra using whole cells in the 96-well format for the quantification of CYP genes expressed in Pichia pastoris. Very little interference from the hemoproteins of P. pastoris enabled CYP quantification even at relatively low expression levels. P. pastoris strains carrying a single copy or three copies of both hCPR and CYP2D6 integrated into the chromosomal DNA were used to establish the method in 96-well format, allowing to detect quantities of CYP2D6 as low as 6 nmol gCDW(-1 ) and 12 pmol per well. Finally, the established method was successfully demonstrated and used to screen P. pastoris clones expressing Candida CYP52A13.