Mechanistic Insights into Interactions between Bacterial Class I P450 Enzymes and Redox Partners

Zhang, Wei; Du, Lei; Li, Fengwei; Zhang, Qizhou; Qu, Zepeng; Han, Lei; Zhong, Li; Sun, Jiangman; Qi, Fengxia; Yao, Qiuping; Sun, Yue; Geng, Ce; Li, Shengying

doi:10.1021/acscatal.8b02913

Cited by 86 publications

(83 citation statements)

References 98 publications

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“…By analyzing the reactivity profiles of 896 reactions, plastidic-type FdR and Fe 2 S 2 Fdx were found to be the favored types of redox partners by Class I P450 systems. Based on the empirically derived rules, the optimal cognate Fdx of PikC from Streptomyces venezuelae ATCC 15439 was predicted and confirmed in vitro to be SveFdx1948 (76). This work has provided information about the P450-preferred redox partners, and we envision that the findings will benefit future practical applications of P450 enzymes.…”

Section: Redox Partner Engineeringmentioning

confidence: 86%

“…To determine whether there are any principles for guiding the screening of optimal redox partners for a given Class I bacterial P450, Zhang et al (76) constituted a reaction matrix network based on 16 Fdxs, eight FdRs, and six P450s toward seven substrates. By analyzing the reactivity profiles of 896 reactions, plastidic-type FdR and Fe 2 S 2 Fdx were found to be the favored types of redox partners by Class I P450 systems.…”

Section: Redox Partner Engineeringmentioning

confidence: 99%

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Engineering cytochrome P450 enzyme systems for biomedical and biotechnological applications

et al. 2019

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Cytochrome P450 enzymes (P450s) are broadly distributed among living organisms and play crucial roles in natural product biosynthesis, degradation of xenobiotics, steroid biosynthesis, and drug metabolism. P450s are considered as the most versatile biocatalysts in nature because of the vast variety of substrate structures and the types of reactions they catalyze. In particular, P450s can catalyze regio- and stereoselective oxidations of nonactivated C–H bonds in complex organic molecules under mild conditions, making P450s useful biocatalysts in the production of commodity pharmaceuticals, fine or bulk chemicals, bioremediation agents, flavors, and fragrances. Major efforts have been made in engineering improved P450 systems that overcome the inherent limitations of the native enzymes. In this review, we focus on recent progress of different strategies, including protein engineering, redox-partner engineering, substrate engineering, electron source engineering, and P450-mediated metabolic engineering, in efforts to more efficiently produce pharmaceuticals and other chemicals. We also discuss future opportunities for engineering and applications of the P450 systems.

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Section: Redox Partner Engineeringmentioning

confidence: 86%

Section: Redox Partner Engineeringmentioning

confidence: 99%

Engineering cytochrome P450 enzyme systems for biomedical and biotechnological applications

et al. 2019

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“…Future studies will improve the catalytic performance (e.g., coupling efficiency) of TxtC biocatalysts. Encouragingly, during the review of the present work, the group of Challis reported the rapid conversion of 3 into 2 and then 1 by TxtC reconstituted with spinach ferredoxin and ferredoxin reductase, which suggested the evaluation of additional redox partners for creating more active, self‐sufficient biocatalysts . Furthermore, the crystal structures of TxtC can aid engineering efforts to develop biocatalysts for hydroxylating structurally diverse aromatic DKPs.…”

Section: Discussionmentioning

confidence: 75%

A Promiscuous Cytochrome P450 Hydroxylates Aliphatic and Aromatic C−H Bonds of Aromatic 2,5‐Diketopiperazines

et al. 2019

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Cytochrome P450 enzymes generally functionalize inert C−H bonds, and thus, they are important biocatalysts for chemical synthesis. However, enzymes that catalyze both aliphatic and aromatic hydroxylation in the same biotransformation process have rarely been reported. A recent biochemical study demonstrated the P450 TxtC for the biosynthesis of herbicidal thaxtomins as the first example of this unique type of enzyme. Herein, the detailed characterization of substrate requirements and biocatalytic applications of TxtC are reported. The results reveal the importance of N‐methylation of the thaxtomin diketopiperazine (DKP) core on enzyme reactions and demonstrate the tolerance of the enzyme to modifications on the indole and phenyl moieties of its substrates. Furthermore, hydroxylated, methylated, aromatic DKPs are synthesized through a biocatalytic route comprising TxtC and the promiscuous N‐methyltransferase Amir_4628; thus providing a basis for the broad application of this unique P450.

