Dual-Dynamic Chemistries-Based Fast-Reprocessing and High-Performance Covalent Adaptable Networks.

Covalent adaptable networks (CANs) possess multiple functions including reprocessing (or recyclability), self-healing, welding, shape shifting, 3D printing, etc., due to the network rearrangement from dynamic bonds, and favorable performance from their cross-linked feature, and they are supposed to be as sustainable alternatives to thermosets. However, the thermal and mechanical properties, and stability of CANs are often sacrificed for rapid network rearrangement. In this paper, fast-reprocessing CANs with high performance are facilely constructed by in situ polymerization and dynamic cross-linking of styrene (St), maleic anhydride (MA), and acetal diol (BHAD). The rigid and hydrophobic polymer backbone endow the materials with high glass transition temperatures, mechanical performance, and water resistance. Besides, carboxylic group-catalyzed dual dynamic ester and acetal-based networks exhibit faster stress relaxation and realize extrusion reprocessing. This work provides an ingenious and simple strategy of construction of CANs combining rapid network rearrangement and excellent comprehensive performance, which is beneficial for the application of CANs.

Improving production of Streptomyces griseus trypsin for enzymatic processing of insulin precursor.

BACKGROUND: Trypsin has many applications in food and pharmaceutical manufacturing. Although commercial trypsin is usually extracted from porcine pancreas, this source carries the risks of infectivity and immunogenicity. Microbial Streptomyces griseus trypsin (SGT) is a prime alternative because it possesses efficient hydrolysis activity without such risks. However, the remarkable hydrolysis efficiency of SGT causes autolysis, and five autolysis sites, R21, R32, K122, R153, and R201, were identified from its autolysate. RESULTS: The tbcf (K101A, R201V) mutant was screened by a directed selection approach for improved activity in flask culture (60.85 ± 3.42 U mL-1, increased 1.5-fold). From the molecular dynamics simulation, in the K101A/R201V mutant the distance between the catalytical residues D102 and H57 was shortened to 6.5 Å vs 7.0 Å in the wild type, which afforded the improved specific activity of 1527.96 ± 62.81 U mg-1. Furthermore, the production of trypsin was increased by 302.8% (689.47 ± 6.78 U mL-1) in a 3-L bioreactor, with co-overexpression of chaperones SSO2 and UBC1 in Pichia pastoris. CONCLUSIONS: SGT protein could be a good source of trypsin for insulin production. As a result of the hydrolysates analysis and direct selection, the activity of the tbcf (K101A, R201V) mutant increased 1.5-fold. Furthermore, the production of trypsin was improved threefold by overexpressing chaperone protein in Pichia pastoris. Future studies should investigate the application of SGT to insulin and pharmaceutical manufacturing.

Rational engineering of multiple module pathways for the production of L-phenylalanine in Corynebacterium glutamicum.

Microbial production of L-phenylalanine (L-Phe) from renewable sources has attracted much attention recently. In the present study, Corynebacterium glutamicum 13032 was rationally engineered to produce L-Phe from inexpensive glucose. First, all the L-Phe biosynthesis pathway genes were investigated and the results demonstrated that in addition to AroF and PheA, the native PpsA, TktA, AroE and AroA, and the heterologous AroL and TyrB were also the key enzymes for L-Phe biosynthesis. Through combinational expression of these key enzymes, the L-Phe production was increased to 6.33 ± 0.13 g l(-1) which was about 1.48-fold of that of the parent strain C. glutamicum (pXM-pheA (fbr)-aroF (fbr)) (fbr, feedback-inhibition resistance). Furthermore, the production of L-Phe was improved to 9.14 ± 0.21 g l(-1) by modifying the glucose and L-Phe transport systems and blocking the acetate and lactate biosynthesis pathways. Eventually, the titer of L-Phe was enhanced to 15.76 ± 0.23 g l(-1) with a fed-batch fermentation strategy. To the best of our knowledge, this was the highest value reported in rationally engineered C. glutamicum 13032 strains. The results obtained will also contribute to rational engineering of C. glutamicum for production of other valuable aromatic compounds.

Small RNA regulators in bacteria: powerful tools for metabolic engineering and synthetic biology.

Small RNAs, a large class of ancient posttranscriptional regulators, have recently attracted considerable attention. A plethora of small RNAs has been identified and characterized, many of which belong to the major small noncoding RNA (sRNA) or riboswitch families. It has become increasingly clear that most small RNAs play critical regulatory roles in many processes and are, therefore, considered to be powerful tools for metabolic engineering and synthetic biology. In this review, we describe recent achievements in the identification, characterization, and application of small RNAs. We give particular attention to advances in the design and synthesis of novel sRNAs and riboswitches for metabolic engineering. In addition, a novel strategy for hierarchical control of global metabolic pathways is proposed.

