Frontispiece: Whole‐Cell Biotransformation of Benzene to Phenol Catalysed by Intracellular Cytochrome P450BM3 Activated by External Additives

Karasawa, Masayuki; Stanfield, Joshua Kyle; Yanagisawa, Sota; Okada, Shoji; Watanabe, Yoshihito

doi:10.1002/anie.201883861

Cited by 2 publications

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“…As the extensive applications of these hemoproteins in the fields of artificial meat alternatives, [ 10,47 ] high‐cell‐density fermentation, [ 24–26 ] and biocatalysts, [ 13–16 ] it is imperative to investigate their biochemical properties thoroughly. First, the absorption spectrum of these hemoproteins was detected between 350 and 700 nm (Figure 5b).…”

Section: Resultsmentioning

confidence: 99%

“…[ 10 ] In addition, Vitreoscilla hemoglobin (V‐Hb), with strong oxygen transfer capacity, is a powerful tool in metabolic engineering to enhance cell growth and product synthesis. [ 11,12 ] Furthermore, the cytochrome P450‐BM3 monooxygenase derived from Bacillus megaterium can catalyze regio‐ and stereo‐selective hydroxylation reactions, making it an efficient biocatalyst to synthesize various valuable compounds such as drugs (steroidal C7β alcohols), [ 13 ] the natural product (phenol), [ 14 ] and chemical intermediates (cyclooctanone and hydroquinone). [ 15,16 ]…”

Section: Introductionmentioning

confidence: 99%

“…[ 26 ] As for whole‐cell P450s catalysis, E. coli is a commonly utilized host to heterologously express soluble P450s. [ 13–16 ] However, P. pastoris has good tolerance to organic solvents and more significant potential for producing hydrophobic compounds than E. coli and S. cerevisiae . [ 27,28 ] Notably, a previous study found that among four commonly used microbial hosts ( E. coli , S. cerevisiae , P. pastoris , and Yarrowia lipolytica ), P. pastoris exhibited the highest catalytic activity and stability for human cytochrome P450 2D6.…”

Section: Introductionmentioning

confidence: 99%

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Biosynthesis of High‐Active Hemoproteins by the Efficient Heme‐SupplyPichia PastorisChassis

Yu,

Zhao,

Zhou

et al. 2023

Advanced Science

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Microbial synthesis of valuable hemoproteins has become a popular research topic, and Pichia pastoris is a versatile platform for the industrial production of recombinant proteins. However, the inadequate supply of heme limits the synthesis of high‐active hemoproteins. Here a strategy for enhancing intracellular heme biosynthesis to improve the titers and functional activities of hemoproteins is reported. After selecting a suitable expressional strategy for globins, the efficient heme‐supply P. pastoris chassis is established by removing the spatial segregation during heme biosynthesis, optimizing precursor synthesis, assembling rate‐limiting enzymes using protein scaffolds, and inhibiting heme degradation. This robust chassis produces several highly active hemoproteins, including porcine myoglobin, soy hemoglobin, Vitreoscilla hemoglobin, and P450‐BM3, which can be used in the development of artificial meat, high‐cell‐density fermentation, and whole‐cell catalytic synthesis of high‐value‐added compounds. Furthermore, the engineered chassis strain has great potential for producing and applying other hemoproteins with high activities in various fields.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Biosynthesis of High‐Active Hemoproteins by the Efficient Heme‐SupplyPichia PastorisChassis

Yu,

Zhao,

Zhou

et al. 2023

Advanced Science

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Oleic acid based experimental evolution of Bacillus megaterium yielding an enhanced P450 BM3 variant

2022

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Background Unlike most other P450 cytochrome monooxygenases, CYP102A1 from Bacillus megaterium (BM3) is both soluble and fused to its redox partner forming a single polypeptide chain. Like other monooxygenases, it can catalyze the insertion of oxygen unto the carbon-hydrogen bond which can result in a wide variety of commercially relevant products for pharmaceutical and fine chemical industries. However, the instability of the enzyme holds back the implementation of a BM3-based biocatalytic industrial processes due to the important enzyme cost it would prompt. Results In this work, we sought to enhance BM3’s total specific product output by using experimental evolution, an approach not yet reported to improve this enzyme. By exploiting B. megaterium’s own oleic acid metabolism, we pressed the evolution of a new variant of BM3, harbouring 34 new amino acid substitutions. The resulting variant, dubbed DE, increased the conversion of the substrate 10-pNCA to its product p-nitrophenolate 1.23 and 1.76-fold when using respectively NADPH or NADH as a cofactor, compared to wild type BM3. Conclusions This new DE variant, showed increased organic cosolvent tolerance, increased product output and increased versatility in the use of either nicotinamide cofactors NADPH and NADH. Experimental evolution can be used to evolve or to create libraries of evolved BM3 variants with increased productivity and cosolvent tolerance. Such libraries could in turn be used in bioinformatics to further evolve BM3 more precisely. The experimental evolution results also supports the hypothesis which surmises that one of the roles of BM3 in Bacillus megaterium is to protect it from exogenous unsaturated fatty acids by breaking them down.

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Frontispiece: Whole‐Cell Biotransformation of Benzene to Phenol Catalysed by Intracellular Cytochrome P450BM3 Activated by External Additives

Cited by 2 publications

References 0 publications

Biosynthesis of High‐Active Hemoproteins by the Efficient Heme‐SupplyPichia PastorisChassis

Biosynthesis of High‐Active Hemoproteins by the Efficient Heme‐SupplyPichia PastorisChassis

Oleic acid based experimental evolution of Bacillus megaterium yielding an enhanced P450 BM3 variant

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