Semi-rational engineering of cytochrome CYP153A from Marinobacter aquaeolei for improved ω-hydroxylation activity towards oleic acid

Duan, Yan; Ba, Lina; Gao, Jianwei; Gao, Xianxing; Zhu, Dunming; Jong, René M. de; Mink, Daniel; Kaluzna, Iwona; Lin, Zhanglin

doi:10.1007/s00253-016-7634-1

Cited by 14 publications

(9 citation statements)

References 40 publications

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“…Activity SM, ISM w-hydroxylation of oleic acid, NNK [111] Laccase (Pycnoporusc innabarinus)A ctivity ISM oxidation of sinapic acid [112] Alcohol Limonene epoxide hydrolase enantioselectivity,activity,thermostability CAST/ISMm ultiparameter optimization [119] Nitrilase (Pyrococcus abyssi)a ctivity,enantioselectivity ISM dynamic kinetic resolution [122] N-oligosaccharyltransferase PglB (Campylobacter jejuni) activity ISM application in vaccine [123] Halohydrind ehalogenase (Arthrobacterium radiobacter HheC) enantioselectivity,activity CAST/ISMD C-analyzerapplied [124] Nitrile hydratase regioselectivity ISM in conjunction with mutability landscapes [125] Tagatose epimerase (Pseudomonas cichorii)…”

Section: Commentmentioning

confidence: 99%

The Crucial Role of Methodology Development in Directed Evolution of Selective Enzymes

et al. 2020

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Directed evolution of stereo‐, regio‐, and chemoselective enzymes constitutes a unique way to generate biocatalysts for synthetically interesting transformations in organic chemistry and biotechnology. In order for this protein engineering technique to be efficient, fast, and reliable, and also of relevance to synthetic organic chemistry, methodology development was and still is necessary. Following a description of early key contributions, this review focuses on recent developments. It includes optimization of molecular biological methods for gene mutagenesis and the design of efficient strategies for their application, resulting in notable reduction of the screening effort (bottleneck of directed evolution). When aiming for laboratory evolution of selectivity and activity, second‐generation versions of Combinatorial Active‐Site Saturation Test (CAST) and Iterative Saturation Mutagenesis (ISM), both involving saturation mutagenesis (SM) at sites lining the binding pocket, have emerged as preferred approaches, aided by in silico methods such as machine learning. The recently proposed Focused Rational Iterative Site‐specific Mutagenesis (FRISM) constitutes a fusion of rational design and directed evolution. On‐chip solid‐phase chemical gene synthesis for rapid library construction enhances library quality notably by eliminating undesired amino acid bias, the future of directed evolution?

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Section: Commentmentioning

confidence: 99%

The Crucial Role of Methodology Development in Directed Evolution of Selective Enzymes

et al. 2020

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“…Of the two families, the soluble bacterial CYP153s have received more attention, probably due in part to the inherent difficulties often encountered with the heterologous expression and purification of membrane associated fungal CYPs, to which the CYP52 family belongs. Some CYP153s have also been used in the terminal hydroxylation of fatty acids to produce ω-hydroxy fatty acids (ω-HFAs) [7], with further improvement towards this class of substrates through structure guided and semi-rational evolution [8][9][10].…”

Section: Introductionmentioning

confidence: 99%

Deconstruction of the CYP153A6 Alkane Hydroxylase System: Limitations and Optimization of In Vitro Alkane Hydroxylation

Kochius¹,

Marwijk²,

Ebrecht³

et al. 2018

Catalysts

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Some of the most promising results for bacterial alkane hydroxylation to alcohols have been obtained with the cytochrome P450 monooxygenase CYP153A6. CYP153A6 belongs to the class I CYPs and is generally expressed from an operon that also encodes the ferredoxin (Fdx) and ferredoxin reductase (FdR) which transfer electrons to CYP153A6. In this study, purified enzymes (CYP, Fdx, FdR and dehydrogenases for cofactor regeneration) were used to deconstruct the CYP153A6 system into its separate components, to investigate the factors limiting octane hydroxylation in vitro. Proteins in the cytoplasm (cell-free extract) were found to better enhance and stabilize hydroxylase activity compared to bovine serum albumin (BSA) and catalase. Optimization of the CYP:Fdx:FdR ratio also significantly improved both turnover frequencies (TFs) and total turnover numbers (TTNs) with the ratio of 1:1:60 giving the highest values of 3872 h−1 and 45,828 moloctanol molCYP−1, respectively. Choice and concentration of dehydrogenase for cofactor regeneration also significantly influenced the reaction. Glucose dehydrogenase concentrations had to be as low as possible to avoid fast acidification of the reaction medium, which in the extreme caused precipitation of the CYP and other proteins. Cofactor regeneration based on glycerol failed, likely due to accumulation of dihydroxyacetone. Scaling the reactions up from 1 mL in vials to 60 mL in shake flasks and 120 mL in bioreactors showed that mixing and shear forces will be important obstacles to overcome in preparative scale reactions.

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“…In this sense, combination of the pNP-ester derivative hydrolysis with a prior agar screening assay led to the identification of a variant of the Rhizopus oryzae lipase displaying a 100% stability improvement towards octanal and other aldehydes (Di Lorenzo et al, 2007). Also, a NADPH oxidation assay monitoring the absorbance at 340 nm followed by GC analysis was applied to determine the ω-hydroxylation activity towards oleic acid of the cytochrome P450 monooxygenase from Marinobacter aquaeolei (CYP153A) (Duan et al, 2016). Alternatively, the activity of a P450-BM3 variants library for terminal palmitic acid oxidation was analyzed by LM-CS, using isotope labeled palmitic acid (Brühlmann et al, 2014).…”

Section: Library Screeningmentioning

confidence: 99%

Protein engineering of enzymes involved in lipid modification

Oroz‐Guinea

Zorn

Brundiek³

2018

Lipid Modification by Enzymes and Engineered Microbes

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This chapter provides an overview of the most important protein engineering tools and strategies applied to enzymes involved in lipid modification. Thus, application of several methodologies is discussed, including directed evolution, rational protein design, construction of chimeric enzymes and de novo design, together with the utility of bioinformatic tools along the protein engineering process. Additionally, different approaches for the creation of semi-rational libraries and the potential use of different high-throughput screenings for the detection of improved protein variants are described. The examples provided cover the recent achievements in the utilization of these techniques on a broad variety of enzymes. This comprises not only engineered lipases with altered fatty acid chain length selectivity, fatty acid specificity and improved performance in esterification reactions, but also successfully altered phospholipases, lipoxygenases, P450 monooxygenases, decarboxylating enzymes and fatty acid hydratases.

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Semi-rational engineering of cytochrome CYP153A from Marinobacter aquaeolei for improved ω-hydroxylation activity towards oleic acid

Cited by 14 publications

References 40 publications

The Crucial Role of Methodology Development in Directed Evolution of Selective Enzymes

The Crucial Role of Methodology Development in Directed Evolution of Selective Enzymes

Deconstruction of the CYP153A6 Alkane Hydroxylase System: Limitations and Optimization of In Vitro Alkane Hydroxylation

Protein engineering of enzymes involved in lipid modification

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