伴有辅酶原位再生的胞外酶耦合法制备（R）-苯基乙二醇

经5轮诱变筛选,从近平滑假丝酵母（Candida parapsilosis CICC1676）中分离得到产NADH依赖型羰基还原酶（Carbonyl reductase,CR）菌株CP-9。所产羰基还原酶（CRCp-9）经两步快速纯化获得纯化倍数为11．5倍,比活力为1．84 U/mg的酶液,其还原反应的最适pH值为6．5,最适温度为40℃。该酶转化β-羟基苯乙酮制备手性化合物（R）-苯基乙二醇,因此是（R）-专一性羰基还原酶。该酶与NADH普适性再生酶-甲酸脱氢酶（For-mate dehydrogenase,FDH）在胞外相耦联,构建伴有辅酶再生与反复利用的CR/FDH双酶催化制备立体醇体系,底物β-羟基苯乙酮转化率达95．4％,产物（R）-苯基乙二醇得率为93％,辅酶的总转化数（Total turn number, TTN）达267,产物e．e．值为98．6％,批次耦合反应生产能力达0．8 g/L/h,较单酶催化有较大提高,与细胞转化法相比也具有较好的生产能力。因此,伴有辅酶再生的胞外酶耦合催化具有潜在的制备手性醇化合物的工业应用价值。     

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     

Enzymatic synthesis of amoxicillin: avoiding limitations of the mechanistic approach for reaction kinetics

A recurrent doubt that occurs to the enzyme‐kinetics modeler is, When should I stop adding parameters to my mechanistic model in order to fit a non‐conventional behavior? This problem becomes more and more involving when the complexity of the reaction network increases. This work intends to show how the use of artificial neural networks may circumvent the need of including an overwhelming number of parameters in the rate equations obtained through the classical, mechanistic approach. We focus on the synthesis of amoxicillin by the reaction of p‐OH‐phenylglycine methyl ester and 6‐aminopenicillanic acid, catalyzed by penicillin G acylase immobilized on glyoxyl‐agarose, at 25°C and pH 6.5. The reaction was carried on a batch reactor. Three kinetic models of this system were compared: a mechanistic, a semi‐empiric, and a hybrid–neural network (NN). A semi‐empiric, simplified model with a reasonable number of parameters was initially built‐up. It was able to portray many typical process conditions. However, it either underestimated or overestimated the rate of synthesis of amoxicillin when substrates' concentrations were low. A more complex, full‐scale mechanistic model that could span all operational conditions was intractable for all practical purposes. Finally, a hybrid model, that coupled artificial neural networks (NN) to mass‐balance equations was established, that succeeded in representing all situations of interest. Particularly, the NN could predict with accuracy reaction rates for conditions where the semi‐empiric model failed, namely, at low substrate concentrations, a situation that would occur, for instance, at the end of a fed‐batch industrial process. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 622–631, 2002.     

Host cell and expression engineering for development of an <Emphasis Type="Italic">E. coli</Emphasis> ketoreductase catalyst: Enhancement of formate dehydrogenase activity for regeneration of NADH

Background  

Enzymatic NADH or NADPH-dependent reduction is a widely applied approach for the synthesis of optically active organic compounds. The overall biocatalytic conversion usually involves in situ regeneration of the expensive NAD(P)H. Oxidation of formate to carbon dioxide, catalyzed by formate dehydrogenase (EC 1.2.1.2; FDH), presents an almost ideal process solution for coenzyme regeneration that has been well established for NADH. Because isolated FDH is relatively unstable under a range of process conditions, whole cells often constitute the preferred form of the biocatalyst, combining the advantage of enzyme protection in the cellular environment with ease of enzyme production. However, the most prominent FDH used in biotransformations, the enzyme from the yeast Candida boidinii, is usually expressed in limiting amounts of activity in the prime host for whole cell biocatalysis, Escherichia coli. We therefore performed expression engineering with the aim of enhancing FDH activity in an E. coli ketoreductase catalyst. The benefit resulting from improved NADH regeneration capacity is demonstrated in two transformations of technological relevance: xylose conversion into xylitol, and synthesis of (S)-1-(2-chlorophenyl)ethanol from o-chloroacetophenone.     

