L-tyrosine is one of the 20 amino acids that make up proteins and is an essential amino acid for mammals, often used as a nutritional supplement. The conventional methods for synthesizing L-tyrosine have some problems such as the production of many by-products, high requirements for production conditions, and environmental pollution. In this study, we designed and constructed a multi-enzyme cascade for the synthesis of L-tyrosine with alanine, glutamate, ammonium chloride, and phenol as substrates. Initially, the sources of glutamate oxidase, alanine aminotransferase, and tyrosine phenol lyase were screened and analyzed, which was followed by the identification of the rate-limiting enzyme in the reaction process. A colorimetric screening method was established, and the rate-limiting enzyme DbAlaA was engineered to enhance its activity by 40.0%. Subsequently, the reaction conditions, including temperature, pH, cell concentration, and surfactant and coenzyme dosages, were optimized. After optimization, the yield of L-tyrosine reached 9.93 g/L, with a alanine conversion rate of 54.90%. Finally, a feed-batch fermentation strategy was adopted, and the yield of L-tyrosine reached 56.07 g/L after 24 h, with a alanine conversion rate of 65.22%. This study provides a reference for the whole-cell catalytic synthesis of L-tyrosine and its industrialization.
Tyrosine/biosynthesis* ; Escherichia coli/metabolism* ; Tyrosine Phenol-Lyase/genetics* ; Multienzyme Complexes/metabolism* ; Fermentation

Objective:To investigate the efficacy, safety and possible mechanisms of celecoxib as an adjunctive treatment for schizophrenia.Methods:90 schizophrenic inpatients at the second affiliated hospital of Xinxiang Medical College from April 2019 to October 2020 were recruited and randomly assigned to a placebo group or the celecoxib adjunctive treatment group using a random number table. In the placebo group, 46 patients (29 males, 17 females; aged 21-34, mean age 27.46±6.50 years) completed a 6-week follow-up. In the celecoxib group, 44 patients (32 males, 12 females; aged 21-39, mean age 30.52±8.69 years) completed a 6-week follow-up. The Positive and Negative Syndrome Scale (PANSS) was used to assess psychiatric symptoms in both groups. Changes in PANSS score at the end of the treatment were compared to evaluate the efficacy of celecoxib. Metabolic indicators such as weight, body mass index, waist circumference and plasm glucolipid, as well as cardiovascular indicators like blood pressure, electrocardiogram and routine blood tests, and adverse events were collected for the safety evaluation. Serum tumor necrosis factor-α (TNF-α), Interleukin-4 (IL-4) and interferon-γ (IFN-γ) were also tested. Pearson correlation analysis was used to explore the relationship between cytokine levels, PANSS score, PANSS reduction rate [(pre-treatment score-post-treatment score)/pre-treatment score×100%], and the safety measurements in the two groups, analyzing the role of inflammation in celecoxib adjunctive therapy.Results:The change of PANSS positive score at the end of the 6th week was significantly higher in the celecoxib adjuvant treatment group than in the placebo group (-8.00±6.12 vs -4.78±5.19, H=-0.55, P=0.009). The weight changes, body mass index, total cholesterol, and triglycerides over 6 weeks were significantly lower in the celecoxib group compared to the placebo group ( F=-7.37, -7.30, 2.56, -2.54; all P<0.05). No serious adverse events were found in celecoxib adjuvant therapy. In the placebo group, baseline TNF-α levels were positively correlated with baseline negative symptoms and PANSS reduction rate ( r=0.260 and 0.330, both P<0.05), and negatively correlated with the 6-week weight ( r=-0.311, P<0.05); baseline IL-4 levels were positively correlated with the 6-week PANSS total score and the 6-week PANSS negative score ( r=0.320 and 0.397, both P<0.05), and negatively correlated with PANSS reduction rate and 6-week blood glucose ( r=-0.316 and -0.331, both P<0.05); Six-week IFN-γ levels were negatively correlated with low-density lipoprotein levels ( r=-0.306, P<0.05). And no such correlation was found in celecoxib adjuvant group. Conclusion:Celecoxib adjunctive therapy can improve positive symptoms of schizophrenia without causing adverse reactions. Inflammatory state is related to schizophrenia symptoms, treatment efficacy and metabolic abnormalities.