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“…Generalizable strategies that support the functional expression of plant cytochrome P450 enzymes for biosynthesis pathway optimization in S. cerevisiae have been developed [43]. Considering that the appropriate coupling between P450 enzymes and CPRs including their relative expression is critical for the overall catalytic efficiency [44], the performance of HPO could be further enhanced by using promoters with different strengths to optimize the relative expression level of HPO and ATR1. On the other hand, it is generally accepted that the optimal redox partners for a P450 enzyme should be homogeneous ones [45].…”

Section: Discussionmentioning

confidence: 99%

Metabolic engineering Saccharomyces cerevisiae for de novo production of the sesquiterpenoid (+)-nootkatone

et al. 2020

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Background: (+)-Nootkatone is a highly valued sesquiterpenoid compound, exhibiting a typical grapefruit aroma and various desired biological activities for use as aromatics and pharmaceuticals. The high commercial demand of (+)-nootkatone is predominately met by chemical synthesis, which entails the use of environmentally harmful reagents. Efficient synthesis of (+)-nootkatone via biotechnological approaches is thus urgently needed to satisfy its industrial demand. However, there are only a limited number of studies that report the de novo synthesis of (+)-nootkatone from simple carbon sources in microbial cell factories, and with relatively low yield. Results: As the direct precursor of (+)-nootkatone biosynthesis, (+)-valencene was first produced in large quantities in Saccharomyces cerevisiae by overexpressing (+)-valencene synthase CnVS of Callitropsis nootkatensis in combination with various mevalonate pathway (MVA) engineering strategies, including the expression of CnVS and farnesyl diphosphate synthase (ERG20) as a fused protein, overexpression of a truncated form of the rate-limiting enzyme 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (tHMG1), and downregulating the squalene synthase enzyme (ERG9). These approaches altogether brought the production of (+)-valencene to 217.95 mg/L. Secondly, we addressed the (+)-valencene oxidation by overexpressing the Hyoscyamus muticus premnaspirodiene oxygenase (HPO) variant (V482I/A484I) and cytochrome P450 reductase (ATR1) from Arabidopsis thaliana. However, (+)-valencene was predominantly oxidized to β-nootkatol and only minor amounts of (+)-nootkatone (9.66 mg/L) were produced. We further tackled the oxidation of β-nootkatol to (+)-nootkatone by screening various dehydrogenases. Our results showed that the short-chain dehydrogenase/reductase (SDR) superfamily dehydrogenases ZSD1 of Zingiber zerumbet and ABA2 of Citrus sinensis were capable of effectively catalyzing β-nootkatol oxidation to (+)-nootkatone. The yield of (+)-nootkatone increased to 59.78 mg/L and 53.48 mg/L by additional overexpression of ZSD1 and ABA2, respectively. Conclusion: We successfully constructed the (+)-nootaktone biosynthesis pathway in S. cerevisiae by overexpressing the (+)-valencene synthase CnVS, cytochrome P450 monooxygenase HPO, and SDR family dehydrogenases combined with the MVA pathway engineering, providing a solid basis for the whole-cell production of (+)-nootkatone. The two effective SDR family dehydrogenases tested in this study will serve as valuable enzymatic tools in further optimizing (+)-nootkatone production.

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Mechanistic Insights into Interactions between Bacterial Class I P450 Enzymes and Redox Partners

Cited by 86 publications

References 98 publications

Engineering cytochrome P450 enzyme systems for biomedical and biotechnological applications

Engineering cytochrome P450 enzyme systems for biomedical and biotechnological applications

A Promiscuous Cytochrome P450 Hydroxylates Aliphatic and Aromatic C−H Bonds of Aromatic 2,5‐Diketopiperazines

Metabolic engineering Saccharomyces cerevisiae for de novo production of the sesquiterpenoid (+)-nootkatone

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