Improvement of the 25-hydroxyvitamin D3 production in a CYP109A2-expressing Bacillus megaterium system

Abdulmughni, Ammar; Erichsen, Björn; Hensel, Jürgen; Hannemann, Frank; Bernhardt, Rita

doi:10.1016/j.jbiotec.2020.09.027

Cited by 5 publications

(6 citation statements)

References 20 publications

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“…Cytochrome P450 monooxygenase CYP109A2 from B. megaterium DSM319 was previously shown to convert VD3 to 25‐hydroxyvitamin D3 (25(OH)VD3) [21,23]. Our current study reveals that this enzyme is also able to convert TES to 16β‐OH‐TES in a highly regio‐ and stereoselective manner.…”

Section: Discussionmentioning

confidence: 56%

“…The bacterial strain Bacillus megaterium DSM319 [20] contains three different P450s with wild-type steroid hydroxylation activities, that is, CYP106A1, CYP109E1, and CYP109A2. The latter two enzymes, which share a sequence identity of 45%, have both been subject to catalytic and structural analysis [21][22][23][24][25][26]. CYP109E1 was found to convert testosterone (TES) to 16b-hydroxytestosterone (16b-OH-TES) with high regio-and stereoselectivity, but is also able to hydroxylate cholesterol and vitamin D3 (VD3) at carbons C24 and C25, albeit with low regioselectivity [22,24,26].…”

Section: Introductionmentioning

confidence: 99%

“…Less functional and structural data are available for CYP109A2. Like CYP109E1, CYP109A2 is able to hydroxylate VD3, but with a higher regioselectivity, resulting in the formation of C25-hydroxylated VD3 (25-OH-VD3) as the main product [21,23]. However, it is unknown whether CYP109A2 also displays hydroxylating activities with other steroids.…”

Section: Introductionmentioning

confidence: 99%

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Regio‐ and stereoselective steroid hydroxylation by CYP109A2 from Bacillus megaterium explored by X‐ray crystallography and computational modeling

et al. 2023

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The P450 monooxygenase CYP109A2 from Bacillus megaterium DSM319 was previously found to convert vitamin D3 (VD3) to 25‐hydroxyvitamin D3. Here, we show that this enzyme is also able to convert testosterone in a highly regio‐ and stereoselective manner to 16β‐hydroxytestosterone. To reveal the structural determinants governing the regio‐ and stereoselective steroid hydroxylation reactions catalyzed by CYP109A2, two crystal structures of CYP109A2 were solved in similar closed conformations, one revealing a bound testosterone in the active site pocket, albeit at a nonproductive site away from the heme‐iron. To examine whether the closed crystal structures nevertheless correspond to a reactive conformation of CYP109A2, docking and molecular dynamics (MD) simulations were performed with testosterone and vitamin D3 (VD3) present in the active site. These MD simulations were analyzed for catalytically productive conformations, the relative occurrences of which were in agreement with the experimentally determined stereoselectivities if the predicted stability of each carbon‐hydrogen bond was taken into account. Overall, the first‐time determination and analysis of the catalytically relevant 3D conformation of CYP109A2 will allow for future small molecule ligand screening in silico, as well as enabling site‐directed mutagenesis toward improved enzymatic properties of this enzyme.

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Section: Discussionmentioning

confidence: 56%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Regio‐ and stereoselective steroid hydroxylation by CYP109A2 from Bacillus megaterium explored by X‐ray crystallography and computational modeling

et al. 2023

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“…Moreover, nisin treatment contributes to increased permeability of the cell membrane CYP109E1 through whole-cell bioconversion, suggesting that the CYP109E1 I85A variant achieves improved selectivity for 25-hydroxylation of VD 3 . In addition, after a 48-h incubation under optimized conditions, 282.7 mg/L calcifediol was produced by CYP109A2 T103A , a more efficient variant obtained by protein engineering of CYP109A2 [66].…”

Section: Conversion Of Vd 3 To Calcifediol and Calcitriol By Engineer...mentioning

confidence: 99%

Bioconversion of vitamin D3 to bioactive calcifediol and calcitriol as high-value compounds

