• KSBB Journal
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• 제11권5호
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• pp.613-621
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• 1996

Wild-type 곰팡이인 Tolypoc/adium inflatum의 고정상배양을 통해 탄소원, 질소원, 아미노산 등이 peptide 항생제 계통 의 면역억제제인 cyclosporin A(CyA) 생합성에 미치는 영향을 조사하였다. 질소 원으로서 ammonium sulfate가 첨가된 경우에, f fructose 또는maltose 등을 탄소원으로 이용하는 배 지가 glucose가 첨가된 배지에 비해 월등히 높은 C CyA 생산성을 보였으나. ammonium sulfate의 부 재시에는 탄소원들의 종류에 관계없이 CyA 생산성 이 매우 낮게 나타났다. 질소원의 경우는 무기 질소 원인 ammonium sulfate가 CyA 생산에 가장 훌륭 한 성분으로 작용했으며, 그밖에 ammonium phos p phate, ammonium citrate 역시 CyA 생산을 어느 정도 증가시키는 효과를 보였다. 최척 ammonIum s sulfate의 농도는 109/L로 밝혀 졌으며 배양초기 에 a ammonium sulfate릎 첨가해 주는 경우가 배양중간 에 첨가하는 경우보다 CyA 생산에 더 효율적인 것 으로 나타났다. 아미노산의 경우 L-valine에 의한 C CyA 생합성 증진 효과가 가장 투렷하게 나타났다. 최적 L-valine 농도는 109/L이였으며 L-valine이 배양 초반부터 배지 중에 존재하는 것이 CyA 생합 성에 가장 유리한 것으로 나타났다.

• Journal of Microbiology and Biotechnology
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• 제29권8호
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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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• 제26권4호
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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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• 제28권11호
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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.

• 약학회지
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• 제27권3호
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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.

• 한국미생물·생명공학회지
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• 제37권2호
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• pp.140-146
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• 2009

Cyclosporin A 분자의 구성 아미노산 가운데 분지 아미노산인 valine, leucine, isoleucine, threonine 등에 대한 첨가 효과를 확인한 결과, L-valine 첨가구는 비첨가구보다 cyclosporin A 역가는 3.7배, cyclosporin A 구성비율은 약 40%로 가장 크게 증가하였다. 그리고 L-valine의 최적 농도는 0.75%로서 비첨가구에 비하여 cyclosporin A 역가는 3배, cyclosporin A 구성비율은 30% 증가하였다. 그러나 L-valine 1% 단독 첨가구보다 L-valine 0.5%, L-leucine 0.5% 혼합구가 cyclosporin A 역가에서는 약 16% 우수하였고, cyclosporin A 구성비율에서는 약 5% 증가하였다. 또한 다양한 유기질소원의 첨가 효과를 확인한 결과, meat peptone인 Bacto-peptone 첨가구는 유기질소원 비첨가구보다 cyclosporin A의 역가는 약 2배, cyclosporin A 구성비율은 약 13% 증가하였고, Bacto-peptone의 최적농도는 1%인 것으로 확인되었다. 그러나 Casiton, Tryptone, NZ-amine 등 우유 유래 유기질소원 첨가구의 경우, 비첨가구보다 cyclosporin A 역가가 오히려 약 10-20% 감소하는 현상을 보여 cyclosporin A 생합성을 억제하는 것으로 확인되었다. 본 연구에서 최적화된 배지 조건에서, 모균주인 T. inflatum ATCC 34921와 UV 조사로 얻어진 4종의 cyclosporin A 고생산 변이주 4종의 생산성을 비교한 결과, 변이주 YHC-004의 cyclosporin A 역가는 3,430 mg/L로 모균주인 ATCC 34921보다 약 7.1배로 증가하였지만, 모균주와 변이주 4종의 cyclosporin A 구성비율은 약 93-94% 범위에서 비슷한 값을 보였다.

