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Oxetanocin A (OXT-A) is a potent antitumour, antiviral and antibacterial compound. Biosynthesis of OXT-A has been linked to a plasmid-borne Bacillus megaterium gene cluster that contains four genes: oxsA, oxsB, oxrA and oxrB. Here we show that both the oxsA and oxsB genes are required for the production of OXT-A. Biochemical analysis of the encoded proteins, a cobalamin (Cbl)-dependent S-adenosylmethionine (AdoMet) radical enzyme, OxsB, and an HD-domain phosphohydrolase, OxsA, reveals that OXT-A is derived from a 2'-deoxyadenosine phosphate in an OxsB-catalysed ring contraction reaction initiated by hydrogen atom abstraction from C2'. Hence, OxsB represents the first biochemically characterized non-methylating Cbl-dependent AdoMet radical enzyme. X-ray analysis of OxsB reveals the fold of a Cbl-dependent AdoMet radical enzyme, a family of enzymes with an estimated 7,000 members. Overall, this work provides a framework for understanding the interplay of AdoMet and Cbl cofactors and expands the catalytic repertoire of Cbl-dependent AdoMet radical enzymes.

A low production rate for calcium carbonate with microbial solidification technology at low temperatures often restricts its application. For this reason, adding urea to the medium and the domestication of Bacillus megaterium at low temperature were proposed to produce more calcium carbonate based on an analysis of growth characteristics, urease activity, and the production rates for calcium carbonate under different conditions. Sand solidification tests were conducted to demonstrate improvements caused by the methods. The results showed that the higher the temperature, the faster the growth of Bacillus megaterium and the stronger the urease activity. Growth was fastest and urease activity strongest at a pH of 8. Adding urea to the medium and the domestication of B. megaterium at low temperature can both improve the production rate, effectively increasing calcium carbonate precipitation at low temperature. Combining the two methods resulted in greater improvement of the production rate for calcium carbonate. The two methods were also found to improve the effect of sand solidification. Therefore, our study provides a solid foundation for the actual engineering application of bio-cementation technology at low temperature.

Transition metal—catalyzed transfers of carbenes, nitrenes, and oxenes are powerful methods for functionalizing C=C and C—H bonds. Nature has evolved a diverse toolbox for oxene transfers, as exemplified by the myriad monooxygenation reactions catalyzed by cytochrome P450 enzymes. The isoelectronic carbene transfer to olefins, a widely used C—C bond—forming reaction in organic synthesis, has no biological counterpart. Here we report engineered variants of cytochrome P450 BM3 that catalyze highly diastereo- and enantioselective cyclopropanation of styrenes from diazoester reagents via putative carbene transfer. This work highlights the capacity to adapt existing enzymes for the catalysis of synthetically important reactions not previously observed in nature.

Fusion of multiple enzymes to multifunctional constructs has been recognized as a viable strategy to improve enzymatic properties at various levels such as stability, activity and handling. In this study, the genes coding for cytochrome P450 BM3 from B. megaterium and formate dehydrogenase from Pseudomonas sp. were fused to enable both substrate oxidation catalyzed by P450 BM3 and continuous cofactor regeneration by formate dehydrogenase within one construct. The order of the genes in the fusion as well as the linkers that bridge the enzymes were varied. The resulting constructs were compared to individual enzymes regarding substrate conversion, stability and kinetic parameters to examine whether fusion led to any substantial improvements of enzymatic properties. Most noticeably, an activity increase of up to threefold was observed for the fusion constructs with various substrates which were partly attributed to the increased diflavin reductase activity of the P450 BM3. We suggest that P450 BM3 undergoes conformational changes upon fusion which resulted in altered properties, however, no NADPH channeling was detected for the fusion constructs.

This review highlights the recent investigations of class IV polyhydroxyalkanoate (PHA) synthases, the newest classification of PHA synthases. Class IV synthases are prevalent in organisms of the Bacillus genus and are composed of a catalytic subunit PhaC (approximately 40 kDa), which has a PhaC box sequence ([GS]-X-C-X-[GA]-G) at the active site, and a second subunit PhaR (approximately 20 kDa). The representative PHA-producing Bacillus strains are Bacillus megaterium and Bacillus cereus; the nucleotide sequence of phaC and the genetic organization of the PHA biosynthesis gene locus are somewhat different between these two strains. It is generally considered that class IV synthases favor short-chain-length monomers such as 3-hydroxybutyrate (C4) and 3-hydroxyvalerate (C5) for polymerization, but can polymerize some unusual monomers as minor components. In Escherichia coli expressing PhaRC from B. cereus YB-4, the biosynthesized PHA undergoes synthase-catalyzed alcoholytic cleavage using endogenous and exogenous alcohols. This alcoholysis is thought to be shared among class IV synthases, and this reaction is useful not only for the regulation of PHA molecular weight but also for the modification of the PHA carboxy terminus. The novel properties of class IV synthases will open up the possibility for the design of new PHA materials.

