• Journal of Microbiology and Biotechnology
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• 제10권6호
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• pp.789-796
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• 2000

In order to analyze the effects of tktA, $aroF^{FBR}$, and aroL expression in a tryptophan-producing Escherichia coli, a series of plasmids carrying the genes were constructed. Introduction of tktA, $aroF^{FBR}$, and aroL into the E. coli strain resulted in approximately 10-20 fold increase in the activities of transketolase, the feedback inhibition-resistant 3-deoxy-D-arabinoheptulsonate-7-phosphate synthase, and shikimate kinase. Expression of $aroF^{FBR}$ in the aroB mutant strain of E. coli resulted in the accumulation of 10 mM of 3-deoxy-D-arabinoheptulsonate-7-phosphate (DAHP) in the medium. Simultaneous expression of tktA and $aroF^{FBR}$ in the strain further increased the amount of excreted DAHP to 20 mM. In contrast, the mutant strain which has no gene introduced accumulated 0.5 mM of DAHP. However, the expression of tktA and $aroF^{FBR}$ in a tryptophan-producing E. coli strain did not lead to the increased production of tryptophan, but instead, a significant amount of shikimate, which is an intermediate in the tryptophan biosynthetic pathway, was excreted to the growth medium. Despite the fact that additional expression of shikimate kinase in the strain could possibly remove 90% of excreted shikimate to 0.1 mM, the amount of tryptophan produced was still unchanged. Removing shikimate using a cloned aroL gene caused the excretion of glutamate, which suggests disturbed central carbon metabolism. However, when cultivated in a complex medium, the strain expressing tktA, $aroF^{FBR}$, and aroL produced more tryptophan than the parental strain. These data indicate that additional rate-limiting steps are present in the tryptophan biosynthetic pathway, and the carbon flow to the terminal pathway is strictly regulated. Expressing tktA in E. coli cells appeared to impose a great metabolic burden to the cells as evidenced by retarded cell growth in the defined medium. Recombinant E. coli strains harboring plasmids which carry the tktA gene showed a tendency to segregate their plasmids almost completely within 24h.

• 미생물학회지
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• 제32권2호
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• pp.109-114
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• 1994

대장균에서 두 가지 다른 아미노산의 생합성에 관여하는 serC 유전자와 araA유전자는 혼합 operon을 이루고 있다. SerC-aroA 혼합 operon의 발현 조절 현상을 serC-aroA-lacZ fusion plasmid pWH2를 이용하여 측정하였다. serC-aroA 혼합 operon의 발현은 L-tyrosine, L=phenulalanine 및 L-tryptophan 등 방향족 아미노산들에 의하여 억제되었다. 방향족 아미노산에 의한 억제 효과는 $tyrR^-$ 균주 혹은 $trpT^-$ 균주에서는 감소하였다. 또한, 방향족 아미노산은 cyclic AMP에 의한 이 operon의 발현 상승 효과를 감소시키기도 하였다. 이들 결과로부터 대장균 serC-aroA 혼합 operon의 발현은 방향종 아미노산들에 의해 억제된다고 추정하였다.

• Journal of Microbiology and Biotechnology
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• 제33권10호
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• pp.1370-1375
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• 2023

In this study, we aimed to enhance the accumulation of chorismate (CHR) and anthranilate (ANT), key intermediates in the shikimate pathway, by modifying a shikimate over-producing recombinant strain of Corynebacterium glutamicum [19]. To achieve this, we utilized a CRISPR-driven genome engineering approach to compensate for the deletion of shikimate kinase (AroK) as well as ANT synthases (TrpEG) and ANT phosphoribosyltransferase (TrpD). In addition, we inhibited the CHR metabolic pathway to induce CHR accumulation. Further, to optimize the shikimate pathway, we overexpressed feedback inhibition-resistant Escherichia coli AroG and AroH genes, as well as C. glutamicum AroF and AroB genes. We also overexpressed QsuC and substituted shikimate dehydrogenase (AroE). In parallel, we optimized the carbon metabolism pathway by deleting the gntR family transcriptional regulator (IolR) and overexpressing polyphosphate/ATP-dependent glucokinase (PpgK) and glucose kinase (Glk). Moreover, acetate kinase (Ack) and phosphotransacetylase (Pta) were eliminated. Through our CRISPR-driven genome re-design approach, we successfully generated C. glutamicum cell factories capable of producing up to 0.48 g/l and 0.9 g/l of CHR and ANT in 1.3 ml miniature culture systems, respectively. These findings highlight the efficacy of our rational cell factory design strategy in C. glutamicum, which provides a robust platform technology for developing high-producing strains that synthesize valuable aromatic compounds, particularly those derived from the shikimate pathway metabolites.

• Journal of Microbiology and Biotechnology
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• 제31권9호
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• pp.1305-1310
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• 2021

Shikimate is a key high-demand metabolite for synthesizing valuable antiviral drugs, such as the anti-influenza drug, oseltamivir (Tamiflu). Microbial-based strategies for shikimate production have been developed to overcome the unstable and expensive supply of shikimate derived from traditional plant extraction processes. In this study, a microbial cell factory using Corynebacterium glutamicum was designed to overproduce shikimate in a fed-batch culture system. First, the shikimate kinase gene (aroK) responsible for converting shikimate to the next step was disrupted to facilitate the accumulation of shikimate. Several genes encoding the shikimate bypass route, such as dehydroshikimate dehydratase (QsuB), pyruvate kinase (Pyk1), and quinate/shikimate dehydrogenase (QsuD), were disrupted sequentially. An artificial operon containing several shikimate pathway genes, including aroE, aroB, aroF, and aroG were overexpressed to maximize the glucose uptake and intermediate flux. The rationally designed shikimate-overproducing C. glutamicum strain grown in an optimized medium produced approximately 37.3 g/l of shikimate in 7-L fed-batch fermentation. Overall, rational cell factory design and culture process optimization for the microbial-based production of shikimate will play a key role in complementing traditional plant-derived shikimate production processes.