Efficient synthesis of l-tert-leucine through reductive amination using leucine dehydrogenase and formate dehydrogenase coexpressed in recombinant E. coli

Enantiopure l-tert-leucine (l-Tle) was synthesized through reductive amination of trimethylpyruvate catalyzed by cell-free extracts of recombinant Escherichia coli coexpressing leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH). The leudh gene from Lysinibacillus sphaericus CGMCC 1.1677 encoding LeuDH was cloned and coexpressed with NAD⁺-dependent FDH from Candida boidinii for NADH regeneration. The batch reaction conditions for the synthesis of l-Tle were systematically optimized. Two substrate feeding modes (intermittent and continuous) were addressed to alleviate substrate inhibition and thus improve the space-time yield. The continuous feeding process was conveniently performed in water at an overall substrate concentration up to 1.5 M, with both conversion and ee of >99% and space-time yield of 786 g L⁻¹ d⁻¹, respectively. Furthermore, the preparation was successfully scaled up to a 1 L scale, demonstrating the developed procedure showed a great industrial potential for the production of enantiopure l-Tle.     

Biocatalytic process optimization based on mechanistic modeling of cholic acid oxidation with cofactor regeneration

Reduction and oxidation of steroids in the human gut are catalyzed by hydroxysteroid dehydrogenases of microorganisms. For the production of 12-ketochenodeoxycholic acid (12-Keto-CDCA) from cholic acid the biocatalytic application of the 12α-hydroxysteroid dehydrogenase of Clostridium group P, strain C 48-50 (HSDH) is an alternative to chemical synthesis. However, due to the intensive costs the necessary cofactor (NADP(+) ) has to be regenerated. The alcohol dehydrogenase of Thermoanaerobacter ethanolicus (ADH-TE) was applied to catalyze the reduction of acetone while regenerating NADP(+) . A mechanistic kinetic model was developed for the process development of cholic acid oxidation using HSDH and ADH-TE. The process model was derived by identifying the parameters for both enzymatic models separately using progress curve measurements of batch processes over a broad range of concentrations and considering the underlying ordered bi-bi mechanism. Both independently derived kinetic models were coupled via mass balances to predict the production of 12-Keto-CDCA with HSDH and integrated cofactor regeneration with ADH-TE and acetone as co-substrate. The prediction of the derived model was suitable to describe the dynamics of the preparative 12-Keto-CDCA batch production with different initial reactant and enzyme concentrations. These datasets were used again for parameter identification. This led to a combined model which excellently described the reaction dynamics of biocatalytic batch processes over broad concentration ranges. Based on the identified process model batch process optimization was successfully performed in silico to minimize enzyme costs. By using 0.1 mM NADP(+) the HSDH concentration can be reduced to 3-4 μM and the ADH concentration to 0.4-0.6 μM to reach the maximal possible conversion of 100 mM cholic acid within 48 h. In conclusion, the identified mechanistic model offers a powerful tool for a cost-efficient process design.     

The effect of electrochemical regeneration upon the enzymatic catalysis of a thermodynamically unfavorable reaction

