[Heterologous expression of bacterial tyrosinase and its applications in biological hair dyeing and DOPA modification of hydrolyzed silk fibroin].

Tyrosinase is a copper-containing polyphenol oxidase widely applied in the food, cosmetics, pharmaceutical, and other industries. Currently, the production of commercial tyrosinase primarily relies on extraction from fungi, which has high costs, low purity, low specific activity, and poor stability. The objective of this study is to obtain highly expressed bacterial tyrosinase with potential for industrial applications. The bacterial tyrosinases from five different sources were heterologously expressed in Escherichia coli BL21(DE3), and the tyrosinases TyrBm and TyrVs derived from Bacillus megaterium and Verrucomicrobium spinosum were obtained with the enzyme activities of (16.1±0.2) U/mL and (48.6±0.9) U/mL, respectively. After protein purification, we compared the enzymatic properties of TyrBm and TyrVs, which revealed that TyrVs exhibited better thermal stability and higher substrate specificity than TyrBm. On the basis of characterizing TyrVs with high catalytic performance, we established a biological hair dyeing system based on TyrVs catalysis to achieve in-situ catalytic hair dyeing. The color washing fastness test measured the ∆E value less than 7.38±0.64 after simulated 14-day cleaning. To facilitate the rapid separation of catalytic products and enzymes, we successfully constructed an immobilized enzyme TyrVs-CipA dependent on self-assembly label CipA and applied this enzyme in the DOPA modification of hydrolyzed silk fibroin (HSF). The immobilized enzyme continuously catalyzed HSF for more than seven cycles, resulting in a single DOPA modification degree exceeding 70.00%. Further investigations demonstrated that DOPA modification enhances the scavenging activity of HSF towards DPPH and O2- radicals by 507.80% and 78.23%, respectively. This study provides a technical foundation for the development of environmentally friendly biological hair dye based on tyrosinase and biomaterials for tissue engineering.

Probing the function of C-terminal region of recombinant α-amylase BmaN1 from Bacillus megaterium NL3.

The α-amylase BmaN1 from Bacillus megaterium NL3 is a member of GH13_45 subfamily that has a conserved C-terminal region of approximately 30 residues. This region features a motif of five aromatic amino acids predicted to play a role in starch binding. This study aimed to unravel the role of the C-terminal region in starch hydrolysis. The full-length and C-terminally truncated forms of BmaN1 (BmaN1∆C) were expressed in Escherichia coli ArcticExpress (DE3), resulting in proteins with molecular weights of 56 kDa and 49 kDa, respectively. They exhibited comparable enzymatic activity in the hydrolysis of soluble starch, displaying versatility across a wide range of pH values, temperatures, and NaCl concentrations. BmaN1 and BmaN1∆C activities were inhibited by acarbose and were reduced by SDS and EDTA. In terms of binding and degrading the starch granules, BmaN1∆C showed lower affinity and activity in comparison to BmaN1. Our study indicates that the C-terminal region of BmaN1 significantly enhances its binding affinity and degrading the raw starches.IMPORTANCEα-Amylase (EC 3.2.1.1) stands as an endo-acting enzyme, essential for catalyzing the hydrolysis of α-1,4 glycosidic bonds within starch molecules. The relevance of α-amylases in biotechnological applications is substantial, constituting approximately 30% of the global enzyme market. Among these enzymes, BmaN1 was the first α-amylase identified to possess distinct catalytic residues within the GH13 family. BmaN1 from B. megaterium NL3 belongs to the GH13_45 subfamily. This subfamily is characterized by a conserved C-terminal region consisting of approximately 30 residues that contains a motif of five aromatic residues predicted to be involved in starch binding. Our study shows that the C-terminal effectively contributes to binding and degrading the raw starch granules. This pioneering research on BmaN1 expands our understanding of α-amylases and holds promise for innovative biotechnological advancements.

Microbially induced calcium carbonate precipitation through CO2 sequestration via an engineered Bacillus subtilis.

