Fed-batch bioconversion of indene to cis-indandiol

Amanullah, Ashraf; Hewitt, Christopher J.; Nienow, Alvin W.; Lee, C; Chartrain, Michel; Buckland, BC; Drew, Stephen W.; Woodley, John M.

doi:10.1016/s0141-0229(02)00183-7

Cited by 14 publications

(10 citation statements)

References 21 publications

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“…The efficiency of oxygenase-based whole-cell biocatalysts in terms of volumetric productivity steadily increased during the past decades. Typically, the productivity was limited by substrate and product toxicity (Amanullah et al, 2002b;Held et al, 1998;Schmid et al, 2001;Wubbolts et al, 1996), product degradation and catabolite repression (Meyer et al, 2005;O'Connor et al, 1995;O'Leary et al, 2002;Santos et al, 2000), or by a low overall stability of recombinant biocatalysts (Bühler et al, 2003b;Panke et al, 1999;Staijen et al, 2000). By far the highest productivity (850 U/L tot , 1,220 U/L aq ) was reported in 1996 for the production of nontoxic toluene-cis-glycol from toluene catalyzed by mutant P. putida UV4 (Lilly and Woodley, 1996).…”

Section: Volumetric Productivitymentioning

confidence: 99%

“…Typically, such toxic effects of organic chemicals are circumvented by regulated substrate addition (Amanullah et al, 2002b;Carragher et al, 2001;Hack et al, 2000) and in situ product removal (Lye and Woodley, 1999;Schügerl and Hubbuch, 2005;Stark and von Stockar, 2003;Witholt et al, 1992). Alternatives include preadaptation of microorganisms using low concentrations of organic solvents (Da Silveira et al, 2003;Ramos-Gonzalez et al, 2001;Silveira et al, 2004) and the use of solvent tolerant strains as biocatalysts (Wery et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

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The efficiency of recombinantEscherichia coli as biocatalyst for stereospecific epoxidation

Park

Bühler

Habicher

et al. 2006

Biotechnol. Bioeng.

103

117

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Styrene is efficiently converted into (S)-styrene oxide by growing Escherichia coli expressing the styrene monooxygenase genes styAB of Pseudomonas sp. strain VLB120 in an organic/aqueous emulsion. Now, we investigated factors influencing the epoxidation activity of recombinant E. coli with the aim to improve the process in terms of product concentration and volumetric productivity. The catalytic activity of recombinant E. coli was not stable and decreased with reaction time. Kinetic analyses and the independence of the whole-cell activity on substrate and biocatalyst concentrations indicated that the maximal specific biocatalyst activity was not exploited under process conditions and that substrate mass transfer and enzyme inhibition did not limit bioconversion performance. Elevated styrene oxide concentrations, however, were shown to promote acetic acid formation, membrane permeabilization, and cell lysis, and to reduce growth rate and colony-forming activity. During biotransformations, when cell viability was additionally reduced by styAB overexpression, such effects coincided with decreasing specific epoxidation rates and metabolic activity. This clearly indicated that biocatalyst performance was reduced as a result of product toxicity. The results point to a product toxicity-induced biological energy shortage reducing the biocatalyst activity under process conditions. By reducing exposure time of the biocatalyst to the product and increasing biocatalyst concentrations, volumetric productivities were increased up to 1,800 micromol/min/liter aqueous phase (with an average of 8.4 g/L(aq) x h). This represents the highest productivity reported for oxygenase-based whole-cell biocatalysis involving toxic products.

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Section: Volumetric Productivitymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The efficiency of recombinantEscherichia coli as biocatalyst for stereospecific epoxidation

Park

Bühler

Habicher

et al. 2006

Biotechnol. Bioeng.

103

117

View full text Add to dashboard Cite

show abstract

“…Fed-Batch Mode. When a bioconversion is limited by substrate availability, whether due to low aqueous solubility, slow dissolution rate, or inhibition/toxicity, the slow addition (feeding) of the substrate into the reaction medium is a common solution (20,(32)(33)(34)(35)(36)(37)(38). Substrate feeding is also a good option for processes that require a low aqueous concentration of substrate for a better yield or higher ee.…”

Section: Reactor Operationmentioning

confidence: 99%

Substrate Supply for Effective Biocatalysis

Kim¹,

Pollard²,

Woodley³

2007

Biotechnology Progress

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Using biocatalysis for some chemical synthesis steps has unique advantages such as achieving higher product selectivity under ambient process conditions. However, a common limitation with such systems is the inhibition or toxicity posed by the starting substrate as well as limited aqueous solubility in many cases. In this review, we discuss the supply of substrate to bioconversions. The delivery of substrate via an auxiliary, which may be water-miscible, or a second phase such as a water-immiscible organic solvent, adsorbing resin, or a gas, is examined through recent examples in the field. Finally, guidelines for experimental planning and process considerations are suggested to facilitate the choice of substrate delivery method and accelerate process development.

