Production and secretion of recombinant spider silk in Bacillus megaterium.

BACKGROUND: Silk proteins have emerged as versatile biomaterials with unique chemical and physical properties, making them appealing for various applications. Among them, spider silk, known for its exceptional mechanical strength, has attracted considerable attention. Recombinant production of spider silk represents the most promising route towards its scaled production; however, challenges persist within the upstream optimization of host organisms, including toxicity and low yields. The high cost of downstream cell lysis and protein purification is an additional barrier preventing the widespread production and use of spider silk proteins. Gram-positive bacteria represent an attractive, but underexplored, microbial chassis that may enable a reduction in the cost and difficulty of recombinant silk production through attributes that include, superior secretory capabilities, frequent GRAS status, and previously established use in industry. RESULTS: In this study, we explore the potential of gram-positive hosts by engineering the first production and secretion of recombinant spider silk in the Bacillus genus. Using an industrially relevant B. megaterium host, it was found that the Sec secretion pathway enables secretory production of silk, however, the choice of signal sequence plays a vital role in successful secretion. Attempts at increasing secreted titers revealed that multiple translation initiation sites in tandem do not significantly impact silk production levels, contrary to previous findings for other gram-positive hosts and recombinant proteins. Notwithstanding, targeted amino acid supplementation in minimal media was found to increase production by 135% relative to both rich media and unaltered minimal media, yielding secretory titers of approximately 100 mg/L in flask cultures. CONCLUSION: It is hypothesized that the supplementation strategy addressed metabolic bottlenecks, specifically depletion of ATP and NADPH within the central metabolism, that were previously observed for an E. coli host producing the same recombinant silk construct. Furthermore, this study supports the hypothesis that secretion mitigates the toxicity of the produced silk protein on the host organism and enhances host performance in glucose-based minimal media. While promising, future research is warranted to understand metabolic changes more precisely in the Bacillus host system in response to silk production, optimize signal sequences and promoter strengths, investigate the mechanisms behind the effect of tandem translation initiation sites, and evaluate the performance of this system within a bioreactor.

Bioconversion of glycerol into polyhydroxyalkanoates through an atypical metabolism shift using Priestia megaterium during fermentation processes: A statistical analysis of carbon and nitrogen source concentrations.

Polyhydroxyalkanoates (PHA) are bioplastics which are well known as intracellular energy storage compounds and are produced in a large number of prokaryotic species. These bio-based inclusions are biodegradable, biocompatible and environmental friendly. Industrial production of, short chain and medium chain length PHA, involves the use of microorganisms and their enzymes. Priestia megaterium previously known as Bacillus megaterium is a well-recognized bacterium for producing short chain length PHA. This study focuses to characterize this bacterium for the production of medium chain length PHA, and a novel blend of both types of monomers having enhanced properties and versatile applications. Statistical analyses and simulations were used to demonstrate that cell dry weight can be derived as a function of OD600 and PHA content. Optimization of growth conditions resulted in the maximum PHA production as: 0. 05 g. g-x. H-1, where the rate of PHA production was 0.28 g L-1. H-1 and PHA concentration was 4.94 g. L-1. This study also demonstrated FTIR to be a semi quantitative tool for PHA production. Moreover, conversion of scl-PHA to mcl-PHA with reference to time intermissions using GC-FID are shown.

Biosynthesis of resveratrol by an endophytic Priestia megaterium PH3 via the phenylpropane pathway.

