• KSBB Journal
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• v.11 no.5
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• pp.613-621
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• 1996

The effects of important medium components such as carbon, nitrogen sources and amino acids on the production of cyclosporin A(CyA) were investigated in an immobilized fungal cell fermentation using Tolypocladium inflatum. As carbon sources in the synthetic medium, fructose and maltose stimulated CyA production remarkably compared to glucose when ammonium sulfate was supplemented as a nitrogen source. In the absence of ammonium sulfate in the medium, however, CyA biosynthesis was reduced considerably without regard to C-sources tested. Ammonium sulfate was found to be the best N-source, and also ammonium phosphate and ammonium citrate showed some positive effects on CyA production. Optimum concentration of ammonium sulfate was 10g/L, and supplementation of ammonium sulfate at the start of fermentation was found to be the most efficacious for maximal production of CyA. Among the constituent amino acids of cyclic peptide, CyA, L-valine had the most significant effect on the biosynthesis of CyA, and maximum CyA production was observed when 10 g/L of L-valine was initially added.

• Journal of Microbiology and Biotechnology
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• v.29 no.8
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• pp.1288-1298
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• 2019

Bacterial ATP synthases drive ATP synthesis by a rotary mechanism, and play a vital role in physiology and cell metabolism. Corynebacterium glutamicum is well known as an industrial workhorse for amino acid production, and its ATP synthase operon contains eight structural genes and two adjacent genes, cg1360 and cg1361. So far, the physiological functions of Cg1360 (GenBank CAF19908) and Cg1361 (GenBank CAF19909) remain unclear. Here, we showed that Cg1360 was a hydrophobic protein with four transmembrane helices (TMHs), while no TMH was found in Cg1361. Deletion of cg1360, but not cg1361, led to significantly reduced cell growth using glucose and acetic acid as carbon sources, reduced F1 portions in the membrane, reduced ATP-driven proton-pumping activity and ATPase activity, suggesting that Cg1360 plays an important role in ATP synthase function. The intracellular ATP concentration in the ${\Delta}cg1360$ mutant was decreased to 72% of the wild type, while the NADH and NADPH levels in the ${\Delta}cg1360$ mutant were increased by 29% and 26%, respectively. However, the ${\Delta}cg1361$ mutant exhibited comparable intracellular ATP, NADH and NADPH levels with the wild-type strain. Moreover, the effect of cg1360 deletion on L-valine production was examined in the L-valine-producing V-10 strain. The final production of L-valine in the $V-10-{\Delta}cg1360$ mutant reached $9.2{\pm}0.3g/l$ in shake flasks, which was 14% higher than that of the V-10 strain. Thus, Cg1360 can be used as an effective engineering target by altering energy metabolism for the enhancement of amino acid production in C. glutamicum.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.4
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• pp.523-528
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• 2013

This study investigates the effects of three branched-chain amino acids (BCAA; valine, leucine, and isoleucine) on the in vitro ruminal fermentation of wheat straw using batch cultures of mixed ruminal microorganisms. BCAA were added to the buffered ruminal fluid at a concentration of 0, 2, 4, 7, or 10 mmol/L. After 72 h of anaerobic incubation, pH, volatile fatty acids (VFA), and ammonia nitrogen ($NH_3$-N) in the ruminal fluid were determined. Dry matter (DM) and neutral detergent fiber (NDF) degradability were calculated after determining the DM and NDF in the original material and in the residue after incubation. The addition of valine, leucine, or isoleucine increased the total VFA yields ($p{\leq}0.001$). However, the total VFA yields did not increase with the increase of BCAA supplement level. Total branched-chain VFA yields linearly increased as the supplemental amount of BCAA increased (p<0.001). The molar proportions of acetate and propionate decreased, whereas that of butyrate increased with the addition of valine and isoleucine (p<0.05). Moreover, the proportions of propionate and butyrate decreased (p<0.01) with the addition of leucine. Meanwhile, the molar proportions of isobutyrate were increased and linearly decreased (p<0.001) by valine and leucine, respectively. The addition of leucine or isoleucine resulted in a linear (p<0.001) increase in the molar proportions of isovalerate. The degradability of NDF achieved the maximum when valine or isoleucine was added at 2 mmol/L. The results suggest that low concentrations of BCAA (2 mmol/L) allow more efficient regulation of ruminal fermentation in vitro, as indicated by higher VFA yield and NDF degradability. Therefore, the optimum initial dose of BCAA for in vitro ruminal fermentation is 2 mmol/L.