BACKGROUND: Microbially induced calcium carbonate precipitation has been extensively researched for geoengineering applications as well as diverse uses within the built environment. Bacteria play a crucial role in producing calcium carbonate minerals, via enzymes including carbonic anhydrase-an enzyme with the capability to hydrolyse CO2, commonly employed in carbon capture systems. This study describes previously uncharacterised carbonic anhydrase enzyme sequences capable of sequestering CO2 and subsequentially generating CaCO3 biominerals and suggests a route to produce carbon negative cementitious materials for the construction industry. RESULTS: Here, Bacillus subtilis was engineered to recombinantly express previously uncharacterised carbonic anhydrase enzymes from Bacillus megaterium and used as a whole cell catalyst allowing this novel bacterium to sequester CO2 and convert it to calcium carbonate. A significant decrease in CO2 was observed from 3800 PPM to 820 PPM upon induction of carbonic anhydrase and minerals recovered from these experiments were identified as calcite and vaterite using X-ray diffraction. Further experiments mixed the use of this enzyme (as a cell free extract) with Sporosarcina pasteurii to increase mineral production whilst maintaining a comparable level of CO2 sequestration. CONCLUSION: Recombinantly produced carbonic anhydrase successfully sequestered CO2 and converted it into calcium carbonate minerals using an engineered microbial system. Through this approach, a process to manufacture cementitious materials with carbon sequestration ability could be developed.

Tuning the peroxidase activity of artificial P450 peroxygenase by engineering redox-sensitive residues.

Cytochrome P450 monooxygenases (P450s) are well recognized as versatile bio-oxidation catalysts. However, the catalytic functions of P450s are highly dependent on NAD(P)H and redox partner proteins. Our group has recently reported the use of a dual-functional small molecule (DFSM) for generating peroxygenase activity of P450BM3, a long-chain fatty acid hydroxylase from Bacillus megaterium. The DFSM-facilitated P450BM3 peroxygenase system exhibited excellent peroxygenation activity and regio-/enantioselectivity for various organic substrates, such as styrenes, thioanisole, small alkanes, and alkylbenzenes. Very recently, we demonstrated that the DFSM-facilitated P450BM3 peroxygenase could be switched to a peroxidase by engineering the redox-sensitive tyrosine residues in P450BM3. Given the great potential of P450 peroxidase for C-H oxyfunctionalization, we herein report scrutiny of the effect of mutating redox-sensitive residues on peroxidase activity by deeply screening all redox-sensitive residues of P450BM3, namely methionines, tryptophans, cysteines, and phenylalanines. As a result, six beneficial mutations at positions M212, F81, M112, F173, M177, and F77 were screened out from 78 constructed mutants, and significantly enhanced the peroxidase activity of P450BM3 in the presence of Im-C6-Phe, a typical DFSM molecule. Further combination of the beneficial mutations resulted in a more than 100-fold improvement in peroxidase activity compared with that of the combined parent enzyme and DFSM, comparable to or better than most natural peroxidases. In addition, mutations of redox-sensitive residues even dramatically increased, by more than 300-fold, the peroxidase activity of the starting F87A enzyme in the absence of the DFSM, despite the far lower apparent catalytic turnover number compared with the DFSM-P450 system. This study provides new insights and a potential strategy for regulating the catalytic promiscuity of P450 enzymes for multiple functional oxidations.

A new physical and biological strategy to reduce the content of zearalenone in infected wheat kernels: the effect of cold needle perforation, microorganisms, and purified enzyme.