Objective:To investigate the efficacy, safety and possible mechanisms of celecoxib as an adjunctive treatment for schizophrenia.Methods:90 schizophrenic inpatients at the second affiliated hospital of Xinxiang Medical College from April 2019 to October 2020 were recruited and randomly assigned to a placebo group or the celecoxib adjunctive treatment group using a random number table. In the placebo group, 46 patients (29 males, 17 females; aged 21-34, mean age 27.46±6.50 years) completed a 6-week follow-up. In the celecoxib group, 44 patients (32 males, 12 females; aged 21-39, mean age 30.52±8.69 years) completed a 6-week follow-up. The Positive and Negative Syndrome Scale (PANSS) was used to assess psychiatric symptoms in both groups. Changes in PANSS score at the end of the treatment were compared to evaluate the efficacy of celecoxib. Metabolic indicators such as weight, body mass index, waist circumference and plasm glucolipid, as well as cardiovascular indicators like blood pressure, electrocardiogram and routine blood tests, and adverse events were collected for the safety evaluation. Serum tumor necrosis factor-α (TNF-α), Interleukin-4 (IL-4) and interferon-γ (IFN-γ) were also tested. Pearson correlation analysis was used to explore the relationship between cytokine levels, PANSS score, PANSS reduction rate [(pre-treatment score-post-treatment score)/pre-treatment score×100%], and the safety measurements in the two groups, analyzing the role of inflammation in celecoxib adjunctive therapy.Results:The change of PANSS positive score at the end of the 6th week was significantly higher in the celecoxib adjuvant treatment group than in the placebo group (-8.00±6.12 vs -4.78±5.19, H=-0.55, P=0.009). The weight changes, body mass index, total cholesterol, and triglycerides over 6 weeks were significantly lower in the celecoxib group compared to the placebo group ( F=-7.37, -7.30, 2.56, -2.54; all P<0.05). No serious adverse events were found in celecoxib adjuvant therapy. In the placebo group, baseline TNF-α levels were positively correlated with baseline negative symptoms and PANSS reduction rate ( r=0.260 and 0.330, both P<0.05), and negatively correlated with the 6-week weight ( r=-0.311, P<0.05); baseline IL-4 levels were positively correlated with the 6-week PANSS total score and the 6-week PANSS negative score ( r=0.320 and 0.397, both P<0.05), and negatively correlated with PANSS reduction rate and 6-week blood glucose ( r=-0.316 and -0.331, both P<0.05); Six-week IFN-γ levels were negatively correlated with low-density lipoprotein levels ( r=-0.306, P<0.05). And no such correlation was found in celecoxib adjuvant group. Conclusion:Celecoxib adjunctive therapy can improve positive symptoms of schizophrenia without causing adverse reactions. Inflammatory state is related to schizophrenia symptoms, treatment efficacy and metabolic abnormalities.

Objective:This study evaluated the metabolic phenotype in offspring of maternal immune activation(MIA) rats, to determine whether it applies to the study of the mechanism of metabolic syndrome associated with schizophrenia, and to analyze its possible pathogenesis.Methods:Polyinosinic-polycytidylic acid (Poly I:C) or saline was injected via tail vein at 9.5 days of pregnancy, and the offspring were defined as MIA group and control group. Behavior, blood routine, blood glucose, blood lipid and liver function were detected. Besides, inflammatory cytokine levels were analyzed by quantitative PCR. The independent -sample t-test was used for the comparison between groups. Results:The results showed that the rats of the MIA group had an increased anxiety level, memory impairment, and sensory gating dysfunction in early adulthood. However, the rats of the MIA group already showed the risk of metabolic disorders in their adolescence, including the increased proportion of monocytes ( t=2.50, df=14, P=0.028), elevated LDL-C level ( t=3.34, df=14, P=0.005), and increased atherosclerosis index ( t=2.23, df=14, P=0.043). The rats of the MIA group showed abnormal liver morphology and decreased globulin ( t=3.61, df=14, P=0.003) and total protein levels ( t=3.40, df=14, P=0.004), suggesting possible impaired liver function. In addition, the rats of the MIA group showed systemic inflammation of the brain and liver. Conclusion:These results demonstrate that behavioral disorders and metabolic disorders coexist in the rats of the MIA group, and metabolic disorders appear earlier than significant behavioral abnormalities, suggesting the possible value of this model in the study of schizophrenia complicated by metabolic syndrome. In addition, systemic inflammation of the brain and liver may be a possible mechanism for behavioral disorders and metabolic disorders in the offspring of MIA.

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.

Objective:This study evaluated the metabolic phenotype in offspring of maternal immune activation(MIA) rats, to determine whether it applies to the study of the mechanism of metabolic syndrome associated with schizophrenia, and to analyze its possible pathogenesis.Methods:Polyinosinic-polycytidylic acid (Poly I:C) or saline was injected via tail vein at 9.5 days of pregnancy, and the offspring were defined as MIA group and control group. Behavior, blood routine, blood glucose, blood lipid and liver function were detected. Besides, inflammatory cytokine levels were analyzed by quantitative PCR. The independent -sample t-test was used for the comparison between groups. Results:The results showed that the rats of the MIA group had an increased anxiety level, memory impairment, and sensory gating dysfunction in early adulthood. However, the rats of the MIA group already showed the risk of metabolic disorders in their adolescence, including the increased proportion of monocytes ( t=2.50, df=14, P=0.028), elevated LDL-C level ( t=3.34, df=14, P=0.005), and increased atherosclerosis index ( t=2.23, df=14, P=0.043). The rats of the MIA group showed abnormal liver morphology and decreased globulin ( t=3.61, df=14, P=0.003) and total protein levels ( t=3.40, df=14, P=0.004), suggesting possible impaired liver function. In addition, the rats of the MIA group showed systemic inflammation of the brain and liver. Conclusion:These results demonstrate that behavioral disorders and metabolic disorders coexist in the rats of the MIA group, and metabolic disorders appear earlier than significant behavioral abnormalities, suggesting the possible value of this model in the study of schizophrenia complicated by metabolic syndrome. In addition, systemic inflammation of the brain and liver may be a possible mechanism for behavioral disorders and metabolic disorders in the offspring of MIA.

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

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

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*