We constructed plasmid pMTac to overexpress 3-ketosteroid-Δ1-dehydrogenase (KSDD) in Mycobacterium neoaurum JC-12 for improving androst-1,4-diene-3,17-dione (ADD) production. To construct pMTac, pACE promoter on pMF41 was replaced by tac promoter, and then four recombinants were constructed, which were M. neoaurum JC-12/pMF41-gfp, M. neoaurum JC-12/pMTac-gfp, M. neoaurum JC-12/pMF41-ksdd and M. neoaurum JC-12/pMTac-ksdd. Fluorescence detection results show that much more green fluorescent protein (GFP) was expressed in M. neoaurum JC-12/pMTac-ksdd than M. neoaurum JC-12/pMF41-ksdd. The activity of KSDD was 2.41 U/mg in M. neoaurum JC-12/pMTac-ksdd, 6.53-fold as that of M. neoaurum JC-12 and 4.36-fold as that of M. neoaurum JC-12/pMF41-ksdd. In shake flask fermentation, ADD production of M. neoaurum JC-12/pMTac-ksdd was 5.94 g/L, increased about 22.2% compared to the original strain M. neoaurum JC-12 and 12.7% to M. neoaurum JC-12/pMF41-ksdd. AD (4-androstene-3,17-dione) production of JC-12/pMTac-ksdd was 0.17 g/L, decreased 81.5% compared to M. neoaurum JC-12 and 71.2% to M neoaurum JC-12/pMF41-ksdd. In the 5 L fermenter, 20 g/L phytosterols was used as substrate, ADD production of M. neoaurum JC-12/pMTac-ksdd was improved to 10.28 g/L. pMTac is favorable for expressing KSDD in M. neoaurum JC-12, and overexpression of KSDD has beneficial effect on ADD producing, and it is the highest level ever reported using fermentation method in M. neoaurum.
Androstadienes ; metabolism ; Fermentation ; Industrial Microbiology ; Mycobacterium ; Oxidoreductases ; genetics ; metabolism ; Phytosterols ; metabolism ; Plasmids

Objective To explore the role of PLC/PIP2 signal pathway in the changes of mouse thymus CD4 + CD25 + NRP1 + Treg and TGF-β1 after different doses of X-ray irradiation Methods 36 ICR mice were randomly divided into 6 groups according to the irradiation doses of 0,0.5,1.0,2.0,4.0 and 6.0 Gy,respectively.Flow cytometry was used to detect the expression of NRP1 + Treg,and ELISA was used to detect the expression of TGF-β1 in mouse thymocytes at 16 h post-irradiation.The EL-4 cells were irradiated by X-rays at the dose of 4.0 Gy after co-cultured with the PMA and TMB-8 for 2 hours.Flow cytometry was used to detect the expression of NRP1 + Treg,and ELISA was used to detect the changes of TGF-β1 at 48 h post-irradiation.Results The NRP1 + Treg appeared a transient decrease at both 0.5 and 1.0 Gy irradiation and reached its valley value at 1.0 Gy (t =6.96,P ＜ 0.01),then showed a dosedependent increase and reached its peak at 6.0 Gy (t =6.70,P ＜ 0.01).The TGF-β1 level decreased after 0.5 Gy X-rays (t =12.53,P ＜0.01),then increased at a dose-dependent manner and reached its peak at 4.0 Gy (t =10.40-19.56,P ＜ 0.01).Compared with the sham-irradiation,NRP1 + Treg was decreased significantly after PMA treatment (t =3.06,P ＜ 0.01),while it was up-regulated significantly after irradiation in the presence of PMA (t =8.27,P ＜ 0.01).TGF-β1 was reduced in the presence of PMA with or without irradiation (t =10.46-39.69,P ＜ 0.01).NRP1 + Treg and TGF-β1 were increased significantly after TMB-8 treatment (t =5.53-44.26,P ＜ 0.01).Conclusions NRP1 + Treg cells and TGF-β1 were up-regulated after a high dose radiation,and the PLC/PIP2 signal pathway may participate in the regulation.

