Synthesis of optically pure <Emphasis Type="Italic">S</Emphasis>-sulfoxide by <Emphasis Type="Italic">Escherichia coli</Emphasis> transformant cells coexpressing the P450 monooxygenase and glucose dehydrogenase genes

A cytochrome P450 monooxygenase (P450SMO) from Rhodococcus sp. can catalyze asymmetric oxygenation of sulfides to S-sulfoxides. However, P450SMO-catalyzed biotransformations require a constant supply of NAD(P)H, the expense of which constitutes a great hindrance for this enzyme application. In this study, we investigated the asymmetric oxygenation of sulfide to S-sulfoxide using E. coli cells, which co-express both the P450SMO gene from Rhodococcus sp. and the glucose dehydrogenase (GDH) gene from Bacillus subtilis, as a catalyst. The results showed that the catalytic performance of co-expression systems was markedly improved compared to the system lacking GDH. When using recombinant E. coli BL21 (pET28a-P450-GDH) whole cell as a biocatalyst, NADPH was efficiently regenerated when glucose was supplemented in the reaction system. A total conversion of 100% was achieved within 12 h with 2 mM p-chlorothioanisole substrate, affording 317.3 mg/L S-sulfoxide obtained. When the initial sulfide concentration was increased to 5 mM, the substrate conversion was also increased nearly fivefold: S-sulfoxide amounted to 2.5 mM (396.6 mg/L) and the ee value of sulfoxide product exceeded 98%. In this system, the effects of glucose concentration and substrate concentration were further investigated for efficient biotransformation. This system is highly advantageous for the synthesis of optically pure S-sulfoxide.     

Engineering of recombinant E. coli cells co‐expressing P450pyrTM monooxygenase and glucose dehydrogenase for highly regio‐ and stereoselective hydroxylation of alicycles with cofactor recycling

E. coli (P450pyrTM‐GDH) with dual plasmids, pETDuet containing P450pyr triple mutant I83H/M305Q/A77S (P450pyrTM) and ferredoxin reductase (FdR) genes and pRSFDuet containing glucose dehydrogenase (GDH) and ferredoxin (Fdx) genes, was engineered to show a high activity (12.7 U g^?1 cdw) for the biohydroxylation of N‐benzylpyrrolidine 1 and a GDH activity of 106 U g^?1 protein. The E. coli cells were used as efficient biocatalysts for highly regio‐ and stereoselective hydroxylation of alicyclic substrates at non‐activated carbon atom with enhanced productivity via intracellular recycling of NAD(P)H. Hydroxylation of N‐benzylpyrrolidine 1 with resting cells in the presence of glucose showed excellent regio‐ and stereoselectivity, giving (S)‐N‐benzyl‐3‐hydroxypyrrolidine 2 in 98% ee as the sole product in 9.8 mM. The productivity is much higher than that of the same biohydroxylation using E. coli (P450pyrTM)b without expressing GDH. E. coli (P450pyrTM‐GDH) was found to be highly regio‐ and stereoselective for the hydroxylation of N‐benzylpyrrolidin‐2‐one 3 , improving the regioselectivity from 90% of the wild‐type P450pyr to 100% and giving (S)‐N‐benzyl‐4‐hydroxylpyrrolidin‐2‐one 4 in 99% ee as the sole product. A high activity of 15.5 U g^?1 cdw was achieved and (S)‐ 4 was obtained in 19.4 mM. E. coli (P450pyrTM‐GDH) was also found to be highly regio‐ and stereoselective for the hydroxylation of N‐benzylpiperidin‐2‐one 5 , increasing the ee of the product (S)‐N‐benzyl‐4‐hydroxy‐piperidin‐2‐one 6 to 94% from 33% of the wild‐type P450pyr. A high activity of 15.8 U g^?1 cdw was obtained and (S)‐ 6 was produced in 3.3 mM as the sole product. E. coli (P450pyrTM‐GDH) represents the most productive system known thus far for P450‐catalyzed hydroxylations with cofactor recycling, and the hydroxylations with E. coli (P450pyrTM‐GDH) provide with simple and useful syntheses of (S)‐ 2 , (S)‐ 4 , and (S)‐ 6 that are valuable pharmaceutical intermediates and difficult to prepare. Biotechnol. Bioeng. 2013; 110: 363–373. © 2012 Wiley Periodicals, Inc.     