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“…Enzyme synthesis and cofactor regeneration are related to host metabolism, which in turn may be affected by the toxicity of organic substrates and products. Such toxicity can efficiently be attenuated by regulated substrate feeding (1,11) and in situ product removal (7,38,61,66,74). Yet, microbial cells, especially in a nongrowing state, often lose biooxidation activity over time, which may be due to oxygenase inactivation and/or the metabolic burden imposed by redox-coenzyme withdrawal and reactive oxygen species formed via uncoupled oxygen reduction.…”

mentioning

confidence: 99%

NADH Availability Limits Asymmetric Biocatalytic Epoxidation in a Growing Recombinant Escherichia coli Strain

Bühler

Park

Blank

et al. 2008

Appl Environ Microbiol

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Styrene can efficiently be oxidized to (S)-styrene oxide by recombinant Escherichia coli expressing the styrene monooxygenase genes styAB from Pseudomonas sp. strain VLB120. Targeting microbial physiology during whole-cell redox biocatalysis, we investigated the interdependency of styrene epoxidation, growth, and carbon metabolism on the basis of mass balances obtained from continuous two-liquid-phase cultures. Full induction of styAB expression led to growth inhibition, which could be attenuated by reducing expression levels. Operation at subtoxic substrate and product concentrations and variation of the epoxidation rate via the styrene feed concentration allowed a detailed analysis of carbon metabolism and bioconversion kinetics. Fine-tuned styAB expression and increasing specific epoxidation rates resulted in decreasing biomass yields, increasing specific rates for glucose uptake and the tricarboxylic acid (TCA) cycle, and finally saturation of the TCA cycle and acetate formation. Interestingly, the biocatalysis-related NAD(P)H consumption was 3.2 to 3.7 times higher than expected from the epoxidation stoichiometry. Possible reasons include uncoupling of styrene epoxidation and NADH oxidation and increased maintenance requirements during redox biocatalysis. At epoxidation rates of above 21 mol per min per g cells (dry weight), the absence of limitations by O 2 and styrene and stagnating NAD(P)H regeneration rates indicated that NADH availability limited styrene epoxidation. During glucose-limited growth, oxygenase catalysis might induce regulatory stress responses, which attenuate excessive glucose catabolism and thus limit NADH regeneration. Optimizing metabolic and/or regulatory networks for efficient redox biocatalysis instead of growth (yield) is likely to be the key for maintaining high oxygenase activities in recombinant E. coli.Oxygenases catalyze regio-and enantioselective oxyfunctionalization reactions and have a considerable potential in the area of asymmetric organic synthesis (2, 35). These enzymes typically depend on coenzymes such as NAD(P)H and are unstable outside cells, and they often consist of multiple components. Thus, for synthetic applications, their use in whole cells is favored over the use of isolated enzymes (7,18).The productivity of oxygenase-based whole-cell biocatalysts is influenced by various factors, such as maximal enzyme activity, enzyme level, substrate mass transfer, substrate and/or product inhibition/toxicity, and cofactor regeneration (7,13,54,68). Enzyme synthesis and cofactor regeneration are related to host metabolism, which in turn may be affected by the toxicity of organic substrates and products. Such toxicity can efficiently be attenuated by regulated substrate feeding (1, 11) and in situ product removal (7,38,61,66,74). Yet, microbial cells, especially in a nongrowing state, often lose biooxidation activity over time, which may be due to oxygenase inactivation and/or the metabolic burden imposed by redox-coenzyme withdrawal and reactive oxygen species formed via...

show abstract

Fed-batch bioconversion of indene to cis-indandiol

Cited by 14 publications

References 21 publications

The efficiency of recombinantEscherichia coli as biocatalyst for stereospecific epoxidation

The efficiency of recombinantEscherichia coli as biocatalyst for stereospecific epoxidation

Substrate Supply for Effective Biocatalysis

NADH Availability Limits Asymmetric Biocatalytic Epoxidation in a Growing Recombinant Escherichia coli Strain

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