• Journal of Microbiology and Biotechnology
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• v.28 no.11
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• pp.1916-1927
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• 2018

Corynebacterium glutamicum is an excellent platform for the production of amino acids, and is widely used in the fermentation industry. Most industrial strains are traditionally obtained by repeated processes of random mutation and selection, but the genotype of these strains is often unclear owing to the absence of genomic information. As such, it is difficult to improve the growth and amino acid production of these strains via metabolic engineering. In this study, we generated a complete genome map of an industrial L-valine-producing strain, C. glutamicum XV. In order to establish the relationship between genotypes and physiological characteristics, a comparative genomic analysis was performed to explore the core genome, structural variations, and gene mutations referring to an industrial L-leucine-producing strain, C. glutamicum CP, and the widely used C. glutamicum ATCC 13032. The results indicate that a 36,349 bp repeat sequence in the CP genome contained an additional copy each of lrp and brnFE genes, which benefited the export of L-leucine. However, in XV, the kgd and panB genes were disrupted by nucleotide insertion, which increase the availability of precursors to synthesize L-valine. Moreover, the specific amino acid substitutions in key enzymes increased their activities. Additionally, a novel strategy is proposed to remodel central carbon metabolism and reduce pyruvate consumption without having a negative impact on cell growth by introducing the CP-derived mutant $H^+$/citrate symporter. These results further our understanding regarding the metabolic networks in these strains and help to elucidate the influence of different genotypes on these processes.

• YAKHAK HOEJI
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• v.27 no.3
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• pp.185-205
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• 1983

Penicillins and cephalosporins are biosynthesized from L-.alpha.-aminoadipic acid, L-cysteine and L-valine. A tripeptide, LLD-$\delta$-($\alpha$-aminoadipyl)cysteinylvaline(LLD-ACV) was isolated from fermentation broths of Cephalosporium acremonium as well as of Penicillium chrysogenum and it was proved that the LL-$\delta$-($\alpha$-aminoadipyl cysteine was formed first in mycelia, to which valine would be connected to give LLD-ACV. However, several points are still unsolved; first, what mechanism is involved in the configurational change from L-valine to D-valine, second, what kind of cyclization mechanism gives a $\betha$-lactam ring and a thiazolidine ring and third, what is the pathways for the ring expansion from penicillins to cephalosporins. At present, it seems clear that LLD-ACV is cyclized to give isopenicillin N, which is transformed to penicillin N and further to cepbalosporin C. Other hydrophobic penicillins, including benzyl penicillin and penicillin V, are formed from isopenicillin N by acyl-exchange reactions catalyzed by penicillin transferase, rather than by acylation reaction on 6-aminopenicillanic acid(6-APA), which was isolated from the fermentation broth of P. chrysogenum and which would be formed by hydrolysis of $\delta-(\alpha$-amincadipyl)amido moiety at the C-6 position in isopenicillin N or penicillin N by penicillin acylase. Acylation of 6-APA is catalyzed also by penicillin acylase, but the reaction is proved not to be involved in penicillin biosynthesis. Understanding the biosynthesis of penicillins and cephalsoporins would provide solutions to increase in fermentation yields of penicillins, especially of cephalosporins and a solution to biological production of 7-aminocepbalosporanic acid (7-ACA) which is of importance in pharmaceutical industry. Still regulation mechanisms in penicillin and cephalosporin biosynthesis are unveiled at all.

• Microbiology and Biotechnology Letters
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• v.37 no.2
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• pp.140-146
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• 2009

Cyclosporin, an immunosuppressant, is a representative group of biologically active secondary metabolites produced by the fungus Tolypocladium inflatum. The amount and ratio of cyclosporin derivatives in the culture broth are an important factors for the production of cyclosporin A and the purification in the industrial process. Therefore, we studied the effect of amino acids and complex organic nitrogen sources using Tolypocladium inflatum mutants on the productivity of cyclosporin A and the ratio of cyclosporin derivatives. Overproducing mutant YHC-004 having seven times higher productivity than mother strain's could be obtained through the artificial mutation by UV irradiation. The concentration and kind of organic nitrogens and amino acids shows the profound effect on the productivity of cyclosporin A and ratio of cyclosporin derivatives. As a result, it was possible to raise the productivity and the ratio of cyclosporin A up to 3,430 mg/L and 93% respectively, but on the other hand the other cyclosporin derivatives decreased less than 2% in the culture broth.