Cytochrome P450‐catalyzed O‐dealkylation coupled with photochemical NADPH regeneration

Cytochrome P450 monooxygenases are multifunctional enzymes with potential applications in chemoenzymatic synthesis of complex chemicals as well as in studies of metabolism and xenobiotics. Widespread application of cytochrome P450s, however, is encumbered by the critical need for redox equivalents in their catalytic function. To overcome this limitation, we studied visible light‐driven regeneration of NADPH for P450‐catalyzed O‐dealkylation reaction; we used eosin Y as a photosensitizing dye, triethanolamine as an electron donor, and [Cp*Rh(bpy)H₂O] as an electron mediator. We analyzed catalytic activity of cell‐free synthesized P450 BM3 monooxygenase variant (Y51F/F87A, BM3m2) in the presence of key components for NADPH photoregeneration. The P450‐catalyzed O‐dealkylation reaction sustainably maintained its turnover with the continuous supply of photoregenerated NADPH. Visible light‐driven, non‐enzymatic NADPH regeneration provides a new route for efficient, sustainable utilization of P450 monooxygenases. Biotechnol. Bioeng. 2013; 110: 383–390. © 2012 Wiley Periodicals, Inc.     

Design of a cytochrome P450BM3 reaction system linked by two‐step cofactor regeneration catalyzed by a soluble transhydrogenase and glycerol dehydrogenase

A cytochrome P450BM3‐catalyzed reaction system linked by a two‐step cofactor regeneration was investigated in a cell‐free system. The two‐step cofactor regeneration of redox cofactors, NADH and NADPH, was constructed by NAD⁺‐dependent bacterial glycerol dehydrogenase (GLD) and bacterial soluble transhydrogenase (STH) both from Escherichia coli. In the present system, the reduced cofactor (NADH) was regenerated by GLD from the oxidized cofactor (NAD⁺) using glycerol as a sacrificial cosubstrate. The reducing equivalents were subsequently transferred to NADP⁺ by STH as a cycling catalyst. The resultant regenerated NADPH was used for the substrate oxidation catalyzed by cytochrome P450BM3. The initial rate of the P450BM3‐catalyzed reaction linked by the two‐step cofactor regeneration showed a slight increase (approximately twice) when increasing the GLD units 10‐fold under initial reaction conditions. In contrast, a 10‐fold increase in STH units resulted in about a 9‐fold increase in the initial reaction rate, implying that transhydrogenation catalyzed by STH was the rate‐determining step. In the system lacking the two‐step cofactor regeneration, 34% conversion of 50 μM of a model substrate (p‐nitrophenoxydecanoic acid) was attained using 50 μM NADPH. In contrast, with the two‐step cofactor regeneration, the same amount of substrate was completely converted using 5 μM of oxidized cofactors (NAD⁺ and NADP⁺) within 1 h. Furthermore, a 10‐fold dilution of the oxidized cofactors still led to approximately 20% conversion in 1 h. These results indicate the potential of the combination of GLD and STH for use in redox cofactor recycling with catalytic quantities of NAD⁺ and NADP⁺. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

High-level expression of recombinant glucose dehydrogenase and its application in NADPH regeneration

Two glucose dehydrogenase (E.C. 1.1.1.47) genes, gdh223 and gdh151, were cloned from Bacillus megaterium AS1.223 and AS1.151, and were inserted into pQE30 to construct the expression vectors, pQE30-gdh223 and pQE30-gdh151, respectively. The transformant Escherichia coli M15 with pQE30-gdh223 gave a much higher glucose dehydrogenase activity than that with the plasmid pQE30-gdh151. Thus it was used to optimize the expression of glucose dehydrogenase. An proximately tenfold increase in GDH activity was achieved by the optimization of culture and induction conditions, and the highest productivity of glucose dehydrogenase (58.7 U/ml) was attained. The recombinant glucose dehydrogenase produced by E. coli M15 (pQE30-gdh223) was then used to regenerate NADPH. NADPH was efficiently regenerated in vivo and in vitro when 0.1 M glucose was supplemented concomitantly in the reaction system. Finally, this coenzyme-regenerating system was coupled with a NADPH-dependent bioreduction for efficient synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate from ethyl 4-chloro-3-oxobutanoate.     

