Modular optimization of multi-gene pathways for fumarate production

Microbial fumarate production from renewable feedstock is a promising and sustainable alternative to petroleum-based chemical synthesis. Here, we report a modular engineering approach that systematically removed metabolic pathway bottlenecks and led to significant titer improvements in a multi-gene fumarate metabolic pathway. On the basis of central pathway architecture, yeast fumarate biosynthesis was re-cast into three modules: reduction module, oxidation module, and byproduct module. We targeted reduction module and oxidation module to the cytoplasm and the mitochondria, respectively. Combinatorially tuning pathway efficiency by constructing protein fusions RoMDH-P160A and KGD2-SUCLG2 and optimizing metabolic balance by controlling genes RoPYC, RoMDH-P160A, KGD2-SUCLG2 and SDH1 expression strengths led to significantly improved fumarate production (20.46 g/L). In byproduct module, synthetizing DNA-guided scaffolds and designing sRNA switchs enabled further production improvement up to 33.13 g/L. These results suggest that modular pathway engineering can systematically optimize biosynthesis pathways to enable an efficient production of fumarate.     

Inhibitory activity of surfactin,produced by different Bacillus subtilis subsp. subtilis strains,against Listeria monocytogenes sensitive and bacteriocin-resistant strains

Three surfactin-producing Bacillus subtilis strains, C4, M1 and G2III, previously isolated from honey and intestines from the Apis mellifera L. bee, were phylogenetically characterized at sub-species level as B. subtilis subsp. subtilis using gyrA gene sequencing. The antagonistic effect of surfactin was studied against seven different Listeria monocytogenes strains, 6 of which were resistant to bacteriocins. Surfactin showed anti-Listeria activity against all 7 strains and a dose of 0.125 mg/mL of surfactin was enough to inhibit this pathogen. Surfactin sintetized by B. subtilis subsp. subtilis C4 inhibited the pathogen in lower concentrations, 0.125 mg/mL, followed by G2III and M1 with 0.25 and 1 mg/mL, respectively. In particular, a dose of 0.125 mg/mL reduced the viability of L. monocytogenes 99/287 RB6, a bacteriocin-resistant strain, to 5 log orders. Surfactin assayed maintained anti-Listeria activity within a pH range of between 2 and 10, after heat treatment (boiling for 10 min and autoclaving at 121 °C for 15 min) and after treatment with proteolytic enzymes. These results suggest that surfactin can be used as a new tool for prevention and the control of L. monocytogenes in different environments, for example, in the food industry.     

Metabolic engineering of Escherichia coli for production of salvianic acid A via an artificial biosynthetic pathway

Salvianic acid A, a valuable derivative from L-tyrosine biosynthetic pathway of the herbal plant Salvia miltiorrhiza, is well known for its antioxidant activities and efficacious therapeutic potential on cardiovascular diseases. Salvianic acid A was traditionally isolated from plant root or synthesized by chemical methods, both of which had low efficiency. Herein, we developed an unprecedented artificial biosynthetic pathway of salvianic acid A in E. coli, enabling its production from glucose directly. In this pathway, 4-hydroxyphenylpyruvate was converted to salvianic acid A via D-lactate dehydrogenase (encoding by d-ldh from Lactobacillus pentosus) and hydroxylase complex (encoding by hpaBC from E. coli). Furthermore, we optimized the pathway by a modular engineering approach and deleting genes involved in the regulatory and competing pathways. The metabolically engineered E. coli strain achieved high productivity of salvianic acid A (7.1 g/L) with a yield of 0.47 mol/mol glucose.     

Direct ethanol production from N-acetylglucosamine and chitin substrates by Mucor species

Chitin, which is a polymer of β-(1–4) linked N-acetyl-d-glucosamine (GlcNAc) residues, is one of the most abundant renewable resources in nature, after cellulose. In this study, we found some native Mucor strains, which can use GlcNAc and chitin substrates as carbon sources for growth and ethanol production. One of these strains, M. circinelloides NBRC 6746 produced 18.6 ± 0.6 g/l of ethanol from 50 g/l of GlcNAc after 72 h and the maximum ethanol production rate was 0.75 ± 0.1 g/l/h. Furthermore, M. circinelloides NBRC 4572 produced 6.00 ± 0.22 and 0.46 ± 0.04 g/l of ethanol from 50 g/l of colloidal chitin and chitin powder after 16 and 12 days, respectively. We also found an extracellular chitinolytic enzyme producing strain M. ambiguus NBRC 8092, and successfully improved ethanol productivity of NBRC 4572 from colloidal chitin using crude chitinolytic enzyme derived from NBRC 8092. The ethanol titer reached 9.44 ± 0.10 g/l after 16 days. These results were the first bioethanol production from GlcNAc and chitin substrates by native organisms, and also suggest that these Mucor strains have great potential for the simultaneous saccharification and fermentation (SSF) of chitin biomass.     

