Deoxycytidine production by a metabolically engineered Escherichia coli strain.

BACKGROUND: Rational engineering studies for deoxycytidine production were initiated due to low intracellular levels and tight regulation. To achieve high-level production of deoxycytidine, a useful precursor of decitabine, genes related to feed-back inhibition as well as the biosynthetic pathway were engineered. Additionally, we predicted the impact of individual gene expression levels on a complex metabolic network by microarray analysis. Based on these findings, we demonstrated rational metabolic engineering strategies capable of producing deoxycytidine. RESULTS: To prepare the deoxycytidine producing strain, we first deleted 3 degradation enzymes in the salvage pathway (deoA, udp, and deoD) and 4 enzymes involved in the branching pathway (dcd, cdd, codA and thyA) to completely eliminate degradation of deoxycytidine. Second, purR, pepA and argR were knocked out to prevent feedback inhibition of CarAB. Third, to enhance influx to deoxycytidine, we investigated combinatorial expression of pyrG, T4 nrdCAB and yfbR. The best strain carried pETGY (pyrG-yfbR) from the possible combinatorial plasmids. The resulting strain showed high deoxycytidine yield (650 mg/L) but co-produced byproducts. To further improve deoxycytidine yield and reduce byproduct formation, pgi was disrupted to generate a sufficient supply of NADPH and ribose. Overall, in shake-flask cultures, the resulting strain produced 967 mg/L of dCyd with decreased byproducts. CONCLUSIONS: We demonstrated that deoxycytidine could be readily achieved by recombineering with biosynthetic genes and regulatory genes, which appeared to enhance the supply of precursors for synthesis of carbamoyl phosphate, based on transcriptome analysis. In addition, we showed that carbon flux rerouting, by disrupting pgi, efficiently improved deoxycytidine yield and decreased byproduct content.

Disruption of genes for the enhanced biosynthesis of α-ketoglutarate in Corynebacterium glutamicum.

The development of microbial strains for the enhanced production of α-ketoglutarate (α-KG) was investigated using a strain of Corynebacterium glutamicum that overproduces of l-glutamate, by disrupting three genes involved in the α-KG biosynthetic pathway. The pathways competing with the biosynthesis of α-KG were blocked by knocking out aceA (encoding isocitrate lyase, ICL), gdh (encoding glutamate dehydrogenase, l-gluDH), and gltB (encoding glutamate synthase or glutamate-2-oxoglutarate aminotransferase, GOGAT). The strain with aceA, gltB, and gdh disrupted showed reduced ICL activity and no GOGAT and l-gluDH activities, resulting in up to 16-fold more α-KG production than the control strain in flask culture. These results suggest that l-gluDH is the key enzyme in the conversion of α-KG to l-glutamate; therefore, prevention of this step could promote α-KG accumulation. The inactivation of ICL leads the carbon flow to α-KG by blocking the glyoxylate pathway. However, the disruption of gltB did not affect the biosynthesis of α-KG. Our results can be applied in the industrial production of α-KG by using C. glutamicum as producer.

Deoxycytidine production by metabolically engineered Corynebacterium ammoniagenes.

Corynebacterium ammoniagenes N424 was metabolically modified to isolate overproducers of deoxycytidine. Inosine auxotrophy (ino) was initially introduced to prevent the flow of PRPP (phosphoribosyl pyrophosphate) into the purine biosynthetic pathway by random mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine. Following that, mutants possessing hydroxyurea resistance (HU(r)) were isolated to increase the activity of ribonucleoside diphosphate reductase, which catalyzes the reduction of ribonucleoside diphosphate to deoxyribonucleoside diphosphate. Then, in order to block the flow of dCTP into the TMP biosynthetic pathway via dUTP, thymine auxotrophy (thy(-)) was introduced into the mutant IH30 with ino(-) and Hlf. The resulting mutant IM7, possessing the characteristics of ino(-), HU(r), and thy(-), was deficient in dCTP deaminase and produced significantly higher amounts of deoxycytidine (81.3 mg/L) compared to its mother strain IH30 (6.2 mg/L). Deoxycytidine productivity was further enhanced by isolating the mutant IU19, which was resistant to 5-fluorouracil, an inhibitor of carbamoyl phosphate synthase. This enzyme catalyzed the synthesis of carbamoyl phosphate from glutamine, HCO (3)(-), and ATP. 5-Fluorouracil also inhibited aspartate trans-carbamoylase, catalyzeing the condensation of carbamoyl phosphate and aspartate. Finally, 5-fluorocytosine resistance (FC(r)) was introduced into the mutant strain IU19 to relieve the repression caused by accumulation of pyrimidine nucleosides. The mutant strain IC14-C6 possessing all the five characteristics described above produced 226.3 mg/L of deoxycytidine, which was at least 2,000 fold higher compared to the wild type, and accumulated only a negligible amount of other pyrimidines under shake flask fermentation.