Characterization of a flavin-containing monooxygenase from <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> and its application to production of indigo and indirubin

Objective

To examine the role of a gene encoding flavin-containing monooxygenase (cFMO) from Corynebacterium glutamicum ATCC13032 when cloned and expressed in Escherichia coli for the production of indigo pigments.

Results

The blue pigments produced by recombinant E. coli were identified as indigo and indirubin. The cFMO was purified as a fused form with maltose-binding protein (MBP). The enzyme was optimal at 25 °C and pH 8. From absorption spectrum analysis, the cFMO was classified as a flavoprotein. FMO activity was strongly inhibited by 1 mM Cu²⁺ and recovered by adding 1–10 mM EDTA. The enzyme catalyzed the oxidation of TMA, thiourea, and cysteamine, but not glutathione or cysteine. MBP-cFMO had an indole oxygenase activity through oxygenation of indole to indoxyl. The recombinant E. coli produced 685 mg indigo l^?1 and 103 mg indirubin l^?1 from 2.5 g l-tryptophan l^?1.

Conclusion

The results suggest the cFMO can be used for the microbial production of both indigo and indirubin.

     

Biotransformation of a crizotinib intermediate using a mutant alcohol dehydrogenase of Lactobacillus kefir coupled with glucose dehydrogenase

Abstract

(S)-1-(2, 6-dichloro-3-fluorophenyl) ethanol, the key chiral intermediate of crizotinib, was prepared from 1-(2, 6-dichloro-3-fluorophenyl) ethanone using the alcohol dehydrogenases from Lactobacillus kefir (ADH-LK) with a tetrad mutant (ADH-LKM, F147L/Y190P/V196L/A202W), coupled with glucose dehydrogenase (GDH). In the present study, ADH-LKM and GDH were successfully heterologous expressed in recombinant Escherichia coli. During the regeneration of NADPH with GDH, 150?g/L substrate was totally transformed into target chiral alcohol with an enantiomeric excess value of 99.9% after 12?h at 30?°C (pH 7.0). Our study demonstrates the potential for industrial green production of the key chiral intermediate of crizotinib.     

Multi‐enzymatic one‐pot reduction of dehydrocholic acid to 12‐keto‐ursodeoxycholic acid with whole‐cell biocatalysts

Ursodeoxycholic acid (UDCA) is a bile acid of industrial interest as it is used as an agent for the treatment of primary sclerosing cholangitis and the medicamentous, non‐surgical dissolution of gallstones. Currently, it is prepared industrially from cholic acid following a seven‐step chemical procedure with an overall yield of <30%. In this study, we investigated the key enzymatic steps in the chemo‐enzymatic preparation of UDCA—the two‐step reduction of dehydrocholic acid (DHCA) to 12‐keto‐ursodeoxycholic acid using a mutant of 7β‐hydroxysteroid dehydrogenase (7β‐HSDH) from Collinsella aerofaciens and 3α‐hydroxysteroid dehydrogenase (3α‐HSDH) from Comamonas testosteroni. Three different one‐pot reaction approaches were investigated using whole‐cell biocatalysts in simple batch processes. We applied one‐biocatalyst systems, where 3α‐HSDH, 7β‐HSDH, and either a mutant of formate dehydrogenase (FDH) from Mycobacterium vaccae N10 or a glucose dehydrogenase (GDH) from Bacillus subtilis were expressed in a Escherichia coli BL21(DE3) based host strain. We also investigated two‐biocatalyst systems, where 3α‐HSDH and 7β‐HSDH were expressed separately together with FDH enzymes for cofactor regeneration in two distinct E. coli hosts that were simultaneously applied in the one‐pot reaction. The best result was achieved by the one‐biocatalyst system with GDH for cofactor regeneration, which was able to completely convert 100 mM DHCA to >99.5 mM 12‐keto‐UDCA within 4.5 h in a simple batch process on a liter scale. Biotechnol. Bioeng. 2013; 110: 68–77. © 2012 Wiley Periodicals, Inc.     

