The Oxidoreductase DsbA1 negatively influences 2,4-diacetylphloroglucinol biosynthesis by interfering the function of Gcd in Pseudomonas fluorescens 2P24.

BACKGROUND: The polyketide antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG), produced by Pseudomonas fluorescens 2P24, is positively regulated by the GacS-GacA two-component system. RESULTS: Here we reported on the characterization of DsbA1 (disulfide oxidoreductase) as novel regulator of biocontrol activity in P. fluorescens. Our data showed that mutation of dsbA1 caused the accumulation of 2,4-DAPG in a GacA-independent manner. Further analysis indicated that DsbA1 interacts with membrane-bound glucose dehydrogenase Gcd, which positively regulates the production of 2,4-DAPG. Mutation of cysteine (C)-235, C275, and C578 of Gcd, significantly reduced the interaction with DsbA1, enhanced the activity of Gcd and increased 2,4-DAPG production. CONCLUSIONS: Our results suggest that DsbA1 regulates the 2,4-DAPG concentration via fine-tuning the function of Gcd in P. fluorescens 2P24.

Engineering CRISPR interference system to enhance the production of pyrroloquinoline quinone in Klebsiella pneumonia.

Pyrroloquinoline quinone (PQQ) is a cofactor of glucose dehydrogenase (GDH) and thus participates in glucose utilization. In Klebsiella pneumoniae, glucose utilization involves PQQ-dependent direct oxidation pathway (DOP) and phosphoenolpyruvate-dependent transport system (PTS). It is challenging to overproduce PQQ, as its biosynthesis remains unclear. Here, we report that PQQ production can be enhanced by stimulating the metabolic demand for it. First, we developed CRISPR interference (CRISPRi) system to block PTS and thereby intensify DOP. In shake-flask cultivation, the strain with CRISPRi system (simultaneously inhibiting four PTS-related genes) produced 225·65 nmol l-1 PQQ, which was 2·14 times that of wild type. In parallel, an exogenous soluble glucose dehydrogenase (sGDH) was overexpressed in K. pneumoniae. In the shake-flask cultivation, this sGDH-overexpressing strain accumulated 140·05 nmol l-1 PQQ, which was 1·33 times that of wild type. To combine the above two strategies, we engineered a strain harbouring both CRISPRi vector and sGDH-overexpressing vector. In the shake-flask cultivation, this two-plasmid strain generated 287·01 nmol l-1 PQQ, which was 2·72 times that of wild type. In bioreactor cultivation, this two-plasmid strain produced 2206·1 nmol l-1 PQQ in 57 h, which was 7·69 times that in shake-flask cultivation. These results indicate that PQQ production can be enhanced by intensifying DOP, as the apo-enzyme GDH is intrinsically coupled with cofactor PQQ. This study provides a strategy for the production of cofactors whose biosynthesis mechanisms remain ambiguous. SIGNIFICANCE AND IMPACT OF THE STUDY: Pyrroloquinoline quinone (PQQ) is an economically important chemical, which typically serves as a cofactor of glucose dehydrogenase (GDH) and thus participates in glucose metabolism. Klebsiella pneumoniae can naturally synthesize PQQ, but current yield constrains its commercialization. In this study, the PQQ level was improved by stimulating metabolic demand for PQQ, instead of overexpressing PQQ synthetic genes, as the synthetic mechanism remains ambiguous.

Functional Characterization of the ycjQRS Gene Cluster from Escherichia coli: A Novel Pathway for the Transformation of d-Gulosides to d-Glucosides.

A combination of bioinformatics, steady-state kinetics, and NMR spectroscopy has revealed the catalytic functions of YcjQ, YcjS, and YcjR from the ycj gene cluster in Escherichia coli K-12. YcjS was determined to be a 3-keto-d-glucoside dehydrogenase with a kcat = 22 s-1 and kcat/ Km = 2.3 × 104 M-1 s-1 for the reduction of methyl α-3-keto-d-glucopyranoside at pH 7.0 with NADH. YcjS also exhibited catalytic activity for the NAD+-dependent oxidation of d-glucose, methyl ß-d-glucopyranoside, and 1,5-anhydro-d-glucitol. YcjQ was determined to be a 3-keto-d-guloside dehydrogenase with kcat = 18 s-1 and kcat/ Km = 2.0 × 103 M-1 s-1 for the reduction of methyl α-3-keto-gulopyranoside. This is the first reported dehydrogenase for the oxidation of d-gulose. YcjQ also exhibited catalytic activity with d-gulose and methyl ß-d-gulopyranoside. The 3-keto products from both dehydrogenases were found to be extremely labile under alkaline conditions. The function of YcjR was demonstrated to be a C4 epimerase that interconverts 3-keto-d-gulopyranosides to 3-keto-d-glucopyranosides. These three enzymes, YcjQ, YcjR, and YcjS, thus constitute a previously unrecognized metabolic pathway for the transformation of d-gulosides to d-glucosides via the intermediate formation of 3-keto-d-guloside and 3-keto-d-glucoside.

