Engineering the "Missing Link" in Biosynthetic (-)-Menthol Production: Bacterial Isopulegone Isomerase.
Engineering the "Missing Link" in Biosynthetic (-)-Menthol Production: Bacterial Isopulegone Isomerase.
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DOI：
10.1021/acscatal.7b04115
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发表时间：
2018-03-02
期刊：
影响因子：
12.9
通讯作者：
Scrutton NS
中科院分区：
文献类型：
--
作者：
Currin A;Dunstan MS;Johannissen LO;Hollywood KA;Vinaixa M;Jervis AJ;Swainston N;Rattray NJW;Gardiner JM;Kell DB;Takano E;Toogood HS;Scrutton NS
The realization of a synthetic biology approach to microbial (1R,2S,5R)-(−)-menthol (1) production relies on the identification of a gene encoding an isopulegone isomerase (IPGI), the only enzyme in the Mentha piperita biosynthetic pathway as yet unidentified. We demonstrate that Δ5-3-ketosteroid isomerase (KSI) from Pseudomonas putida can act as an IPGI, producing (R)-(+)-pulegone ((R)-2) from (+)-cis-isopulegone (3). Using a robotics-driven semirational design strategy, we identified a key KSI variant encoding four active site mutations, which confer a 4.3-fold increase in activity over the wild-type enzyme. This was assisted by the generation of crystal structures of four KSI variants, combined with molecular modeling of 3 binding to identify key active site residue targets. The KSI variant was demonstrated to function efficiently within cascade biocatalytic reactions with downstream Mentha enzymes pulegone reductase and (−)-menthone:(−)-menthol reductase to generate 1 from 3. This study introduces the use of a recombinant IPGI, engineered to function efficiently within a biosynthetic pathway for the production of 1 in microorganisms.
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DOI：
10.1016/j.bbrc.2012.05.010
发表时间：
2012-06-08
影响因子：
3.1
作者：
Kim, Su-Hwa;Lee, Sanghoon;Jeon, Ju-Hong
通讯作者：
Jeon, Ju-Hong
DOI：
10.1126/science.1191652
发表时间：
2010-10-01
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Ajikumar PK;Xiao WH;Tyo KE;Wang Y;Simeon F;Leonard E;Mucha O;Phon TH;Pfeifer B;Stephanopoulos G
通讯作者：
Stephanopoulos G
影响因子：
2.4
作者：
Currin, Andrew;Swainston, Neil;Kell, Douglas B.
通讯作者：
Kell, Douglas B.
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH
影响因子：
3.9
作者：
KJONAAS, RB;VENKATACHALAM, KV;CROTEAU, R
通讯作者：
CROTEAU, R