Redox-switch regulatory mechanism of thiolase from Clostridium acetobutylicum

Kim, Sangwoo; Jang, Yu‐Sin; Ha, Sung-Chul; Ahn, Joo‐Myung; Kim, Eun-Jung; Lim, Jae Hong; Cho, Changhee; Ryu, Yong Shin; Lee, Sung Kuk; Lee, Sang Yup; Kim, Kyungjin

doi:10.1038/ncomms9410

Cited by 60 publications

(45 citation statements)

References 57 publications

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“…The mutant site of the thiolase in the BKM19 strain is valine 5 which is in the β1 of the secondary structure. This site is far from the catalytic cysteine loop and the regulatory determinant region proposed in our previous study . However, the mutant thiolase ( thlA V5A ) showed a 15% higher activity than that of wild‐type thiolase ( thlA WT ).…”

Section: Discussionmentioning

confidence: 66%

“…Compared with other thiolases, the C. acetobutylicum thiolase possesses a flexible regulatory determinant region (RDR) that undergoes a large structural change upon formation of the disulfide bond. Moreover, mutant thl V77Q/N153Y/A286K , which is regulated by non‐redox switch modulation due to a rigid RDR, exhibited a higher enzyme activity than that of the wild‐type enzyme and showed enhanced butanol production in batch fermentation . Interestingly, the position of the 5 th residue (valine) is located distal from the active site of the enzyme, indicating that this mutation might not have a direct effect on enzyme catalysis (Fig.…”

Section: Resultsmentioning

confidence: 98%

“…Two catalytic residues, Cys88 and Cys378, in thiolase are involved in the formation of a disulfide bond causing a difference in the mode of conformational changes. The mutant thiolase (V77Q/N153Y/A286K) incapable of forming a disulfide bond exhibited a higher enzyme activity than that of the wild‐type enzyme and showed enhanced butanol production in batch fermentation . Furthermore, the optimized thiolase (R133G, H156N, and G222V) identified through screening of a random mutant library showed significantly increased thiolase activity when CoA‐SH was added .…”

Section: Discussionmentioning

confidence: 99%

“…5B). In previous studies, C. acetobutylicum ATCC 824 overexpressing the thlA WT gene showed that a metabolic shift from the acidogenic to solventogenic phase was hampered during fermentation [33,34] and showed production of similar amounts of solvents and acids, 4.5 g/L n-butanol (a yield of 0.11 g/g glucose), 0.3 g/L of ethanol, 7.0 g/L butyric acid and 5.4 g/L acetic acid (Fig. 4A).…”

Section: Variations Of the Thla Gene In The Bkm19 Strainmentioning

confidence: 99%

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Genome analysis of a hyper acetone‐butanol‐ethanol (ABE) producing Clostridium acetobutylicum BKM19

Cho

Choe

Jang

et al. 2017

Biotechnology Journal

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Previously the development of a hyper acetone-butanol-ethanol (ABE) producing Clostridium acetobutylicum BKM19 strain capable of producing 30.5% more total solvent by random mutagenesis of its parental strain PJC4BK, which is a buk mutant C. acetobutylicum ATCC 824 strain is reported. Here, BKM19 and PJC4BK strains are re-sequenced by a high-throughput sequencing technique to understand the mutations responsible for enhanced solvent production. In comparison with the C. acetobutylicum PJC4BK, 13 single nucleotide variants (SNVs), one deletion and one back mutation SNV are identified in the C. acetobutylicum BKM19 genome. Except for one SNV found in the megaplasmid, all mutations are found in the chromosome of BKM19. Among them, a mutation in the thlA gene encoding thiolase is further studied with respect to enzyme activity and butanol production. The mutant thiolase (thlA ) is showed a 32% higher activity than that of the wild-type thiolase (thlA ). In batch fermentation, butanol production is increased by 26% and 23% when the thlA gene is overexpressed in the wild-type C. acetobutylicum ATCC 824 and in its derivative, the thlA-knockdown TKW-A strain, respectively. Based on structural analysis, the mutation in thiolase does not have a direct effect on the regulatory determinant region (RDR). However, the mutation at the 5 residue seems to influence the stability of the RDR, and thus, increases the enzymatic activity and enhances solvent production in the BKM19 strain.