• Journal of Microbiology and Biotechnology
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• v.20 no.7
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• pp.1086-1091
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• 2010

This study explored the use of gellan gum as an immobilization matrix for the production of cyclosporin A (CyA) by immobilized spores and mycelia of Tolypocladium inflatum MTCC 557. Different carriers, such as gellan gum, sodium alginate, celite beads, and silica, were tested as immobilization carriers, along with the role of the carrier concentration, biomass weight, number of spore-inoculated beads, and repeated utilization of the immobilized fungus. The maximum CyA production was 274 mg/l when using gellan gum [1% (w/v)], and a mycelial weight of 7.5% (w/v) supported the maximum production of CyA. Additionally, the addition of a combination of $_L$-valine (6 g/l) and $_L$-leucine (5 g/l) after 48 h of fermentation produced 1,338 mg/l of CyA when using gellan gum. The immobilized mycelia beads were found to remain stable for four repetitive cycles, indicating their potential for semicontinuous CyA production.

• KSBB Journal
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• v.18 no.6
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• pp.455-460
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• 2003

This study was designed to find out the rapid fermentation of soy sauce from koji hydrolyzates using air bubble column reactor packed with coimmobilized mixed culture system. Continuous ripid production was performed by coimmobilized Z. rouxiii BH-90 and C. versatilis BH-91. Coimmobilized cells of Zygosaccharomyces rouxii BH-90 and Candida versatilis BH-91 mixture cells in the column reactor produced 2.8％ ethyl alcohol and 18mg/L 4-ethylguaiacol over 96 hours under the optimal conditions. Coimmobilized cells produced 2.30∼2.4% ethyl alcohol during 30 days, and decreased gradually from 40 days to 70 days. Also coimmobilized cells produced 4-ethylguaiacol at the constant rate of 16∼18mg/L and decreased gradually after 40 days. Final product of soy sauce contained 2.4% ethyl alcohol and 18mg/L 4-ethylguaiacol. However, amino acid compositions of soy sauce were consisted of predominantly glutamic acid, leucin, arginine, aspartic acid, Iysine and valine, which were more than 50% of total amino acid.

• Microbiology and Biotechnology Letters
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• v.19 no.1
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• pp.76-81
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• 1991

- The effect of redox potential (ORP) on lysine production by a leucine auxotrophic regulatory mutant of Corynebacterium glutclmicum on molasses medium was investigated in a 2-1 jar fermentor at pH 6.9 and $32^{\circ}C$. At a dilution rate of D=O.l $h ^1$, a maximum yield of Yr,,s=0.24 was obtained in either carbon- or leucine-limited chemostat where the redox potential was between -60 mV and - 100 mV. This level of redox potential corresponded to moderate oxygen deficiency. Under a high oxygen deficient condition of the redox potential of - 130 rnV (oxygen-limited chemostat), all the kinetic parameters such as $Y_[p/s}, q_s\; and \; q_p$ were decreased significantly and significant amounts of byproducts including glycine, alanine and valine were accumulated in the culture, indicating that the control of redox potential is important in lysine fermentation. At the redox potential of - 40 mV, on the other hand, large quantities of arginine (up to 0.38g/l) and glutamic acid (up to 0.12 g/l) were produced. A maximum lysine productivity of 2.41 g/l/h was achieved at - 66 mV under a carbon-limited condition.

• Microbiology and Biotechnology Letters
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• v.26 no.1
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• pp.45-49
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• 1998

Two kinds of Mutants of Corynebacterium glutamicum, which were resistant to branched chain amino acid analogues, were obtained for L-leucine production; C. glutamicum LT26 resistant to 4-azaleucine and $\alpha$-amino-$eta$-hydroxyvaleric acid, and from which C. glutamicum LT3811-70 resistant to DL-4-thiaisoleucine were derived. Accumulation of L-leucine in the culture broths of these mutant strains, C. glutamicum LT26 and LT3811-70, were much higher than those of their parent strains even though they were non-auxotrophic mutants. Enzymatic analyses were performed to measure the activities of $\alpha$-acetohydroxy acid synthase (AHAS) and $\alpha$-isopropylmalate synthase (IPMS), which were the key enzymes for the L-isoleucine, L-valine and L-leucine biosynthetic pathways branching from a common precursor. In C. glutamicum LT26 and LT3811-70, AHAS and IPMS were found to be derepressed and desensitized to L-leucine. In addition, in C. glutamicum LT3811-70, IPMS was further more derepressed by L-leucine and AHAS was more desensitized by L-isoleucine and L-valine compared to its parent strain, C. gIEitamicum LT26.