Objective:A case-control association analysis was performed to investigate if the single nucleotide polymorphisms (SNPs) of N-cadherin(CDH2) gene is implicated in schizophrenia in a Han Chinese population.Methods:A total of 528 patients with paranoid schizophrenia and 528 healthy controls were recruited from northern Henan province to analyze 25 SNPs located in CDH2 gene.The clinical symptoms of 267 first-episode schizophrenia patients were evaluated with positive and negative syndrome scale (PANSS), and the correlation between CDH2 gene and clinical symptoms was analyzed by SNPStats software online.Results:Allele frequencies of rs9951577 and rs1231268 were significantly correlated with schizophrenia( P<0.05), genotype frequency of rs1639387 was significantly correlated with schizophrenia( P=0.044). After gender classification, SNPs rs1789470 and rs28365328 were found to be significantly correlated with schizophrenia in female patients ( P=0.044, 0.019). In addition, the study found that CDH2 was correlated with the clinical characteristics of schizophrenia( P<0.05), and the negative factor score of patients between GG type rs1231268 and the other two genotypes (AG+ AA) ((21.12±8.41) vs (18.87±7.52)) was statistically significant ( P<0.05). Conclusion:CDH2 gene may be one of the susceptibility genes to SZ, and has definite correlation with clinical negative symptoms.

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.
Escherichia coli/genetics* ; Formate Dehydrogenases ; Hydroxybutyrates ; Threonine Dehydratase

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.
Aminobutyrates ; Escherichia coli/genetics* ; Leucine Dehydrogenase/genetics* ; Threonine Dehydratase

L-asparaginase hydrolyzes L-asparagine to produce L-aspartic acid and ammonia. It is widely distributed in microorganisms, plants and serum of some rodents, and has important applications in the pharmaceutical and food industries. However, the poor thermal stability, low catalytic efficiency and low yield hampered the further application of L-asparaginase. In this paper, rational design and 5' untranslated region (5'UTR) design strategies were used to increase the specific enzyme activity and protein expression of L-asparaginase derived from Rhizomucor miehei (RmAsnase). The results showed that among the six mutants constructed through homology modeling combined with sequence alignment, the specific enzyme activity of the mutant A344E was 1.5 times higher than the wild type. Subsequently, a food-safe strain Bacillus subtilis 168/pMA5-A344E was constructed, and the UTR strategy was used for the construction of recombinant strain B. subtilis 168/pMA5 UTR-A344E. The enzyme activity of B. subtilis 168/pMA5 UTR-A344E was 7.2 times higher than that of B. subtilis 168/pMA5-A344E. The recombinant strain B. subtilis 168/pMA5 UTR-A344E was scaled up in 5 L fermenter, and the final yield of L-asparaginase was 489.1 U/mL, showing great potential for industrial application.
Asparaginase/genetics* ; Bacillus subtilis/genetics* ; Industrial Microbiology ; Protein Engineering ; Rhizomucor/enzymology* ; Sequence Alignment

5-aminolevulinic acid (5-ALA) plays an important role in the fields of medicine and agriculture. 5-ALA can be produced by engineered Escherichia coli and Corynebacterium glutamicum. We systematically engineered the C4 metabolic pathway of C. glutamicum to further improve its ability to produce 5-ALA. Firstly, the hemA gene encoding 5-ALA synthase (ALAS) from Rhodobacter capsulatus and Rhodopseudomonas palustris were heterologously expressed in C. glutamicum, respectively. The RphemA gene of R. palustris which showed relatively high enzyme activity was selected. Screening of the optimal ribosome binding site sequence RBS5 significantly increased the activity of RphemA. The ALAS activity of the recombinant strain reached (221.87±3.10) U/mg and 5-ALA production increased by 14.3%. Subsequently, knocking out genes encoding α-ketoglutarate dehydrogenase inhibitor protein (odhI) and succinate dehydrogenase (sdhA) increased the flux of succinyl CoA towards the production of 5-ALA. Moreover, inhibiting the expression of hemB by means of sRNA reduced the degradation of 5-ALA, while overexpressing the cysteine/O-acetylserine transporter eamA increased the output efficiency of intracellular 5-ALA. Shake flask fermentation using the engineered strain C. glutamicum 13032/∆odhI/∆sdhA-sRNAhemB- RBS5RphemA-eamA resulted in a yield of 11.90 g/L, which was 57% higher than that of the original strain. Fed-batch fermentation using the engineered strain in a 5 L fermenter produced 25.05 g/L of 5-ALA within 48 h, which is the highest reported-to-date yield of 5-ALA from glucose.
Aminolevulinic Acid/metabolism* ; Corynebacterium glutamicum/metabolism* ; Fermentation ; Metabolic Engineering ; Rhodobacter capsulatus/enzymology* ; Rhodopseudomonas/enzymology*