With the aim of reintroducing wheat grains naturally contaminated with mycotoxins into the food value chain, a decontamination strategy was developed in this study. For this purpose, in a first step, the whole wheat kernels were pre-treated using cold needle perforation. The pore size was evaluated by scanning electron microscopy and the accessibility of enzymes and microorganisms determined using fluorescent markers in the size range of enzymes (5 nm) and microorganisms (10 µm), and fluorescent microscopy. The perforated wheat grains, as well as non-perforated grains as controls, were then incubated with selected microorganisms (Bacillus megaterium Myk145 and B. licheniformis MA572) or with the enzyme ZHD518. The two bacilli strains were not able to significantly reduce the amount of zearalenone (ZEA), neither in the perforated nor in the non-perforated wheat kernels in comparison with the controls. In contrast, the enzyme ZHD518 significantly reduced the initial concentration of ZEA in the perforated and non-perforated wheat kernels in comparison with controls. Moreover, in vitro incubation of ZHD518 with ZEA showed the presence of two non-estrogenic degradation products of ZEA: hydrolysed zearalenone (HZEA) and decarboxylated hydrolysed ZEA (DHZEA). In addition, the physical pre-treatment led to a reduction in detectable mycotoxin contents in a subset of samples. Overall, this study emphasizes the promising potential of combining physical pre-treatment approaches with biological decontamination solutions in order to address the associated problem of mycotoxin contamination and food waste reduction.

Sequence-Guided Redesign of an Omega-Transaminase from Bacillus megaterium for the Asymmetric Synthesis of Chiral Amines.

ω-Transaminases (ω-TAs) are attractive biocatalysts asymmetrically catalyzing ketones to chiral amines. However, poor non-native catalytic activity and substrate promiscuity severely hamper its wide application in industrial production. Protein engineering efforts have generally focused on reshaping the substrate-binding pockets of ω-TAs. However, hotspots around the substrate tunnel as well as distant sites outside the pockets may also affect its activity. In this study, the ω-TA from Bacillus megaterium (BmeTA) was selected for engineering. The tunnel mutation Y164F synergy with distant mutation A245T which was acquired through a multiple sequence alignment showed improved soluble expression, a 3.7-fold higher specific activity and a 19.9-fold longer half-life at 45 °C. Molecule Dynamics simulation explains the mechanism of improved catalytic activity, enhanced thermostability and improved soluble expression of BmeTAY164F/A245T(2 M). Finally, the resting cells of 2 M were used for biocatalytic processes. 450 mM of S-methoxyisopropylamine (S-MOIPA) was obtained with an ee value of 97.3 % and a conversion rate of 90 %, laying the foundation for its industrial production. Mutant 2 M was also found to be more advantageous in catalyzing the transamination of various ketones. These results demonstrated that sites that are far away from the active center also play an important role in the redesign of ω-TAs.

Detection of Sulfoquinovosidase Activity in Cell Lysates Using Activity-Based Probes.

The sulfolipid sulfoquinovosyl diacylglycerol (SQDG), produced by plants, algae, and cyanobacteria, constitutes a major sulfur reserve in the biosphere. Microbial breakdown of SQDG is critical for the biological utilization of its sulfur. This commences through release of the parent sugar, sulfoquinovose (SQ), catalyzed by sulfoquinovosidases (SQases). These vanguard enzymes are encoded in gene clusters that code for diverse SQ catabolic pathways. To identify, visualize and isolate glycoside hydrolase CAZY-family 31 (GH31) SQases in complex biological environments, we introduce SQ cyclophellitol-aziridine activity-based probes (ABPs). These ABPs label the active site nucleophile of this enzyme family, consistent with specific recognition of the SQ cyclophellitol-aziridine in the active site, as evidenced in the 3D structure of Bacillus megaterium SQase. A fluorescent Cy5-probe enables visualization of SQases in crude cell lysates from bacteria harbouring different SQ breakdown pathways, whilst a biotin-probe enables SQase capture and identification by proteomics. The Cy5-probe facilitates monitoring of active SQase levels during different stages of bacterial growth which show great contrast to more traditional mRNA analysis obtained by RT-qPCR. Given the importance of SQases in global sulfur cycling and in human microbiota, these SQase ABPs provide a new tool with which to study SQase occurrence, activity and stability.