The association between enzymatic and electrochemical reactions, enzymatic electrocatalysis, had proven to be a very powerful tooth in both analytical and synthetic fields. However, most of the combinations studied have involved enzymatic catalysis of irreversible or quasi-irreversible reaction. In the present work, we have investigated the possibility of applying enzymatic electrocatalysis to a case where the electrochemical reaction drives a thermodynamically unfavorable reversible reaction. Such thermodynamically unfavorable reactions include most of the oxidations catalyzed by dehydrogenases. Yeast alcohol dehydrogenase (E.C. 1.1.1.1) was chosen as a model enzyme because the oxidation of ethanol is thermodynamically very unfavorable and because its kinetics are well known. The electrochemical reaction was the oxidation of NADH which is particularly attractive as a method of cofactor regeneration. Both the electrochemical and enzymatic reactions occur in the same batch reactor in such a way that electrical energy is the only external driving force. Two cases were experimentally and theoretically developed with the enzyme either in solution or immobilized onto the electrode's surface. In both cases, the electrochemical reaction could drive the enzymatic reaction by NADH consumption in solution or directly in the enzyme's microenvironment. However even for a high efficiency of NADH consumption, the rate of enzymatic catalysis was limited by product (acetaldedehyde) inhibition. Extending this observation to the subject of organic synthesis catalyzed by dehydrogenases, we concluded that thermodynamically unfavorable reaction and can only be used in a process if efficient NAD regeneration and product elimination are simultaneously carried out within the reactor.     

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.     

Physiological and quantitative proteomic analyses unraveling potassium deficiency stress response in alligator weed (<Emphasis Type="Italic">Alternanthera philoxeroides</Emphasis> L.) root

Key message

KEG is involved in mediating the proteasome-dependent degradation of FDH, a stress-responsive enzyme. The UPS may function to suppress FDH mediated stress responses under favorable growth conditions.

Abstract

Formate dehydrogenase (FDH) has been studied in bacteria and yeasts for the purpose of industrial application of NADH co-factor regeneration. In plants, FDH is regarded as a universal stress protein involved in responses to various abiotic and biotic stresses. Here we show that FDH abundance is regulated by the ubiquitin proteasome system (UPS). FDH is ubiquitinated in planta and degraded by the 26S proteasome. Interaction assays identified FDH as a potential substrate for the RING-type ubiquitin ligase Keep on Going (KEG). KEG is capable of attaching ubiquitin to FDH in in vitro assays and the turnover of FDH was increased when co-expressed with a functional KEG in planta, suggesting that KEG contributes to FDH degradation. Consistent with a role in regulating FDH abundance, transgenic plants overexpressing KEG were more sensitive to the inhibitory effects of formate. In addition, FDH is a phosphoprotein and dephosphorylation was found to increase the stability of FDH in degradation assays. Based on results from this and previous studies, we propose a model where KEG mediates the ubiquitination and subsequent degradation of phosphorylated FDH and, in response to unfavourable growth conditions, reduction in FDH phosphorylation levels may prohibit turnover allowing the stabilized FDH to facilitate stress responses.

     

Metabolomics for biotransformations: Intracellular redox cofactor analysis and enzyme kinetics offer insight into whole cell processes

For redox reactions catalyzed by microbial cells the analysis of involved cofactors is of special interest since the availability of cofactors such as NADH or NADPH is often limiting and crucial for the biotransformation efficiency. The measurement of these cofactors has usually been carried out using spectrophotometric cycling assays. Today LC‐MS/MS methods have become a valuable tool for the identification and quantification of intracellular metabolites. This technology has been adapted to measure all four nicotinamide cofactors (NAD, NADP, NADH, and NADPH) during a whole cell biotransformation process catalyzed by recombinant Escherichia coli cells. The cells overexpressing an alcohol dehydrogenase from Lactobacillus brevis were used for the reduction of methyl acetoacetate (MAA) with substrate‐coupled cofactor regeneration by oxidation of 2‐propanol. To test the reliability of the measurement the data were evaluated using a process model. This model was derived using the measured concentrations of reactants and cofactors for initiation as well as the kinetic constants from in vitro measurements of the isolated enzyme. This model proves to be highly effective in the process development for a whole cell redox biotransformation in predicting both the right concentrations of cofactors and reactants in a batch and in a CSTR process as well as the right in vivo expression level of the enzyme. Moreover, a sensitivity analysis identifies the cofactor regeneration reaction as the limiting step in case for the reduction of MAA to the corresponding product (R)‐methyl 3‐hydroxybutyrate. Using the combination of in vitro enzyme kinetic measurements, measurements of cofactors and reactants and an adequate model initiated by intracellular concentrations of all involved reactants and cofactors the whole cell biotransformation process can be understood quantitatively. Biotechnol. Bioeng. 2009; 104: 251–260 © 2009 Wiley Periodicals, Inc.     

