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2-Phenylethanol (2- PE) is an aromatic alcohol with wide applications, but there is still no efficient microbial cell factory for 2-PE based on Escherichia coli. In this study, we constructed a metabolically engineered E. coli capable of de novo synthesis of 2-PE from glucose. Firstly, the heterologous styrene-derived and Ehrlich pathways were individually constructed in an L-Phe producer. The results showed that the Ehrlich pathway was better suited to the host than the styrene-derived pathway, resulting in a higher 2-PE titer of ∼0.76 ± 0.02 g/L after 72 h of shake flask fermentation. Furthermore, the phenylacetic acid synthase encoded by feaB was deleted to decrease the consumption of 2-phenylacetaldehyde, and the 2-PE titer increased to 1.75 ± 0.08 g/L. As phosphoenolpyruvate (PEP) is an important precursor for L-Phe synthesis, both the crr and pykF genes were knocked out, leading to ∼35% increase of the 2-PE titer, which reached 2.36 ± 0.06 g/L. Finally, a plasmid-free engineered strain was constructed based on the Ehrlich pathway by integrating multiple ARO10 cassettes (encoding phenylpyruvate decarboxylases) and overexpressing the yjgB gene. The engineered strain produced 2.28 ± 0.20 g/L of 2-PE with a yield of 0.076 g/g glucose and productivity of 0.048 g/L/h. To our best knowledge, this is the highest titer and productivity ever reported for the de novo synthesis of 2-PE in E. coli. In a 5-L fermenter, the 2-PE titer reached 2.15 g/L after 32 h of fermentation, suggesting that the strain has the potential to efficiently produce higher 2-PE titers following further fermentation optimization.

Epoxy resin, amine curing agent (IPDA) and polytetramethylene ether glycol (PTG2000) were used to prepare a new type of highly cross-linked and uniform thermoset epoxy resin microspheres (Ems) through reaction-induced phase separation. Its structure, particle size and thermal properties were characterized. The results showed that the epoxy resin microsphere was an active microsphere containing hydroxyl and amino groups on the surface, with certain monodispersity and a high glass transition temperature (Tg) (178.33 °C). Ems were further successfully used to prepare waterborne polyurethane/epoxy resin microsphere (WPU/Ems) composite films by in situ polymerization. When the composite film contained 4 wt% Ems, the tensile strength increased by 2.5-fold and the elongation-at-break hardly changed compared with pure WPU. Also, the thermal stability of the composite films was significantly improved with the addition of Ems. Fourier transform infrared spectroscopy and scanning electron microscopy results proved that WPU was grafted onto Ems with specific association. Ems were well dispersed in the WPU matrix and acted as chemical cross-linkers. Ems and WPU formed strong interfacial interaction through covalent grafting, which significantly improved the mechanical properties and thermal stability of the prepared composites.

A facile method to prepare low-density polyethylene (LDPE)/polystyrene (PS) interpolymer resin particles by swelling suspension polymerization without addition of extra swelling agent was developed. The polymerization temperature, polymerization time, and initiator concentration were investigated. Fourier transform infrared spectroscopy analysis confirmed that the LDPE/PS interpolymer resin particles were successfully prepared and a small amount of PS-g-LDPE existed in the resin. Scanning electron microscopy revealed that PS was uniformly distributed in the LDPE matrix, indicating excellent compatibility between PS and LDPE. The mechanical properties of LDPE/PS interpolymer resin were intermediate between PS and LDPE polymers.

