Regio‐ and Stereoselective Biohydroxylations with a Recombinant Escherichia coli Expressing P450pyr Monooxygenase of Sphingomonas Sp. HXN‐200

Zhang, Wei; Tang, Weng Lin; Wang, Zunsheng; Li, Zhi

doi:10.1002/adsc.201000266

Cited by 23 publications

(18 citation statements)

References 70 publications

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“…P450pyr10 mutants were generated that showed excellent enantioselectivity for the hydroxylation of N ‐benzylpyrrolidine 11. Nevertheless, it is a challenge to develop an enzyme for the nonterminal hydroxylation of alkanes with excellent regio‐ and enantioselectivity: Many monooxygenases, such as sMMO, alkB, and P450pyr, showed terminal selectivity;4c, 10e, 12 P450BM‐3 and P450cyp102A3 showed ω‐1, ω‐2, and ω‐3 selectivity;7a, 13 engineered sMMO and alkB mutants demonstrated some subterminal selectivity;4d, 14 engineered P450BM‐3 mutants gave enhanced subterminal selectivity,15 with 89 % regioselectivity and 65 % enantioselectivity as the best results for the subterminal hydroxylation of n ‐octane; and engineered P450cam mutants showed subterminal hydroxylation of n ‐butane16 and propylbenzene (78 % regioselectivity),6a but no product ee values were reported.…”

Section: Methodsmentioning

confidence: 99%

Engineering of P450pyr Hydroxylase for the Highly Regio‐ and Enantioselective Subterminal Hydroxylation of Alkanes

Yang

Liu

2014

Angew Chem Int Ed

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Terminal-selective cytochrome P450pyr has been successfully engineered through directed evolution for the subterminal hydroxylation of alkanes with excellent regio- and enantioselectivity. A sensitive colorimetric high-throughput screening (HTS) assay was developed for the measurement of both the regioselectivity and the enantioselectivity of a hydroxylation reaction. By using the HTS assay and iterative saturation mutagenesis, sextuple-mutant P450pyrSM1 was created for the hydroxylation of n-octane (1) to give (S)-2-octanol (2) with 98 % ee and >99 % subterminal selectivity. The engineered P450 is the first enzyme for this type of highly selective alkane hydroxylation, being useful for the C-H activation and functionalization of alkanes and the preparation of enantiopure alcohols. Molecular modeling provided structure-based understanding of the fully altered regioselectivity and the excellent enantioselectivity. Another sextuple-mutant P450pyrSM2 catalyzed the hydroxylation of propylbenzene (3) to afford (S)-1-phenyl-2-propanol (4) with 95 % ee and 98 % subterminal selectivity.

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Section: Methodsmentioning

confidence: 99%

Engineering of P450pyr Hydroxylase for the Highly Regio‐ and Enantioselective Subterminal Hydroxylation of Alkanes

Yang

Liu

2014

Angew Chem Int Ed

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“…Initially, we tried to introduce a third plasmid containing the GDH gene into our previous developed recombinant E. coli (Zhang et al, 2010) containing the P450 gene in one plasmid and the genes of Fdx and FdR in another plasmid. However, the constructed strain with the three‐plasmids did not show any hydroxylation activity.…”

Section: Resultsmentioning

confidence: 99%

“…Cytochrome P450 monooxygenase is an important class of monooxygenases (Bell et al, 2010; Jung et al, 2011; Kille et al, 2011; Munro et al, 2007; Turner, 2009; Whitehouse et al, 2008; Zhang et al, 2010), with P450 BM3 (Münzer et al, 2005; Peters et al, 2003) and P450cam (Bell et al, 2001; Jones et al, 1996; Stevenson et al, 1996) as the prominent examples. We recently discovered a novel P450pyr monooxygenase from Sphingomonas sp.…”

Section: Introductionmentioning

confidence: 99%

Engineering of recombinant E. coli cells co‐expressing P450pyrTM monooxygenase and glucose dehydrogenase for highly regio‐ and stereoselective hydroxylation of alicycles with cofactor recycling