• Journal of Microbiology and Biotechnology
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• 제20권7호
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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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• 제18권6호
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• pp.455-460
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• 2003

간장의 속성 발효를 위한 방법으로 간장 발효에 간여하는 Z. rouxii BH-90과 C. versatilis BH-91 혼합 균체를 동시 고정화하여 이를 air bubble column reactor에 충진하여 국 고온 분해액을 주입하면서 연속적으로 발효를 시도하였다. 균체의 고정화 담체로는 실리카겔： 3% 알긴산소다 (1:1의 비율) 혼합액을 사용하여 Z. rouxii BH-90과 C. versatilis BH-91을 동시 고정화하여 air bubble column reactor에 충진하여 발효시킨 결과 발효 96시간 경과시 ethyl alcohol이 2.4% 생성되었으며 4-ethylguaiacol은 18mg/L가 생성되었다. Air bubble column reactor에서 Z. rouxii BH-90과 C. versatilis BH-91 혼합균체의 동시 고정화 beads의 연속적 속성 발효시 시작일로부터 30일까지는 ethyl alcohol이 2.5∼2.8%로 거의 일정하게 생성되었고, 40∼70일에는 약간 감소하였다. 그리고 동시 고정화 균체의 발효에서는 40일까지는 4-ethylguaiacol의 함량이 16∼18mg/L로 거의 일정하게 생성되었으나 발효 45일 이후 70일까지는 약간 감소하였다. 발효종료 후의 제품을 분석한 결과 동시 고정화 균체의 발효액에서는 2.4%의 ethyl alcohol이 생성되었고, 18mg/L의 4-ethylguaiacol이 생산되었다. 유리 아미노산의 함량은 glutamic acid, leucine, arginine, aspartic acid, Iysin 및 valine이 전체 아미노산의 50% 이상을 차지하고 있었다.

• 한국미생물·생명공학회지
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• 제19권1호
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• pp.76-81
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• 1991

2l 발효조에서 pH6.9, 온도 $32^{\circ}C$일 때 당밀배지를 이용하여 Corynebacterium glutamicum의 영양요구성 유사체 내성변이주에 의한 라이신 발효시 산화환원 전위 (ORP)가 라이신 발효속도의 특성에 미치는 영향을 조사하였다. 희석률이 0.1$h ^1$일때 탄소원이 제한되건 로이신이 제한되건 산소가 제한되지 않는 한 최대의 대당수율 24를 보였으며, 이 때의 산화환원 전윈 값은 -60mV와 -100mV 범위에 해당하였다. 산화화원 전위 값이 -130mV의 매우 낮은 용존산소 조건하에서는 대당수율 밀 $q_s, q_p$ 등의 발효 반응속도 상수값들이 크게 감소하였으며 glvcine, alanine, valine을 포함하는 발효 부산물의 축적량이 매우 높아졌다.

• 한국미생물·생명공학회지
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• 제26권1호
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• pp.45-49
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• 1998

Corynebacterium glutamicum ATCC13032를 모균주로 한 아미노산 유사체들에 저항성을 지닌 돌연변이 균주들로부터 두 종류의 L-로이신 생산균주를 개발하였다. 그 중 하나인 C. glutamicum LT26은 4-azaleucine과 $\alpha$-amino-$eta$-hydroxyvaleric acid에 저항성을 지니는 균주이며, 다른 한 균주는 C. glutamicum LT3811-70로서 C. glutamicum LT26을 모균주로한 DL-4-tiaisoleucine 저항성 돌연변이 균주이다. 이들 두 돌연변이 균주들의 배양액내에서의 L-로이신의 축적은 이들이 비영양요구성 균주임에도 불구하고 모균주보다 획기적으로 높았으며 이를 해명하고자 L-이소로이신과 L-발린 그리고 L-로이신 생합성 과정의 주반응 효소인 $\alpha$-acetohydroxy acid synthase(AHAS)와 $\alpha$-isopropylmalate synthase(IPMS)의 분석을 수행하였다. C. glutamicum LT26과 LT3811-70에서 AHAS와 IPMS는 모두 L-로이신에 대해 효소활성저해와 효소합성저해가 거의 해제되었고, C. glutamicum LT3811-70 균주의 경우 모균주인 C. glutamicum LT26 균주보다 IPMS의 L-로이신에 대해 효소 합성저해가, AHAS는 L-이소로이신과 L-발린등에 대해 효소활성저해가 10% 이상 더 해제되었음을 알 수 있었다.