Construction of a two-strain system for asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to (S)-4-chloro-3-hydroxybutanoate ethyl ester

Escherichia coli M15 (pQE30-car0210) was constructed to express carbonyl reductase (CAR) by cloning the car gene from Candida magnoliae and inserting it into pQE30. By cultivating E. coli M15 (pQE30-car0210) and M15 (pQE30-gdh0310), 8.2-fold and 12.3-fold enhancements in specific enzymatic activity over the corresponding original strain were achieved, respectively. After separate cultivations, these two strains were then mixed together at appropriate ratio to construct a novel two-strain system, in which M15 (pQE30-car0210) expressed CAR for ethyl 4-chloro-3-oxobutanoate (COBE) bioreduction and M15 (pQE30-gdh0310) expressed glucose dehydrogenase (GDH) for nicotinamide adenine dinucleotide phosphate (NADPH) regeneration. In this complex system, the effects of substrate concentration, the biomass ratio between two strains as well as reaction temperature were investigated for efficient bioreduction. The results showed that the bioreduction reaction could be completed effectively without any addition of GDH or NADPH/NADP⁺. An optical purity of 99% (enantiometric efficiency) was obtained, and the yield of (S)-4-chloro-3-hydroxybutanoate ethyl ester reached 96.6% when initial concentration of COBE was 36.9 mM. The coupling reactions between two different strains were further explored by determining the profile of NADPH in the reaction broth.     

Bioreduction with Efficient Recycling of NADPH by Coupled Permeabilized Microorganisms

The glucose dehydrogenase (GDH) from Bacillus subtilis BGSC 1A1 was cloned and functionally expressed in Escherichia coli BL21(pGDH1) and XL-1 Blue(pGDH1). Controlled permeabilization of recombinant E. coli BL21 and XL-1 Blue with EDTA-toluene under optimized conditions resulted in permeabilized cells with specific activities of 61 and 14 U/g (dry weight) of cells, respectively, for the conversion of NADP⁺ to NADPH upon oxidation of glucose. The permeabilized recombinant strains were more active than permeabilized B. subtilis BGSC 1A1, did not exhibit NADPH/NADH oxidase activity, and were useful for regeneration of both NADH and NADPH. Coupling of permeabilized cells of Bacillus pumilus Phe-C3 containing an NADPH-dependent ketoreductase and an E. coli recombinant expressing GDH as a novel biocatalytic system allowed enantioselective reduction of ethyl 3-keto-4,4,4-trifluorobutyrate with efficient recycling of NADPH; a total turnover number (TTN) of 4,200 mol/mol was obtained by using E. coli BL21(pGDH1) as the cofactor-regenerating microorganism with initial addition of 0.005 mM NADP⁺. The high TTN obtained is in the practical range for producing fine chemicals. Long-term stability of the permeabilized cell couple and a higher product concentration were demonstrated by 68 h of bioreduction of ethyl 3-keto-4,4,4-trifluorobutyrate with addition of 0.005 mM NADP⁺ three times; 50.5 mM (R)-ethyl 3-hydroxy-4,4,4-trifluorobutyrate was obtained with 95% enantiomeric excess, 84% conversion, and an overall TTN of 3,400 mol/mol. Our method results in practical synthesis of (R)-ethyl 3-hydroxy-4,4,4-trifluorobutyrate, and the principle described here is generally applicable to other microbial reductions with cofactor recycling.     

Improvement of P450(BM-3) whole-cell biocatalysis by integrating heterologous cofactor regeneration combining glucose facilitator and dehydrogenase in E. coli

Escherichia coli BL21, expressing a quintuple mutant of P450_BM-3, oxyfunctionalizes α-pinene in an NADPH-dependent reaction to α-pinene oxide, verbenol, and myrtenol. We optimized the whole-cell biocatalyst by integrating a recombinant intracellular NADPH regeneration system through co-expression of a glucose facilitator from Zymomonas mobilis for uptake of unphosphorylated glucose and a NADP⁺-dependent glucose dehydrogenase from Bacillus megaterium that oxidizes glucose to gluconolactone. The engineered strain showed a nine times higher initial α-pinene oxide formation rate corresponding to a sixfold higher yield of 20 mg g⁻¹ cell dry weight after 1.5 h. The initial total product formation rate was 1,000 μmol h⁻¹ μmol⁻¹ P450 leading to a total of 32 mg oxidized products per gram cell of dry weight after 1.5 h. The physiological functioning of the heterologous cofactor regeneration system was illustrated by a sevenfold increased α-pinene oxide yield in the presence of glucose compared to glucose-free conditions.     