Microbially induced calcite precipitation (MICP) has long been the focus of material scientists, environmental microbiologists and civil engineers because of its potential to yield biosynthesized binders that can serve as alternatives to cement or resins. Several microbial strains play crucial roles in this process and catalyse pathways for the formation of minerals, which are believed to substantially reduce the environmental impact of building materials and activities. Among the studied strains, Bacillus megaterium is not as common as Sporosarcina species. The latter microorganisms are well known to drive the fastest ureolytic-driven MICP process, i.e., precipitation of CaCO 3 after urea breakdown into carbonate and CaCl 2 addition to the system. This paper sheds light on the activities of B. megaterium , which possesses dual enzymatic capabilities for MICP and is equipped with both the enzymes urease and carbonic anhydrase. We postulate that, depending on the growth conditions, B. megaterium can activate either of these genes to ultimately induce CaCO 3 precipitation. Herein, experiments are carried out in open and closed systems. C 13 -labelled urea is employed to identify the carbon source in the precipitated CaCO 3 . The results from Fourier transform infrared spectroscopy (FTIR) revealed the precipitation of calcite. In the presence of urea and CO 2 at atmospheric levels, B. megaterium activates the ureolytic pathway to perform urea hydrolysis. However, at increased CO 2 levels, more precisely, at levels greater than 470 times the atmospheric level, carbonic anhydrase is activated, catalysing the hydration of the molecule to produce HCO 3 − . When C 13 -labelled urea was utilized, only 6% of the precipitated CaCO 3 mineral was linked to ureolysis, and it was found that the remaining 94% was formed due to the mineralization of CO 2 . Overall, in this work, we aim to introduce the process conditions and protocols that favour the sequestration of atmospheric CO 2 as CaCO 3 via the metabolic activities of B. megaterium .

The investigation on the synergistic role of urease (UA) and carbonic anhydrase (CA) in biomineralization of calcium carbonate in Bacillus megaterium suggested that the precipitation of CaCO 3 is significantly faster in bacterial culture than in crude enzyme solutions. Calcite precipitation is significantly reduced when both the enzymes are inhibited in comparison with those of the individual enzyme inhibitions indicating that both UA and CA are crucial for efficient mineralization. Carbonic anhydrase plays a role in hydrating carbon dioxide to bicarbonate, while UA aids in maintaining the alkaline pH that promotes calcification process.

N -oxidation of N -heterocycles is essential in the synthesis of natural products but challenging due to low efficacy and poor regioselectivity. In this study, the N -oxidation selective potential of P450BM3 from Bacillus megaterium for N -heterocyclic compounds is investigated. Here, twelve amino acids located in the active center, including A74, L75, V78, A82, F87, I263, A264, A328, P329, A330, I401, and L437, are investigated by site-saturation mutation. As a result, F87, A264, L75, V78, A328, I401, and L437 are identified as hotspot residues. Subsequently, the combinatorial active-site saturation test/iterative saturation mutagenesis strategy is performed. Using quinoline as a model substrate, the mutant F87G/A264G/A328L exhibits N -oxidation selectivity of up to 99.0%, with a conversion rate of 99.3%. Molecular dynamics simulations uncover a “push-pull” molecular mechanism elucidating the pivotal role of steric factors in determining substrate recognition and N -oxidation selectivity. This study provides an efficient N -oxide synthesis method and insights into P450BM3’s molecular mechanisms. N -oxidation of N -heterocycles is essential in the synthesis of natural products and intermediates for pharmaceuticals and pesticides but challenging due to low efficacy and poor regioselectivity. Here, the authors employ structure-guided directed evolution of Bacillus megaterium P450BM3 to obtain variants with expanded N -oxidation selectivity for the synthesis of N -heterocyclic N -oxides.

The aim of the present research was the efficient degradation of industrial textile wastewater dyes using a very active cloned laccase enzyme. For this purpose, potent laccase-producing bacteria were isolated from soil samples collected from wastewater-replenished textile sites in Punjab, Pakistan. The laccase gene from locally isolated strain LI-81, identified as Bacillus megaterium, was cloned into vector pET21a, which was further transformed into E. coli BL21 codon plus. The optimized conditions for the increased production of laccase include fermentation in a 2% glucose, 5% yeast extract and 250 mg/L CuSO4 medium with pH 7.5; inoculation with 5% inoculum; induction with 0.1 mM IPTG at 0.5 O.D.; and incubation for 36 h at 37 °C. The crude enzyme produced was employed for the removal of commercially used textile dyes. The dyes were quickly precipitated under optimized reaction conditions. Rose bengal, brilliant green, brilliant blue G, Coomassie brilliant blue R and methylene blue were precipitated at rates of 10.69, 54.47, 84.04, 78.99 and 7.40%, respectively. The FTIR and UV–Vis spectroscopic analyses of dyes before and after confirmed the chemical changes brought about by the cloned laccase that led to the dye removal.

Background Cytochrome P450 monooxygenase constitutes a significant group of oxidative enzymes that can introduce an oxygen atom in a high regio- and stereo-selectivity mode. We used the Bacillus megaterium cytochrome P450 BM3 (CYP450 BM3) and its variants namely mutant 13 (M13) and mutant 15 (M15) for the hydroxylation of diverse class of flavonoids. Results Among 20 flavonoids, maximum seven flavonoids were hydroxylated by the variants while none of these molecules were accepted by CYP450 BM3 in in vitro reaction. Moreover, M13 exhibited higher conversion of substrates than M15 and CYP450 BM3 enzymes. We found that M13 carried out regiospecific 3ʹ-hydroxylation reaction of naringenin with the highest conversion among all the tested flavonoids. The apparent K m and k cat values of M13 for naringenin were 446 µM and 1.955 s −1 , respectively. In whole-cell biotransformation experiment with 100 µM of naringenin in M9 minimal medium with 2 % glucose in shake flask culture, M13 showed 2.14- and 13.96-folds higher conversion yield in comparison with M15 (16.11 %) and wild type (2.47 %). The yield of eriodictyol was 46.95 µM [~40.7 mg (13.5 mg/L)] in a 3-L volume lab scale fermentor at 48 h in the same medium exhibiting approximately 49.81 % conversion of the substrate. In addition, eriodictyol exhibited higher antibacterial and anticancer potential than naringenin, flavanone and hesperetin. Conclusions We elucidated that eriodictyol being produced from naringenin using recombinant CYP450 BM3 and its variants from B. megaterium , which shows an approach for the production of important hydroxylated compounds of various polyphenols that may span pharmaceutical industries.