• 미생물학회지
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• 제28권2호
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• pp.169-173
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• 1990

E. coli를 이용하여 L-phenylalanine을 대량 생산하기 위한 재조합 플라스미드 pMW10, pMW11과 pMW 12를 구성하였다. L- Phenylalanine 생산을 위한 유전자 $aroF^{FR}$, $pheA^{FR}$은 E. coli MWEC 101-5 균주로부터 분리하였다. 재조합 플라스미드를 함유하고 있는 E.coli 대사 조절변이주들의 L-phenylalanine 생산생과 안전성을 조사하여 $aroF^{FR}$, $pheA^{FR}$유전자들의 효율을 알아보았다. MWEC 101-5/pMW 11 균주에서는 24.3 g/I의 L-phenylalanine이 생산되었으나, 플라스미드의 안정성은 73.8%였다. 본 균주의 prephemte dehydratase, 고유 활동도는 E. coli K-12에 비하여 26배 증가된 것이다.

• Journal of Microbiology and Biotechnology
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• 제29권6호
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• pp.923-932
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• 2019

Current strategies of strain improvement processes are mainly focused on enhancing the synthetic pathways of the products. However, excessive metabolic flux often creates metabolic imbalances, which lead to growth retardation and ultimately limit the yield of the product. To solve this problem, we applied a dynamic regulation strategy to produce $\text\tiny{L}$-phenylalanine ($\text\tiny{L}$-Phe) in Escherichia coli. First, we constructed a series of Phe-induced promoters that exhibited different strengths through modification of the promoter region of tyrP. Then, two engineered promoters were separately introduced into a Phe-producing strain xllp1 to dynamically control the expression level of one pathway enzyme AroK. Batch fermentation results of the strain xllp3 showed that the titer of Phe reached 61.3 g/l at 48 h, representing a titer of 1.36-fold of the strain xllp1 (45.0 g/l). Moreover, the $\text\tiny{L}$-Phe yields on glucose of xllp3 (0.22 g/g) were also greatly improved, with an increase of 1.22-fold in comparison with the xllp1 (0.18 g/g). In summary, we successfully improved the titer of Phe by using dynamic regulation of one key enzyme and this strategy can be applied for improving the performance of strains producing other aromatic amino acids and derived compounds.

• Journal of Microbiology and Biotechnology
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• 제25권9호
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• pp.1442-1448
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• 2015

The flavonoid apigenin and its O-methyl derivative, genkwanin, have various biological activities and can be sourced from some vegetables and fruits. Microorganisms are an alternative for the synthesis of flavonoids. Here, to synthesize genkwanin from tyrosine, we first synthesized apigenin from p-coumaric acid using four genes (4CL, CHS, CHI, and FNS) in Escherichia coli. After optimization of different combinations of constructs, the yield of apigenin was increased from 13 mg/l to 30 mg/l. By introducing two additional genes (TAL and POMT7) into an apigenin-producing E. coli strain, we were able to synthesize 7-O-methyl apigenin (genkwanin) from tyrosine. In addition, the tyrosine content in E. coli was modulated by overexpressing aroG and tyrA. The engineered E. coli strain synthesized approximately 41 mg/l genkwanin.

• 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.

• 생명과학회지
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• 제7권3호
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• pp.172-175
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• 1997

Transgenic hybrid poplar subclones containing herbicide glyphosate resistant gene (aroA) were treated with ozone at the concentration of 100 nL L$^{-1}$ for 6 hr for 5 consecutive days. The foreign gene expression in leaves of all treated plants was reduced both at transcriptional and translational levels confirmed by Northern and Western blot analysis, respectively, as compared to non-treated control plants. These results indicated that the expression of foreign gene in transgenic plants could be affected by the environmental stresses. Thus, the performance of transgenic plants cultivated on field conditions may be lower than they are expected.

• Journal of Microbiology and Biotechnology
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• 제24권8호
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• pp.1109-1117
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• 2014

Chlorogenic acid and hydroxylcinnamoyl shikimates are major dietary phenolics as well as antioxidants, with recently discovered biological, activities including protection against chemotheraphy side effects and prevention of cardiovascular disease and cancer. Certain fruits and vegetables produce these compounds, although a microbial system can also be utilized for synthesis of chlorogenic acid and hydroxylcinnamoyl shikimates. In this study, we engineered Escherichia coli to produce chlorogenic acid and p-coumaroyl shikimates from glucose. For the synthesis of chlorogenic acid, two E. coli strains were used; one strain for the synthesis of caffeic acid from glucose and the other strain for the synthesis of chlorogenic acid from caffeic acid and quinic acid. The final yield of chlorogenic acid using this approach was approximately 78 mg/l. To synthesize p-coumaroyl shikimates, wild-type E. coli as well as several mutants were tested. Mutant E. coli carrying deletions in three genes (tyrR, pheA, and aroL) produced 236 mg/l of p-coumaroyl shikimates.