Recent Developments in NAD(P)H Regeneration for Enzymatic Reductions in One- and Two-Phase Systems

This review discusses recent achievements in the field of cofactor regeneration for the nicotinamide cofactors NADH and NADPH. The examples discussed include alcohol dehydrogenases, formate dehydrogenase, glucose dehydrogenase and a hydrogenase. For the reaction either one-phase systems or two-phase systems in combination with an organic solvent are discussed. For the enantioselective reduction of 2-octanone to (R)-2-octanol it could be shown that enzyme coupled NADPH regeneration with glucose dehydrogenase and glucose results in shorter reaction times and higher yields when compared to the substrate coupled regeneration with 2-propanol.

ADH: alcohol dehydrogenase; LDH: Lactose dehydrogenase; GDH: Glucose dehydrogenase; FDH: Formate dehydrogenase; LB-ADH: alcohol dehydrogenase from Lactobacillus brevis; HL-ADH: alcohol dehydrogenase from horse liver; TB-ADH: alcohol dehydrogenase from Thermoanaerobicum brockii; PS-GDH: Glucose dehydrogenase from Pseudomonas species; [BMIM][PF₆]: Butyl-methyl-imidazoliumhexafluorophosphate     

Simultaneous synthesis of 2‐phenylethanol and L‐homophenylalanine using aromatic transaminase with yeast Ehrlich pathway

2‐Phenylethanol is a widely used aroma compound with rose‐like fragrance and L ‐homophenylalanine is a building block of angiotensin‐converting enzyme (ACE) inhibitor. 2‐phenylethanol and L ‐homophenylalanine were synthesized simultaneously with high yield from 2‐oxo‐4‐phenylbutyric acid and L ‐phenylalanine, respectively. A recombinant Escherichia coli harboring a coupled reaction pathway comprising of aromatic transaminase, phenylpyruvate decarboxylase, carbonyl reductase, and glucose dehydrogenase (GDH) was constructed. In the coupled reaction pathway, the transaminase reaction was coupled with the Ehrlich pathway of yeast; (1) a phenylpyruvate decarboxylase (YDR380W) as the enzyme to generate the substrate for the carbonyl reductase from phenylpyruvate (i.e., byproduct of the transaminase reaction) and to shift the reaction equilibrium of the transaminase reaction, and (2) a carbonyl reductase (YGL157W) to produce the 2‐phenylethanol. Selecting the right carbonyl reductase showing the highest activity on phenylacetaldehyde with narrow substrate specificity was the key to success of the constructing the coupling reaction. In addition, NADPH regeneration was achieved by incorporating the GDH from Bacillus subtilis in the coupled reaction pathway. Based on 40 mM of L ‐phenylalanine used, about 96% final product conversion yield of 2‐phenylethanol was achieved using the recombinant E. coli. Biotechnol. Bioeng. 2009;102: 1323–1329. © 2008 Wiley Periodicals, Inc.     

Increased NADPH availability in <Emphasis Type="Italic">Escherichia coli</Emphasis>: improvement of the product per glucose ratio in reductive whole-cell biotransformation

A basic requirement for the efficiency of reductive whole-cell biotransformations is the reducing capacity of the host. Here, the pentose phosphate pathway (PPP) was applied for NADPH regeneration with glucose as the electron-donating co-substrate using Escherichia coli as host. Reduction of the prochiral β-keto ester methyl acetoacetate to the chiral hydroxy ester (R)-methyl 3-hydroxybutyrate (MHB) served as a model reaction, catalyzed by an R-specific alcohol dehydrogenase. The main focus was maximization of the reduced product per glucose yield of this pathway-coupled cofactor regeneration with resting cells. With a strain lacking the phosphoglucose isomerase, the yield of the reference strain was increased from 2.44 to 3.78 mol MHB/mol glucose. Even higher yields were obtained with strains lacking either phosphofructokinase I (4.79 mol MHB/mol glucose) or phosphofructokinase I and II (5.46 mol MHB/mol glucose). These results persuasively demonstrate the potential of NADPH generation by the PPP in whole-cell biotransformations.     

