The molecular basis and enzyme engineering strategies for improvement of coupling efficiency in cytochrome P450s

Meng, Shuaiqi; Ji, Yu; Zhu, Leilei; Dhoke, Gaurao V.; Davari, Mehdi D.; Schwaneberg, Ulrich

doi:10.1016/j.biotechadv.2022.108051

Cited by 19 publications

(30 citation statements)

References 128 publications

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“…In general, E.coli BL21 and Rosetta strains are the most commonly used strains for heterologous expression. The pET-28a vector harboring the P450 AZC1 gene was transformed into E.coli BL21 DE3 and E.coli Rosetta DE3 plysS strains and four different temperatures (18,20,25, and 30 °C) were tested. Recombinant protein expression (24 h.) in TB media was induced with IPTG (0.1 mM) and the media was supplemented with ALA (0.5 mM) and FeCl 3 (0.5 mM).…”

Section: Enhanced Expression Of the P450 Azc1mentioning

confidence: 99%

“…Although P450s are powerful tools for industry, owing to accepting various substrates and catalyzing a diverse range of reactions, there are several drawbacks to their large‐scale industrial applications. These limitations are low catalytic activity towards non‐natural substrates, low stability in organic solvents, requirement of expensive nicotinamide cofactor and its efficient regeneration, low substrate solubility in aqueous media, and low coupling efficiency [7,12,13,19,20] . In addition, efficient heterologous expression of P450s as active protein and complicated protein purification process are other limitations that hinder widespread industrial application of P450s [21]…”

Section: Introductionmentioning

confidence: 99%

“…These limitations are low catalytic activity towards non-natural substrates, low stability in organic solvents, requirement of expensive nicotinamide cofactor and its efficient regeneration, low substrate solubility in aqueous media, and low coupling efficiency. [7,12,13,19,20] In addition, efficient heterologous expression of P450s as active protein and complicated protein purification process are other limitations that hinder widespread industrial application of P450s. [21] Most of the P450 monooxygenases require an external electron transfer protein for catalytic activity and use NAD(P)H as an electron donor.…”

Section: Introductionmentioning

confidence: 99%

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Expression and Characterization of a New Self‐Sufficient P450 Monooxygenase (P450 AZC1) from Azorhizobium caulinodans

Yıldırım,

Ozic,

Ensari

2023

ChemBioChem

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Oxyfunctionalization of non-activated carbon bonds by P450 monooxygenases has drawn great industrial attraction. Selfsufficient P450s containing catalytic heme and reductase domains in a single polypeptide chain offer many advantages since they do not require external electron transfer partners.Here, we report the first P450 enzyme identified and expressed from Azorhizobium caulinodans. Firstly, expression conditions of P450 AZC1 were optimized for enhanced expression in E.coli. The highest P450 content was obtained in E.coli Rosetta DE3 plysS when it was incubated in TB media supplemented with 0.75 mM IPTG, 0.5 mM ALA, and 0.75 mM FeCl 3 at 25 °C for 24 hours. Subsequently, the purified enzyme showed a broad substrate spectrum including fatty acids, linear and cyclic alkanes, aromatics, and pharmaceuticals. Finally, P450 AZC1 showed optimal activity at pH 6.0 and 40 °C and a broad pH and temperature profile, making it a promising candidate for industrial applications.

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Section: Enhanced Expression Of the P450 Azc1mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Expression and Characterization of a New Self‐Sufficient P450 Monooxygenase (P450 AZC1) from Azorhizobium caulinodans

Yıldırım,

Ozic,

Ensari

2023

ChemBioChem

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“…As increases in activity can easily come at the cost of desired properties like efficiency or regioselectivity, a more holistic approach in designing these parameters simultaneously has been suggested. [321] With the number of reports on ML-based protein engineering steadily increasing, examples of ML-engineered enzymes with non-proteogenic parts begin to enter the literature. A ML algorithm specifically trained on the NADPH-dependent family of alcohol-forming fatty acyl reductases (FARs) predicted mutations that resulted in a twofold increase in activity.…”

Section: Examplesmentioning

confidence: 99%

“…Despite these successes, the complex structure and mechanism of P450s have largely prevented the design of mutants fit for industrial applications. As increases in activity can easily come at the cost of desired properties like efficiency or regioselectivity, a more holistic approach in designing these parameters simultaneously has been suggested [321] …”

Section: Examplesmentioning

confidence: 99%

Curse and Blessing of Non‐Proteinogenic Parts in Computational Enzyme Engineering

Korbeld

Fürst

2023

ChemBioChem

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Enzyme engineering aims to improve or install a new function in biocatalysts for applications ranging from chemical synthesis to biomedicine. For decades, computational techniques have been developed to predict the effect of protein changes and design new enzymes. However, these techniques may have been optimized to deal with proteins composed of the standard amino acid alphabet, while the function of many enzymes relies on non‐proteogenic parts like cofactors, nucleic acids, and post‐translational modifications. Enzyme systems containing such molecules might be handled or modeled improperly by computational tools, and thus be unsuitable, or require additional tweaking, parameterization, or preparation. In this review, we give an overview of common and recent tools and workflows available to computational enzyme engineers. We highlight the various pitfalls that come with including non‐proteogenic compounds in computations and outline potential ways to address common issues. Finally, we showcase successful examples from the literature that computationally engineered such enzymes.

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Systems engineering Escherichia coli for efficient production p‐coumaric acid from glucose

Qiu,

Wang,

Zuo

et al. 2024

Biotech & Bioengineering

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P‐coumaric acid (p‐CA), a pant metabolite with antioxidant and anti‐inflammatory activity, is extensively utilized in biomedicine, food, and cosmetics industry. In this study, a synthetic pathway (PAL) for p‐CA was designed, integrating three enzymes (AtPAL2, AtC4H, AtATR2) into a higher l‐phenylalanine‐producing strain Escherichia coli PHE05. However, the lower soluble expression and activity of AtC4H in the PAL pathway was a bottleneck for increasing p‐CA titers. To overcome this limitation, the soluble expression of AtC4H was enhanced through N‐terminal modifications. And an optimal mutant, AtC4HL373T/G211H, which exhibited a 4.3‐fold higher kcat/Km value compared to the wild type, was developed. In addition, metabolic engineering strategies were employed to increase the intracellular NADPH pool. Overexpression of ppnk in engineered E. coli PHCA20 led to a 13.9‐folds, 1.3‐folds, and 29.1% in NADPH content, the NADPH/NADP+ ratio and p‐CA titer, respectively. These optimizations significantly enhance p‐CA production, in a 5‐L fermenter using fed‐batch fermentation, the p‐CA titer, yield and productivity of engineered strain E. coli PHCA20 were 3.09 g/L, 20.01 mg/g glucose, and 49.05 mg/L/h, respectively. The results presented here provide a novel way to efficiently produce the plant metabolites using an industrial strain.

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The molecular basis and enzyme engineering strategies for improvement of coupling efficiency in cytochrome P450s

Cited by 19 publications

References 128 publications

Expression and Characterization of a New Self‐Sufficient P450 Monooxygenase (P450 AZC1) from Azorhizobium caulinodans

Expression and Characterization of a New Self‐Sufficient P450 Monooxygenase (P450 AZC1) from Azorhizobium caulinodans

Curse and Blessing of Non‐Proteinogenic Parts in Computational Enzyme Engineering

Systems engineering Escherichia coli for efficient production p‐coumaric acid from glucose

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