Resveratrol (RES) is a secondary metabolite synthesized by plants in response to environmental stress and pathogen infection, which is of great significance for the industrial production of RES by fermentation culture. In this study, we aimed to explore the biosynthesis pathway of RES and its key enzymes in the Priestia megaterium PH3, which was isolated and screened from peanut fruit. Through Liquid Chromatography-Mass Spectrometry (LC-MS) analysis, we quantified the RES content and distribution in the culture medium and determined that Priestia megaterium PH3 mainly secreted RES extracellularly. Furthermore, the highest production of RES was observed in YPD, yielding an impressive 127.46 ± 6.11 µg/L. By optimizing the fermentation conditions, we achieved a remarkable RES yield of 946.82 ± 24.74 µg/L within just 2 days, which represents the highest reported yield for a natural isolate produced in such a short time frame. Our investigation revealed that the phenylpropane pathway is responsible for RES synthesis in this bacterium, with cinnamate 4-hydroxylase (C4H) identified as the main rate-limiting enzyme. Overall, our findings highlight the robust RES production capabilities of Priestia megaterium PH3, offering novel insights and potential applications for bacterial fermentation in RES production. KEY POINTS: • RES synthesized by the bacterium was confirmed through the phenylpropane pathway. • The key rate-limiting enzyme for biosynthesis-RES is C4H. • RES reached 946.82 ± 24.74 µg/L after fermentation for 2 days.

The Versatile Biocatalyst of Cytochrome P450 CYP102A1: Structure, Function, and Engineering.

Wild-type cytochrome P450 CYP102A1 from Bacillus megaterium is a highly efficient monooxygenase for the oxidation of long-chain fatty acids. The unique features of CYP102A1, such as high catalytic activity, expression yield, regio- and stereoselectivity, and self-sufficiency in electron transfer as a fusion protein, afford the requirements for an ideal biocatalyst. In the past three decades, remarkable progress has been made in engineering CYP102A1 for applications in drug discovery, biosynthesis, and biotechnology. The repertoire of engineered CYP102A1 variants has grown tremendously, whereas the substrate repertoire is avalanched to encompass alkanes, alkenes, aromatics, organic solvents, pharmaceuticals, drugs, and many more. In this article, we highlight the major advances in the past five years in our understanding of the structure and function of CYP102A1 and the methodologies used to engineer CYP102A1 for novel applications. The objective is to provide a succinct review of the latest developments with reference to the body of CYP102A1-related literature.

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

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 16ß-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.

Integrative transcriptome and metabolome revealed the molecular mechanism of Bacillus megaterium BT22-mediated growth promotion in Arabidopsis thaliana.

Plant growth-promoting rhizobacteria (PGPR) can promote plant growth and protect plants from pathogens, which contributes to sustainable agricultural development. Several studies have reported their beneficial characteristics in facilitating plant growth and development and enhancing plant stress resistance through different mechanisms. However, there is still a challenge to study the molecular mechanism of plant response to PGPR. We integrated the transcriptome and metabolome of Arabidopsis thaliana (Arabidopsis) to understand its responses to the inoculation with an isolated PGPR strain (BT22) of Bacillus megaterium. Fresh shoot weight, dry shoot weight and leaf number of Arabidopsis were increased by BT22 treatment, showing a positive growth-promoting effect. According multi-omics analysis, 878 differentially expressed genes (296 up-regulated, 582 down-regulated) and 139 differentially expressed metabolites (66 up-regulated, 73 down-regulated) response to BT22 inoculation. GO enrichment results indicate that the up-regulated genes mainly enriched in the regulation of growth and auxin response pathways. In contrast, the down-regulated genes mainly enriched in wounding response, jasmonic acid and ethylene pathways. BT22 inoculation regulated plant hormone signal transduction of Arabidopsis, including auxin and cytokinin response genes AUX/IAA, SAUR, and A-ARR related to cell enlargement and cell division. The contents of nine flavonoids and seven phenylpropanoid metabolites were increased, which help to induce systemic resistance in plants. These results suggest that BT22 promoted Arabidopsis growth by regulating plant hormone homeostasis and inducing metabolome reprogramming.

Enhanced metabolism of 2,3',4,4',5-pentachlorobiphenyl (CB118) by bacterial cytochrome P450 monooxygenase mutants of Bacillus megaterium.