Oxidation of indole by cytochrome P450 enzymes

Indole is a product of tryptophan catabolism by gut bacteria and is absorbed into the body in substantial amounts. The compound is known to be oxidized to indoxyl and excreted in urine as indoxyl (3-hydroxyindole) sulfate. Further oxidation and dimerization of indoxyl leads to the formation of indigoid pigments. We report the definitive identification of the pigments indigo and indirubin as products of human cytochrome P450 (P450)-catalyzed metabolism of indole by visible, (1)H NMR, and mass spectrometry. P450 2A6 was most active in the formation of these two pigments, followed by P450s 2C19 and 2E1. Additional products of indole metabolism were characterized by HPLC/UV and mass spectrometry. Indoxyl (3-hydroxyindole) was observed as a transient product of P450 2A6-mediated metabolism; isatin, 6-hydroxyindole, and dioxindole accumulated at low levels. Oxindole was the predominant product formed by P450s 2A6, 2E1, and 2C19 and was not transformed further. A stable end product was assigned the structure 6H-oxazolo[3,2-a:4, 5-b']diindole by UV, (1)H NMR, and mass spectrometry, and we conclude that P450s can catalyze the oxidative coupling of indoles to form this dimeric conjugate. On the basis of these results, we propose that the P450/NADPH-P450 reductase system can catalyze oxidation of indole to a variety of products.     

Asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (<Emphasis Type="Italic">R</Emphasis>)-4-chloro-3-hydroxybutanoate with two co-existing,recombinant<Emphasis Type="Italic"> Escherichia coli</Emphasis> strains

Two recombinant strains, E. coli M15 (pQE30-alr0307) and E. coli M15 (pQE30-gdh0310), which were constructed to express, respectively, an NADPH-dependent aldehyde reductase gene and a glucose dehydrogenase gene, were mixed in an appropriate ratio and used for the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (R)-4-chloro-3-hydroxybutanoate. The former strain acted as catalyst and the latter functioned in NADPH regeneration. The biotransformation was completed effectively without any addition of glucose dehydrogenase or NADP⁺/NADPH. An optical purity of 99% (ee) was obtained and the product yield reached 90.5% from 28.5 mM substrate. Revisions requested 27 July 2004/23 September 2004; Revisions received 21 September 2004/29 November 2004     

Biocatalytic production of 5-hydroxy-2-adamantanone by P450cam coupled with NADH regeneration

5-Hydroxy-2-adamantanone is a versatile starting material for the synthesis of various adamantane derivatives. In this study, we investigated the biocatalytic production of 5-hydroxy-2-adamantanone using P450cam monooxygenase coupled with NADH regeneration. We constructed Escherichia coli cells that expressed P450cam and its redox partners, putidaredoxin and putidaredoxin reductase, and cells that co-expressed this P450cam multicomponent system with a glucose dehydrogenase (Gdh) to regenerate NADH using glucose. Two types of cells – wet cells that did not receive any treatment after washing with glycerol-containing buffer, and freeze-dried cells that were lyophilized after the washing – were prepared as whole-cell catalysts. When wet cells were reacted with 2-adamantanone, E. coli cells expressing only the P450cam multicomponent system efficiently produced 5-hydroxy-2-adamantanone in the presence of glucose. However, the co-expression of this P450cam system with Gdh did not further enhance the amount of this product. These results indicate that enough amounts of NADH for P450cam catalysis would be supplied by endogenous glucose metabolism in the E. coli host. In contrast, when freeze-dried cells were used, only the cells co-expressing the P450cam multicomponent system with Gdh efficiently catalyzed the oxidation in the presence of glucose. These results suggest that the exogenous Gdh compensated loss of NADH regeneration by the endogenous glucose metabolism that would be damaged by the lyophilization process. Furthermore, we attempted to produce 5-hydroxy-2-adamantanone with repeated additions of the substrate using wet cells expressing only the P450cam multicomponent system and freeze-dried cells co-expressing this P450cam system with Gdh. These whole-cell catalysts attained high-yield production; the wet cells and the freeze-dried cells produced 36 mM (5.9 g/l) and 21 mM (3.5 g/l) of 5-hydroxy-2-adamantanone, respectively.     