Catalytic mechanism and application of formate dehydrogenase

NAD⁺-dependent formate dehydrogenase (FDH) is an abundant enzyme that plays an important role in energysupply of methylotrophic microorganisms and in response to stress in plants. FDH belongs to the superfamily of D-specific 2-hydroxy acid dehydrogenases. FDH is widely accepted as a model enzyme to study the mechanism of hydride ion transfer in the active center of dehydrogenases because the reaction catalyzed by the enzyme is devoid of proton transfer steps and implies a substrate with relatively simple structure. FDH is also widely used in enzymatic syntheses of optically active compounds as a versatile biocatalyst for NAD(P)H regeneration consumed in the main reaction. This review covers the late developments in cloning genes of FDH from various sources, studies of its catalytic mechanism and physiological role, and its application for new chiral syntheses.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1537–1554.Original Russian Text Copyright © 2004 by Tishkov, Popov.     

Effect of different levels of NADH availability on metabolite distribution in <Emphasis Type="Italic">Escherichia coli</Emphasis> fermentation in minimal and complex media

A range of intracellular NADH availability was achieved by combining external and genetic strategies. The effect of these manipulations on the distribution of metabolites in Escherichia coli was assessed in minimal and complex medium under anoxic conditions. Our in vivo system to increase intracellular NADH availability expressed a heterologous NAD⁺-dependent formate dehydrogenase (FDH) from Candida boidinii in E. coli. The heterologous FDH pathway converted 1 mol formate into 1 mol NADH and carbon dioxide, in contrast to the native FDH where cofactor involvement was not present. Previously, we found that this NADH regeneration system doubled the maximum yield of NADH from 2 mol to 4 mol NADH/mol glucose consumed. In the current study, we found that yields of greater than 4 mol NADH were achieved when carbon sources more reduced than glucose were combined with our in vivo NADH regeneration system. This paper demonstrates experimentally that different levels of NADH availability can be achieved by combining the strategies of feeding the cells with carbon sources which have different oxidation states and regenerating NADH through the heterologous FDH pathway. The general trend of the data is substantially similar for minimal and complex media. The NADH availability obtained positively correlates with the proportion of reduced by-products in the final culture. The maximum theoretical yield for ethanol is obtained from glucose and sorbitol in strains overexpressing the heterologous FDH pathway.     

Immobilization of formate dehydrogenase from Candida boidinii through cross-linked enzyme aggregates

We employed a cross-linked enzyme aggregate (CLEA) method to immobilize formate dehydrogenase (FDH) from Candida boidinii. The optimal conditions for the preparation of CLEAs were determined by examining effects of various parameters: the nature and amount of cross-linking reagent, additive concentration, cross-linking time, and pH during CLEA preparation. The recovered activities of CLEAs were significantly dependent on the concentration of glutaraldehyde; however, the recovered activity was not severely influenced by the content of dextran polyaldehyde as a mild cross-linker. Bovine serum albumin (BSA) was also used as a proteic feeder and enhanced the activity recovery by 130%. The highest recovered activity of CLEA was 18% for formate oxidation reaction and 25% for CO₂ reduction reaction. The residual activity of CLEA prepared with dextran polyaldehyde (Dex-CLEA) was over 95% after 10 cycles of reuse. The thermal stability of Dex-CLEA was increased by a factor of 3.6 more than that of the free enzyme. CLEAs of FDH could be utilized efficiently for both NADH regeneration and CO₂ reduction.     

