Development of a bioconversion process for production of cis-1S,2R-indandiol from indene by recombinant Escherichia coli constructs.
Recombinant Escherichia coli cells expressing the toluene dioxygenase (TDO) genes from Pseudomonas putida convert indene to cis-1S,2R-indandiol, a potentially important intermediate for the chemical synthesis of the HIV-1 protease inhibitor, Crixivan. A bioconversion process was developed through optimization of medium composition and reaction conditions at the shake-flask and 23-1 fermentor scales. A cis-1,2-indandiol productivity of approx. 1000 mg/l was achieved with construct TDO123, which represents a 50-fold increase over the initial titer. Varying the bioconversion conditions did not change the enantiomeric excess (e.e.) for the 1S,2R enantiomer from about 30%, suggesting that toluene dioxygenase intrinsically converts indene to 1S,2R- and 1R,2S-indandiols at a ratio of 2:1. Further inclusion of the Pseudomonas dehydrogenase gene in construct D160-1 led to the production of chirally pure cis-1S,2R-indandiol (e.e. > 99%) as a result of the selective degradation of the 1R,2S enantiomer, with the overall yield (650 mg/l) proportionally reduced. A single stage process was developed for D160-1 and scaled up to the 23-1 fermentor, achieving a cis-1S,2R-indandiol titer of 1200 mg/l.
Reddy
J
J
Bioprocess R&D, Merck & Co. Inc., Rahway, NJ 07065, USA.
Lee
C
C
Neeper
M
M
Greasham
R
R
Zhang
J
J
D003001:Cloning, Molecular; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007189:Indans / Q000378:metabolism*; D007192:Indenes / Q000378:metabolism*; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D016958:Pseudomonas putida / Q000201:enzymology; D013997:Time Factors; D014169:Transformation, Bacterial
D003470:Culture Media; D007189:Indans; D007192:Indenes; C016515:1-indanol; D010105:Oxygenases; C023172:toluene dioxygenase