Formation of the indigo precursor indican in genetically engineered tobacco plants and cell cultures

The production of the blue dye indigo in plants has been assumed to be a possible route to the introduction of novel coloration into flowers or fibres. As the human cytochrome P450 mono-oxygenase 2A6 (CYP2A6) can form indigo in bacterial cultures, we investigated whether the expression of the corresponding cDNA in transgenic plants could lead to indigo formation. In a first attempt, we generated tobacco cell suspension cultures expressing the cDNA encoding human CYP2A6. Supplementation of the medium with indole led to the generation of indican (3-hydroxyindole-β- d -glucoside), a metabolite usually exclusively present in indigoferous dye plants. Hence, the recombinant CYP2A6 converted indole to the reactive metabolite 3-hydroxyindole (indoxyl), whereas rapid glucosylation is obviously conducted by ubiquitous plant glucosyl transferases (GTs). Interestingly, of nine additionally tested plant cell suspension cultures from various plant families, five were also capable of the formation of indican after indole supplementation, although this metabolism was more pronounced in transgenic tobacco cell suspension cultures expressing CYP2A6 cDNA. To evaluate whether indican or even indigo could be produced in whole plants, we generated transgenic tobacco plants harbouring active CYP2A6 together with an indole synthase (BX1) from maize. The genetically engineered tobacco plants accumulated indican, but did not develop a blue coloration. Although the de novo formation of indican in transgenic tobacco plants hampered indigo formation, it supports the contention that biosynthetic pathways can be efficiently mimicked by metabolic engineering.     

P450BM3 fused to phosphite dehydrogenase allows phosphite-driven selective oxidations

To facilitate the wider application of the NADPH-dependent P450_BM3, we fused the monooxygenase with a phosphite dehydrogenase (PTDH). The resulting monooxygenase-dehydrogenase fusion enzyme acts as a self-sufficient bifunctional catalyst, accepting phosphite as a cheap electron donor for the regeneration of NADPH.

The well-expressed fusion enzyme was purified and analyzed in comparison to the parent enzymes. Using lauric acid as substrate for P450_BM3, it was found that the fusion enzyme had similar substrate affinity and hydroxylation selectivity while it displayed a significantly higher activity than the non-fused monooxygenase. Phosphite-driven conversions of lauric acid at restricted NADPH concentrations confirmed multiple turnovers of the cofactor. Interestingly, both the fusion enzyme and the native P450_BM3 displayed enzyme concentration dependent activity and the fused enzyme reached optimal activity at a lower enzyme concentration. This suggests that the fusion enzyme has an improved tendency to form functional oligomers.

To explore the constructed phosphite-driven P450_BM3 as a biocatalyst, conversions of the drug compounds omeprazole and rosiglitazone were performed. PTDH-P450_BM3 driven by phosphite was found to be more efficient in terms of total turnover when compared with P450_BM3 driven by NADPH. The results suggest that PTDH-P450_BM3 is an attractive system for use in biocatalytic and drug metabolism studies.

     

Enhanced production of GDP-<Emphasis Type="SmallCaps">l</Emphasis>-fucose by overexpression of NADPH regenerator in recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Biosynthesis of guanosine 5′-diphosphate-l-fucose (GDP-l-fucose) requires NADPH as a reducing cofactor. In this study, endogenous NADPH regenerating enzymes such as glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (Icd), and NADP⁺-dependent malate dehydrogenase (MaeB) were overexpressed to increase GDP-l-fucose production in recombinant Escherichia coli. The effects of overexpression of each NADPH regenerating enzyme on GDP-l-fucose production were investigated in a series of batch and fed-batch fermentations. Batch fermentations showed that overexpression of G6PDH was the most effective for GDP-l-fucose production. However, GDP-l-fucose production was not enhanced by overexpression of G6PDH in the glucose-limited fed-batch fermentation. Hence, a glucose feeding strategy was optimized to enhance GDP-l-fucose production. Fed-batch fermentation with a pH-stat feeding mode for sufficient supply of glucose significantly enhanced GDP-l-fucose production compared with glucose-limited fed-batch fermentation. A maximum GDP-l-fucose concentration of 235.2 ± 3.3 mg l⁻¹, corresponding to a 21% enhancement in the GDP-l-fucose production compared with the control strain overexpressing GDP-l-fucose biosynthetic enzymes only, was achieved in the pH-stat fed-batch fermentation of the recombinant E. coli overexpressing G6PDH. It was concluded that sufficient glucose supply and efficient NADPH regeneration are crucial for NADPH-dependent GDP-l-fucose production in recombinant E. coli.