A new Bacillus megaterium whole-cell catalyst for the hydroxylation of the pentacyclic triterpene 11-keto-β-boswellic acid (KBA) based on a recombinant cytochrome P450 system

Bleif, Sabrina; Hannemann, Frank; Zapp, Josef; Hartmann, David; Jauch, Johann; Bernhardt, Rita

doi:10.1007/s00253-011-3467-0

Cited by 55 publications

(44 citation statements)

References 35 publications

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“…Strains producing biomolecules, such as phytohormone, 2-pentylfuran, pyruvate, oxetanocin, vitamin B12, and polyhydroxyalkanoate (PHA) which are of industrial importance have been investigated [9][10][11][12]. This bacterium produces proteins of economical importance, namely, agarase, chitosanase, penicillin G acylase, amylases, glucose dehydrogenase, neutral protease, and the family of P-450 cytochrome monooxygenases [13][14][15]. Bacillus megaterium is also amenable for recombinant DNA technology for the improvement of specific enzymes and for protein production [5].…”

Section: Introductionmentioning

confidence: 99%

Rapid Identification of Polyhydroxyalkanoate Accumulating Members of Bacillales Using Internal Primers forphaCGene ofBacillus megaterium

et al. 2013

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Bacillus megaterium is gaining recognition as an experimental model and biotechnologically important microorganism. Recently, descriptions of new strains of B. megaterium and closely related species isolated from diverse habitats have increased. Therefore, its identification requires several tests in combination which is usually time consuming and difficult to do. We propose using the uniqueness of the polyhydroxyalkanoate synthase C gene of B. megaterium in designing primers that amplify the 0.9 kb region of the phaC for its identification. The PCR method was optimized to amplify 0.9 kb region of phaC gene. After optimization of the PCR reaction, two methods were investigated in detail. Method I gave an amplification of a single band of 0.9 kb only in B. megaterium and was demonstrated by several strains of B. megaterium isolated from different habitats. The use of Method I did not result in the amplification of the phaC gene with other members of Bacillales. The specificity for identification of B. megaterium was confirmed using sequencing of amplicon and RT-PCR. Method II showed multiple banding patterns of nonspecific amplicons among polyhydroxyalkanoate accumulating members of Bacillales unique to the respective species. These methods are rapid and specific for the identification of polyhydroxyalkanoate accumulating B. megaterium and members of Bacillales.

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Section: Introductionmentioning

confidence: 99%

Rapid Identification of Polyhydroxyalkanoate Accumulating Members of Bacillales Using Internal Primers forphaCGene ofBacillus megaterium

et al. 2013

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“…Besides native hosts such as Bacillus sp. (Bleif et al 2012), Pseudomonas sp. (Kuhn et al 2012), Streptomyces sp.…”

Section: Whole-cell Processesmentioning

confidence: 99%

Guidelines for development and implementation of biocatalytic P450 processes

Lundemo

Woodley

2015

Appl Microbiol Biotechnol

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Biocatalytic reactions performed by cytochrome P450 monooxygenases are interesting in pharmaceutical research since they are involved in human drug metabolism. Furthermore, they are potentially interesting as biocatalysts for synthetic chemistry because of the exquisite selectivity of the chemistry they undertake. For example, selective hydroxylation can be undertaken on a highly functionalized molecule without the need for functional group protection. Recent progress in the discovery of novel P450s as well as protein engineering of these enzymes strongly encourages further development of their application, including use in synthetic processes. The biological characteristics of P450s (e.g., cofactor dependence) motivate the use of whole-cell systems for synthetic processes, and those processes implemented in industry are so far dominated by growing cells and native host systems. However, for an economically feasible process, the expression of P450 systems in a heterologous host with sufficient biocatalyst yield (g/g cdw) for non-growing systems or space-time yield (g/L/h) for growing systems remains a major challenge. This review summarizes the opportunities to improve P450 whole-cell processes and strategies in order to apply and implement them in industrial processes, both from a biological and process perspective. Indeed, a combined approach of host selection and cell engineering, integrated with process engineering, is suggested as the most effective route to implementation.

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“…An exception is CYP154C5 from Nocardia farcinica, which was identified as potent 16α-hydroxylase for testosterone and similar steroids [87]. Moreover, CYP106A2, which was identified in the 1970s to be responsible for the ability of B. megaterium ATCC 13368 to convert progesterone into 15β-hydroxyprogesterone [88][89][90], is also able of catalyzing a 15β-hydroxylation of other 3-oxo-Δ4-steroids such as 11-deoxycorticosterone, testosterone, and 11-deoxycortisol [74,75,80,91]. Later on it was found that di-and triterpenes can also be highly selectively hydroxylated by CYP106A2 [73, 80, 83, see above].…”

Section: Steroidsmentioning

confidence: 99%

“…The aforementioned CYP106A2 from this organism belongs to the few described microbial P450s that can transform triterpenes. Recent studies showed that this P450 is able to hydroxylate the pentacyclic triterpene 11-keto-β-boswellic acid (KBA) to 15α-hydroxy-KBA, depicting an interesting reaction because this position is not accessible via synthetic chemical methods [80].…”

Section: Triterpenoidsmentioning

confidence: 99%

Terpene Hydroxylation with Microbial Cytochrome P450 Monooxygenases

Janocha

Schmitz

Bernhardt

2015

Biotechnology of Isoprenoids

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Terpenoids comprise a highly diverse group of natural products. In addition to their basic carbon skeleton, they differ from one another in their functional groups. Functional groups attached to the carbon skeleton are the basis of the terpenoids' diverse properties. Further modifications of terpene olefins include the introduction of acyl-, aryl-, or sugar moieties and usually start with oxidations catalyzed by cytochrome P450 monooxygenases (P450s, CYPs). P450s are ubiquitously distributed throughout nature, involved in essential biological pathways such as terpenoid biosynthesis as well as the tailoring of terpenoids and other natural products. Their ability to introduce oxygen into nonactivated C-H bonds is unique and makes P450s very attractive for applications in biotechnology. Especially in the field of terpene oxidation, biotransformation methods emerge as an attractive alternative to classical chemical synthesis. For this reason, microbial P450s depict a highly interesting target for protein engineering approaches in order to increase selectivity and activity, respectively. Microbial P450s have been described to convert industrial and pharmaceutically interesting terpenoids such as ionones, limone, valencene, resin acids, and triterpenes (including steroids) as well as vitamin D3. Highly selective and active mutants have been evolved by applying classical site-directed mutagenesis as well as directed evolution of proteins. As P450s usually depend on electron transfer proteins, mutagenesis has also been applied to improve the interactions between P450s and their respective redox partners. This chapter provides an overview of terpenoid hydroxylation reactions catalyzed by bacterial P450s and highlights the achievements made by protein engineering to establish productive hydroxylation processes.

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A new Bacillus megaterium whole-cell catalyst for the hydroxylation of the pentacyclic triterpene 11-keto-β-boswellic acid (KBA) based on a recombinant cytochrome P450 system

Cited by 55 publications

References 35 publications

Rapid Identification of Polyhydroxyalkanoate Accumulating Members of Bacillales Using Internal Primers forphaCGene ofBacillus megaterium

Rapid Identification of Polyhydroxyalkanoate Accumulating Members of Bacillales Using Internal Primers forphaCGene ofBacillus megaterium

Guidelines for development and implementation of biocatalytic P450 processes

Terpene Hydroxylation with Microbial Cytochrome P450 Monooxygenases

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