Bacterial cytochrome P450 monooxygenase P450BM3 is a promising enzyme to provide novel substrate specificity and enhanced enzymatic activity. The wild type (WT) has been shown to metabolize the widely distributed polychlorinated biphenyl (PCB) 2,3',4,4',5-pentachlorobiphenyl (CB118) to hydroxylated metabolites. However, this reaction requires the coexistence of perfluoroalkyl carboxylic acids (PFCAs). To locate P450BM3 mutants metabolizing CB118 without PFCAs, mutations were selected from amino acids comprising the substrate-binding cavity and the substrate entrance. The mutant A264G showed enhanced hydroxylation activities compared to the WT for the production of five hydroxylated metabolites. Perfluorooctanoic acid addition provided the highest activity, as found in the WT. The docking model of A264G and CB118 indicated that the enlargement of the space above the heme brought CB118 close to the heme, resulting in high activity. In contrast, the mutants L188Q, QG, LVQ, and GVQ, which contain the L188Q mutation, showed higher activity than WT even without PFCAs. Docking models revealed that the closed form found in substrate binding was induced by the L188Q mutation in the substrate non-binding state of the mutants. These mutants are promising for bioremediation of PCBs using enhanced metabolizing activities.

Bacterial spore germination receptors are nutrient-gated ion channels.

Bacterial spores resist antibiotics and sterilization and can remain metabolically inactive for decades, but they can rapidly germinate and resume growth in response to nutrients. Broadly conserved receptors embedded in the spore membrane detect nutrients, but how spores transduce these signals remains unclear. Here, we found that these receptors form oligomeric membrane channels. Mutations predicted to widen the channel initiated germination in the absence of nutrients, whereas those that narrow it prevented ion release and germination in response to nutrients. Expressing receptors with widened channels during vegetative growth caused loss of membrane potential and cell death, whereas the addition of germinants to cells expressing wild-type receptors triggered membrane depolarization. Therefore, germinant receptors act as nutrient-gated ion channels such that ion release initiates exit from dormancy.

Study on the mechanism of biochar affecting the effectiveness of phosphate solubilizing bacteria.

Low phosphorus utilization and phosphorus fertilizer pollution are serious issues primarily affecting soil health. To investigate the effects of biochar on the growth, phosphorus solubilization, and metabolites of phosphorus-solubilizing bacteria (PSB), rice husk biochar (RH) and rice straw biochar (RS) were incubated with Bacillus megatherium (BM1) and Bacillus mucilaginosus (BM2), respectively. The highest phosphorus solubilization was observed in BM2 following the addition of RS. The dissolved amount of phosphorus was 244.99 mg/L, which was 43.86% higher than that of the control group. Hence, biochar can improve the phosphorus solubilization capacity of PSB by affecting the organic acid and polysaccharide contents, and phosphatase activity secreted by the PSB, as the porous structure and surface characteristics of biochar ensured the adsorption of PSB. This study can help improve the functional activity of PSB and provide basis for improving the utilization of soil phosphorus, which in turn, aid in the development of biochar-based microbial fertilizers.

A non-K+-solubilizing PGPB (Bacillus megaterium) increased K+ deprivation tolerance in Oryza sativa seedlings by up-regulating root K+ transporters.

Potassium is one of the principal macronutrients required by all plants, but its mobility is restricted between soil compartments. Numerous studies have shown that Plant Growth Promoting Bacteria (PGPB) can facilitate nutrient uptake. The present work examined the effects of the PGPB (Bacillus megaterium) on rice plants subjected to potassium deprivation. To study only direct effects of B. megaterium, we first checked its lack of capacity to solubilize soil K. Rice plants were provided with 1.5 mM K (100%) or 0.015 mM K (1%) and growth related parameters, nutrient concentrations and gene expression of K+ transporters were determined. After two weeks, the 1% K treatment reduced growth of non-inoculated plants by about 50% compared with the 100% K treatment. However, there was no effect of reduced K nutrition on growth of inoculated plants. The reduction in growth in non-inoculated plants was accompanied by a similar reduction in K+ concentration in both roots and leaves and an overall 80% reduction of the plant potassium concentrations. In inoculated plants a 50% reduction occurred only in leaves. The expression of the K+ transporters HKT1;1, 1;2, 1;5, 2;2, 2;3 and 2;4 was up-regulated by the inoculation of B. megaterium under K deprivation conditions, explaining their higher K tissue concentrations and growth. Thus, the bacterial strain improved plant potassium nutrition without affecting K+ availability in the soil. The results demonstrate the potential of this bacteria for using as a biofertilizer to reduce the amount of potassium fertilizers to be applied in the field.

