Development of a bioconversion process for production of cis -1 S ,2 R -indandiol from indene by recombinant Escherichia coli constructs

Abstract

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.

Biodegradation of Aromatic Compounds by Escherichia coli

“…Despite their difficulty of handling, these biocatalysts are of great biotechnological value because of their regio-and stereoselectivity. A number of E. coli biocatalysts have been designed to express aromatic ring mono-and/or dioxygenases for the biotransformation of particular aromatic compounds to cis-dihydrodiols, epoxides, and catechols that have diverse environmental and pharmaceutical applications (224,258,348). The biosynthesis of epoxides, versatile chemical building blocks in the manufacture of optically active compounds, illustrates this point.…”

Regioselectivity and Enantioselectivity of Naphthalene Dioxygenase during Arene cis -Dihydroxylation: Control by Phenylalanine 352 in the α Subunit

“…The use of dioxygenases to initiate biocatalytic routes for the production of pharmaceuticals and natural products has received significant attention of late (9,12,25,42), and the possibility of generating new synthons with opposite stereochemistry is an attractive alternative to asymmetric chemical synthesis (7).…”

Recombinant Chlorobenzene Dioxygenase from Pseudomonas sp. P51: A Biocatalyst for Regioselective Oxidation of Aromatic Nitriles

“…[29,38,39] However, in most expression systems used so far, such as pDTG601, [40] pKST11, [41] pDTG141, [42] p1/1, [39] pLAC365, pTAC365, [43] and pTCB144, [14] the dioxygenase genes were cloned downstream of promoters such as the lac or tac promoter. Such promoters are known to be not tightly regulated and the leakiness of these regulatory systems have been shown before.…”