Fed-batch bioconversion of indene to cis-indandiol

• Published in: Enzyme and microbial technology Vol. 31; no. 7; pp. 954 - 967
• Main Authors: Amanullah, A, Hewitt, C.J, Nienow, A.W, Lee, C, Chartrain, M, Buckland, B.C, Drew, S.W, Woodley, J.M
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Inc 02-12-2002; Elsevier Science
• Subjects: Biological and medical sciences; cis-Indandiol; Fed-batch bioconversion; Flow cytometry; Fundamental and applied biological sciences. Psychology; HIV protease inhibitor; Indene; Rhodococcus; HIV protease inhibitor; Flow cytometry; Rhodococcus; cis-Indandiol; Indene; Fed-batch bioconversion
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/S0141-0229(02)00183-7

The bioconversion of indene to
cis-(1
S,2
R) indandiol, a key intermediate in the synthesis of Merck’s HIV protease inhibitor, CRIXIVAN™ can be achieved during the growth of a
Rhodococcus strain. In a previous study, we reported on the application of multi-parameter flow cytometry for the measurement of indene toxicity to the strain, and found that concentrations up to 0.25
g/l of indene (0.037
g
indene/g dry cell weight) in batch bioconversions did not influence cell physiology. Using this information, this study reports on the implementation of a single phase indene fed-batch bioconversion. Cytoplasmic membrane (membrane) integrity and membrane polarisation of a large number of cells were measured during such bioconversions using multi-parameter flow cytometry and compared to a control in order to assess any toxic effects of indene feeding. The results indicate that indene supply at a rate of 0.1
g/l/h is feasible without any deleterious effects on cell physiology. The delay in indene metabolism was significantly shorter, with lower concentrations of by-product formation, when it was added to the culture in the stationary phase than when it was added at the beginning of the exponential phase of the fermentation.
cis-Indandiol production rates could be enhanced from 20
mg/l/h, in a previously reported silicone oil two-liquid phase system, up to 200
mg/l/h by a combination of suitable indene feeding rates in the stationary phase and operating with a high biomass concentration to limit the effects of toxicity. In addition, the yield of
cis-indandiol on indene (g/g) was higher at 0.48 in the single phase system compared to 0.20 in the two-liquid phase system. However, the final concentration of
cis-indandiol was considerably lower, possibly as a result of higher dehydrogenase activity resulting in an increased transformation of
cis-indandiol to 1-keto-2-hydroxy indan. This study has demonstrated that it is feasible to feed indene directly in the stationary phase of the bioconversion using high biomass concentrations to obtain enhanced
cis-indandiol formation rates as well as yields based on indene utilisation compared to a two-phase silicone oil system.