Degradation of alkane hydrocarbons by Priestia megaterium ZS16 and sediments consortia with special reference to toxicity and oxidative stress induced by the sediments in the vicinity of an oil refinery.

Petroleum hydrocarbon is a critical ecological issue with impact on ecosystems through bioaccumulation. It poses significant risks to human health. Due to the extent of alkane hydrocarbon pollution in some environments, biosurfactants are considered as a new multifunctional technology for the efficient removal of petroleum-based contaminants. To this end, Yamuna river sediments were collected at different sites in the vicinity of Mathura oil refinery, UP (India). They were analysed by atomic absorption spectrophotometry and gas chromatography-mass spectrometry (GC-MS) for heavy metals and organic pollutants. Heptadecane, nonadecane, oleic acid ester and phthalic acid were detected. In total 107 bacteria were isolated from the sediments and screened for biosurfactant production. The most efficient biosurfactant producing strain was tested for its capability to degrade hexadecane efficiently at different time intervals (0 h, 7 d, 14 d and 21 d). FT-IR analysis defined the biosurfactant as lipopeptide. 16S rRNA gene sequencing identified the bacterium as Priestia megaterium. The strain lacks resistance to common antibiotics thus making it an important candidate for remediation. The microbial consortia present in the sediments were also investigated for their capability to degrade C16, C17 and C18 alkane hydrocarbons. By using gas chromatography-mass spectrophotometry the metabolites were identified as 1-docosanol, dodecanoic acid, 7-hexadecenal, (Z)-, hexadecanoic acid, docosanoic acid, 1-hexacosanal, 9-octadecenoic acid, 3-octanone, Z,Z-6,28-heptatriactontadien-2-one, heptacosyl pentafluoropropionate, 1,30-triacontanediol and decyl octadecyl ester. Oxidative stress in Vigna radiata L. roots was observed by using Confocal Laser Scanning Microscopy. A strong reduction in seed germination and radicle and plumule length was observed when Vigna radiata L. was treated with different concentrations of sediment extracts, possibly due to the toxic effects of the pollutants in the river sediments. Thus, this study is significant since it considers the toxicological effects of hydrocarbons and to degrade them in an environmentally friendly manner.

Characterization of a novel detergent-resistant type I pullulanase from Bacillus megaterium Y103 and its application in laundry detergent.

This study aims to find a moderate pullulanase for detergent industry. The pulY103B gene (2217 bp) from Bacillus megaterium Y103 was cloned and expressed in Escherichia coli. PulY103B contained four conserved regions of glycoside hydrolase family (GH) 13 and the typical sequence of type I pullulanase. The optimal reaction conditions of PulY103B were pH 6.5 and 40 °C. In addition, it remained stable below 40 °C and over 80% of activity was retained at pH ranging from 6.0 to 8.5. The best substrate for the enzyme was pullulan. Furthermore, it exhibited activity toward wheat starch (36.5%) and soluble starch (33.4%) but had no activity toward amylose and glycogen. Maltotriose and maltohexaose were major pullulan hydrolysis products. Soluble starch and amylopectin were mainly hydrolyzed into maltotetraose. These results indicated that PulY103B is a novel type I pullulanase with transglycosylation activity via formation of α-1,4-glucosidic linkages. Moreover, PulY103B was strongly stimulated by nonionic detergents [viz, Tween 20 (10%), Tween 80 (1%), Triton X-100 (20%)] and commercial liquid detergents (3.0 g/L). Wash performance tests demonstrated that the mixture of PulY103B and detergent removed starch-based stains better than using detergent alone (p < 0.05). Therefore, this pullulanase has big potential as a detergent additive.