Enhancing the catalytic efficiency of M32 carboxypeptidase by semi-rational design and its applications in food taste improvement.

BACKGROUND: Carboxypeptidase is an exopeptidase that hydrolyzes amino acids at the C-terminal end of the peptide chain and has a wide range of applications in food. However, in industrial applications, the relatively low catalytic efficiency of carboxypeptidases is one of the main limiting factors for industrialization. RESULTS: The study has enhanced the catalytic efficiency of Bacillus megaterium M32 carboxypeptidase (BmeCPM32) through semi-rational design. Firstly, the specific activity of the optimal mutant, BmeCPM32-M2, obtained through single-site mutagenesis and combinatorial mutagenesis, was 2.2-fold higher than that of the wild type (187.9 versus 417.8 U mg-1), and the catalytic efficiency was 2.9-fold higher (110.14 versus 325.75 s-1 mmol-1). Secondly, compared to the wild type, BmeCPM32-M2 exhibited a 1.8-fold increase in half-life at 60 °C, with no significant changes in its enzymatic properties (optimal pH, optimal temperature). Finally, BmeCPM32-M2 significantly increased the umami intensity of soy protein isolate hydrolysate by 55% and reduced bitterness by 83%, indicating its potential in developing tasty protein components. CONCLUSION: Our research has revealed that the strategy based on protein sequence evolution and computational residue mutation energy led to an improved catalytic efficiency of BmeCPM32. Molecular dynamics simulations have revealed that a smaller substrate binding pocket and increased enzyme-substrate affinity are the reasons for the enhanced catalytic efficiency. Furthermore the number of hydrogen bonds and solvent and surface area may contribute to the improvement of thermostability. Finally, the de-bittering effect of BmeCPM32-M2 in soy protein isolate hydrolysate suggests its potential in developing palatable protein components. © 2024 Society of Chemical Industry.

High Cell Density Cultivation Combined with high Specific Enzyme Activity: Cultivation Protocol for the Production of an Amine Transaminase from Bacillus megaterium in E. coli.

High cell density cultivation is an established method for the production of various industrially important products such as recombinant proteins. However, these protocols are not always suitable for biocatalytic processes as the focus often lies on biomass production rather than high specific activities of the enzyme inside the cells. In contrast, a range of shake flask protocols are well known with high specific activities but rather low cell densities. To overcome this gap, we established a tailor-made fed-batch protocol combining both aspects: high cell density and high specific activities of heterologously produced enzyme. Using the example of an industrially relevant amine transaminase from Bacillus megaterium, we describe a strategy to optimize the cultivation yield based on the feed rate, IPTG concentration, and post-induction temperature. By adjusting these key parameters, we were able to increase the specific activity by 2.6-fold and the wet cell weight by even 17-fold compared to shake flasks. Finally, we were able to verify our established protocol by transferring it to another experimenter. With that, our optimization strategy can serve as a template for the production of high titers of heterologously produced, active enzymes and might enable the availability of these catalysts for upscaling biocatalytic processes.

Genetic fusion of P450 BM3 and formate dehydrogenase towards self-sufficient biocatalysts with enhanced activity.

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.

A Promiscuous Bacterial P450: The Unparalleled Diversity of BM3 in Pharmaceutical Metabolism.

CYP102A1 (BM3) is a catalytically self-sufficient flavocytochrome fusion protein isolated from Bacillus megaterium, which displays similar metabolic capabilities to many drug-metabolizing human P450 isoforms. BM3's high catalytic efficiency, ease of production and malleable active site makes the enzyme a desirable tool in the production of small molecule metabolites, especially for compounds that exhibit drug-like chemical properties. The engineering of select key residues within the BM3 active site vastly expands the catalytic repertoire, generating variants which can perform a range of modifications. This provides an attractive alternative route to the production of valuable compounds that are often laborious to synthesize via traditional organic means. Extensive studies have been conducted with the aim of engineering BM3 to expand metabolite production towards a comprehensive range of drug-like compounds, with many key examples found both in the literature and in the wider industrial bioproduction setting of desirable oxy-metabolite production by both wild-type BM3 and related variants. This review covers the past and current research on the engineering of BM3 to produce drug metabolites and highlights its crucial role in the future of biosynthetic pharmaceutical production.

