Controlled regioselectivity of fatty acid oxidation by whole cells producing cytochrome P450BM-3 monooxygenase under varied dissolved oxygen concentrations

Schneider, Sebastian; Wubbolts, Marcel; Oesterhelt, G.; Sanglard, Dominique

doi:10.1002/(sici)1097-0290(19990805)64:3<333::aid-bit9>3.0.co;2-2

Cited by 37 publications

(17 citation statements)

References 22 publications

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“…OD 600, optical density at 600 nm. (14,19,29,35,37). Full-scale biofilm reactors have already been used in bioremediation and wastewater treatment, including various reactors for optimal gas-liquid contact (25,27).…”

Section: Figmentioning

confidence: 99%

Enhanced Benzaldehyde Tolerance in Zymomonas mobilis Biofilms and the Potential of Biofilm Applications in Fine-Chemical Production

Li¹,

Webb

Kjelleberg

et al. 2006

Appl Environ Microbiol

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Biotransformation plays an increasingly important role in the industrial production of fine chemicals due to its high product specificity and low energy requirement. One challenge in biotransformation is the toxicity of substrates and/or products to biocatalytic microorganisms and enzymes. Biofilms are known for their enhanced tolerance of hostile environments compared to planktonic free-living cells. Zymomonas mobilis was used in this study as a model organism to examine the potential of surface-associated biofilms for biotransformation of chemicals into value-added products. Z. mobilis formed a biofilm with a complex three-dimensional architecture comprised of microcolonies with an average thickness of 20 m, interspersed with water channels. Microscopic analysis and metabolic activity studies revealed that Z. mobilis biofilm cells were more tolerant to the toxic substrate benzaldehyde than planktonic cells were. When exposed to 50 mM benzaldehyde for 1 h, biofilm cells exhibited an average of 45% residual metabolic activity, while planktonic cells were completely inactivated. Three hours of exposure to 30 mM benzaldehyde resulted in sixfold-higher residual metabolic activity in biofilm cells than in planktonic cells. Cells inactivated by benzaldehyde were evenly distributed throughout the biofilm, indicating that the resistance mechanism was different from mass transfer limitation. We also found that enhanced tolerance to benzaldehyde was not due to the conversion of benzaldehyde into less toxic compounds. In the presence of glucose, Z. mobilis biofilms in continuous cultures transformed 10 mM benzaldehyde into benzyl alcohol at a steady rate of 8.11 g (g dry weight)؊1 day ؊1 with a 90% molar yield over a 45-h production period.Microbes in nature are commonly in surface-associated, matrix-enclosed structures referred to as biofilms (7,28). Bacteria in biofilms can differ profoundly in terms of phenotypic characteristics (31, 33) or adaptive responses to stress (17, 38) from their planktonic counterparts, which can provide survival advantages and protection in a range of environmental conditions (16,23). Biofilms are also commonly found on medical implants and are responsible for many persistent infections due to their increased resistance to antimicrobial agents (13).While deleterious in clinical settings, biofilms are of great interest in biotechnology. For example, biofilms have been used as biocatalysts in bioremediation and wastewater treatment processes (21,23,27) and in fermentative vinegar (9) and ethanol (20) production from agricultural materials, as well as in biosynthesis of polymers (42). To our knowledge, the use of biofilms for transformation of fine chemicals into value-added products has not been reported previously.Biotransformation using viable cells capable of cofactor regeneration is an important approach to fine-chemical production (30, 34). A common obstacle in biocatalytic fine-chemical production is the potential toxic effects of substrates and/or products on cells during the process. S...

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Section: Figmentioning

confidence: 99%

Enhanced Benzaldehyde Tolerance in Zymomonas mobilis Biofilms and the Potential of Biofilm Applications in Fine-Chemical Production

Li¹,

Webb

Kjelleberg

et al. 2006

Appl Environ Microbiol

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“…4 In-chain positions in branched fatty acids, that have been reported to be hydroxylated by cytochrome P450s (indicated with grey circles, for details see Table 7) Fig. 5 In-chain positions in fluorinated fatty acids, that have been reported to be hydroxylated by cytochrome P450s (indicated with grey circles, for details see Table 7) in a higher productivity (3.5 U g −1 cdw, 4 mM of products) at cost of selectivity, as numerous over-oxidation products were identified [118]. For improving the solubility of C12:0 in an aqueous system cyclodextrins and co-solvents were employed.…”

Section: Challenges In Productivity Of Cyp102a1mentioning

confidence: 99%

Regioselective Biocatalytic Hydroxylation of Fatty Acids by Cytochrome P450s

2017

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“…E. coli cells expressing P450 BM-3 have been used as whole-cell catalysts to produce mixtures of chiral 12-, 13-, and 14-hydroxypentadecanoic acid with high optical purity. 23 Furthermore, P450 BM-3 can efficiently be purified on a preparative scale using a single-step purification method. 24 Here we describe a screening platform for a broad range of aromatic and O-heterocyclic substrates, which, in terms of sensitivity and linear detection range, are superior to previously reported systems.…”

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confidence: 99%

Sensitive Assay for Laboratory Evolution of Hydroxylases toward Aromatic and Heterocyclic Compounds

Wong¹,

Wu²,

Roccatano³

et al. 2005

SLAS Discovery

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Powerful directed evolution methods have been developed for tailoring proteins to our needs in industrial applications. Here, the authors report a medium-throughput assay system designed for screening mutant libraries of oxygenases capable of inserting a hydroxyl group into a C-H bond of aromatic or O-heterocyclic compounds and for exploring the substrate profile of oxygenases. The assay system is based on 4-aminoantipyrine (4-AAP), a colorimetric phenol detection reagent. By using 2 detection wavelengths (509 nm and 600 nm), the authors achieved a linear response from 50 to 800 µM phenol and standard deviations below 11% in 96-well plate assays. The monooxygenase P450 BM-3 and its F87A mutant were used as a model system for medium-throughput assay development, identification of novel substrates (e.g., phenoxytoluene, phenylallyether, and coumarone), and discovery of P450 BM-3 F87A mutants with 8-fold improvement in 3-phenoxytoluene hydroxylation activity. This activity increase was achieved by screening a saturation mutagenesis library of amino acid position Y51 using the 4-AAP protocol in the 96-well format. (Journal of Biomolecular Screening 2005:246-252)

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Controlled regioselectivity of fatty acid oxidation by whole cells producing cytochrome P450BM-3 monooxygenase under varied dissolved oxygen concentrations

Cited by 37 publications

References 22 publications

Enhanced Benzaldehyde Tolerance in Zymomonas mobilis Biofilms and the Potential of Biofilm Applications in Fine-Chemical Production

Enhanced Benzaldehyde Tolerance in Zymomonas mobilis Biofilms and the Potential of Biofilm Applications in Fine-Chemical Production

Regioselective Biocatalytic Hydroxylation of Fatty Acids by Cytochrome P450s

Sensitive Assay for Laboratory Evolution of Hydroxylases toward Aromatic and Heterocyclic Compounds

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