Recruiting a Phosphite Dehydrogenase/Formamidase-Driven Antimicrobial Contamination System in Bacillus subtilis for Nonsterilized Fermentation of Acetoin.

Microbial contamination, especially in large-scale processes, is partly a life-or-death issue for industrial fermentation. Therefore, the aim of this research was to create an antimicrobial contamination system in Bacillus subtilis 168 (an ideal acetoin producer for its safety and acetoin synthesis potential). First, introduction of the formamidase (FmdA) from Helicobacter pylori and the phosphite dehydrogenase (PtxD) from Pseudomonas stutzeri enabled the engineered Bacillus subtilis to simultaneously assimilate formamide and phosphite as nitrogen (N) and phosphorus (P) sources. Thus, the engineered B. subtilis became the dominant population in a potentially contaminated system, while contaminated microbes were starved of key nutrients. Second, stepwise metabolic engineering via chromosome-based overexpression of the relevant glycolysis and acetoin biosynthesis genes led to a 1.12-fold increment in acetoin titer compared with the starting host. Finally, with our best acetoin producer, 25.56 g/L acetoin was synthesized in the fed-batch fermentation, with a productivity of 0.33 g/L/h and a yield of 0.37 g/g under a nonsterilized and antibiotic-free system. More importantly, our work fulfills many key criteria of sustainable chemistry since sterilization is abolished, contributing to the simplified fermentation operation with lower energy consumption and cost.

Combinatorial synthetic pathway fine-tuning and cofactor regeneration for metabolic engineering of Escherichia coli significantly improve production of D-glucaric acid.

D-glucaric acid (GA) has been identified as among promising biotechnological alternatives to oil-based chemicals. GA and its derivatives are widely used in food additives, dietary supplements, drugs, detergents, corrosion inhibitors and biodegradable materials. The increasing availability of a GA market is improving the cost-effectiveness and efficiency of various biosynthetic pathways. In this study, an engineered Escherichia coli strain GA10 was constructed by systematic metabolic engineering. This involved redirecting metabolic flux into the GA biosynthetic pathways, blocking the conversion pathways of d-glucuronic acid (GlcA) and GA into by-products, introducing an in situ NAD+ regeneration system and fine-tuning the activity of the key enzyme, myo-inositol oxygenase (Miox). Subsequently, the culture medium was optimized to achieve the best performance of the GA10 strain. GA was produced at 5.35 g/L (extracellular and intracellular), with a maximized yield of ∼0.46 mol/mol on d-glucose and glycerol, by batch fermentation. This work demonstrates efficient biosynthetic pathways of GA in E. coli by metabolic engineering and should accelerate the application of GA biosynthetic pathways in industrial processes.

Co-immobilization of multiple enzymes by self-assembly and chemical crosslinking for cofactor regeneration and robust biocatalysis.

Artificial multienzyme biocatalysts have played a crucial role in biosynthesis because they allow for conducting complex reactions. Here, we reorted a facile approach to fabricate multienzyme nanodevices with high catalytic activity and stability based on protein assembly and chemical crosslinking. The self-assembled partner SpyCatcher and SpyTag were genetically fused with 2,3-butanediol hydrogenase and formate hydrogenase to produce KgBDH-SC (SpyCatcher-fused 2,3-butanediol hydrogenase) and FDH-ST (SpyTag-fused formate hydrogenase), respectively. After assembling the two fusion proteins, the complexes were then immobilized on the functionalized silicon dioxide nanoparticles by glutaraldehyde, forming KgBDH-SC-ST-FDH-SiO2 with the capability of reducing 2-hydroxyacetophenone to (R)-1-phenyl-1,2-ethanediol with cofactor regeneration. Under the optimal conditions, the created co-immobilized enzymes performed 49% activity recovery compared with the mixture of free enzymes as well as showed 2.9-fold higher catalytic activity than the traditional random co-immobilized enzymes. Moreover, KgBDH-SC-ST-FDH-SiO2 showed better pH stability and organic solvents stability than the free enzymes, and remained 52.5% overall catalytic activity after 8 cycles. Finally, the co-immobilized enzymes can reduce 60 mM HAP for fabrication of (R)-PED with cofactor regeneration in the phosphate buffer reaction system, affording 83.9% yield and above 99% optical purity.

Recombinant expression and characterization of a novel cold-adapted type I pullulanase for efficient amylopectin hydrolysis.