Modulation of Transaminase Activity by Encapsulation in Temperature-Sensitive Poly(N-acryloyl glycinamide) Hydrogels.

Smart hydrogels hold much potential for biocatalysis, not only for the immobilization of enzymes, but also for the control of enzyme activity. We investigated upper critical solution temperature-type poly N-acryloyl glycinamide (pNAGA) hydrogels as a smart matrix for the amine transaminase from Bacillus megaterium (BmTA). Physical entrapment of BmTA in pNAGA hydrogels results in high immobilization efficiency (>89 %) and high activity (97 %). The temperature-sensitiveness of pNAGA is preserved upon immobilization of BmTA and shows a gradual deswelling upon temperature reduction. While enzyme activity is mainly controlled by temperature, deactivation tended to be higher for immobilized BmTA (≈62-68 %) than for free BmTA (≈44 %), suggesting a deactivating effect due to deswelling of the pNAGA gel. Although the deactivation in response to hydrogel deswelling is not yet suitable for controlling enzyme activity sufficiently, it is nevertheless a good starting point for further optimization.

Rapid detection of pesticide in milk, cereal and cereal based food and fruit juices using paper strip-based sensor.

The study was aimed to validate paper strip sensors for the detection of pesticide residues in milk, cereal-based food, and fruit juices in comparison with GC-MS/MS under field conditions. The detection limit of pesticide using rapid paper strip sensor for organophosphate, carbamate, organochlorine, fungicide, and herbicide group ranges from 1 to 10, 1-50, 250-500, 1-50, and 1 ppb, respectively in milk and milk product, cereal-based food and fruit juices. Among 125 samples of milk samples collected from the market 33 milk samples comprising 31 raw milk and 2 pasteurized milk found positive for pesticide using the strip-based sensor. In cereal based food and fruit juice samples, 6 cereal flours and 4 fruit juices were found positive for pesticide residues. The pesticide positive samples were further evaluated quantitatively using GC-MS/MS wherein 7 samples comprised of raw milk, pasteurized milk, rice flour, wheat flour, maize flour, apple juice, and pomegranate juice have shown the presence of chlorpyrifos, chlorpyrifos-methyl, α-endosulfan, ß-endosulfan DDD and DDT at trace level as well as at above MRL level. It is envisaged that the developed paper strip sensor can be a potential tool in the rapid and cost-effective screening of a large number of food samples for pesticide residues.

Ene-reductase transformation of massoia lactone to δ-decalactone in a continuous-flow reactor.

The demand for natural food flavorings increases every year. Biotransformation has become an attractive approach to obtain natural products. In this work, enantiomerically pure (R)-(+)-δ-decalactone was obtained by reduction of the C=C double bond of natural massoia lactone in a continuous-flow reactor. Of 13 different ene-reductases isolated, purified and tested, OYE3 was found to be the most efficient biocatalyst. The selected biocatalyst, either in the form of purified enzyme, cell lysate, whole cells or immobilized cells, was tested in the batch system as well as in the packed-bed flow bioreactor. The biotransformation performed in batch mode, using Ca2+-alginate immobilized cells of Escherichia coli BL21(DE3)/pET30a-OYE3, furnished the desired product with complete conversion in 30 min. The process was intensified using a continuous-flow reactor-membrane filtration system (flow 0.1 mL/min, substrate concentration 10 mM, pH 7, 24 °C) with cell lysate as biocatalyst combined with a cofactor regeneration system, which allowed obtaining > 99% bioconversion of massoia lactone.

Synthesis of Multi-Protein Complexes through Charge-Directed Sequential Activation of Tyrosine Residues.