The mechanism of anthracene degradation by tryptophan -2,3-dioxygenase (T23D) in Comamonas testosteroni.

It is well known that anthracene is a persistent organic pollutant. Among the four natural polycyclic aromatic hydrocarbons (PAHs) degrading strains, Comamonas testosterone (CT1) was selected as the strain with the highest degradation efficiency. In the present study, prokaryotic transcriptome analysis of CT1 revealed an increase in a gene that encodes tryptophane-2,3-dioxygenase (T23D) in the anthracene and erythromycin groups compared to CK. Compared to the wild-type CT1 strain, anthracene degradation by the CtT23D knockout mutant (CT-M1) was significantly reduced. Compared to Escherichia coli (DH5α), CtT23D transformed DH5α (EC-M1) had a higher degradation efficiency for anthracene. The recombinant protein rT23D oxidized tryptophan at pH 7.0 and 37 °C with an enzyme activity of 2.42 ± 0.06 µmol min-1·mg-1 protein. In addition, gas chromatography-mass (GC-MS) analysis of anthracene degradation by EC-M1 and the purified rT23D revealed that 2-methyl-1-benzofuran-3-carbaldehyde is an anthracene metabolite, suggesting that it is a new pathway.

Enhanced production of L-phenylalanine in Corynebacterium glutamicum due to the introduction of Escherichia coli wild-type gene aroH.

Metabolic engineering is a powerful tool which has been widely used for producing valuable products. For improving L-phenylalanine (L-Phe) accumulation in Corynebacterium glutamicum, we have investigated the target genes involved in the biosynthetic pathways. The genes involved in the biosynthesis of L-Phe were found to be strictly regulated genes by feedback inhibition. As a result, overexpression of the native wild-type genes aroF, aroG or pheA resulted in a slight increase of L-Phe. In contrast, overexpression of aroF (wt) or pheA (fbr) from E. coli significantly increased L-Phe production. Co-overexpression of aroF (wt) and pheA (fbr) improved the titer of L-Phe to 4.46 ± 0.06 g l⁻¹. To further analyze the target enzymes in the aromatic amino acid synthesis pathway between C. glutamicum and E. coli, the wild-type gene aroH from E. coli was overexpressed and evaluated in C. glutamicum. As predicted, upregulation of the wild-type gene aroH resulted in a remarkable increase of L-Phe production. Co-overexpression of the mutated pheA (fbr) and the wild-type gene aroH resulted in the production of L-Phe up to 4.64 ± 0.09 g l⁻¹. Based on these results we conclude that the wild-type gene aroH from E. coli is an appropriate target gene for pathway engineering in C. glutamicum for the production of aromatic amino acids.

Construction of collagen II/hyaluronate/chondroitin-6-sulfate tri-copolymer scaffold for nucleus pulposus tissue engineering and preliminary analysis of its physico-chemical properties and biocompatibility.

To construct a novel scaffold for nucleus pulposus (NP) tissue engineering, The porous type II collagen (CII)/hyaluronate (HyA)-chondroitin-6-sulfate (6-CS) scaffold was prepared using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) cross-linking system. The physico-chemical properties and biocompatibility of CII/HyA-CS scaffolds were evaluated. The results suggested CII/HyA-CS scaffolds have a highly porous structure (porosity: 94.8 +/- 1.5%), high water-binding capacity (79.2 +/- 2.8%) and significantly improved mechanical stability by EDC/NHS crosslinking (denaturation temperature: 74.6 +/- 1.8 and 58.1 +/- 2.6 degrees C, respectively, for the crosslinked scaffolds and the non-crosslinked; collagenase degradation rate: 39.5 +/- 3.4 and 63.5 +/- 2.0%, respectively, for the crosslinked scaffolds and the non-crosslinked). The CII/HyA-CS scaffolds also showed satisfactory cytocompatibility and histocompatibility as well as low immunogenicity. These results indicate CII/HyA-CS scaffolds may be an alternative material for NP tissue engineering due to the similarity of its composition and physico-chemical properties to those of the extracellular matrices (ECM) of native NP.

Metabolic engineering of Escherichia coli for production of 2-phenylethanol from renewable glucose.

2-Phenylethanol (2-PE) is an important aromatic alcohol with a rose-like odor and has wide applications. The present work aims to construct a synthetic pathway for 2-PE synthesis from glucose in Escherichia coli. First, the genes adh1 (encoding alcohol dehydrogenase) and kdc (encoding phenylpyruvate decarboxylase) from Saccharomyces cerevisiae S288c and Pichia pastoris GS115 were investigated in E. coli, respectively, and single overexpression of adh1 or kdc significantly increased 2-PE accumulation. When co-overexpressing adh1 and kdc, 2-PE was increased up to 130 from 57 mg/L. Furthermore, by optimizing coordinated expression of the four committed genes aroF, pheA, adh1 and kdc, 2-PE was improved to 285 mg/L which was the highest production of 2-PE by the recombinant E. coli system. In addition, our results also demonstrated that the tyrB gene, which encodes aromatic-amino-acid transaminase, plays an important role on 2-PE synthesis.