Dynamic mechanistic modeling of the multienzymatic one‐pot reduction of dehydrocholic acid to 12‐keto ursodeoxycholic acid with competing substrates and cofactors

Ursodeoxycholic acid (UDCA) is a bile acid which is used as pharmaceutical for the treatment of several diseases, such as cholesterol gallstones, primary sclerosing cholangitis or primary biliary cirrhosis. A potential chemoenzymatic synthesis route of UDCA comprises the two‐step reduction of dehydrocholic acid to 12‐keto‐ursodeoxycholic acid (12‐keto‐UDCA), which can be conducted in a multienzymatic one‐pot process using 3α‐hydroxysteroid dehydrogenase (3α‐HSDH), 7β‐hydroxysteroid dehydrogenase (7β‐HSDH), and glucose dehydrogenase (GDH) with glucose as cosubstrate for the regeneration of cofactor. Here, we present a dynamic mechanistic model of this one‐pot reduction which involves three enzymes, four different bile acids, and two different cofactors, each with different oxidation states. In addition, every enzyme faces two competing substrates, whereas each bile acid and cofactor is formed or converted by two different enzymes. First, the kinetic mechanisms of both HSDH were identified to follow an ordered bi–bi mechanism with EBQ‐type uncompetitive substrate inhibition. Rate equations were then derived for this mechanism and for mechanisms describing competing substrates. After the estimation of the model parameters of each enzyme independently by progress curve analyses, the full process model of a simple batch‐process was established by coupling rate equations and mass balances. Validation experiments of the one‐pot multienzymatic batch process revealed high prediction accuracy of the process model and a model analysis offered important insight to the identification of optimum reaction conditions. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:375–386, 2015     

Kinetic Modeling of Amino Acid Oxidation Catalyzed by a New D‐Amino Acid Oxidase from Arthrobacter protophormiae

Amino acid oxidases, which enantiospecifically catalyze the oxidative deamination of either D‐ or L‐amino acids, belong to the class of oxidoreductases functioning with a tightly bound cofactor. This cofactor favors industrial applications of D‐amino acid oxidases (D‐AAO). Hence, the enzyme is very important for the industrial application in the purification and determination of certain amino acids. In developing the enzyme‐catalyzed reaction for large‐scale production, modeling of the reaction kinetics plays an important role. Therefore, the subject of this study was the kinetics of the oxidative deamination, a very complex reaction system, which is catalyzed by D‐AAO from Arthrobacter protophormiae using its natural substrate D‐methionine and the aromatic amino acid 3,4‐dihydroxyphenyl‐D‐alanine (D‐DOPA). The kinetic parameters determined by the measurement of the initial rate and nonlinear regression were verified in batch reactor experiments by comparing calculated and experimental concentration‐time curves. It was found that the enzyme is highly specific towards D‐methionine (K_m = 0.24 mM) and not as specific to D‐DOPA as a substrate (K_m = 9.33 mM). The enzyme activity towards D‐methionine ( = 3.01 U/mL) was approx. seven times higher than towards D‐DOPA ( = 20.01 U/mL). The enzyme exhibited no activity towards L‐methionine and L‐DOPA. Batch and repetitive batch experiments were performed with both substrates in the presence and in the absence of catalase for hydrogen peroxide decomposition. Their comparison made it possible to conclude that hydrogen peroxide has no negative influence on the enzyme activity.     

The effect of NAPRTase overexpression on the total levels of NAD,the NADH/NAD+ ratio,and the distribution of metabolites in Escherichia coli