Generalizable Protein Biosensors Based on Synthetic Switch Modules.

Allosteric protein switches are key controllers of information and energy processing in living organisms and are desirable engineered control tools in synthetic systems. Here we present a generally applicable strategy for construction of allosteric signaling systems with inputs and outputs of choice. We demonstrate conversion of constitutively active enzymes into peptide-operated synthetic allosteric ON switches by insertion of a calmodulin domain into rationally selected sites. Switches based on EGFP, glucose dehydrogenase, NanoLuciferase, and dehydrofolate reductase required minimal optimization and demonstrated a dynamic response ranging from 1.8-fold in the former case to over 200-fold in the latter case. The peptidic nature of the calmodulin ligand enables incorporation of such synthetic switch modules into higher order sensory architectures. Here, a ligand-mediated increase in proximity of the allosteric switch and the engineered activator peptide modulates biosensor's activity. Created biosensors were used to measure concentrations of clinically relevant drugs and biomarkers in plasma, saliva, and urine with accuracy comparable to that of the currently used clinical diagnostic assays. The approach presented is generalizable as it allows rapid construction of efficient protein switches that convert binding of a broad range of analytes into a biochemical activity of choice enabling construction of artificial signaling and metabolic circuits of potentially unlimited complexity.

Cloning and expression of d-glucoside 3-dehydrogenase from Rhizobium sp. S10 in Escherichia coli and its application for d-gulose production.

The novel isolated Rhizobium sp. S10 was identified as d-glucoside 3-dehydrogenase (G3DH) producing microbe. Therefore, the gene encoding for G3DH from Rhizobium sp. S10 was cloned and overexpressed in Escherichia coli strain JM109 as a soluble enzyme complex. The recombinant G3DH (rG3DH) was purified with relatively high specific activity of 38.54 U/mg compared to the previously characterized and cloned G3DHs. The purified rG3DH showed the highest activity at pH 7.0, 40 °C toward cellobiose. It can also oxidize a broad range of mono-disaccharides including saccharide derivatives. The glycosides oxidizing activity combined with chemical reaction, could produce d-gulose from lactitol via 3-ketolactitol.

The gluconate shunt is an alternative route for directing glucose into the pentose phosphate pathway in fission yeast.

Glycolysis and the pentose phosphate pathway both play a central role in the degradation of glucose in all domains of life. Another metabolic route that can facilitate glucose breakdown is the gluconate shunt. In this shunt glucose dehydrogenase and gluconate kinase catalyze the two-step conversion of glucose into the pentose phosphate pathway intermediate 6-phosphogluconate. Despite the presence of these enzymes in many organisms, their only established role is in the production of 6-phosphogluconate for the Entner-Doudoroff pathway. In this report we performed metabolic profiling on a strain of Schizosaccharomyces pombe lacking the zinc-responsive transcriptional repressor Loz1 with the goal of identifying metabolic pathways that were altered by cellular zinc status. This profiling revealed that loz1Δ cells accumulate higher levels of gluconate. We show that the altered gluconate levels in loz1Δ cells result from increased expression of gcd1 By analyzing the activity of recombinant Gcd1 in vitro and by measuring gluconate levels in strains lacking enzymes of the gluconate shunt we demonstrate that Gcd1 encodes a novel NADP+-dependent glucose dehydrogenase that acts in a pathway with the Idn1 gluconate kinase. We also find that cells lacking gcd1 and zwf1, which encode the first enzyme in the pentose phosphate pathway, have a more severe growth phenotype than cells lacking zwf1 We propose that in S. pombe Gcd1 and Idn1 act together to shunt glucose into the pentose phosphate pathway, creating an alternative route for directing glucose into the pentose phosphate pathway that bypasses hexokinase and the rate-limiting enzyme glucose-6-phosphate dehydrogenase.