Tryptophan-96 in cytochrome P450 BM3 plays a key role in enzyme survival.

Flavocytochrome P450 from Bacillus megaterium (P450BM3 ) is a natural fusion protein containing reductase and heme domains. In the presence of NADPH and dioxygen the enzyme catalyses the hydroxylation of long-chain fatty acids. Analysis of the P450BM3 structure reveals chains of closely spaced tryptophan and tyrosine residues that might serve as pathways for high-potential oxidizing equivalents to escape from the heme active site when substrate oxidation is not possible. Our investigations of the total number of enzyme turnovers before deactivation have revealed that replacement of selected tryptophan and tyrosine residues with redox inactive groups leads to a twofold reduction in enzyme survival time. Tryptophan-96 is critical for prolonging enzyme activity, suggesting a key protective role for this residue.

The antibacterial mechanism of phenylacetic acid isolated from Bacillus megaterium L2 against Agrobacterium tumefaciens.

Background: Agrobacterium tumefaciens T-37 can infect grapes and other fruit trees and cause root cancer. Given the pollution and damage of chemical agents to the environment, the use of biological control has become an important area of focus. Bacillus megaterium L2 is a beneficial biocontrol strain isolated and identified in the laboratory, which has a good antibacterial effect on a variety of plant pathogens. The antibacterial metabolites of L2 were separated and purified to obtain a bioactive compound phenylacetic acid (PAA). Methods: The potential antibacterial mechanism of PAA against A. tumefaciens T-37 strain was determined by relative conductivity, leakage of nucleic acids, proteins, and soluble total sugars, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and reactive oxygen species (ROS). Results: PAA showed good antibacterial activity against strain A. tumefaciens T-37 with IC50 of 0.8038 mg/mL. Our data suggested that after treatment with PAA, the relative conductivity, nucleic acid, protein, and total soluble sugar of T-37 were increased significantly compared with the chloramphenicol treatment group and the negative treatment group. The total protein synthesis of T-37 cells was inhibited, the consumption of phosphorus decreased with the increase of incubation time, and the content of ROS was significantly higher than that in the negative treatment group. Meanwhile, the activity of two key enzymes (MDH and SDH) involved in the tricarboxylic acid cycle (TCA cycle) decreased. In addition, T-37 cells were found to be damaged by scanning electron microscopy observation. Our results showed that PAA can destroy cell membrane integrity, damage cell structures, affect cell metabolism, and inhibit protein synthesis to exert an antibacterial effect. Conclusions: We concluded that the mechanism of action of the PAA against strain T-37 might be described as PAA exerting antibacterial activity by affecting cell metabolism, inhibiting protein synthesis, and destroying cell membrane integrity and cell ultrastructure. Therefore, PAA has a promising application prospect in the prevention and treatment of root cancer disease caused by A. tumefaciens.

Novel mechanism and degradation kinetics of allethrin using Bacillus megaterium strain HLJ7 in contaminated soil/water environments.