Improved production of alkaline polygalacturonate lyase by homologous overexpression pelA in Bacillus subtilis

A polygalacturonate lyase (PGL), PelA, was purified from the culture broth of Bacillus subtilis 7-3-3, with a molecular weight, optimal temperature, and pH of approximately 45 kDa, 55 °C, and 9.4, respectively. The PGL gene (pelA) was homologously overexpressed in B. subtilis 7-3-3 to increase the gene copies and enhance the PGL production. The resulting PGL activity was 2138 U mL^?1 at 44 h, and the productivity reached 48.58 U (mL h)^?1 through the homologous overexpression of strain B-pN-pelA in a 7.5 L fermentor, the highest PGL production compared to those reported in literature to the best of our knowledge. Crude enzyme has high PGL and PGase activity, which can remove 50.58% of pectin in unpretreatment ramie fibers at 50 °C for 4 h. Meanwhile, the enzyme system with a low level hemicellulase and almost no cellulase will further help in enhancing the efficiency of degumming besides maintaining tenacity of plant fiber. The B. subtilis B-pN-pelA shows high genetic stability and has great potential in the textile industry.     

Comparison of lipopeptide biosurfactants production by Bacillus subtilis strains in submerged and solid state fermentation systems using a cheap carbon source: Some industrial applications of biosurfactants

Biosurfactants, in general has the potential to aid in the recovery of subsurface organic contaminants (environmental remediation) or crude oils (oil recovery). However, high production and purification costs limit its use in these high-volume applications. In the present study, the efficiency of two Bacillus subtilis strains viz., DM-03 and DM-04 for the production of biosurfactants in two fermentation systems viz., solid state fermentation (SSF) and submerged fermentation (SmF) was compared. Both the B. subtilis strains produced appreciable and equal amount of crude lipopeptide biosurfactants (B. subtilis DM-03: 80.0 ± 9 mg/gds in SmF and 67.0 ± 6 mg/gds in SSF; B. subtilis DM-04: 23.0 ± 5.0 mg/gds in SmF and 20.0 ± 2.5 mg/gds in SSF) in the two different fermentation systems using potato peels as cheap carbon source. These thermostable lipopeptide biosurfactants produced by B. subtilis strains either in SSF or in SmF, exhibited strong emulsifying property and could release appreciable amount of oil from saturated sand pack column. Further, it was shown by biochemical analysis, RP-HPLC profile and IR spectra that there is no qualitative and qualitative differences in the composition of crude biosurfactants produced either in SmF or in SSF system.     

Engineering high-level production of fatty alcohols by Saccharomyces cerevisiae from lignocellulosic feedstocks

Fatty alcohols in the C12-C18 range are used in personal care products, lubricants, and potentially biofuels. These compounds can be produced from the fatty acid pathway by a fatty acid reductase (FAR), yet yields from the preferred industrial host Saccharomyces cerevisiae remain under 2% of the theoretical maximum from glucose. Here we improved titer and yield of fatty alcohols using an approach involving quantitative analysis of protein levels and metabolic flux, engineering enzyme level and localization, pull-push-block engineering of carbon flux, and cofactor balancing. We compared four heterologous FARs, finding highest activity and endoplasmic reticulum localization from a Mus musculus FAR. After screening an additional twenty-one single-gene edits, we identified increasing FAR expression; deleting competing reactions encoded by DGA1, HFD1, and ADH6; overexpressing a mutant acetyl-CoA carboxylase; limiting NADPH and carbon usage by the glutamate dehydrogenase encoded by GDH1; and overexpressing the Δ9-desaturase encoded by OLE1 as successful strategies to improve titer. Our final strain produced 1.2 g/L fatty alcohols in shake flasks, and 6.0 g/L in fed-batch fermentation, corresponding to ~ 20% of the maximum theoretical yield from glucose, the highest titers and yields reported to date in S. cerevisiae. We further demonstrate high-level production from lignocellulosic feedstocks derived from ionic-liquid treated switchgrass and sorghum, reaching 0.7 g/L in shake flasks. Altogether, our work represents progress towards efficient and renewable microbial production of fatty acid-derived products.     