Site-selective protein-protein coupling has long been a goal of chemical biology research. In recent years, that goal has been realized to varying degrees through a number of techniques, including the use of tyrosinase-based coupling strategies. Early publications utilizing tyrosinase from Agaricus bisporus(abTYR) showed the potential to convert tyrosine residues into ortho-quinone functional groups, but this enzyme is challenging to produce recombinantly and suffers from some limitations in substrate scope. Initial screens of several tyrosinase candidates revealed that the tyrosinase from Bacillus megaterium (megaTYR) is an enzyme that possesses a broad substrate tolerance. We use the expanded substrate preference as a starting point for protein design experiments and show that single point mutants of megaTYR are capable of activating tyrosine residues in various sequence contexts. We leverage this new tool to enable the construction of protein trimers via a charge-directed sequential activation of tyrosine residues (CDSAT).

Optimisation of Cytochrome P450 BM3 Assisted by Consensus-Guided Evolution.

Cytochrome P450 enzymes have attracted much interest over the years given their ability to insert oxygen into saturated carbon-hydrogen bonds, a difficult feat to accomplish by traditional chemistry. Much of the activity in this field has centered on the bacterial enzyme CYP102A1, or BM3, from Bacillus megaterium, as it has shown itself capable of hydroxylating/acting upon a wide range of substrates, thereby producing industrially relevant pharmaceuticals, fine chemicals, and hormones. In addition, unlike most cytochromes, BM3 is both soluble and fused to its natural redox partner, thus facilitating its use. The industrial use of BM3 is however stifled by its instability and its requirement for the expensive NADPH cofactor. In this work, we added several mutations to the BM3 mutant R966D/W1046S that enhanced the turnover number achievable with the inexpensive cofactors NADH and NBAH. These new mutations, A769S, S847G, S850R, E852P, and V978L, are localized on the reductase domain of BM3 thus leaving the oxidase domain intact. For NBAH-driven reactions by new mutant NTD5, this led to a 5.24-fold increase in total product output when compared to the BM3 mutant R966D/W1046S. For reactions driven by NADH by new mutant NTD6, this enhanced total product output by as much as 2.3-fold when compared to the BM3 mutant R966D/W1046S. We also demonstrated that reactions driven by NADH with the NTD6 mutant not only surpassed total product output achievable by wild-type BM3 with NADPH but also retained the ability to use this latter cofactor with greater total product output as well.

Co-immobilized Alcohol Dehydrogenase and Glucose Dehydrogenase with Resin Extraction for Continuous Production of Chiral Diaryl Alcohol.

Ni2+-functionalized porous ceramic/agarose composite beads (Ni-NTA Cerose) can be used as carrier materials to immobilize enzymes harboring a metal affinity tag. Here, a 6×His-tag fusion alcohol dehydrogenase Mu-S5 and glucose dehydrogenase from Bacillus megaterium (BmGDH) were co-immobilized on Ni-NTA Cerose to construct a packed bed reactor (PBR) for the continuous synthesis of the chiral intermediate (S)-(4-chlorophenyl)-(pyridin-2-yl) methanol ((S)-CPMA) NADPH recycling, and in situ product adsorption was achieved simultaneously by assembling a D101 macroporous resin column after the PBR. Using an optimum enzyme activity ratio of 2:1 (Mu-S5: BmGDH) and hydroxypropyl-ß-cyclodextrin as co-solvent, a space-time yield of 1560 g/(L·d) could be achieved in the first three days at a flow rate of 5 mL/min and substrate concentration of 10 mM. With simplified selective adsorption and extraction procedures, (S)-CPMA was obtained in 84% isolated yield.

Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of L-tert-leucine.