Escherichia coli (E. coli) maintains its total NADH/NAD+ intracellular pool by synthesizing NAD through the de novo pathway and the pyridine nucleotide salvage pathway. The salvage pathway recycles intracellular NAD breakdown products and preformed pyridine compounds from the environment, such as nicotinic acid (NA). The enzyme nicotinic acid phosphoribosyltransferase (NAPRTase; EC 2.4.2.11), encoded by the pncB gene, catalyzes the formation of nicotinate mononucleotide (NAMN), a direct precursor of NAD, from NA. This reaction is believed to be the rate-limiting step in the NAD salvage pathway. The current study investigates the effect of overexpressing the pncB gene from Salmonella typhimurium on the total levels of NAD, the NADH/NAD+ ratio, and the production of different metabolites in E. coli under anaerobic chemostat conditions and anaerobic tube experiments. In addition, this paper studies the effect of combining the overexpression of the pncB gene with an NADH regeneration strategy that increases intracellular NADH availability, as we have previously shown. (The effect of increasing NADH availability on the redistribution of metabolic fluxes in Escherichia coli chemostat cultures, Metabolic Eng. 4, 230-237; Metabolic engineering of Escherichia coli: Increase of NADH availability by overexpressing an NAD(+)-dependent formate dehydrogenase, Metabolic Eng. 4, 217-229.) Overexpression of the pncB gene in chemostat experiments increased the total NAD levels, decreased the NADH/NAD+ ratio, and did not significantly redistribute the metabolic fluxes. However, under anaerobic tube conditions, overexpression of the pncB gene led to a significant shift in the metabolic patterns as evidenced by a decrease in lactate production and an increase as high as two-fold in the ethanol-to-acetate (Et/Ac) ratio. These results suggest that under chemostat conditions the total level of NAD is not limiting and the metabolic rates are fixed by the system at steady state. On the other hand, under transient conditions (such as those in batch cultivation) the increase in the total level of NAD can increase the rate of NADH-dependent pathways (ethanol) and therefore change the final distribution of metabolites. The effect of combining overexpression of the pncB gene with the substitution of the native cofactor-independent formate dehydrogenase (FDH) with an NAD(+)-dependent FDH was also investigated under anaerobic tube conditions. This manipulation produced a metabolic pattern that combines a high Et/Ac ratio similar to that obtained with the new FDH with an intermediate lactate level similar to that obtained with the overexpression of the pncB gene. It was found that addition of the pncB gene to the FDH system does not increase further the production of reduced metabolites because the system for NADH regeneration already reached the maximum theoretical yield of approximately 4 mol NADH/mol of glucose.     

Effect of different levels of NADH availability on metabolic fluxes of Escherichia coli chemostat cultures in defined medium

Escherichia coli overexpressing a NAD(+)-dependent formate dehydrogenase (FDH) from Candida boidinii was grown in chemostat culture on various carbon sources at 0.05 h(-1) dilution rate, under anaerobic conditions using defined medium and compared to a control without the heterologous FDH pathway. Metabolic fluxes, NADH/NAD(+) ratios and NAD(H/(+)) levels were determined under a range of intracellular NADH availability. The effect of NADH manipulation on the distribution of metabolic fluxes in E. coli was assessed under steady-state conditions. The heterologous FDH pathway converts 1 mol of formate into 1 mol of NADH and carbon dioxide, in contrast with the native FDH where no cofactor involvement is present. Previously, we found that this NADH regeneration system doubled the maximum yield of NADH from 2 to 4 mol NADH/mol glucose consumed and reached 4.6 mol NADH/mol of substrate when sorbitol was used as a carbon source in a complex medium. In the current study, it was found that higher NADH yields and NADH/NAD(+) ratios were achieved with our in vivo NADH regeneration system compared to a control lacking the new FDH pathway in the three carbon sources (glucose, gluconate and sorbitol) examined suggesting a more reduced intracellular environment. The total NAD(H/(+)) amounts were very similar for all the combinations studied. It was also found that the ethanol to acetate ratio increased with increased NADH availability. This ratio increased from 1.05 for the control strain in glucose to 9.45 for the strain expressing the heterologous NAD(+)-dependent FDH in sorbitol.     