Milk-clotting enzymes produced by culture of Bacillus subtilis natto

Factors affecting the production of milk-clotting enzyme (MCE) by Bacillus subtilis (natto) Takahashi, a ready available commercial natto starter, were studied. Remarkable milk-clotting activity (MCA), 685.7 SU/ml or 12,000 SU/g, was obtained when the bacteria were cultivated in the medium containing sucrose (50 g/L) and basal salts at pH 6, 37 °C with shaking at 175 rpm for 1 day. The MCA and MCA/PA ratio of the crude enzyme obtained are comparable with those of Pfizer microbial rennin and Mucor rennin. The crude enzyme showed excellent pH and thermal stability; it retained 96% of MCA after incubation for 40 min at 40 °C and retained more than 80% of its activity between pH 4 and pH 7 for more than 30 min at 30 °C. The MCE of B. subtilis (natto) Takahashi has potential as calf rennet substitutes.     

Effective control of black Sigatoka disease using a microbial fungicide based on Bacillus subtilis EA-CB0015 culture

Black Sigatoka disease caused by the fungus Mycosphaerella fijiensis Morelet is the most devastating disease of bananas worldwide. Its management is reliant on protectant and systemic fungicides despite their environmental concerns. This study evaluated the effect of a microbial fungicide (MF) based on Bacillus subtilis EA-CB0015 and its metabolites for the control of black Sigatoka disease on banana plants in greenhouse and field conditions. The MF applied at 1.5 L/ha and 3.0 L/ha provided control of the disease comparable to the protectant fungicide chlorothalonil in greenhouse. In the field, the MF applied in solution with water at 0.15 L/ha and 1.5 L/ha every 11 days during 10 weeks reduced black Sigatoka disease severity in 20.2% and 28.1% respectively; reductions comparable to those obtained with the protectant fungicides chlorothalonil (1.5 L/ha) and mancozeb (3.8 L/ha). The MF incorporated into different programs with systemic fungicides reduced disease level up to 42.9% with no significant differences with the conventional program. To determine which component of the MF is responsible for the activity against M. fijiensis, greenhouse and in vitro tests were set up to evaluate individually the spores, vegetative cells and secondary metabolites of B. subtilis EA-CB0015. All components reduced the severity of the disease and the germination of ascospores. For both trials the activity of the metabolites was higher and comparable to the activity obtained with the MF, indicating that the efficacy of the MF depends mainly on the metabolites and in lesser extent to B. subtilis EA-CB0015 cells.     

Microencapsulation of Bacillus subtilis B99-2 and its biocontrol efficiency against Rhizoctonia solani in tomato

Biological control is a promising approach to protecting plants from disease. Bacillus subtilis has been widely used in agriculture for promoting plant growth and biocontrol. However, their short shelf life limits the application of biological pesticides. The objectives of this study were to develop a microencapsulation procedure of B. subtilis B99-2 using maltodextrin and gum arabic as wall materials to determine the optimum conditions of spray-drying in microencapsulation, evaluate storage stability of microcapsules, and assess their biocontrol efficiency against Rhizoctonia solani in tomato under field conditions. We microencapsulated the Bacillus thallus by spray-drying with various concentrations of the wall material. Maltodextrin was found to be an efficient wall material, especially at concentrations higher than 80%, while gum arabic did not affect the bacterial survival rate. The mean survival rate of B. subtilis was more than 90%, when spray drying was performed at 145 °C, with a feed flow rate of 550 mL h⁻¹, and a spray pressure of 0.15 MPa. B. subtilis microcapsule survival rate was 87.53% after 540 d of storage, which was a longer shelf life than that of wettable powders. Moreover, its biocontrol efficacy reached 79.91% when a dosage of 300 g hm⁻² was used, the microcapsule showed higher control efficacy than Thiram wettable powder against R. solani in tomato under field conditions. All these characteristics indicated that B. subtilis microcapsules have the potential to become a successful biocontrol product.     

