Efficient functional analysis system for cyanobacterial or plant cytochromes P450 involved in sesquiterpene biosynthesis

Harada, Hisashi; Shindo, Kazutoshi; Iki, Kanoko; Teraoka, Ayuko; Okamoto, Sho; Yu, Fengnian; Hattan, Jun-ichiro; Utsumi, Ryutaro; Misawa, Norihiko

doi:10.1007/s00253-010-3062-9

Cited by 23 publications

(22 citation statements)

References 39 publications

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“…F3=H is a cytochrome P450 protein and has never been expressed in E. coli before. Functional expression of plant cytochrome P450 in E. coli is a challenge due to various factors, such as solubility and cofactor incorporation (22)(23)(24). Therefore, one of the major objectives in the present study was the soluble expression of the fusion protein of truncated flavonoid 3=-hydroxylase (tF3=H) and truncated P450 reductase (tCPR) in E. coli.…”

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confidence: 99%

Efficient Synthesis of Eriodictyol from l -Tyrosine in Escherichia coli

Zhu

et al. 2014

Appl Environ Microbiol

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The health benefits of flavonoids for humans are increasingly attracting attention. Because the extraction of high-purity flavonoids from plants presents a major obstacle, interest has emerged in biosynthesizing them using microbial hosts. Eriodictyol is a flavonoid with anti-inflammatory and antioxidant activities. Its efficient synthesis has been hampered by two factors: the poor expression of cytochrome P450 and the low intracellular malonyl coenzyme A (malonyl-CoA) concentration in Escherichia coli. To address these issues, a truncated plant P450 flavonoid, flavonoid 3=-hydroxylase (tF3=H), was functionally expressed as a fusion protein with a truncated P450 reductase (tCPR) in E. coli. This allowed the engineered E. coli to produce eriodictyol from L-tyrosine by simultaneously coexpressing the fusion protein with tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). In addition, metabolic engineering was employed to enhance the availability of malonyl-CoA so as to achieve a new metabolic balance and rebalance the relative expression of genes to enhance eriodictyol accumulation. This approach made the production of eriodictyol 203% higher than that in the control strain. By using these strategies, the production of eriodictyol from L-tyrosine reached 107 mg/liter. The present work offers an approach to the efficient synthesis of other hydroxylated flavonoids from L-tyrosine or even glucose in E. coli.

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confidence: 99%

Efficient Synthesis of Eriodictyol from l -Tyrosine in Escherichia coli

Zhu

et al. 2014

Appl Environ Microbiol

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“…8B). 63,64 The cyclase forms monocyclic germacrene A and co-expression of the cyclase with the P450 results in a new bicyclic oxygenated product. It is proposed that P450 mediated epoxidation is followed by intramolecular cyclisation.…”

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Unrivalled diversity: the many roles and reactions of bacterial cytochromes P450 in secondary metabolism

et al. 2018

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The cytochromes P450 (P450s) are a superfamily of heme-containing monooxygenases that perform diverse catalytic roles in many species, including bacteria. The P450 superfamily is widely known for the hydroxylation of unactivated C-H bonds, but the diversity of reactions that P450s can perform vastly exceeds this undoubtedly impressive chemical transformation. Within bacteria, P450s play important roles in many biosynthetic and biodegradative processes that span a wide range of secondary metabolite pathways and present diverse chemical transformations. In this review, we aim to provide an overview of the range of chemical transformations that P450 enzymes can catalyse within bacterial secondary metabolism, with the intention to provide an important resource to aid in understanding of the potential roles of P450 enzymes within newly identified bacterial biosynthetic pathways. 1.Introduction to P450s 2.Chemistry of P450 enzymes 3.Bacterial biosynthesis pathways involving P450s 3.1Terpene metabolism 3.1. Battersby (1925Battersby ( -2018 to the molecular understanding of biosynthesis over the course of their careers.

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“…CPR is an essential redox partner for functional expression of CYP, and previous studies of CYP expression have employed endogenous CPR in eukaryotic hosts such as yeast (Katsuyama et al 2007), or have co-expressed heterologous (Harada et al 2011;Kim et al 2009;Leonard and Koffas 2007;Zhu et al 2014) or homologous CPR (Harada et al 2011;Hotze et al 1995;Leonard et al 2006). Moreover, genetic distances between the sources of the CPR and CYP reportedly influenced the enzyme activity (Kim et al 2009).…”

Section: Discussionmentioning

confidence: 99%

“…Addition of hemin instead of ALA yielded lower production, and the use of nutrition-rich TB medium instead of LB medium decreased the production (data not shown). Ammonium iron (II) sulfate was used as an iron source for heme production in previous studies (Harada et al 2011); therefore, we determined the effects of ammonium iron (II) sulfate in preliminary experiment. The supplement of 100 µM ammonium iron (II) sulfate had a tendency to increase the productivity, although no statistically significant difference was observed (data not shown).…”

Section: The Optimum Culture Conditionsmentioning

confidence: 99%

“…In particular, in vitro and in vivo assays with E. coli expression systems have been exploited to characterize the roles of CYP74 in allene oxide and aldehyde biosynthesis, CYP79 and CYP83 in glucosinolate biosynthesis, and CYP71 in the hydroxylation of sesquiterpenes (Brash 2009;Harada et al 2011;Nafisi et al 2006). E. coli expression systems have been used for large-scale preparation and purification of IFS and pcoumarate 3-hydroxylase (CYP98A).…”

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Functional expression of cytochrome P450 in <i>Escherichia coli</i>: An approach to functional analysis of uncharacterized enzymes for flavonoid biosynthesis

Uchida

Akashi

Aoki

2015

Plant Biotechnology

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Biochemical analyses of metabolic enzymes are performed using in vitro assays with enzymes and substrates. Enzyme samples are generally prepared from heterologous cell expression systems, which are also used as tools to obtain substrates that are not commercially available or cannot be easily isolated from natural sources. Cytochrome P450 (CYP) comprises a widely distributed family of monooxygenases that are commonly used for biosynthesis of natural products. Since CYP activity requires an electron transport system with membrane bound CYP reductase (CPR), it has been believed that CYP is not easily expressed in Escherichia coli, despite multiple advantages as a heterologous system compared with other systems that employ eukaryotic yeast and insect cells. In this study, we demonstrated simple and efficient methods for functional expression of CYPs in E. coli using commercially available vectors in which the transmembrane-domain truncated CYP was co-expressed with CPR as a discrete polypeptide, and we also used them to identify CYP81E11, CYP81E12, and CYP81E18 of soybean as isoflavone 2′-hydroxylase. Culture conditions were optimized for the bioconversion of I2′H, and the highest production was 161 mg l −1 of medium under optimized conditions. Subsequently, six other CYPs that are involved in flavonoid biosynthesis were tested for their applicability in the E. coli expression system. Establishment of the present method may facilitate functional expression of CYPs for preparation of CYP products, including substrates for enzymatic reactions, valuable natural products, and their unnatural derivatives.

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Efficient functional analysis system for cyanobacterial or plant cytochromes P450 involved in sesquiterpene biosynthesis

Cited by 23 publications

References 39 publications

Efficient Synthesis of Eriodictyol from l -Tyrosine in Escherichia coli

Efficient Synthesis of Eriodictyol from l -Tyrosine in Escherichia coli

Unrivalled diversity: the many roles and reactions of bacterial cytochromes P450 in secondary metabolism

Functional expression of cytochrome P450 in <i>Escherichia coli</i>: An approach to functional analysis of uncharacterized enzymes for flavonoid biosynthesis

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