Hyeon-Cheol Lee

High NADPH/NADP+ ratio improves thymidine production by a metabolically engineered Escherichia coli strain.

Thymidine is a commercially important precursor in the production of antiviral drugs, including azidothymidine for the treatment of AIDS. In order to produce thymidine in a large scale, we previously developed a thymidine-overproducing Escherichia coli strain BLdtug24 by engineering pathways. To further enhance thymidine yield, we increased the availability of a cofactor (NADPH) in thymidine biosynthesis by disrupting phosphoglucose isomerase in BLdtug24 to construct BLdtugp24. Additionally, NAD(+) kinase or soluble transhydrogenase was overexpressed in BLdtugp24, which can reroute glucose metabolic flow from the EMP pathway to the PP pathway, to construct BLdtugp34N or BLdtugp34U, respectively. In chemostat cultures, BLdtugp24 had an increased NADPH availability and a 4-fold enhancement in thymidine yield for glucose compared with BLdtug24. BLdtugp34N and BLdtugp34U had increased thymidine yields for glucose by 1.2- and 2-fold compared with BLdtugp24, respectively. The NADPH/NADP(+) ratios at steady-state had overall positive correlations with thymidine yields in these strains. Real-time RT-PCR analysis revealed that the transcriptional regulations of NAD(P)H-related enzymes and transhydrogenases affected the redox balance and shifted reaction equilibrium toward increasing NADPH. In fed-batch fermentation, BLdtugp34U with the highest NADPH/NADP(+) ratio in chemostat experiment produced 1.9gl(-1) of thymidine with 29.7mgl(-1)h(-1) of thymidine productivity.

Fermentative Production of Thymidine by a Metabolically Engineered Escherichia coli Strain

ABSTRACT Thymidine is an important precursor in the production of various antiviral drugs, including azidothymidine for the treatment of AIDS. Since thymidine-containing nucleotides are synthesized only by the de novo pathway during DNA synthesis, it is not easy to produce a large amount of thymidine biologically. In order to develop a host strain to produce thymidine, thymidine phosphorylase, thymidine kinase, and uridine phosphorylase genes were deleted from an Escherichia coli BL21 strain to develop BLdtu. Since the genes coding for the enzymes related to the nucleotide salvage pathway were disrupted, BLdtu was unable to utilize thymidine or thymine, and thymidine degradation activity was completely abrogated. We additionally expressed T4 thymidylate synthase, T4 nucleotide diphosphate reductase, bacteriophage PBS2 TMP phosphohydrolase, E. coli dCTP deaminase, and E. coli uridine kinase in the BLdtu strain to develop a thymidine-producing strain (BLdtu24). BLdtu24 produced 649.3 mg liter−1 of thymidine in a 7-liter batch fermenter for 24 h, and neither thymine nor uridine was detected. However, the dUTP/dTTP ratio was increased in BLdtu24, which could lead to increased double-strand breakages and eventually to cell deaths during fermentation. To enhance thymidine production and to prevent cell deaths during fermentation, we disrupted a gene (encoding uracil-DNA N-glycosylase) involved in DNA excision repair to suppress the consumption of dTTP and developed BLdtug24. Compared with the thymidine production in BLdtu24, the thymidine production in BLdtug24 was increased by ∼1.2-fold (740.3 mg liter−1). Here, we show that a thymidine-producing strain with a relatively high yield can be developed using a metabolic engineering approach.