Cold-adapted pullulanase with high catalytic activity and stability is of special interest for its wide application in cold starch hydrolysis, but few pullulanases displaying excellent characteristics at ambient temperature and acidic pH have hitherto been reported. Here, a novel pullulanase from Bacillus methanolicus PB1 was successfully expressed in Escherichia coli BL21 (DE3) and determined to be a cold-adapted type I pullulanase (PulPB1) with maximum activity at 50 °C and pH 5.5. The recombinant PulPB1 showed great stability, its half-life at 50 °C was 137 h. PulPB1 can efficiently hydrolyze pullulan and amylopectin, with activities of 292 and 184 U/mg at 50 °C and pH 5.5, respectively. Moreover, the N-terminal domain of PulPB1 was found to significantly affect the enzymatic performance. Following truncation of the N-terminal domain, the activity towards pullulan decreased markedly from 292 to 141 U/mg and the half-life at 50 °C decreased from 137 to 10 h. Compared to the hydrolysis system with amyloglucosidase alone, the catalytic efficiency showed a 2.4-fold increase on combining PulPB1 with amyloglucosidase for amylopectin hydrolysis at 40 °C. This demonstrates that PulPB1 is promising for development as a superior candidate for cold amylopectin hydrolysis.

Immobilization of Cofactor Self-Sufficient Recombinant Escherichia coli for Enantioselective Biosynthesis of (R)-1-Phenyl-1,2-Ethanediol.

(R)-1-phenyl-1,2-ethanediol is an important synthon for the preparation of ß-adrenergic blocking agents. This study identified a (2R,3R)-butanediol dehydrogenase (KgBDH) from Kurthia gibsonii SC0312, which showed high enantioselectivity for production of (R)-1-phenyl-1,2-ethanediol by reduction of 2-hydroxyacetophenone. KgBDH was expressed in a recombinant engineered strain, purified, and characterized. It showed good catalytic activity at pH 6-8 and better stability in alkaline (pH 7.5-8) than an acidic environment (pH 6.0-7.0), providing approximately 73 and 88% of residual activity after 96 h at pH 7.5 and 8.0, respectively. The maximum catalytic activity was obtained at 45°C; nevertheless, poor thermal stability was observed at >30°C. Additionally, the examined metal ions did not activate the catalytic activity of KgBDH. A recombinant Escherichia coli strain coexpressing KgBDH and glucose dehydrogenase (GHD) was constructed and immobilized via entrapment with a mixture of activated carbon and calcium alginate via entrapment. The immobilized cells had 1.8-fold higher catalytic activity than that of cells immobilized by calcium alginate alone. The maximum catalytic activity of the immobilized cells was achieved at pH 7.5, and favorable pH stability was observed at pH 6.0-9.0. Moreover, the immobilized cells showed favorable thermal stability at 25-30°C and better operational stability than free cells, retaining approximately 55% of the initial catalytic activity after four cycles. Finally, 81% yields (195 mM product) and >99% enantiomeric excess (ee) of (R)-1-phenyl-1,2-ethanediol were produced within 12 h through a fed-batch strategy with the immobilized cells (25 mg/ml wet cells) at 35°C and 180 rpm, with a productivity of approximately 54 g/L per day.

Improving the thermostability and activity of Paenibacillus pasadenensis chitinase through semi-rational design.

Chitinase is a promising biocatalyst for chitin biotransformation in the field of recalcitrant biomass degradation. Excellent catalytic performance is conducive to its commercial utilization. In this work, sequence- and structure-based semi-rational design was performed to evolve the thermostability and activity of a previously identified chitinase PpChi1 from Paenibacillus pasadenensis CS0611. After combinational mutagenesis, the mutant S244C-I319C/T259P with disulfide bond introduction and proline substitution exhibited higher specific activity at higher temperature, 26.3-fold in half-life value at 50 °C, and a 7.9 °C rise in half-inactivation temperature T1/215min compared to the wild-type enzyme. The optimal reaction temperature of the mutant was shifted from 45 °C to 52.5 °C. Molecular dynamic simulation and structure analysis confirmed that these improvements of the mutant were attributed to its stabilized folding form, possibly caused by the decreased entropy of unfolding. This work gives an initial insight into the effect of conserved proline residues in thermostable chitinases and proposes a feasible approach for improving chitinase thermostability to facilitate its application in chitin hydrolysis to valuable oligosaccharides.

Highly enantioselective resolution of racemic 1-phenyl-1,2-ethanediol to (S)-1-phenyl-1,2-ethanediol by Kurthia gibsonii SC0312 in a biphasic system.

The asymmetric resolution of racemic 1-phenyl-1,2-ethanediol (PED) to (S)-PED by Kurthia gibsonii SC0312 (K. gibsonii SC0312) was conducted in a biphasic system comprised of an organic solvent and aqueous phosphate buffer. The impacts of organic solvents on the whole cell catalytic activity, metabolic activity, membrane integrity, and material distribution were first evaluated. The results showed that all organic solvents, except for dibutyl phthalate, showed a detrimental effect on the metabolic activity of the cells, especially for those with low log P values. All organic solvents were capable of changing the membrane permeability and membrane integrity of the cells. Moreover, some organic solvents showed a good extraction of the oxidation product. Finally, a high yield of 47.7 % of (S)-PED was obtained by the asymmetric resolution of racemic PED using K. gibsonii SC0312 in a biphasic system under the optimal conditions: racemic PED 120 mM, temperature 35 °C, reaction time 6 h, 180 rpm, and a volume ratio of dibutyl phthalate to aqueous phosphate buffer of 1:1. The optical purity of (S)-PED increased from 51.3 % to >99 %. This work described an efficient approach to improve reaction efficiency, and constructed a highly effective biphasic reaction system for the fabrication of (S)-PED via K. gibsonii SC0312.