As a common pyrethroid insecticide, allethrin is widely used for various purposes in agriculture and home applications. At present, allethrin residues have been frequently detected worldwide, yet little is known about the kinetics and degradation mechanisms of this insecticide. In this study, a highly efficient allethrin-degrading bacterium, Bacillus megaterium strain HLJ7, was obtained through enrichment culture technology. Strain HLJ7 can remove 96.5% of 50 mg L-1 allethrin in minimal medium within 11 days. The first-order kinetic analysis of degradation demonstrated that the half-life of allethrin degradation by strain HLJ7 was 3.56 days, which was significantly shorter than the 55.89 days of the control. The Box-Behnken design of the response surface method optimized the degradation conditions for strain HLJ7: temperature 32.18 °C, pH value 7.52, and inoculation amount 1.31 × 107 CFU mL-1. Using Andrews equation, the optimal concentration of strain HLJ7 to metabolize allethrin was determined to be 21.15 mg L-1, and the maximum specific degradation rate (qmax), half-rate constant (Ks) and inhibition coefficient (Ki) were calculated to be 1.80 d-1, 1.85 mg L-1 and 68.13 mg L-1, respectively. Gas chromatography-mass spectrometry identified five intermediate metabolites, suggesting that allethrin could be degraded firstly by cleavage of its carboxylester bond, followed by degradation of the five-carbon ring and subsequent metabolism. The results of soil remediation experiments showed that strain HLJ7 has excellent bioremediation potential in the soils. After 15 days of treatment, about 70.8% of the initial allethrin (50 mg kg-1) was removed and converted into nontoxic intermediate metabolites, and its half-life was significantly reduced in the soils. Taken together, these findings shed light on the degradation mechanisms of allethrin and also highlight the promising potentials of B. megaterium HLJ7 in bioremediation of allethrin-comtaminated environment.

Oleic acid based experimental evolution of Bacillus megaterium yielding an enhanced P450 BM3 variant.

BACKGROUND: Unlike most other P450 cytochrome monooxygenases, CYP102A1 from Bacillus megaterium (BM3) is both soluble and fused to its redox partner forming a single polypeptide chain. Like other monooxygenases, it can catalyze the insertion of oxygen unto the carbon-hydrogen bond which can result in a wide variety of commercially relevant products for pharmaceutical and fine chemical industries. However, the instability of the enzyme holds back the implementation of a BM3-based biocatalytic industrial processes due to the important enzyme cost it would prompt. RESULTS: In this work, we sought to enhance BM3's total specific product output by using experimental evolution, an approach not yet reported to improve this enzyme. By exploiting B. megaterium's own oleic acid metabolism, we pressed the evolution of a new variant of BM3, harbouring 34 new amino acid substitutions. The resulting variant, dubbed DE, increased the conversion of the substrate 10-pNCA to its product p-nitrophenolate 1.23 and 1.76-fold when using respectively NADPH or NADH as a cofactor, compared to wild type BM3. CONCLUSIONS: This new DE variant, showed increased organic cosolvent tolerance, increased product output and increased versatility in the use of either nicotinamide cofactors NADPH and NADH. Experimental evolution can be used to evolve or to create libraries of evolved BM3 variants with increased productivity and cosolvent tolerance. Such libraries could in turn be used in bioinformatics to further evolve BM3 more precisely. The experimental evolution results also supports the hypothesis which surmises that one of the roles of BM3 in Bacillus megaterium is to protect it from exogenous unsaturated fatty acids by breaking them down.

Effects of chlorpyrifos on the metabolic profiling of Bacillus megaterium strain RRB.

Many microorganisms have been reported to degrade organic pollutants in the environment and plants, however, the specific information about the effect of organic pollutants on the metabolism of microorganisms is poorly investigated. In the present study, the effect of the pesticide chlorpyrifos on the metabolic profiling of Bacillus megaterium strain RRB was investigated using metabolomics. Our data show that chlorpyrifos acting as an energy source was readily concentrated in the strain RRB from the culture medium. During early cultivation, the shift in energy sources from tryptic soy broth to chlorpyrifos may temporarily cause the strain RRB to enter the starvation stage, where some synthesis-related amino acids and intermediates in the pathways of TCA cycle and pyridoxine metabolism were decreased. The increase of nucleotides and lysine may help the strain RRB cope with the starvation stage. During later cultivation, many metabolites including organic acids, nucleosides and sugar phosphates were gradually accumulated, which indicates that chlorpyrifos could be utilized by the stain RRB to generate metabolites bacteria needed. In addition, arginine acting as a nitrogen-storage amino acid was gradually decreased with later cultivation, suggesting that chlorpyrifos could not provide enough nitrogen for bacteria.