Pham

Gao

2012

Biotech & Bioengineering

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E. coli (P450pyrTM-GDH) with dual plasmids, pETDuet containing P450pyr triple mutant I83H/M305Q/A77S (P450pyrTM) and ferredoxin reductase (FdR) genes and pRSFDuet containing glucose dehydrogenase (GDH) and ferredoxin (Fdx) genes, was engineered to show a high activity (12.7 U g⁻¹ cdw) for the biohydroxylation of N-benzylpyrrolidine 1 and a GDH activity of 106 U g⁻¹ protein. The E. coli cells were used as efficient biocatalysts for highly regio- and stereoselective hydroxylation of alicyclic substrates at non-activated carbon atom with enhanced productivity via intracellular recycling of NAD(P)H. Hydroxylation of N-benzylpyrrolidine 1 with resting cells in the presence of glucose showed excellent regio- and stereoselectivity, giving (S)-N-benzyl-3-hydroxypyrrolidine 2 in 98% ee as the sole product in 9.8 mM. The productivity is much higher than that of the same biohydroxylation using E. coli (P450pyrTM)b without expressing GDH. E. coli (P450pyrTM-GDH) was found to be highly regio- and stereoselective for the hydroxylation of N-benzylpyrrolidin-2-one 3, improving the regioselectivity from 90% of the wild-type P450pyr to 100% and giving (S)-N-benzyl-4-hydroxylpyrrolidin-2-one 4 in 99% ee as the sole product. A high activity of 15.5 U g⁻¹ cdw was achieved and (S)-4 was obtained in 19.4 mM. E. coli (P450pyrTM-GDH) was also found to be highly regio- and stereoselective for the hydroxylation of N-benzylpiperidin-2-one 5, increasing the ee of the product (S)-N-benzyl-4-hydroxy-piperidin-2-one 6 to 94% from 33% of the wild-type P450pyr. A high activity of 15.8 U g⁻¹ cdw was obtained and (S)-6 was produced in 3.3 mM as the sole product. E. coli (P450pyrTM-GDH) represents the most productive system known thus far for P450-catalyzed hydroxylations with cofactor recycling, and the hydroxylations with E. coli (P450pyrTM-GDH) provide with simple and useful syntheses of (S)-2, (S)-4, and (S)-6 that are valuable pharmaceutical intermediates and difficult to prepare.

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Section: Methodsmentioning

confidence: 99%

Engineering of P450pyr Hydroxylase for the Highly Regio‐ and Enantioselective Subterminal Hydroxylation of Alkanes

Yang

Liu

2014

Angewandte Chemie

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Terminal‐selective cytochrome P450pyr has been successfully engineered through directed evolution for the subterminal hydroxylation of alkanes with excellent regio‐ and enantioselectivity. A sensitive colorimetric high‐throughput screening (HTS) assay was developed for the measurement of both the regioselectivity and the enantioselectivity of a hydroxylation reaction. By using the HTS assay and iterative saturation mutagenesis, sextuple‐mutant P450pyrSM1 was created for the hydroxylation of n‐octane (1) to give (S)‐2‐octanol (2) with 98 % ee and >99 % subterminal selectivity. The engineered P450 is the first enzyme for this type of highly selective alkane hydroxylation, being useful for the CH activation and functionalization of alkanes and the preparation of enantiopure alcohols. Molecular modeling provided structure‐based understanding of the fully altered regioselectivity and the excellent enantioselectivity. Another sextuple‐mutant P450pyrSM2 catalyzed the hydroxylation of propylbenzene (3) to afford (S)‐1‐phenyl‐2‐propanol (4) with 95 % ee and 98 % subterminal selectivity.

show abstract

Regio‐ and Stereoselective Biohydroxylations with a Recombinant Escherichia coli Expressing P450_pyr Monooxygenase of Sphingomonas Sp. HXN‐200

Cited by 23 publications

References 70 publications

Engineering of P450pyr Hydroxylase for the Highly Regio‐ and Enantioselective Subterminal Hydroxylation of Alkanes

Engineering of P450pyr Hydroxylase for the Highly Regio‐ and Enantioselective Subterminal Hydroxylation of Alkanes

Engineering of recombinant E. coli cells co‐expressing P450pyrTM monooxygenase and glucose dehydrogenase for highly regio‐ and stereoselective hydroxylation of alicycles with cofactor recycling

Engineering of P450pyr Hydroxylase for the Highly Regio‐ and Enantioselective Subterminal Hydroxylation of Alkanes

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