(S)-N-Boc-3-hydroxypiperidine is an important intermediate of the anticancer drug ibrutinib and is mainly synthesized by the asymmetric reduction catalyzed by ketoreductase coupled with glucose dehydrogenase at present. In this study, the coexpression recombinant strains E. coli/pET28-K-rbs-G with single promoter and E. coli/pETDuet-K-G with double promoters were first constructed for the coexpression of ketoreductase and glucose dehydrogenase in the same cell. Then, the catalytic efficiency of E. coli/pET28-K-rbs-G for synthesizing (S)-N-Boc-3-hydroxypiperidine was found to be higher than that of E. coli/pETDuet-K-G due to the more balanced activity ratio and higher catalytic activity. On this basis, the catalytic conditions of E. coli/pET28-K-rbs-G were further optimized, and finally both the conversion of the reaction and the optical purity of the product were higher than 99%. In the end, the cell-free extract was proved to be a better catalyst than the whole cell with the improved catalytic efficiency of different recombinant strains. This study developed a better coexpression strategy for ketoreductase and glucose dehydrogenase by investigating the effect of activity ratios and forms of the biocatalysts on the catalytic efficiency deeply, which provided a research basis for the efficient synthesis of chiral compounds.

Reprogramming glycolysis for directing glycolytic metabolites to a specific metabolic pathway is expected to be useful for increasing microbial production of certain metabolites, such as amino acids, lipids or considerable secondary metabolites. In this report, a strategy of increasing glycolysis by altering the metabolism of inositol pyrophosphates (IPs) for improving the production of S-adenosyl-l-methionine (SAM) for diverse pharmaceutical applications in yeast is presented. The genes associated with the metabolism of IPs, arg82, ipk1 and kcs1, were deleted, respectively, in the yeast strain Saccharomyces cerevisiae CGMCC 2842. It was observed that the deletions of kcs1 and arg82 increased SAM by 83.3 % and 31.8 %, respectively, compared to that of the control. In addition to the improved transcription levels of various glycolytic genes and activities of the relative enzymes, the levels of glycolytic intermediates and ATP were also enhanced. To further confirm the feasibility, the kcs1 was deleted in the high SAM-producing strain Ymls1ΔGAPmK which was deleted malate synthase gene mls1 and co-expressed the Acetyl-CoA synthase gene acs2 and the SAM synthase gene metK1 from Leishmania infantum, to obtain the recombinant strain Ymls1Δkcs1ΔGAPmK. The level of SAM in Ymls1Δkcs1ΔGAPmK reached 2.89 g L−1 in a 250-mL flask and 8.86 g L−1 in a 10-L fermentation tank, increasing 30.2 % and 46.2 %, respectively, compared to those levels in Ymls1ΔGAPmK. The strategy of increasing glycolysis by deletion of kcs1 and arg82 improved SAM production in yeast.

(R)-Selective ω-transaminase (ω-TA) is a key enzyme for the asymmetric reductive amination of carbonyl compounds to produce chiral amines which are essential parts of many therapeutic compounds. However, its practical industrial applications are hindered by the low catalytic efficiency and poor thermostability of naturally occurring enzymes. In this work, we report the molecular modification of (R)-selective ω-TA from Aspergillus terreus (AtTA) to allow asymmetric reductive amination of 4-hydroxy-2-butanone, producing (R)-3-amino-1-butanol. Based on substrate docking analysis, 4 residues in the substrate tunnel and binding pocket of AtTA were selected as mutation hotspots. The screening procedure was facilitated by the construction of a “small-intelligent” library and the use of thin-layer chromatography for preliminary screening. The resulting mutant AtTA-M5 exhibited a 9.6-fold higher kcat/Km value and 9.4 °C higher $$ {\mathrm{T}}_{1/2}^{10\min } $$ than that of wild-type AtTA. Furthermore, the conversion of 20 and 50 g L−1 4-hydroxy-2-butanone by AtTA-M5 reached 90.8% and 79.1%, suggesting significant potential for production of (R)-3-amino-1-butanol. Under the same conditions, wild-type AtTA achieved less than 5% conversion. Moreover, the key mutation (S215P in AtTA) was validated in 7 other (R)-selective ω-TAs, indicating its general applicability in improving the catalytic efficiency of homologous (R)-selective ω-TAs.