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Section: Discussionmentioning

confidence: 66%

Section: Resultsmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Section: Variations Of the Thla Gene In The Bkm19 Strainmentioning

confidence: 99%

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Genome analysis of a hyper acetone‐butanol‐ethanol (ABE) producing Clostridium acetobutylicum BKM19

Cho

Choe

Jang

et al. 2017

Biotechnology Journal

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“…최근 본 연구진에서는 acetyl-CoA acetyl transferase (THL) [13], 3-hydroxybutyryl-CoA reductase (HBD) [9], crotonase (CRT) [ …”

Section: 의해 처음 보고되었으며 대량생산에 의한 산업화는 20세기mentioning

confidence: 99%

Crystal Structure of Thiolase from Clostridium butyricum

Kim¹,

Kim²

2016

Journal of Life Science

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Thiolase is an enzyme that catalyzes condensation reactions between two acetyl-CoA molecules to produce acetoacetyl-CoA. As thiolase catalyzes is the first reaction in the production of n-butanol, knowledge of the molecular and regulatory mechanism of the enzyme is crucial for synthesizing high-value biofuel. Thiolase from Clostridium butyricum (CbTHL) was expressed, purified, and crystallized. X-ray diffraction data were collected from the crystals, and the 3-dimentional structure of the enzyme was determined at 2.0 Å. The overall structure of thiolase was similar to that of type II biosynthetic thiolases, such as thiolase from C. acetobutylicum (CaTHL). The superposition of this structure with that of CaTHL complexed with CoA revealed the residues that comprise the catalytic and substrate binding sites of CbTHL. The catalytic site of CbTHL contains three conserved residues, Cys88, His349, and Cys379, which may function as a covalent nucleophile, general base, and second nucleophile, respectively. For substrate binding, the way in which CbTHL stabilized the ADP moiety of CoA was unlike that of other thiolases, whereas the stabilization of β-mercaptoethyamine and pantothenic acid moieties of CoA was quite similar to that of other enzymes. The most interesting observation in the CbTHL structure was that the enzyme was regulated through redox-switch modulation, using a reversible disulfide bond.Key words : Clostridium butyricum, disulfide bond, redox-switch modulation, structure, thiolase *Corresponding author *Tel : +82-53-950-5377, Fax : +82-53-955-5522 *E-mail : kkim@knu.ac.kr This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Engineering Clostridium cellulovorans for highly selective n‐butanol production from cellulose in consolidated bioprocessing

Teng

Hou

et al. 2021

Biotech & Bioengineering

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Cellulosic n-butanol from renewable lignocellulosic biomass has gained increased interest. Previously, we have engineered Clostridium cellulovorans, a cellulolytic acidogen, to overexpress the bifunctional butyraldehyde/butanol dehydrogenase gene adhE2 from C. acetobutylicum for n-butanol production from crystalline cellulose. However, butanol production by this engineered strain had a relatively low yield of approximately 0.22 g/g cellulose due to the coproduction of ethanol and acids. We hypothesized that strengthening the carbon flux through the central butyryl-CoA biosynthesis pathway and increasing intracellular NADH availability in C. cellulovorans adhE2 would enhance n-butanol production. In this study, thiolase (thlA CA ) from C. acetobutylicum and 3-hydroxybutyryl-CoA dehydrogenase (hbd CT ) from C. tyrobutyricum were overexpressed in C. cellulovorans adhE2 to increase the flux from acetyl-CoA to butyryl-CoA. In addition, ferredoxin-NAD(P) + oxidoreductase (fnr), which can regenerate the intracellular NAD(P)H and thus increase butanol biosynthesis, was also overexpressed. Metabolic flux analyses showed that mutants overexpressing these genes had a significantly increased carbon flux toward butyryl-CoA, which resulted in increased production of butyrate and butanol.The addition of methyl viologen as an electron carrier in batch fermentation further directed more carbon flux towards n-butanol biosynthesis due to increased reducing equivalent or NADH. The engineered strain C. cellulovorans adhE2-fnr CA -thlA CAhbd CT produced n-butanol from cellulose at a 50% higher yield (0.34 g/g), the highest ever obtained in batch fermentation by any known bacterial strain. The engineered C. cellulovorans is thus a promising host for n-butanol production from cellulosic biomass in consolidated bioprocessing.

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Redox-switch regulatory mechanism of thiolase from Clostridium acetobutylicum

Cited by 60 publications

References 57 publications

Genome analysis of a hyper acetone‐butanol‐ethanol (ABE) producing Clostridium acetobutylicum BKM19

Genome analysis of a hyper acetone‐butanol‐ethanol (ABE) producing Clostridium acetobutylicum BKM19

Crystal Structure of Thiolase from Clostridium butyricum

Engineering Clostridium cellulovorans for highly selective n‐butanol production from cellulose in consolidated bioprocessing

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