Isomaltose Production by Modification of the Fructose-Binding Site on the Basis of the Predicted Structure of Sucrose Isomerase from “Protaminobacter rubrum”

ABSTRACT “Protaminobacter rubrum” sucrose isomerase (SI) catalyzes the isomerization of sucrose to isomaltulose and trehalulose. SI catalyzes the hydrolysis of the glycosidic bond with retention of the anomeric configuration via a mechanism that involves a covalent glycosyl enzyme intermediate. It possesses a 325RLDRD329 motif, which is highly conserved and plays an important role in fructose binding. The predicted three-dimensional active-site structure of SI was superimposed on and compared with those of other α-glucosidases in family 13. We identified two Arg residues that may play important roles in SI-substrate binding with weak ionic strength. Mutations at Arg325 and Arg328 in the fructose-binding site reduced isomaltulose production and slightly increased trehalulose production. In addition, the perturbed interactions between the mutated residues and fructose at the fructose-binding site seemed to have altered the binding affinity of the site, where glucose could now bind and be utilized as a second substrate for isomaltose production. From eight mutant enzymes designed based on structural analysis, the R325Q mutant enzyme exhibiting high relative activity for isomaltose production was selected. We recorded 40.0% relative activity at 15% (wt/vol) additive glucose with no temperature shift; the maximum isomaltose concentration and production yield were 57.9 g liter−1 and 0.55 g of isomaltose/g of sucrose, respectively. Furthermore, isomaltose production increased with temperature but decreased at a temperature of >35°C. Maximum isomaltose production (75.7 g liter−1) was recorded at 35°C, and its yield for the consumed sucrose was 0.61 g g−1 with the addition of 15% (wt/vol) glucose. The relative activity for isomaltose production increased progressively with temperature and reached 45.9% under the same conditions.

Improvement of Coenzyme Q10 Production by Increasing the NADH/NAD+ Ratio in Agrobacterium tumefaciens

Previously screened CoQ10-overproducing Agrobacterium tumefaciens A603-35 showed a relatively high NADH/NAD+ ratio (1.1), as compared to parental strain C58 (0.2) when we increased the expression levels of NADH-generating enzymes. Also, the intracellular NADH/NAD+ ratio showed a positive correlation with the CoQ10 content in A603-35. Overexpression of glyceraldehyde 3-phosphate dehydrogenase in A603-35 shifted the NADH/NAD+ ratio at 48 h from 0.8 to 1.2, and thus the CoQ10 content in flask culture increased from 2.16 to 3.63 mg/g DCW. Due to the addition of hydroxybutyrate to the culture media, the intracellular NADH/NAD+ ratio in A603-35-gapA shifted from 1.2 to 1.4, which led to an increase CoQ10 content (5.27 mg/g DCW).

An engineered genetic selection for ternary protein complexes inspired by a natural three-component hitchhiker mechanism

The bacterial twin-arginine translocation (Tat) pathway is well known to translocate correctly folded monomeric and dimeric proteins across the tightly sealed cytoplasmic membrane. We identified a naturally occurring heterotrimer, the Escherichia coli aldehyde oxidoreductase PaoABC, that is co-translocated by the Tat translocase according to a ternary “hitchhiker” mechanism. Specifically, the PaoB and PaoC subunits, each devoid of export signals, are escorted to the periplasm in a piggyback fashion by the Tat signal peptide-containing subunit PaoA. Moreover, export of PaoA was blocked when either PaoB or PaoC was absent, revealing a surprising interdependence for export that is not seen for classical secretory proteins. Inspired by this observation, we created a bacterial three-hybrid selection system that links the formation of ternary protein complexes with antibiotic resistance. As proof-of-concept, a bispecific antibody was employed as an adaptor that physically crosslinked one antigen fused to a Tat export signal with a second antigen fused to TEM-1 β-lactamase (Bla). The resulting non-covalent heterotrimer was exported in a Tat-dependent manner, delivering Bla to the periplasm where it hydrolyzed β-lactam antibiotics. Collectively, these results highlight the remarkable flexibility of the Tat system and its potential for studying and engineering ternary protein interactions in living bacteria.