Efficient synthesis of 5-hydroxymethyl-2-furancarboxylic acid by Escherichia coli overexpressing aldehyde dehydrogenases.

Catalytic transformation of biomass-derived furans into value-added chemicals and biofuels has received considerable interest recently. In this work, aldehyde dehydrogenases (ALDHs) were identified from Comamonas testosteroni SC1588 for the oxidation of bio-based furans into furan carboxylic acids. Of the whole-cell biocatalysts constructed, Escherichia coli expressing a vanillin dehydrogenase (E. coli_CtVDH1) proved to be the best for the oxidation of 5-hydroxymethylfurfural (HMF). 5-Hydroxymethyl-2-furancarboxylic acid (HMFCA) was obtained in the yield of approximately 92 % within 12 h using this recombinant strain when the HMF concentration was up to 200 mM. In a fed-batch process, 292 mM of HMFCA was produced within 20.5 h, thereby providing a productivity as high as 2.0 g/L h. Other furan carboxylic acids were synthesized in the yields of 83-95%. Besides, the partially purified HMF was smoothly converted into HMFCA by this recombinant strain, with a 90% yield.

Metabolic engineering of a robust Escherichia coli strain with a dual protection system.

Considerable attention has been given to the development of robust fermentation processes, but microbial contamination and phage infection remain deadly threats that need to be addressed. In this study, a robust Escherichia coli BL21(DE3) strain was successfully constructed by simultaneously introducing a nitrogen and phosphorus (N&P) system in combination with a CRISPR/Cas9 system. The N&P metabolic pathways were able to express formamidase and phosphite dehydrogenase in the host cell, thus enabled cell growth in auxotrophic 3-(N-morpholino)propanesulfonic acid medium with formamide and phosphite as nitrogen and phosphorus sources, respectively. N&P metabolic pathways also allowed efficient expression of heterologous proteins, such as green fluorescent protein (GFP) and chitinase, while contaminating bacteria or yeast species could hardly survive in this medium. The host strain was further engineered by exploiting the CRISPR/Cas9 system to enhance the resistance against phage attack. The resultant strain was able to grow in the presence of T7 phage at a concentration of up to 2 × 107 plaque-forming units/ml and produce GFP with a yield of up to 30 µg/109 colony-forming units, exhibiting significant advantages over conventional engineered E. coli. This newly engineered, robust E. coli BL21(DE3) strain therefore shows great potential for future applications in industrial fermentation.

Fungal polysaccharide similar with host Dendrobium officinale polysaccharide: Preparation, structure characteristics and biological activities.

Based on the concept of endophytic fungus, if endophytic fungus can produce the same or similar product as the host plant, which will get rid of the restrictions of farmland, seasonal and pest, the active product could be sustainably obtained. In this study, an endophytic fungus polysaccharide FP showing the similar structure with the host Dendrobium officinale polysaccharide (DOP) was sustainably and cost-effectively obtained under preferred reaction conditions with different C/N ratio. The FP with high yield up to 2.77 ±â€¯0.51 g/L showed same monosaccharide composition with DOP as well as some host-plant-associated polysaccharides in published literatures. The main chain of FP was composed by →3,6)-ß-L-Man-(1→, α-D-Glc-(1→, →4)-α-D-Glc-(1→, →3,6)-ß-D-Gal-(1→, and →6)-ß-D-Gal-(1→, while the side chain was α-D-Glc-(1→. Meanwhile, FP was confirmed as a safe polysaccharide with good antioxidant, antiglycation and immunomodulatory activities. Furthermore, TLR2 and TLR4 were confirmed as the membrane receptors of FP on RAW264.7 cells.

Highly efficient asymmetric reduction of 2-octanone in biphasic system by immobilized Acetobacter sp. CCTCC M209061 cells.

Highly efficient asymmetric reduction of 2-octanone to (R)-2-octanol catalyzed by immobilized Acetobacter sp. CCTCC M209061 cells was achieved in a biphasic system. Bioreduction conducted in aqueous single phase buffer was limited due to poor solubility and toxicity towards cells cause by product accumulation. Introduction of [C4MIM]·Ac accelerated the biotransformation process, giving 99% yield, >99% product e.e. and 1.42-fold higher initial reaction rate in conversion of 10 mM 2-octanone as substrate, compared with 99% yield, 97.3% product e.e. and 1.57 µmol min-1 of initial reaction rate in a aqueous single phase buffer system containing 6 mM 2-octanone as the optimal substrate concentration. Moreover, in the [C4MIM]·Ac-containing buffer/n-tetradecane biphasic system, the optimal substrate concentration was enhanced by 83 times (500 mM) in comparison with that in aqueous single phase buffer, resulting in 53.4% yield (267 mM), 99% product e.e. with 8.9 mM g-1 h-1 space time yield.