Enhancement of thymidine production in E. coli by eliminating repressors regulating the carbamoyl phosphate synthetase operon.

PURPOSE OF WORK: Thymidine is an important precursor in antiviral drugs. We have enhanced thymidine production in E. coli by eliminating the repressors in the transcription of the gene coding for carbamoyl phosphate synthetase. The operon for carbamoyl phosphate synthetase (CarAB) in the thymidine biosynthesis regulatory pathway was derepressed by disrupting three known repressors (purR, pepA and argR). Combinatorial disruption of three repressors increased CarA expression levels in accordance with degree of disruption, which had a positive correlation with thymidine production. By simultaneous disruption of three repressors (BLdtugRPA), CarA expression level was increased by 3-fold compared to the parental strain, leading to an increased thymidine yield from 0.25 to 1.1 g thymidine l(-1). From BLdtugRPA, we established BLdtugRPA24 by transforming two plasmids expressing enzymes in the thymidine biosynthetic pathway and obtained 5.2 g thymidine l(-1) by Ph-stat fed-batch fermentation.

Molecular cloning and sequence analysis of a mannitol dehydrogenase gene and isolation of mdh promoter from Candida magnoliae.

The mdh gene encodes mannitol dehydrogenase (MDH), which catalyzes the conversion of fructose into mannitol. The putative mdh gene of Candida magnoliae was isolated by PCR using the primers deduced from the N-terminal amino acid sequences of an intact MDH and its tryptic peptides, cloned in E. coli, and sequenced. The mdh gene consisted of 852 bp encoding for 283 amino acids. Analysis of the amino acid sequence revealed that MDH consisted of typical NADPH-dependent short chain dehydrogenases/reductases (SDRs). To develop a strong promoter to induce expression of the foreign genes in C. magnolia, the putative promoter was isolated. The reporter protein, GFP, was well-expressed under the control of the putative mdh promoter of 153 bp in C. magnoliae.

Optimization of culture conditions and medium composition for the production of micrococcin GO5 by Micrococcus sp. GO5.

To enhance the production of micrococcin GO5, a bacteriocin produced by Micrococcus sp. GO5, cultivation conditions and medium composition were optimized. The optimal initial pH and temperature for bacteriocin production were 7.0-9.0 and 37 degrees C, respectively. Micrococcus sp. GO5 displayed the highest micrococcin GO5 activity when grown in modified MRS medium that contained lactose or sucrose, rather than glucose, as a carbon source. The maximum bacteriocin activity was obtained in modified MRS medium containing 0.5% tryptone and 1.0% yeast extract as nitrogen sources instead of the other nitrogen sources present in MRS medium. Bacteriocin production was greatly affected by the concentration of K(2)HPO(4); strain GO5 produced eight-fold more bacteriocin in medium containing 2.0-2.5% K(2)HPO(4) than in medium containing 0.2% K(2)HPO(4). The optimal concentration of MgSO(4).7H(2)O for bacteriocin production was 0.5%. The production of micrococcin GO5 was increased 32-fold in shake flask culture and 16-fold in a bioreactor using the optimized medium (TY medium), compared with culturing in MRS medium.

Production, purification, and characterization of micrococcin GO5, a bacteriocin produced by Micrococcus sp. GO5 isolated from kimchi.

