Elliottdjurhuus5376

D-allulose-3-epimerase (DPEase) is the key enzyme for isomerization of D-fructose to D-allulose. In order to improve its thermal stability, short amphiphilic peptides (SAP) were fused to the N-terminal of DPEase. SDS-PAGE analysis showed that the heterologously expressed DPEase folded correctly in Bacillus subtilis, and the protein size was 33 kDa. After incubation at 40 °C for 48 h, the residual enzyme activity of SAP1-DSDPEase was 58%. To make the recombinant B. subtilis strain reusable, cells were immobilized with a composite carrier of sodium alginate (SA) and titanium dioxide (TiO2). The results showed that 2% SA, 2% CaCl2, 0.03% glutaraldehyde solution and a ratio of TiO2 to SA of 14 were optimal for immobilization. Under these conditions, up to 82% of the activity of immobilized cells could be retained. Compared with free cells, the optimal reaction temperature of immobilized cells remained unchanged at 80 °C but the thermal stability improved. After 10 consecutive cycles, the mechanical strength remained unchanged, while 58% of the enzyme activity could be retained, with a conversion rate of 28.8% achieved. This study demonstrated a simple approach for using SAPs to improve the thermal stability of recombinant enzymes. Moreover, addition of TiO2 into SA during immobilization was demonstrated to increase the mechanical strength and reduce cell leakage.Acetic acid is a common inhibitor present in lignocellulosic hydrolysate. Development of acetic acid tolerant strains may improve the production of biofuels and bio-based chemicals using lignocellulosic biomass as raw materials. Current studies on stress tolerance of yeast Saccharomyces cerevisiae have mainly focused on transcription control, but the role of transfer RNA (tRNA) was rarely investigated. We found that some tRNA genes showed elevated transcription levels in a stress tolerant yeast strain. In this study, we further investigated the effects of overexpressing an arginine transfer RNA gene tR(ACG)D and a leucine transfer RNA gene tL(CAA)K on cell growth and ethanol production of S. cerevisiae BY4741 under acetic acid stress. The tL(CAA)K overexpression strain showed a better growth and a 29.41% higher ethanol productivity than that of the control strain. However, overexpression of tR(ACG)D showed negative influence on cell growth and ethanol production. Further studies revealed that the transcriptional levels of HAA1, MSN2, and MSN4, which encode transcription regulators related to stress tolerance, were up-regulated in tL(CAA)K overexpressed strain. This study provides an alternative strategy to develop robust yeast strains for cellulosic biorefinery, and also provides a basis for investigating how yeast stress tolerance is regulated by tRNA genes.The influence of different affinity tags on enzyme characteristics varies. The (S)-carbonyl reductase 2 (SCR2) from Candida parapsilosis can reduce 2-hydroxyacetophenone, which is a valuable prochiral ketones. Different affinity tags, i.e. his-tag, strep-tag and MBP-tag, were attached to the N terminus of SCR2. These tagged SCR2 enzymes, i.e. his6-SCR2, strep-SCR2 and MBP-SCR2, were heterologously expressed in Escherichia coli and purified to study their characteristics towards 2-hydroxyacetophenone reduction. Affinity tags did affect the characteristics of the recombinant SCR2 enzymes. Specifically, affinity tags affect the stability of recombinant SCR2 enzymes 1) At pH 6.0, the remaining enzyme activities of his6-SCR2 and strep-SCR2 were only 95.2% and 90.0% of the untagged SCR2, while that of MBP-SCR2 was 1.2 times of the untagged SCR2 after incubating for 13 h at 30 °C. 2) The half-life of MBP-SCR2 at 50 °C was 26.6%-48.8% longer than those of strep-SCR2, his6-SCR2 and untagged SCR2. Protokylol cost 3) The kcat of MBP-SCR2 was about 1.25-1.45 times of that of small affinity-tagged and untagged SCR2 after storing at -80 °C for 60 d. Structural informatics indicated that the α-helices at the C terminus of MBP-SCR2 contributed to the stability of the N terminus of fusion protein of SCR2. Data from circular dichroism showed that the MBP-tag has some influence on the secondary structure of SCR2, while melting temperature analysis demonstrated that the Tm of the recombinant MBP-SCR2 was about 5 °C higher than that of the untagged SCR2. This study obtained an efficient and stable recombinant SCR2, i.e. the MBP-SCR2. Moreover, this study could serve as a reference for other researchers to evaluate and select appropriate affinity tags for their research.Dopamine is the precursor of a variety of natural antioxidant compounds. In the body, dopamine acts as a neurotransmitter that regulates a variety of physiological functions of the central nervous system. Thus, dopamine is used for the clinical treatment of various types of shock. Dopamine could be produced by engineered microbes, but with low efficiency. In this study, DOPA decarboxylase gene from Sus scrofa (Ssddc) was cloned into plasmids with different copy numbers, and transformed into a previously developed L-DOPA producing strain Escherichia coli T004. The resulted strain was capable of producing dopamine from glucose directly. To further improve the production of dopamine, a sequence-based homology alignment mining (SHAM) strategy was applied to screen more efficient DOPA decarboxylases, and five DOPA decarboxylase genes were selected from 100 candidates. In shake-flask fermentation, the DOPA decarboxylase gene from Homo sapiens (Hsddc) showed the highest dopamine production (3.33 g/L), while the DOPA decarboxylase gene from Drosophila Melanogaster (Dmddc) showed the least residual L-DOPA concentration (0.02 g/L). In 5 L fed-batch fermentations, production of dopamine by the two engineered strains reached 13.3 g/L and 16.2 g/L, respectively. The residual concentrations of L-DOPA were 0.45 g/L and 0.23 g/L, respectively. Finally, the Ssddc and Dmddc