Convenient and Universal Fabrication Method for Antibody-Enzyme Complexes as Sensing Elements Using the SpyCatcher/SpyTag System.

Antibody-enzyme complexes (AECs) are ideal sensing elements, especially when oxidoreductases are used as the enzymes in the complex, with the potential to carry out rapid electrochemical measurements. However, conventional methods for the fabrication of AECs, including direct fusion and chemical conjugation, are associated with issues regarding the generation of insoluble aggregates and production of homogeneous AECs. Here, we developed a convenient and universal method for the fabrication of homogeneous AECs using the SpyCatcher/SpyTag system. We used an anti-epidermal growth factor receptor (EGFR) variable domain of a heavy chain antibody (VHH) and a glucose dehydrogenase (GDH) derived from Aspergillus flavus ( AfGDH) as the model antibody and enzyme, respectively. Both SpyTag-fused VHH and SpyCatcher-fused AfGDH were successfully prepared using an Escherichia coli expression system, whereas anti-EGFR AECs were produced by simply mixing the two fusion proteins. A bivalent AEC, AfGDH with two VHH at both terminals, was also prepared and exhibited an increased affinity. A soluble EGFR was successfully detected in a dose-dependent manner using immobilized anti-EGFR immunoglobulin G (IgG) and bivalent AEC. We also confirmed the universality of this AEC fabricating method by applying it to another VHH. This method results in the convenient and universal preparation of sensing elements with the potential for electrochemical measurement.

Identification and characterization of thermostable glucose dehydrogenases from thermophilic filamentous fungi.

FAD-dependent glucose dehydrogenase (FAD-GDH), which contains FAD as a cofactor, catalyzes the oxidation of D-glucose to D-glucono-1,5-lactone, and plays an important role in biosensors measuring blood glucose levels. In order to obtain a novel FAD-GDH gene homolog, we performed degenerate PCR screening of genomic DNAs from 17 species of thermophilic filamentous fungi. Two FAD-GDH gene homologs were identified and cloned from Talaromyces emersonii NBRC 31232 and Thermoascus crustaceus NBRC 9129. We then prepared the recombinant enzymes produced by Escherichia coli and Pichia pastoris. Absorption spectra and enzymatic assays revealed that the resulting enzymes contained oxidized FAD as a cofactor and exhibited glucose dehydrogenase activity. The transition midpoint temperatures (T m) were 66.4 and 62.5 °C for glycosylated FAD-GDHs of T. emersonii and T. crustaceus prepared by using P. pastoris as a host, respectively. Therefore, both FAD-GDHs exhibited high thermostability. In conclusion, we propose that these thermostable FAD-GDHs could be ideal enzymes for use as thermotolerant glucose sensors with high accuracy.

Gene cloning and expression of a glucoside 3-dehydrogenase from Sphingobacterium faecium ZJF-D6, and used it to produce N-p-nitrophenyl-3-ketovalidamine.

In this study, we report the cloning and expression of a functional glucoside 3-dehydrogenase (G3DH) gene from Sphingobacterium faecium ZJF-D6. This gene is 1686 bp in length and encodes a peptide of 562 amino acids. The G3DH gene was successfully expressed in E. coli, and the recombinant enzyme could oxidize glucosides, galactosides and analogues at C-3 position. The sequence and multiple alignment analysis showed that the enzyme has highest identity with G3DHs from Paraglaciecola polaris LMG 21857, Aliiglaciecola lipolytica E3 and Halomonas sp. alpha-15. The recombinant G3DH was purified on Ni-NTA column and exhibited the highest activity at pH 7.6 and 30 °C. It was sensitive to acid and alkali, and showed well thermostability. The SfG3DH could oxidize a wild range of sugars. When recombinant E. coli BL21 cells were used as catalyst, a high rate of conversion to N-p-nitrophenyl-3-ketovalidamine was achieved, and no p-nitroaniline was detected. This process offers a promising approach to fulfill substrate of 3-ketovalidoxylamine A C-N lyase production.

A novel pyrroloquinoline quinone-dependent 2-keto-D-glucose dehydrogenase from Pseudomonas aureofaciens.