Strain GO5, a bacteriocin-producing bacterium, was isolated from green onion kimchi and identified as Micrococcus sp. The bacteriocin, micrococcin GO5, displayed a broad spectrum of inhibitory activity against a variety of pathogenic and nonpathogenic microorganisms, as tested by the spot-on-lawn method; its activity spectrum was almost identical to that of nisin. Micrococcin GO5 was inactivated by trypsin (whereas nisin was not) and was completely stable at 100 degrees C for 30 min and in the pH range of 2.0 to 7.0. Micrococcin GO5 exhibited a typical mode of bactericidal activity against Micrococcus flavus ATCC 10240. It was purified to homogeneity through ammonium sulfate precipitation, ultrafiltration, and CM-Sepharose column chromatography. The molecular mass of micrococcin GO5 was estimated to be about 5.0 kDa by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in situ activity assay with the indicator organism. The amino acid sequence of micrococcin GO5 lacks lanthionine and beta-methyllanthionine and is rich in hydrophobic amino acids and glycine, providing the basis for the high heat stability of this bacteriocin. The N-terminal amino acid sequence of micrococcin GO5 is Lys-Lys-Ser-Phe-Cys-Gln-Lys, and no homology to bacteriocins reported previously was observed in the amino acid composition or N-terminal amino acid sequence. Based on the physicochemical properties, small molecular size, and inhibition of Listeria monocytogenes, micrococcin GO5 has been placed with the class II bacteriocins, but its broad spectrum of activity differs from that of other bacteriocins in this class.

Purification and characterization of a novel mannitol dehydrogenase from a newly isolated strain of Candida magnoliae.

Mannitol biosynthesis in Candida magnoliae HH-01 (KCCM-10252), a yeast strain that is currently used for the industrial production of mannitol, is catalyzed by mannitol dehydrogenase (MDH) (EC 1.1.1.138). In this study, NAD(P)H-dependent MDH was purified to homogeneity from C. magnoliae HH-01 by ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The relative molecular masses of C. magnoliae MDH, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, were 35 and 142 kDa, respectively, indicating that the enzyme is a tetramer. This enzyme catalyzed both fructose reduction and mannitol oxidation. The pH and temperature optima for fructose reduction and mannitol oxidation were 7.5 and 37 degrees C and 10.0 and 40 degrees C, respectively. C. magnoliae MDH showed high substrate specificity and high catalytic efficiency (k(cat) = 823 s(-1), K(m) = 28.0 mM, and k(cat)/K(m) = 29.4 mM(-1) s(-1)) for fructose, which may explain the high mannitol production observed in this strain. Initial velocity and product inhibition studies suggest that the reaction proceeds via a sequential ordered Bi Bi mechanism, and C. magnoliae MDH is specific for transferring the 4-pro-S hydrogen of NADPH, which is typical of a short-chain dehydrogenase reductase (SDR). The internal amino acid sequences of C. magnoliae MDH showed a significant homology with SDRs from various sources, indicating that the C. magnoliae MDH is an NAD(P)H-dependent tetrameric SDR. Although MDHs have been purified and characterized from several other sources, C. magnoliae MDH is distinguished from other MDHs by its high substrate specificity and catalytic efficiency for fructose only, which makes C. magnoliae MDH the ideal choice for industrial applications, including enzymatic synthesis of mannitol and salt-tolerant plants.

Role of glucose in the bioconversion of fructose into mannitol by Candida magnoliae.

The most efficient substrate for mannitol production by Candida magnoliae HH-01 is fructose; glucose and sucrose can also be converted into mannitol but with lower conversion yields. Mannitol dehydrogenase was purified and characterized; it had the highest activity with fructose as the substrate and used only NADPH. In fed-batch fermentation with glucose, the production of mannitol from fructose ceased when the glucose was exhausted but it was reinitiated with the addition of glucose, implying that glucose plays an important role in NADPH regeneration.

Thermotoga neapolitana adenylate kinase is highly active at 30 degrees C.

The adenylate kinase (AK) gene from Thermotoga neapolitana, a hyperthermophilic bacterium, was cloned and overexpressed in Escherichia coli, and the recombinant enzyme was biochemically characterized. The T. neapolitana AK (TNAK) sequence indicates that this enzyme belongs to the long bacterial AKs. TNAK contains the four cysteine residues that bind Zn(2+) in all Gram-positive AKs and in a few other Zn(2+)-containing bacterial AKs. Atomic emission spectroscopy and titration data indicate a content of 1 mol of Zn(2+)/mol of recombinant TNAK. The EDTA-treated enzyme has a melting temperature (T (m)=93.5 degrees C) 6.2 degrees C below that of the holoenzyme (99.7 degrees C), identifying Zn(2+) as a stabilizing feature in TNAK. TNAK is a monomeric enzyme with a molecular mass of approx. 25 kDa. TNAK displays V (max) and K (m) values at 30 degrees C identical with those of the E. coli AK at 30 degrees C, and displays very high activity at 80 degrees C, with a specific activity above 8000 units/mg. The unusually high activity of TNAK at 30 degrees C makes it an interesting model to test the role of enzyme flexibility in activity.