Engineering precursor and co-factor supply to enhance D-pantothenic acid production in Bacillus megaterium.

High-yielding chemical and chemo-enzymatic methods of D-pantothenic acid (DPA) synthesis are limited by using poisonous chemicals and DL-pantolactone racemic mixture formation. Alternatively, the safe microbial fermentative route of DPA production was found promising but suffered from low productivity and precursor supplementation. In this study, Bacillus megaterium was metabolically engineered to produce DPA without precursor supplementation. In order to provide a higher supply of precursor D-pantoic acid, key genes involved in its synthesis are overexpressed, resulting strain was produced 0.53 ± 0.08 g/L DPA was attained in shake flasks. Cofactor CH2-THF was found to be vital for DPA biosynthesis and was regenerated through the serine-glycine degradation pathway. Enhanced supply of another precursor, ß-alanine was achieved by codon optimization and dosing of the limiting L-asparate-1-decarboxylase (ADC). Co-expression of Pantoate-ß-alanine ligase, ADC, phosphoenolpyruvate carboxylase, aspartate aminotransferase and aspartate ammonia-lyase enhanced DPA concentration to 2.56 ± 0.05 g/L at shake flasks level. Fed-batch fermentation in a bioreactor with and without the supplementation of ß-alanine increased DPA concentration to 19.52 ± 0.26 and 4.78 ± 0.53 g/L, respectively. This present study successfully demonstrated a rational approach combining precursor supply engineering with cofactor regeneration for the enhancement of DPA titer in recombinant B. megaterium.

One-Pot Biosynthesis of 3-Aminopropionic Acid from Fumaric Acid Using Recombinant Bacillus megaterium Containing a Linear Dual-Enzyme Cascade.

3-Aminopropionic acid (3-APA) has wide applications in food, cosmetics, pharmaceuticals, chemical, and polymer industries. This present study aimed to develop an eco-friendly whole-cell biocatalytic process for the bio-production of 3-APA from fumaric acid (FA) using Bacillus megaterium. A dual-enzyme cascade route with aspartate-1-decarboxylases (ADC) from Bacillus subtilis and native aspartate ammonia-lyase (AspA) was developed. Divergent catalytic efficiencies between these two enzymes led to an imbalance between both enzyme reactions. In order to coordinate AspA and ADC expression levels, gene mining, optimization, and duplication strategies were employed. Additionally, culture cultivation conditions and biocatalysis process parameters were optimized. A maximum 3-APA titer was obtained (11.68 ± 0.26 g/L) with a yield of 0.78 g/g under the following optimal conditions: 45 °C, pH 6.0, and 15 g/L FA. This study established a biocatalysis process for the production of 3-APA from FA using the whole cells of the recombinant B. megaterium.

Modulating the Coupling Efficiency of P450 BM3 by Controlling Water Diffusion through Access Tunnel Engineering.

Cytochromes P450 have gained much interest for their broad substrate scope in the catalysis of oxidation reactions for pharmaceuticals, plastics, and hormones. However, achieving high coupling efficiency by the engineering of P450s is still a big challenge. The presence of extra water around the active site is deemed to be related to uncoupling. In this study, the access tunnels of P450 BM3 from Bacillus megaterium are engineered to control water access from bulk solvent to the active site. Nine residues located in tunnels are investigated by site-saturation mutagenesis to reduce water diffusion, thereby improving the coupling efficiency. The recombined variant N319L/T411V/T436A shows improved coupling efficiency (from 31.2 % to 52.6 %). Tunnel polarity analysis and molecular dynamics simulation further indicate that reduced water molecules around the active site lead to higher coupling efficiency. Overall, this study provides valuable insight on improving coupling efficiency by controlling water diffusion through tunnel engineering.