A gene encoding an enzyme similar to a pyrroloquinoline quinone (PQQ)-dependent sugar dehydrogenase from filamentous fungi, which belongs to new auxiliary activities (AA) family 12 in the CAZy database, was cloned from Pseudomonas aureofaciens. The deduced amino acid sequence of the cloned enzyme showed only low homology to previously characterized PQQ-dependent enzymes, and multiple-sequence alignment analysis showed that the enzyme lacks one of the three conserved arginine residues that function as PQQ-binding residues in known PQQ-dependent enzymes. The recombinant enzyme was heterologously expressed in an Escherichia coli expression system for further characterization. The UV-visible (UV-Vis) absorption spectrum of the oxidized form of the holoenzyme, prepared by incubating the apoenzyme with PQQ and CaCl2, revealed a broad peak at approximately 350 nm, indicating that the enzyme binds PQQ. With the addition of 2-keto-d-glucose (2KG) to the holoenzyme solution, a sharp peak appeared at 331 nm, attributed to the reduction of PQQ bound to the enzyme, whereas no effect was observed upon 2KG addition to authentic PQQ. Enzymatic assay showed that the recombinant enzyme specifically reacted with 2KG in the presence of an appropriate electron acceptor, such as 2,6-dichlorophenol indophenol, when PQQ and CaCl2 were added. (1)H nuclear magnetic resonance ((1)H-NMR) analysis of reaction products revealed 2-keto-d-gluconic acid (2KGA) as the main product, clearly indicating that the recombinant enzyme oxidizes the C-1 position of 2KG. Therefore, the enzyme was identified as a PQQ-dependent 2KG dehydrogenase (Pa2KGDH). Considering the high substrate specificity, the physiological function of Pa2KGDH may be for production of 2KGA.

Production of medium chain length polyhydroxyalkanoate in metabolic flux optimized Pseudomonas putida.

BACKGROUND: Pseudomnas putida is a natural producer of medium chain length polyhydroxyalkanoates (mcl-PHA), a polymeric precursor of bioplastics. A two-fold increase of mcl-PHA production via inactivation of the glucose dehydrogenase gene gcd, limiting the metabolic flux towards side products like gluconate was achieved before. Here, we investigated the overproduction of enzymes catalyzing limiting steps of mcl-PHA precursor formation. RESULTS: A genome-based in silico model for P. putida KT2440 metabolism was employed to identify potential genetic targets to be engineered for the improvement of mcl-PHA production using glucose as sole carbon source. Here, overproduction of pyruvate dehydrogenase subunit AcoA in the P. putida KT2440 wild type and the Δgcd mutant strains led to an increase of PHA production. In controlled bioreactor batch fermentations PHA production was increased by 33% in the acoA overexpressing wild type and 121% in the acoA overexpressing Δgcd strain in comparison to P. putida KT2440. Overexpression of pgl-encoding 6-phosphoglucolactonase did not influence PHA production. Transcriptome analyses of engineered PHA producing P. putida in comparison to its parental strains revealed the induction of genes encoding glucose 6-phosphate dehydrogenase and pyruvate dehydrogenase. In addition, NADPH seems to be quantitatively consumed for efficient PHA synthesis, since a direct relationship between low levels of NADPH and high concentrations of the biopolymer were observed. In contrast, intracellular levels of NADH were found increased in PHA producing organisms. CONCLUSION: Production of mcl-PHAs was enhanced in P. putida when grown on glucose via overproduction of a pyruvate dehydrogenase subunit (AcoA) in combination with a deletion of the glucose dehydrogenase (gcd) gene as predicted by in silico elementary flux mode analysis.

Screening of peptide ligands for pyrroloquinoline quinone glucose dehydrogenase using antagonistic template-based biopanning.

We have developed a novel method, antagonistic template-based biopanning, for screening peptide ligands specifically recognizing local tertiary protein structures. We chose water-soluble pyrroloquinoline quinone (PQQ) glucose dehydrogenase (GDH-B) as a model enzyme for this screening. Two GDH-B mutants were constructed as antagonistic templates; these have some point mutations to induce disruption of local tertiary structures within the loop regions that are located at near glucose-binding pocket. Using phage display, we selected 12-mer peptides that specifically bound to wild-type GDH-B but not to the antagonistic templates. Consequently, a peptide ligand showing inhibitory activity against GDH-B was obtained. These results demonstrate that the antagonistic template-based biopanning is useful for screening peptide ligands recognizing the specific local tertiary structure of proteins.

Serratia marcescens quinoprotein glucose dehydrogenase activity mediates medium acidification and inhibition of prodigiosin production by glucose.