genes were integrated into the genome of E. coli T004 to obtain genetically stable dopamine-producing strains. In 5 L fed-batch fermentation, 17.7 g/L of dopamine was produced, which records the highest titer reported to date.Leucine dehydrogenase (LDH) is the key rate-limiting enzyme in the production of L-2-aminobutyric acid (L-2-ABA). In this study, we modified the C-terminal Loop region of this enzyme to improve the specific enzyme activity and stability for efficient synthesis of L-2-ABA. Using molecular dynamics simulation of LDH, we analyzed the change of root mean square fluctuation (RMSF), rationally designed the Loop region with greatly fluctuated RMSF, and obtained a mutant EsLDHD2 with a specific enzyme activity 23.2% higher than that of the wild type. Since the rate of the threonine deaminase-catalyzed reaction converting L-threonine into 2-ketobutyrate was so fast, the multi-enzyme cascade catalysis system became unbalanced. Therefore, the LDH and the formate dehydrogenase were double copied in a new construct E. coli BL21/pACYCDuet-RM. Compared with E. coli BL21/pACYCDuet-RO, the molar conversion rate of L-2-ABA increased by 74.6%. The whole cell biotransformation conditions were optimized and the optimal pH, temperature and substrate concentration were 7.5, 35 °C and 80 g/L, respectively. Under these conditions, the molar conversion rate was higher than 99%. Finally, 80 g and 40 g L-threonine were consecutively fed into a 1 L reaction mixture under the optimal conversion conditions, producing 97.9 g L-2-ABA. Thus, this strategy provides a green and efficient synthesis of L-2-ABA, and has great industrial application potential.Hydroxytyrosol is an important fine chemical and is widely used in food and medicine as a natural antioxidant. Production of hydroxytyrosol through synthetic biology is of important significance. Here we cloned and functionally characterized a hydroxylase encoding gene HpaBC from Escherichia coli BL21, and both subunits of this enzyme can be successfully expressed to convert the tyrosol into hydroxytyrosol. A HpaBC gene integration expression cassette under the tac promoter was constructed, and integrated into the genome of a tyrosol hyper-producing E. coli YMG5A*R using CRISPR-Cas9 technology. Meanwhile, the pathway for production of acetic acid was deleted, resulting in a recombinant strain YMGRD1H1. Shake flask fermentation showed that strain YMGRD1H1 can directly use glucose to produce hydroxytyrosol, reaching a titer of 1.81 g/L, and nearly no by-products were detected. A titer of 2.95 g/L was achieved in a fed-batch fermentation conducted in a 5 L fermenter, which is the highest titer for the de novo synthesis of hydroxytyrosol from glucose reported to date. Production of hydroxytyrosol by engineered E. coli lays a foundation for further construction of hydroxytyrosol cell factories with industrial application potential, adding another example for microbial manufacturing of aromatic compounds.2-Hydroxybutyric acid (2-HBA) is an important intermediate for synthesizing biodegradable materials and various medicines. Chemically synthesized racemized 2-HBA requires deracemization to obtain optically pure enantiomers for industrial application. In this study, we designed a cascade biosynthesis system in Escherichia coli BL21 by coexpressing L-threonine deaminase (TD), NAD-dependent L-lactate dehydrogenase (LDH) and formate dehydrogenase (FDH) for production of optically pure (S)-2-HBA from bulk chemical L-threonine (L-Thr). To coordinate the production rate and the consumption rate of the intermediate 2-oxobutyric acid in the multi-enzyme cascade catalytic reactions, we explored promoter engineering to regulate the expression levels of TD and FDH, and developed a recombinant strain P21285FDH-T7V7827 with a tunable system to achieve a coordinated multi-enzyme expression. The recombinant strain P21285FDH-T7V7827 was able to efficiently produce (S)-2-HBA with the highest titer of 143 g/L and a molar yield of 97% achieved within 16 hours. This titer was approximately 1.83 times than that of the highest yield reported to date, showing great potential for industrial application. Our results indicated that constructing a multi-enzyme-coordinated expression system in a single cell significantly contributed to the biosynthesis of hydroxyl acids.Threonine aldolases catalyze the aldol condensation of aldehydes with glycine to furnish β-hydroxy-α-amino acid with two stereogenic centers in a single reaction. This is one of the most promising green methods for the synthesis of optically pure β-hydroxy-α-amino acid with high atomic economy and less negative environmental impact. Several threonine aldolases from different origins have been identified and characterized. The insufficient -carbon stereoselectivity and the challenges of balancing kinetic versus thermodynamic control to achieve the optimal optical purity and yield hampered the application of threonine aldolases. This review summarizes the recent advances in discovery, catalytic mechanism, high-throughput screening, molecular engineering and applications of threonine aldolases, with the aim to provide some insights for further research in this field.Protein kinase CK2 is a common, evolutionarily conserved and ubiquitous protein kinase. In recent years, increasing evidences have shown that CK2 has a variety of phosphorylated protein substrates, which play important roles in growth, development and various diseases. Therefore, CK2 may participate in these physiological processes by regulating the phosphorylation of these substrates. This article briefly reviewed the structural characteristics of protein kinase CK2 and its physiological functions in growth, development, immunity, formation of tumor and other diseases, in order to provide knowledge basis for further research on the regulatory mechanism of CK2 and potential applications of its inhibitors.