BACKGROUND: Biosynthesis of L-tert-leucine (L-tle), a significant pharmaceutical intermediate, by a cofactor regeneration system friendly and efficiently is a worthful goal all the time. The cofactor regeneration system of leucine dehydrogenase (LeuDH) and glucose dehydrogenase (GDH) has showed great coupling catalytic efficiency in the synthesis of L-tle, however the multi-enzyme complex of GDH and LeuDH has never been constructed successfully. RESULTS: In this work, a novel fusion enzyme (GDH-R3-LeuDH) for the efficient biosynthesis of L-tle was constructed by the fusion of LeuDH and GDH mediated with a rigid peptide linker. Compared with the free enzymes, both the environmental tolerance and thermal stability of GDH-R3-LeuDH had a great improved since the fusion structure. The fusion structure also accelerated the cofactor regeneration rate and maintained the enzyme activity, so the productivity and yield of L-tle by GDH-R3-LeuDH was all enhanced by twofold. Finally, the space-time yield of L-tle catalyzing by GDH-R3-LeuDH whole cells could achieve 2136 g/L/day in a 200 mL scale system under the optimal catalysis conditions (pH 9.0, 30 °C, 0.4 mM of NAD+ and 500 mM of a substrate including trimethylpyruvic acid and glucose). CONCLUSIONS: It is the first report about the fusion of GDH and LeuDH as the multi-enzyme complex to synthesize L-tle and reach the highest space-time yield up to now. These results demonstrated the great potential of the GDH-R3-LeuDH fusion enzyme for the efficient biosynthesis of L-tle.

Heterologous expression of cobalamin dependent class-III enzymes.

The family of cobalamin class-III dependent enzymes is composed of the reductive dehalogenases (RDases) and related epoxyqueuosine reductases. RDases are crucial for the energy conserving process of organohalide respiration. These enzymes have the ability to reductively cleave carbon-halogen bonds, present in a number of environmentally hazardous pollutants, making them of significant interest for bioremediation applications. Unfortunately, it is difficult to obtain sufficient yields of pure RDase isolated from organohalide respiring bacteria for biochemical studies. Hence, robust heterologous expression systems are required that yield the active holo-enzyme which requires both iron-sulphur cluster and cobalamin incorporation. We present a comparative study of the heterologous expression strains Bacillus megaterium, Escherichia coli HMS174(DE3), Shimwellia blattae and a commercial strain of Vibrio natrigenes, for cobalamin class-III dependent enzymes expression. The Nitratireductor pacificus pht-3B reductive dehalogenase (NpRdhA) and the epoxyqueuosine reductase from Streptococcus thermophilus (StoQ) were used as model enzymes. We also analysed whether co-expression of the cobalamin transporter BtuB, supports increased cobalamin incorporation into these enzymes in E. coli. We conclude that while expression in Bacillus megaterium resulted in the highest levels of cofactor incorporation, co-expression of BtuB in E. coli presents an appropriate balance between cofactor incorporation and protein yield in both cases.

A transaldolase-dependent sulfoglycolysis pathway in Bacillus megaterium DSM 1804.

Sulfoquinovose (6-deoxy-6-sulfoglucose, SQ) is a component of sulfolipids found in the photosynthetic membranes of plants and other photosynthetic organisms, and is one of the most abundant organosulfur compounds in nature. Microbial degradation of SQ, termed sulfoglycolysis, constitutes an important component of the biogeochemical sulfur cycle. Two sulfoglycolysis pathways have been reported, with one resembling the Embden-Meyerhof-Parnas (sulfo-EMP) pathway, and the other resembling the Entner-Doudoroff (sulfo-ED) pathway. Here we report a third sulfoglycolysis pathway in the bacterium Bacillus megaterium DSM 1804, in which sulfosugar cleavage is catalyzed by the transaldolase SqvA, which converts 6-deoxy-6-sulfofructose and glyceraldehyde 3-phosphate into fructose -6-phosphate and (S)-sulfolactaldehyde. Variations of this transaldolase-dependent sulfoglycolysis (sulfo-TAL) pathway are present in diverse bacteria, and add to the diversity of mechanisms for the degradation of this abundant organosulfur compound.