Serratia marcescens is a model organism for the study of secondary metabolites. The biologically active pigment prodigiosin (2-methyl-3-pentyl-6-methoxyprodiginine), like many other secondary metabolites, is inhibited by growth in glucose-rich medium. Whereas previous studies indicated that this inhibitory effect was pH dependent and did not require cyclic AMP (cAMP), there is no information on the genes involved in mediating this phenomenon. Here we used transposon mutagenesis to identify genes involved in the inhibition of prodigiosin by glucose. Multiple genetic loci involved in quinoprotein glucose dehydrogenase (GDH) activity were found to be required for glucose inhibition of prodigiosin production, including pyrroloquinoline quinone and ubiquinone biosynthetic genes. Upon assessing whether the enzymatic products of GDH activity were involved in the inhibitory effect, we observed that d-glucono-1,5-lactone and d-gluconic acid, but not d-gluconate, were able to inhibit prodigiosin production. These data support a model in which the oxidation of d-glucose by quinoprotein GDH initiates a reduction in pH that inhibits prodigiosin production through transcriptional control of the prodigiosin biosynthetic operon, providing new insight into the genetic pathways that control prodigiosin production. Strains generated in this report may be useful in large-scale production of secondary metabolites.

A novel reductase from Candida albicans for the production of ethyl (S)-4-chloro-3-hydroxybutanoate.

A novel NADPH-dependent reductase (CaCR) from Candida albicans was cloned for the first time. It catalyzed asymmetric reduction to produce ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE). It contained an open reading frame of 843 bp encoding 281 amino acids. When co-expressed with a glucose dehydrogenase in Escherichia coli, recombinant CaCR exhibited an activity of 5.7 U/mg with ethyl 4-chloro-3-oxobutanoate (COBE) as substrate. In the biocatalysis of COBE to (S)-CHBE, 1320 mM (S)-CHBE was obtained without extra NADP+/NADPH in a water/butyl acetate system, and the optical purity of the (S)-isomer was higher than 99% enantiomeric excess.

Exploring Performance Parameters of Artificial Allosteric Protein Switches.

Biological information processing networks rely on allosteric protein switches that dynamically interconvert biological signals. Construction of their artificial analogues is a central goal of synthetic biology and bioengineering. Receptor domain insertion is one of the leading methods for constructing chimeric protein switches. Here we present an in vitro expression-based platform for the analysis of chimeric protein libraries for which traditional cell survival or cytometric high throughput assays are not applicable. We utilise this platform to screen a focused library of chimeras between PQQ-glucose dehydrogenase and calmodulin. Using this approach, we identified 50 chimeras (approximately 23% of the library) that were activated by calmodulin-binding peptides. We analysed performance parameters of the active chimeras and demonstrated that their dynamic range and response times are anticorrelated, pointing to the existence of an inherent thermodynamic trade-off. We show that the structure of the ligand peptide affects both the response and activation kinetics of the biosensors suggesting that the structure of a ligand:receptor complex can influence the chimera's activation pathway. In order to understand the extent of structural changes in the reporter protein induced by the receptor domains, we have analysed one of the chimeric molecules by CD spectroscopy and hydrogen-deuterium exchange mass spectrometry. We concluded that subtle ligand-induced changes in the receptor domain propagated into the GDH domain and affected residues important for substrate and cofactor binding. Finally, we used one of the identified chimeras to construct a two-component rapamycin biosensor and demonstrated that core switch optimisation translated into improved biosensor performance.

Effect of substrate inhibition and cooperativity on the electrochemical responses of glucose dehydrogenase. Kinetic characterization of wild and mutant types.

Thanks to its insensitivity to dioxygen and to its good catalytic reactivity, and in spite of its poor substrate selectivity, quinoprotein glucose dehydrogenase (PQQ-GDH) plays a prominent role among the redox enzymes that can be used for analytical purposes, such as glucose detection, enzyme-based bioaffinity assays, and the design of biofuel cells. A detailed kinetic analysis of the electrochemical catalytic responses, leading to an unambiguous characterization of each individual steps, seems a priori intractable in view of the interference, on top of the usual ping-pong mechanism, of substrate inhibition and of cooperativity effects between the two identical subunits of the enzyme. Based on simplifications suggested by extended knowledge previously acquired by standard homogeneous kinetics, it is shown that analysis of the catalytic responses obtained by means of electrochemical nondestructive techniques, such as cyclic voltammetry, with ferrocene methanol as a mediator, does allow a full characterization of all individual steps of the catalytic reaction, including substrate inhibition and cooperativity and, thus, allows to decipher the reason that makes the enzyme more efficient when the neighboring subunit is filled with a glucose molecule. As a first practical illustration of this electrochemical approach, comparison of the native enzyme responses with those of a mutant (in which the asparagine amino acid in position 428 has been replaced by a cysteine residue) allowed identification of the elementary steps that makes the mutant type more efficient than the wild type when cooperativity between the two subunits takes place, which is observed at large mediator and substrate concentrations. A route is thus opened to structure-reactivity relationships and therefore to mutagenesis strategies aiming at better performances in terms of catalytic responses and/or substrate selectivity.