Wang

Zeng

Jia

et al. 2022

Biotechnol Biofuels

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Biological catalysis is an important approach for the production of high-value-added compounds, especially for products with complex structures. Limited by the complex steps of chemical synthesis and low yields, the bioconversion of vitamin D3 (VD3) to calcifediol and calcitriol, which are natural steroid products with high added value and significantly higher biological activity compared to VD3, is probably the most promising strategy for calcifediol and calcitriol production, and can be used as an alternative method for chemical synthesis. The conversion efficiency of VD3 to calcifediol and calcitriol has continued to rise in the past few decades with the help of several different VD3 hydroxylases, mostly cytochrome P450s (CYPs), and newly isolated strains. The production of calcifediol and calcitriol can be systematically increased in different ways. Specific CYPs and steroid C25 dehydrogenase (S25DH), as VD3 hydroxylases, are capable of converting VD3 to calcifediol and calcitriol. Some isolated actinomycetes have also been exploited for fermentative production of calcifediol and calcitriol, although the VD3 hydroxylases of these strains have not been elucidated. With the rapid development of synthetic biology and enzyme engineering, quite a lot of advances in bioproduction of calcifediol and calcitriol has been achieved in recent years. Therefore, here we review the successful strategies of promoting VD3 hydroxylation and provide some perspective on how to further improve the bioconversion of VD3 to calcifediol and calcitriol.

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“…In contrast to E. coli, B. megaterium does not produce endotoxins; it has a comparable growth rate with E. coli and, could reach even higher optical density (Bunk et al 2010). B. megaterium reported as a promising host for the production of a wide variety of compounds such as recombinant proteins (Biedendieck 2016), glycosaminoglycans (Nehru et al 2020), PHB (Israni et al 2020) and, vitamins (Abdulmughni et al 2020). B. megaterium is widely, studied host for the bio-catalytic studies, for instance the whole-cell production of Shikimic acid (Ghosh et al 2014) and, 15 β-hydroxycyproterone acetate (Kiss et al 2015).…”

Section: Introductionmentioning

confidence: 99%

Combinatorial approach for improved production of whole-cell 3-aminopropionic acid in recombinant Bacillus megaterium: codon optimization, gene duplication and process optimization

2021

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In this study, we aimed to develop a Bacillus megaterium based whole-cell biocatalyst for the bio-production of 3-aminopropionic acid (3-APA). l-aspartate-α-decarboxylases (ADC) (EC: 4.1.1.11) from Escherichia coli, B. megaterium, Corynebacterium glutamicum, and Bacillus subtilis were expressed in B. megaterium. B. subtilis derived ADC (panD Bs ) exhibited the highest ADC activity of 0.9 ± 0.02 U/mL in recombinant B. megaterium. Combination of codon optimization and gene duplication strategies resulted in 415.56% enhancement of ADC activity compared to panD Bs . The culture growth conditions of B. megaterium (BMD-7) for 3-APA production were optimized as follows: inducer concentration, 0.5% (w/v); time of induction, 3 h; induction temperature, 37 °C and post-induction incubation time, 8 h. Improvement of the whole-cell biocatalytic process efficiency, was dealt by optimization of reaction temperature, reaction pH, metal ion additives and l-aspartic acid concentration. Shake flask level experiments yielded an enhanced 3-APA titer (16.18 ± 0.26 g/L) and a yield of 0.89 g/g under optimized conditions viz., 45 °C, pH 6.0 and 20 g/L of l-aspartic acid. This study demonstrates the potential of B. megaterium for 3-APA production and paves the scope for the development of 3-APA producing strains in near future. Keywords 3-Aminopropionic acid • Biocatalysis • Codon bias • Gene expression • l-Aspartate-α-decarboxylase • l-Aspartic acid * Senthilkumar Sivaprakasam

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Improvement of the 25-hydroxyvitamin D3 production in a CYP109A2-expressing Bacillus megaterium system

Cited by 5 publications

References 20 publications

Regio‐ and stereoselective steroid hydroxylation by CYP109A2 from Bacillus megaterium explored by X‐ray crystallography and computational modeling

Regio‐ and stereoselective steroid hydroxylation by CYP109A2 from Bacillus megaterium explored by X‐ray crystallography and computational modeling

Bioconversion of vitamin D3 to bioactive calcifediol and calcitriol as high-value compounds

Combinatorial approach for improved production of whole-cell 3-aminopropionic acid in recombinant Bacillus megaterium: codon optimization, gene duplication and process optimization

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