Homology modeling was used to identify two particular residues, Glu175 and Ala176, in Pseudomonas stutzeri phosphite dehydrogenase (PTDH) as the principal determinants of nicotinamide cofactor (NAD(+) and NADP(+)) specificity. Replacement of these two residues by site-directed mutagenesis with Ala175 and Arg176 both separately and in combination resulted in PTDH mutants with relaxed cofactor specificity. All three mutants exhibited significantly better catalytic efficiency for both cofactors, with the best kinetic parameters displayed by the double mutant, which had a 3.6-fold higher catalytic efficiency for NAD(+) and a 1000-fold higher efficiency for NADP(+). The cofactor specificity was changed from 100-fold in favor of NAD(+) for the wild-type enzyme to 3-fold in favor of NADP(+) for the double mutant. Isoelectric focusing of the proteins in a nondenaturing gel showed that the replacement with more basic residues indeed changed the effective pI of the protein. HPLC analysis of the enzymatic products of the double mutant verified that the reaction proceeded to completion using either substrate and produced only the corresponding reduced cofactor and phosphate. Thermal inactivation studies showed that the double mutant was protected from thermal inactivation by both cofactors, while the wild-type enzyme was protected by only NAD(+). The combined results provide clear evidence that Glu175 and Ala176 are both critical for nicotinamide cofactor specificity. The rationally designed double mutant might be useful for the development of an efficient in vitro NAD(P)H regeneration system for reductive biocatalysis.

Fosfomycin is a clinically utilized, highly effective antibiotic, which is active against methicillin- and vancomycin-resistant pathogens. Here we report the cloning and characterization of a complete fosfomycin biosynthetic cluster from Streptomyces fradiae and heterologous production of fosfomycin in S. lividans. Sequence analysis coupled with gene deletion and disruption revealed that the minimal cluster consists of fom1-4, fomA-D. A LuxR-type activator that was apparently required for heterologous fosfomycin production was also discovered approximately 13 kb away from the cluster and was named fomR. The genes fomE and fomF, previously thought to be involved in fosfomycin biosynthesis, were shown not to be essential by gene disruption. This work provides new insights into fosfomycin biosynthesis and opens the door for fosfomycin overproduction and creation of new analogs via biomolecular pathway engineering.

ABSTRACT:The in situ regeneration of reduced nicotinamide cofactors (NAD(P)H) is necessary for practical synthesis of many important chemicals. Here, we report the engineering of a highly stable and active mutant phosphite dehydrogenase (12x-A176R PTDH) from Pseudomonas stutzeri and evaluation of its potential as an effective NADPH regeneration system in an enzyme membrane reactor. Two practically important enzymatic reactions including xylose reductase-catalyzed xylitol synthesis and alcohol dehydrogenase-catalyzed (R)-phenylethanol synthesis were used as model systems, and the mutant PTDH was directly compared to the commercially available NADP þ -specific Pseudomonas sp. 101 formate dehydrogenase (mut Pse-FDH) that is widely used for NADPH regeneration. In both model reactions, the two regeneration enzymes showed similar rates of enzyme activity loss; however, the mutant PTDH showed higher substrate conversion and higher total turnover numbers for NADP þ than mut Pse-FDH. The spacetime yields of the product with the mutant PTDH were also up to fourfold higher than those with mut Pse-FDH. In particular, a space-time yield of 230 g L À1 d À1 xylitol was obtained with the mutant PTDH using a charged nanofiltration membrane, representing the highest productivity compared to other existing biological processes for xylitol synthesis based on yeast D-xylose converting strains or similar in vitro enzyme membrane reactor systems.

Hydroxyethylphosphonate is a required intermediate in fosfomycin biosynthesis.

NAD(P)H-dependent oxidoreductases are valuable tools for synthesis of chiral compounds. The expense of the cofactors, however, requires in situ cofactor regeneration for preparative applications. We have attempted to develop an enzymatic system based on phosphite dehydrogenase (PTDH) from Pseudomonas stutzeri to regenerate the reduced nicotinamide cofactors NADH and NADPH. Here we report the use of directed evolution to address one of the main limitations with the wild-type PTDH enzyme, its low stability. After three rounds of random mutagenesis and high-throughput screening, 12 thermostabilizing amino acid substitutions were identified. These 12 mutations were combined by site-directed mutagenesis, resulting in a mutant whose T 50 is 20°C higher and half-life of thermal inactivation at 45°C is >7,000-fold greater than that of the parent PTDH. The engineered PTDH has a half-life at 50°C that is 2.4-fold greater than the Candida boidinii formate dehydrogenase, an enzyme widely used for NADH regeneration. In addition, its catalytic efficiency is slightly higher than that of the parent PTDH. Various mechanisms of thermostabilization were identified using molecular modeling. The improved stability and effectiveness of the final mutant were shown using the industrially important bioconversion of trimethylpyruvate to L-tert-leucine. The engineered PTDH will be useful in NAD(P)H regeneration for industrial biocatalysis.The potential of enzyme-catalyzed transformations in the pharmaceutical and fine-chemical industries has long been considered to have great promise (7,10,23). Despite the development of rapid and efficient methods for creating designer biocatalysts, inherent limitations in the chemistry that these enzymes perform still persist. The majority of biocatalysts used in industry are hydrolytic in action (ϳ65%) and perform rather simple chemistry (4). More complicated enzymatic reactions often require one or more costly cofactors, which, when added in stoichiometric quantities, make the process not economically feasible. Oxidoreductases, for example, catalyze a myriad of regio-, chemo-, and stereospecific reactions on a variety of functional groups, but often require either NADH or NADPH as a cofactor (8, 11).Various in situ regeneration methods have been developed to allow the use of catalytic quantities of NAD(P) ϩ and NAD(P)H (20). The most successful and widely used enzymatic NADH regeneration system is based on the Candida boidinii formate dehydrogenase (FDH) (1). We have attempted to develop an NADH regeneration process using the enzyme phosphite dehydrogenase (PTDH) from Pseudomonas stutzeri (2). This enzyme catalyzes the nearly irreversible oxidation of phosphite to phosphate with the concomitant reduction of NAD ϩ to NADH. The kinetic and practical advantages of using PTDH for NADH regeneration have been recently reported (22).In addition to NADH regeneration, the enzyme PTDH also has considerable potential in regenerating NADPH. The cost of NADPH is significantly higher than that of NADH, and no widel...