Screening of Aspergillus-derived FAD-glucose dehydrogenases from fungal genome database.

Aspergillus-derived FAD-dependent glucose dehydrogenases (FADGDHs) were screened from fungal genomic databases, primarily by searching for putative homologues of the Aspergillus niger-derived glucose oxidase (GOD). Focusing on a GOD active-site motif, putative proteins annotated as belonging to the glucose methanol choline (GMC) oxidoreductase family were selected. Phylogenetic analysis of these putative proteins produced a GOD clade, which includes the A. niger and Penicillium amagasakiens GODs, and a second clade made up of putative proteins showing 30-40% homology with GOD. The genes encoding the proteins from the second clade were functionally expressed in Escherichia coli, resulting in dye-mediated glucose dehydrogenase (GDH) activity but not GOD activity. These results suggest that the putative proteins belonging to the second clade are FADGDHs. The 3D structure models of these FADGDHs were compared with the 3D structure of GOD.

Generation of a Gluconobacter oxydans knockout collection for improved extraction of rare earth elements.

Bioleaching of rare earth elements (REEs), using microorganisms such as Gluconobacter oxydans, offers a sustainable alternative to environmentally harmful thermochemical extraction, but is currently not very efficient. Here, we generate a whole-genome knockout collection of single-gene transposon disruption mutants for G. oxydans B58, to identify genes affecting the efficacy of REE bioleaching. We find 304 genes whose disruption alters the production of acidic biolixiviant. Disruption of genes underlying synthesis of the cofactor pyrroloquinoline quinone (PQQ) and the PQQ-dependent membrane-bound glucose dehydrogenase nearly eliminates bioleaching. Disruption of phosphate-specific transport system genes enhances bioleaching by up to 18%. Our results provide a comprehensive roadmap for engineering the genome of G. oxydans to further increase its bioleaching efficiency.

Designing a highly active soluble PQQ-glucose dehydrogenase for efficient glucose biosensors and biofuel cells.

We report for the first time a soluble PQQ-glucose dehydrogenase that is twice more active than the wild type for glucose oxidation and was obtained by combining site directed mutagenesis, modelling and steady-state kinetics. The observed enhancement is attributed to a better interaction between the cofactor and the enzyme leading to a better electron transfer. Electrochemical experiments also demonstrate the superiority of the new mutant for glucose oxidation and make it a promising enzyme for the development of high-performance glucose biosensors and biofuel cells.

High-level production of heterologous proteins using untreated cane molasses and corn steep liquor in Escherichia coli medium.

To develop an economical industrial medium, untreated cane molasses (UCM) was tested as a carbon source for fermentation culturing of Escherichia coli. To test the industrial application of this medium, we chose a strain co-expressing a carbonyl reductase (PsCR) and a glucose dehydrogenase (BmGDH). Although corn steep liquor (CSL) could be used as an inexpensive nitrogen source to replace peptone, yeast extract could not be replaced in E. coli media. In a volume of 40 ml per 1-l flask, a cell concentration of optical density (OD(600)) 15.1 and enzyme activities of 6.51 U/ml PsCR and 3.32 U/ml BmGDH were obtained in an optimized medium containing 25.66 g/l yeast extract, 3.88 g/l UCM, and 7.1% (v/v) CSL. When 3.88 g/l UCM was added to the medium at 6 h in a fed-batch process, the E. coli concentration increased to OD(600) of 24, and expression of both PsCR and BmGDH were twofold higher than that of a batch process. Recombinant cells from batch or fed-batch cultures were assayed for recombinant enzyme activity by testing the reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (S)-4-chloro-3-hydroxybutanoate (CHBE). Compared to cells from batch cultures, fed-batch cultured cells showed higher recombinant enzyme expression, producing 560 mM CHBE in the organic phase with a molar yield of 92% and an optical purity of the (S)-isomer of >99% enantiomeric excess.