Enhanced solvent production by metabolic engineering of a twin-clostridial consortium

The efficient fermentative production of solvents (acetone, n-butanol, and ethanol) from a lignocellulosic feedstock using a single process microorganism has yet to be demonstrated. Herein, we developed a consolidated bioprocessing (CBP) based on a twin-clostridial consortium composed of Clostridium cellulovorans and Clostridium beijerinckii capable of producing cellulosic butanol from alkali-extracted, deshelled corn cobs (AECC). To accomplish this a genetic system was developed for C. cellulovorans and used to knock out the genes encoding acetate kinase (Clocel_1892) and lactate dehydrogenase (Clocel_1533), and to overexpress the gene encoding butyrate kinase (Clocel_3674), thereby pulling carbon flux towards butyrate production. In parallel, to enhance ethanol production, the expression of a putative hydrogenase gene (Clocel_2243) was down-regulated using CRISPR interference (CRISPRi). Simultaneously, genes involved in organic acids reassimilation (ctfAB, cbei_3833/3834) and pentose utilization (xylR, cbei_2385 and xylT, cbei_0109) were engineered in C. beijerinckii to enhance solvent production. The engineered twin-clostridia consortium was shown to decompose 83.2 g/L of AECC and produce 22.1 g/L of solvents (4.25 g/L acetone, 11.5 g/L butanol and 6.37 g/L ethanol). This titer of acetone-butanol-ethanol (ABE) approximates to that achieved from a starchy feedstock. The developed twin-clostridial consortium serves as a promising platform for ABE fermentation from lignocellulose by CBP.     

Effects of dietary Bacillus subtilis and inulin supplementation on performance,eggshell quality,intestinal morphology and microflora composition of laying hens in the late phase of production

Eighty Lohmann White laying hens were used to investigate the effect of dietary inclusion of Bacillus subtilis and inulin, individually or in combination, on egg production, eggshell quality, tibia traits, Ca retention, and small intestine morphology and microflora composition from 64 to 75 weeks of age. Hens were randomly distributed into 4 treatment groups, with 5 replicates per treatment and 4 hens per replicate. Treatment groups were fed basal diet (control), basal diet plus 1 g/kg B. subtilis (2.3 × 10⁸ cfu/g), basal diet plus 1 g/kg inulin, or basal diet plus a synbiotic combination of 1 g/kg B. subtilis (2.3 × 10⁸ cfu/g) and 1 g/kg inulin. Dietary supplementation of B. subtilis, inulin or synbiotic improved (P<0.05) feed conversion, egg performance, eggshell quality and calcium retention compared with the control. B. subtilis and synbiotic groups exhibited the highest (P<0.05) increase in egg production and egg weight. Inulin and synbiotic groups exhibited the highest (P<0.05) increase in eggshell thickness and eggshell calcium content, and the lowest (P<0.05) eggshell deformations. Unmarketable eggs were 8.4% (P<0.05) of the total eggs produced by the control group compared to 3.5%, 1.7%, and 1.5% for the B. subtilis, inulin and synbiotic groups, respectively. Tibia density, ash, and Ca content increased (P<0.05) by inulin and synbiotic inclusions, compared with the control. B. subtilis, inulin, and their synbiotic combination increased (P<0.05) villus height and crypt depth in all intestinal segments, compared with the control. B. subtilis and inulin modulated the ileal and caecal microflora composition by decreasing (P<0.05) numbers of Clostridium and Coliforms and increasing (P<0.05) numbers of bifidobacteria and lactobacilli, compared with the control. Colonization of the beneficial microflora along with increasing the villi–crypts absorptive area were directly associated with the improvements in performance and eggshell quality. It can be concluded that egg production and eggshell quality of laying hens can be improved (P<0.05) in the late phase of production by dietary inclusion of B. subtilis and inulin.     

Cloning of amidase gene from Rhodococcus erythropolis and expression by distinct promoters in Bacillus subtilis

Amidase was a crucial enzyme responsible for the conversion of acrylamide to acrylic acid in Rhodococcus erythropolis. Its coding gene ami was amplified by PCR using the genomic DNA of R. erythropolis as template. Subsequently, it was ligated to expression plasmids and transformed in Escherichia coli and Bacillus subtilis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that both recombinant E. coli BL21 (DE3) and B. subtilis generated amidase of 56 kDa. The expression mass and enzyme activity suggested that B. subtilis was more suitable as a host when ami gene was under the control of a powerful promoter. To further study the expression effect of different promoters in B. subtilis, five distinct promoters (sacB, amyE, p43, degQ, aprE) and their native signal peptide genes were employed to separately construct five different vectors harboring ami gene. Of the five novel vectors, the amyE promoter along with its native signal peptide gene was most effective. The maximum specific activity of amidase at pH 7.0 and 37 °C was about 8.7 U/mg and the conversion efficiency could approximately reach 90% within 6 h. This result indicated the expression difference of distinct promoters, which provided the basis for the forthcoming research.     