Construction and application of novel feedback-resistant 3-deoxy-d-arabino-heptulosonate-7-phosphate synthases by engineering the N-terminal domain for L-phenylalanine synthesis.

3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHP synthase) encoded by aroF is the first enzyme of the shikimate pathway. In the present study, an AroF variant with a deficiency in residue Ile11 (named AroF*) was shown to be insensitive to l-tyrosine. According to three-dimensional structure analysis, nine AroF variants were constructed with truncation of different N-terminal fragments, and overexpression of the variants AroF(Δ(1-9)) , AroF(Δ(1-10)) , AroF(Δ(1-12)) and, in particular, AroF(Δ(1-11)) significantly increased the accumulation of l-phenylalanine (l-Phe). However, the AroG and AroH variants with similar truncations of the N-terminal fragments decreased the production of l-Phe. By co-overexpressing AroF(Δ(1-11)) and PheA(fbr) , the production of l-Phe was increased from 2.36 ± 0.07 g L(-1) (co-overexpression of the wild-type AroF and PheA(fbr) ) to 4.29 ± 0.06 g L(-1) . The novel variant AroF(Δ(1-11)) showed great potential for the production of aromatic amino acids and their derivatives.

Preparation and characterization of poly(lactic acid)/starch composites toughened with epoxidized soybean oil.

Blends of entirely bio-sourced polymers, namely polylactide (PLA) and starch, have been melt-compounded by lab-scale co-extruder with epoxidized soybean oil (ESO) as a reactive compatibilizer. The starch granules were grafted with the maleic anhydride (MA) to enhance its reactivity with ESO. The ready reactions between the epoxy groups on ESO, the MA groups on MA-grafted starch (MGST) and the end carboxylic acid groups of PLA brought blending components together and formed a compatible compound. An elongation at break (EB) of 140% was obtained in the blend of PLA/MGST/ESO (80/10/10), increased from 5% of a pure PLA. The grafting content of the MA on the starch granules primarily determined the compatibility and properties of the ternary blends, which was also affected by the relative amount of MGST and ESO.

Effect of castor oil enrichment layer produced by reaction on the properties of PLA/HDI-g-starch blends.

Blends of entirely bio-sourced polymers, namely polylactide (PLA) and starch, have been melt-compounded by lab-scale co-extruder with castor oil (CO) as a plasticizer. The enrichment of castor oil on starch had great effect on the properties of the blends. If the castor oil was mainly dispersed in PLA matrix, the properties of the blends were poor, but when the hexamethylenediisocyanate (HDI) was grafted on starch granules the ready reactions between the hydroxyl on CO and the isocyante on the HDI-grafted starch (HGSTs) brought CO molecules enriched on starch particles. DSC analysis shows that the CO layer on starch has a positive effect on the crystallization of PLA in the ternary blend. The accumulation of CO on starch greatly improves the toughness and impact strength of PLA/starch blends. The grafting content of HDI on the starch granules primarily determined the compatibility and properties of the resulted blends.

Collagen II/hyaluronan/chondroitin-6-sulfate tri-copolymer scaffold for nucleus pulposus tissue engineering.

This study aims to investigate the bioactivity of collagen II/hyaluronan/chondroitin-6-sulfate tri-copolymer as bionic scaffold for nucleus pulposus (NP) tissue engineering. Collagen II (C II) (pH 1-2) was mixed with hyaluronan (HyA) and lyophilized to prepare C II/HyA matrices. Chondroitin 6-sulfate (6-CS) was covalently attached to the C II/HyA matrices using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Then, cells were expanded from rabbit NP and seeded in the tri-copolymer scaffold. Cell-scaffold hybrids were maintained for up to 28 days in culture. Cell viability/proliferation, extracellular matrix (ECM)-related gene expression, and the content of sulfated glycosaminoglycans (s-GAG) were evaluated. Our results are as following: when cultured for 28 days, the cell-scaffold hybrids maintained active cell viability/proliferation and exhibited a significantly increased s-GAG content. In addition, rabbit NP cells cultured in the scaffold demonstrated a significantly higher level of C II and aggrecan gene expression and a significantly lower level of Collagen I (C I) gene expression when compared with that of monolayer cells. Histological studies and scanning electron microscopy (SEM) further indicated newly secreted ECM deposits in the scaffolds. In conclusion, the C II/HyA-CS scaffold may be an alternative material for NP tissue engineering due to its satisfactory bioactivity, and it deserves further in vivo investigation.