Innovative poly(butylene succinate) (PBS)/Spirulina composites were fabricated by melt blending. Maleic anhydride-grafted PBS (PBS-g-MAH) was synthesized and used as a compatibilizer in the composites. Extra amount of water was added to Spirulina to ensure that it acted as a plastic during blending with PBS. The tensile strength and Young’s modulus of the composites considerably increased after incorporation of PBS-g-MAH due to better interfacial adhesion between the components and better dispersion of Spirulina in the PBS matrix, which were verified by scanning electron microscopy. Fourier transform infrared spectroscopy analysis also indicated the reaction between PBS-g-MAH and Spirulina, which resulted in improved Spirulina-PBS interaction. Differential scanning calorimetry analysis revealed that the crystallization temperature of the composites increased after addition of PBS-g-MAH, especially for the composites with higher Spirulina loading, while the PBS in compatibilized composites exhibited higher enthalpies. However, the compatibilized composites exhibited slight decreases of degradation temperature accompanied by slightly higher weight loss as indicated by thermal gravimetric analysis.

A stepwise control strategy for enhancing glutathione (GSH) synthesis in yeast based on oxidative stress and energy metabolism was investigated. First, molasses and corn steep liquor were selected and fed as carbon source mixture at a flow rate of 1.5 g/L/h and 0.4 g/L/h, respectively, for increasing cell density in a 10 L fermenter. When the biomass reached 90 g/L, the KMnO4 sustained-release particles, composed of 1.5% KMnO4, 3% stearic acid, 2% polyethylene glycol and 3% agar powder, were prepared and added to the fermentation broth for maintaining the oxidative stress. The results showed that the maximum GSH accumulation of the group fed KMnO4 sustained-release particles was 39.0% higher than that of KMnO4-fed group. In addition to the improved average GSH productivity and average specific production rate, the activities of GSH peroxidase, γ-glutamylcysteine synthetase and GSH reductase, enzymes taking part in GSH metabolism, were also significantly enhanced by KMnO4 sustained-release particles feeding. Finally, 6 g/L sodium citrate fed as an energy adjuvant elevated the intracellular ATP level for further enhancing GSH production. Through the above stepwise strategy, the GSH accumulation reached 5.76 g/L, which was 2.84-fold higher than that of the control group. The stepwise control strategy based on oxidative stress and energy metabolism significantly improved GSH accumulation in yeast.

The polyurethane prepolymer terminated with a double bond was synthesized using isophorone diisocyanate (IPDI), hydroxyl terminated polybutadiene (HTPB), 1,4-butanediol (BDO), and 2-hydroxyethyl acrylate (HEA). Then, a series of innovative UV-curable polyurethane coatings were prepared by blending ene-terminated polyurethane, fluoroacrylate monomer, and multifunctional thiol crosslinker upon UV exposure. The incorporation of fluoroacrylate monomer and multifunctional thiols into polyurethane coatings significantly enhanced the hydrophobic property, mechanical property, pencil hardness, and glossiness of the polyurethane coatings. This method of preparing UV crosslinkable, hydrophobic polyurethane coatings based on thiol-ene chemistry exhibited numerous advantages over other UV photocuring systems.

Arabinose is considered as an ideal feedstock for the microbial production of value-added chemicals due to its abundance in hemicellulosic wastes. In this study, the araBAD operon from Escherichia coli was introduced into succinate-producing Corynebacterium glutamicum, which enabled aerobic production of succinate using arabinose as sole carbon source. The engineered strain ZX1 (pXaraBAD, pEacsAgltA) produced 74.4 mM succinate with a yield of 0.58 mol (mol arabinose) −1 , which represented 69.9% of the theoretically maximal yield. Moreover, this strain produced 110.2 mM succinate using combined substrates of glucose and arabinose. To date, this is the highest succinate production under aerobic conditions in minimal medium.