Production of butyric acid by a cellulolytic actinobacterium Thermobifida fusca on cellulose

Thermobifida fusca not only produces cellulases, hemicellulases and xylanases, but also excretes butyric acid. In order to achieve a high yield of butyric acid, the effect of different carbon sources: mannose, xylose, lactose, cellobiose, glucose, sucrose and acetates, on butyric acid production was studied. The highest yield of butyric acid was 0.67 g/g C (g-butyric acid/g-carbon input) on cellobiose. The best stir speed and aeration rate for butyric acid production were found to be 400 rpm and 2 vvm in a 5-L fermentor. The maximum titer of 2.1 g/L butyric acid was achieved on 9.66 g/L cellulose. In order to test the production of butyric acid on lignocellulosic biomass, corn stover was used as the substrate, on which there was 2.37 g/L butyric acid produced under the optimized conditions. In addition, butyric acid synthesis pathway was identified involving five genes that catalyzed reactions from acetyl-CoA to butanoyl-CoA in T. fusca.     

Purification and characterization of recombinant Bacillus subtilis 168 catalase using a basic polypeptide from ribosomal protein L2

An efficient purification system for purifying recombinant Bacillus subtilis 168 catalase (KatA) expressed in Escherichia coli was developed. The basic region containing 252–273 amino acids derived from E. coli ribosomal protein L2 was used as an affinity tag while the small ubiquitin-like modifier (SUMO) was introduced as one specific protease cleavage site between the target protein and the purification tags. L2 (252–273)–SUMO fusion protein purification method can be effectively applied to purify the recombinant catalase using cation exchange resin. This purification procedure was used to purify the KatA and achieved a purification fold of 30.5, a specific activity of 48,227.2 U/mg and an activity recovery of 74.5%. The enzyme showed a Soret peak at 407 nm. The enzyme kept its activity between pH 5 and 10 and between 30 °C and 60 °C, with the highest activity at pH 8.0 and 37 °C. The enzyme displayed an apparent Km of 39.08 mM for hydrogen peroxide. These results agree well with the previous reports about B. subtilis catalase. L2 (252–273)–SUMO fusion protein purification technique provides a novel and effective fusion expression system for the production of recombinant proteins.     

Production of di-d-fructofranosyl-2,6′:2′,6-anhydride (DFA IV) by recombinant Bacillus subtilis carrying heterogenous levan fructotransferase from Arthrobacter nicotinovorans GS-9

We developed di-d-fructofranosyl-2,6′:2′,6-anhydride (DFA IV) production system with single culture of Bacillus subtilis directly from sucrose. This system can avoid the purification procedure of levan which organic solvent was used for precipitation. The levan fructotransferase (LFTase) gene was cloned from Arthrobacter nicotinovorans GS-9 (AHU1840, FERM P-15285) and expressed in levan producing B. subtilis 168. LFTase activity was detected in the culture supernatant of the transformant with maximal activity of 0.062 U/ml after 15.5 h post induction. Then sucrose was added as substrate and incubated. About 78 h after addition of sucrose, 20.5 g/l of DFA IV was produced from 139.3 g/l of sucrose consumed. The yield of DFA IV from sucrose was 14.7 wt.%.     

Spore production in solid-state fermentation of rice by Clonostachys rosea,a biopesticide for gray mold of strawberries

Gray mold caused by Botrytis cinerea is an important disease of strawberry. Clonostachys rosea is a mycoparasite of B. cinerea that reduces fruit losses when used as a biocontrol agent. Since spore production by C. rosea has not been optimized, we investigated factors affecting sporulation under aseptic conditions on white rice grains. The greatest spore production in glass flasks, 3.4 × 10⁹ spores/g-dry-matter (gDM), occurred with an initial moisture content of 46% (w/w wet basis), inoculated with 1 × 10⁶ spores/gDM and hand shaken every 15 days. However, a lower inoculum density (9 × 10³ spores/gDM) and no shaking also gave acceptable sporulation. In plastic bags 1.1 × 10⁸ spores/gDM were produced in 15 days, suggesting that larger scale production may be feasible: with this spore content, 24 m² of incubator space would produce sufficient spores for the continued treatment of 1 ha of strawberry plants.