Biotransformation of R-2-hydroxy-4-phenylbutyric acid by D-lactate dehydrogenase and Candida boidinii cells containing formate dehydrogenase coimmobilized in a fibrous bed bioreactor

R-2-hydroxy-4-phenylbutyric acid (R-HPBA) is an important intermediate in the manufacture of angiotensin converting enzyme inhibitors. In this work, a recombinant D-lactate dehydrogenase (LDH) was used to transform 2-oxo-4-phenylbutyric acid (OPBA) to R-HPBA, with concomitant oxidation of beta-nicotinamide adenine dinucleotide (NADH) to NAD(+). The cofactor NADH was regenerated by formate dehydrogenase (FDH) present in whole cells of Candida boidinii, which were pre-treated with toluene to make them permeable. The whole cells used in the process were more stable and easier to prepare as compared with the isolated FDH from the cells. Kinetic study showed that the reaction rate was dependent on the concentration of cofactor, NAD(+), and that both R-HPBA and OPBA inhibited the reaction. A novel method for co-immobilization of whole cells and LDH enzyme on cotton cloth was developed using polyethyleneimine (PEI), which induced the formation of PEI-enzyme-cell aggregates and their adsorption onto cotton cloth, leading to multilayer co-immobilization of cells and enzyme with high loading (0.5 g cell and 8 mg LDH per gram of cotton cloth) and activity yield ( > 95%). A fibrous bed bioreactor with co-immobilized cells and enzyme on the cotton cloth was then evaluated for R-HPBA production in fed-batch and repeated batch modes, which gave relatively stable reactor productivity of 9 g/L . h and product yield of 0.95 mol/mol OPBA when the concentrations of OPBA and R-HPBA were less than 10 g/L.     

Hysteretic behavior of nitrate reductase. Evidence of an allosteric binding site for reduced pyridine nucleotides.

In the absence of NADH, at 25 degrees C, partially purified NADH:nitrate reductase undergoes an approximately 50% reduction of its initial activity during 2 h. With the increase of inactivation, the NADH and nitrite concentration time curves become typical "sigmoidal," i.e. the reaction velocity of the nitrate reductase catalyzed reaction goes through a maximum before equilibrium is reached. About 80% of the original activity of nitrate reductase is restored when the enzyme is incubated for 2 min with 200 microM NADH or NADPH. Also other NADH substrate analogues have similar effects in restoring the lost activity. After incubation with the reduced pyridine nucleotides, the sigmoidal appearance of the NADH concentration time curve disappears almost completely. Despite the fact that NADPH increases the activity of the enzyme, NADPH does not show any competition with the NADH-binding site of nitrate reductase and does not produce nitrite in the absence of NADH. It is therefore concluded that there must be an additional allosteric site which binds either NADH or NADPH, or other pyridine nucleotides with the effect of increasing the activity of the enzyme. A kinetic model is presented which simulates the observed experimental findings.     

Stabilization of NAD-dependent formate dehydrogenase from Candida boidinii by site-directed mutagenesis of cysteine residues.

The gene of the NAD-dependent formate dehydrogenase (FDH) from the yeast Candida boidinii was cloned by PCR using genomic DNA as a template. Expression of the gene in Escherichia coli yielded functional FDH with about 20% of the soluble cell protein. To confirm the hypothesis of a thiol-coupled inactivation process, both cysteine residues in the primary structure of the enzyme have been exchanged by site-directed mutagenesis using a homology model based on the 3D structure of FDH from Pseudomonas sp. 101 and from related dehydrogenases. Compared to the wt enzyme, most of the mutants were significantly more stable towards oxidative stress in the presence of Cu(II) ions, whereas the temperature optima and kinetic constants of the enzymatic reaction are not significantly altered by the mutations. Determination of the Tm values revealed that the stability at temperatures above 50 degrees C is optimal for the native and the recombinant wt enzyme (Tm 57 degrees C), whereas the Tm values of the mutant enzymes vary in the range 44-52 degrees C. Best results in initial tests concerning the application of the enzyme for regeneration of NADH in biotransformation of trimethyl pyruvate to Ltert leucine were obtained with two mutants, FDHC23S and FDHC23S/C262A, which are significantly more stable than the wt enzyme.