Improved production of isomaltulose by a newly isolated mutant of Serratia sp. cells immobilized in calcium alginate

Isomaltulose, also known as palatinose, is produced by sucrose isomerase and has been highlighted as a sugar substitute due to a number of advantageous properties. For the massive production of isomaltulose, high resistance to sucrose and stability of sucrose isomerase as well as sucrose conversion yields would be critical factors. We describe a series of screening procedures to isolate the mutant strain of Serratia sp. possessing enhanced isomaltulose production with improved stability. The new Serratia sp. isolated from a series of screening procedures allowed us to produce isomaltulose from 60% sucrose solution, with over 90% conversion yield. Moreover, when this strain was immobilized in calcium alginate beads and placed in a medium containing 60% sucrose, it showed over 70% sucrose conversion yields for 30 cycles of repeated-batch reactions. Thus, improved conversion activity and stability of the newly isolated Serratia sp. strain in the present study would be highly valuable for industries related to isomaltulose production.

Development of a Novel Plasmid-Free Thymidine Producer by Reprogramming Nucleotide Metabolic Pathways

ABSTRACT A novel thymidine-producing strain of Escherichia coli was prepared by genome recombineering. Eleven genes were deleted by replacement with an expression cassette, and 7 genes were integrated into the genome. The resulting strain, E. coli HLT013, showed a high thymidine yield with a low deoxyuridine content. DNA microarrays were then used to compare the gene expression profiles of HLT013 and its isogenic parent strain. Based on microarray analysis, the pyr biosynthesis genes and 10 additional genes were selected and then expressed in HLT013 to find reasonable candidates for enhancing thymidine yield. Among these, phage shock protein A (PspA) showed positive effects on thymidine production by diminishing redox stress. Thus, we integrated pspA into the HLT013 genome, resulting in E. coli strain HLT026, which produced 13.2 g/liter thymidine for 120 h with fed-batch fermentation. Here, we also provide a basis for new testable hypotheses regarding the enhancement of thymidine productivity and the attainment of a more complete understanding of nucleotide metabolism in bacteria.

Activation of macrophage mediated host defense against Salmonella typhimurium by Morus alba L.

Background The innate immune system plays a crucial role in the initiation and subsequent direction of adaptive immune responses, as well as in the removal of pathogens that have been targeted by an adaptive immune response. Objective Morus alba L. was reported to have immunostimulatory properties that might protect against infectious diseases. However, this possibility has not yet been explored. The present study investigated the protective and immune-enhancing ability of M. alba L. against infectious disease and the mechanisms involved. Design To investigate the immune-enhancing effects of M. alba L., we used a bacterial infection model. Results and discussions The lifespan of mice infected with a lethal dose of Salmonella typhimurium (1 × 107 colony forming units – CFU) was significantly extended when they were administered M. alba L. Furthermore, M. alba L. activated macrophages, monocytes, and neutrophils and induced Th1 cytokines (IL-12, IFN-γ, TNF-α) in mice infected with a sublethal dose (1 × 105 CFU) of S. typhimurium. M. alba L. significantly stimulated the uptake of bacteria into peritoneal macrophages as indicated by increased phagocytosis. Peritoneal macrophages derived from C3H/HeJ mice significantly inhibited M. alba L. induced NO production and TNF-α secretion compared with peritoneal macrophages derived from C3H/HeN mice. Conclusions These results suggest that the innate immune activity of M. alba L. against bacterial infection in mice occurs through activation of the TLR4 signaling pathway.

Transcriptional profiling of thymidine-producing strain recombineered from Escherichia coli BL21

DNA microarrays were used to compare the expression profiles of a thymidine overproducing strain (BLT013) and its isogenic parent, Escherichia coli BL21(DE3), when each was grown under well-defined thymidine production conditions with glycerol as carbon source. Here we describe the experimental procedures and methods in detail to reproduce the results and provide resource to be applied to similar engineering approach (available at Gene Expression Omnibus database under GSE69963). Taken together, the microarray data provide a basis for new testable hypotheses regarding enhancement of thymidine productivity and attaining a more complete understanding of nucleotide metabolism in bacteria.