A dual route for anaerobic succinate production was engineered into Corynebacterium glutamicum. The glyoxylate pathway was reconstructed by overexpressing isocitrate lyase, malate synthase and citrate synthase. The engineered strain produced succinate with a yield of 1.34 mol (mol glucose)(-1). Further overexpression of succinate exporter, SucE, increased succinate yield to 1.43 mol (mol glucose)(-1). Metabolic flux analysis revealed that the glyoxylate pathway was further activated by engineering succinate export system. Using an anaerobic fed-batch fermentation process, the final strain produced 926 mM succinate (= 109 g l(-1)) with an overall volumetric productivity of 9.4 mM h(-1) and an average yield of 1.32 mol (mol glucose)(-1).

Biodegradable composites from poly (butylene succinate-co-butylene adipate) (PBSA) and Taihu Lake (Wuxi, China) blue algae were prepared by melt blending. The property and structure of biocomposites were investigated. By adding extra amount of water to blue algae, the formulated blue algae acted as a plastic in the composites during blending, and exhibited a reinforcing effect on the PBSA matrix. With increasing blue algae content, the thermal stability of the composites decreased; the tensile strength at break and elongation at break of the composites reduced, but the Young's modulus of the composites increased. However, the composite with 30% blue algae loading still exhibited good mechanical performance (tensile strength at break of 21.3 MPa, elongation at break of 180%). The fabrication of value-added PBSA/algae composites appeared as an effective approach to reduce the secondary environmental pollution of Taihu blue algae.

Corynebacterium glutamicum with deletions of succinate dehydrogenase complex and all known acetate-producing pathways still accumulates a certain amount of acetate as the by-product under aerobic conditions. In this study, a new approach to reduce acetate accumulation by engineering an acetate assimilation pathway in C. glutamicum was presented. Two enzymes of the acetyl-CoA synthase from Escherichia coli (acs) and Bacillus subtilis (acsA) were introduced into the strain SAX1 (∆sdhABCD∆ldhA∆pta∆cat∆pqoP sod -ppcP sod -pyc) background, resulting strain SAX2 and SAX3. Both modifications resulted in the elimination of acetate formation and overexpression of acsA had a higher succinate yield. Then, overexpressing the native citrate synthase encoded by gltA in the strain SAX3 resulted in a 65 % decrease in pyruvate yield and a 32 % increase in succinate yield (0.66 mol (mol·glucose)−1) in 24 h. In shake-flask batch fermentations, the optimum strain produced 44.8 mM succinate with an average yield of 0.77 mol (mol·glucose)−1 in 48 h.

A novel aerobic succinate production system was strategically designed that allows Escherichia coli to produce and accumulate succinate with high specific productivity under aerobic conditions. Mutations in the tricarboxylic acid cycle (sdhA, iclR) and byproduct formation pathways (poxB, ackA-pta, mgsA) of E. coli were created to construct the glyoxylate cycle and oxidative branch of the TCA cycle for aerobic succinate production. Strain ZJG13 (ΔsdhA, ΔackA-pta, ΔpoxB, ΔmgsA, ΔiclR) exhibited normal growth behavior and accumulated succinate with an average specific productivity of 0.50mmolg CDW(-1)h(-1) during the fermentation. The glyoxylate shunt operon aceKBA was overexpressed by introducing plasmid pT9aceKAB to ZJG13; the resulting strain had minor effect on productivity improvement. To fully understand the effect of the carboxylation reactions on succinate production, three reactions catalyzed by pyruvate carboxylase (PYC), malic enzyme (MAEA) and phosphoenolpyruvate carboxylase (PPC) were analyzed by a Computational Approach for Strain Optimization aiming at high Productivity (CASOP). Based on the CASOP analysis, carboxylation reaction catalyzed by PYC was the most suitable one to obtain high productivity. When pyc was overexpressed in ZJG13, the specific succinate productivity further increased to 0.76mmolg CDW(-1)h(-1). Fed-batch culture of the strain ZJG13/pT184pyc led to a titer of 36.1g/L succinate, with a specific productivity of 2.75mmolg CDW(-1)h(-1) which stands for the highest value among currently reported aerobic bacterial succinate producers. These results indicate that the CASOP strategy is useful as a guiding tool for the rational strain design with high productivity.