• Journal of Microbiology and Biotechnology
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• 제29권3호
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• pp.392-400
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• 2019

Chondroitin, the precursor of chondroitin sulfate, which is an important polysaccharide, has drawn significant attention due to its applications in many fields. In the present study, a heterologous biosynthesis pathway of chondroitin was designed in a GRAS (generally recognized as safe) strain C. glutamicum. CgkfoC and CgkfoA genes with host codon preference were synthesized and driven by promoter Ptac, which was confirmed as a strong promoter via GFPuv reporter assessment. In a lactate dehydrogenase (ldh) deficient host, intracellular chondroitin titer increased from 0.25 to 0.88 g/l compared with that in a wild-type host. Moreover, precursor enhancement via overexpressing precursor synthesizing gene ugdA further improved chondroitin titers to 1.09 g/l. Chondroitin production reached 1.91 g/l with the engineered strain C. glutamicum ${\Delta}L-CgCAU$ in a 5-L fed-batch fermentation with a single distribution $M_w$ of 186 kDa. This work provides an alternative, safe and novel means of producing chondroitin for industrial applications.

• 한국미생물·생명공학회지
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• 제48권4호
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• pp.506-514
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• 2020

4-Hydroxybenzyl alcohol (4-HB alcohol)은 두통, 경련 행동, 현기증과 같은 신경계 질환에 유익한 효과를 나타내며 천마의 주요 생리활성 성분 중의 하나이다. 대사공학을 통해 4-hydroxybenzoate (4-HBA)를 생산하는 균주로부터 4-HB alcohol을 생산하는 재조합 Corynebacterium glutamicum을 개발하였다. 먼저 4-HBA를 생산하는 APS809로부터 염색체 내 NCgl2922 유전자에 Methanocaldococcus jannaschii 유래의 aroK 유전자를 삽입한 APS963을 개발하였다. 4-HBA의 카로복실 산을 4-hydroxybenzaldehyde (4-HB aldehyde)로의 환원을 촉매하는 Nocardia iowensis 유래의 car 유전자를 염색체에서 발현하는 균주를 개발하기 위해 NCgl1112 유전자 일부 단편에 car 유전자가 삽입된 GAS177를 개발하였다. 더 높은 농도의 4-HB alcohol을 생산하기 위해 4-HB alcohol을 aldehyde로 산화를 촉매하는데 관여하는 creG 유전자를 염색체상에서 제거된 GAS255를 개발하였다. 최종적으로 chorismate를 4-HBA로 전환하는 효소의 유전자 ubiCpr을 pcaHG에 삽입된 GAS355를 개발하였으며, 80 g/l 포도당을 함유한 삼각플라스크에서 발효하여 생산성을 평가한 결과, 2.3 g/l 4-HB alcohol이 생산되었으며 부산물로 0.32 g/l 4-HBA, 0.3 g/l 4-HB aldehyde가 축적되었다.

• Journal of Microbiology and Biotechnology
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• 제20권8호
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• pp.1196-1203
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• 2010

In the present work, methanethiol and dimethyldisulfide were investigated as sulfur sources for methionine synthesis in Corynebacterium glutamicum. In silico pathway analysis predicted a high methionine yield for these reduced compounds, provided that they could be utilized. Wild-type cells were able to grow on both methanethiol and dimethyldisulfide as sole sulfur sources. Isotope labeling studies with mutant strains, exhibiting targeted modification of methionine biosynthesis, gave detailed insight into the underlying pathways involved in the assimilation of methanethiol and dimethyldisulfide. Both sulfur compounds are incorporated as an entire molecule, adding the terminal S-$CH_3$ group to O-acetylhomoserine. In this reaction, methionine is directly formed. MetY (O-acetylhomoserine sulfhydrylase) was identified as the enzyme catalyzing the reaction. The deletion of metY resulted in methionine auxotrophic strains grown on methanethiol or dimethyldisulfide as sole sulfur sources. Plasmid-based overexpression of metY in the ${\Delta}$metY background restored the capacity to grow on methanethiol or dimethyldisulfide as sole sulfur sources. In vitro studies with the C. glutamicum wild type revealed a relatively low activity of MetY for methanethiol (63 mU/mg) and dimethyldisulfide (61 mU/mg). Overexpression of metY increased the in vitro activity to 1,780 mU/mg and was beneficial for methionine production, since the intracellular methionine pool was increased 2-fold in the engineered strain. This positive effect was limited by a depletion of the metY substrate O-acetylhomoserine, suggesting a need for further metabolic engineering targets towards competitive production strains.