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 predictive factors of early neurological deterioration (END) in patients with single subcortical infarction (SSI).Methods:Consecutive patients with SSI within 12 h of onset admitted to the Department of Neurology, Xuzhou Municipal Hospital Affiliated to Xuzhou Medical University from January 2016 to December 2021 were included retrospectively. The clinical and imaging data of the END and non-END groups were collected and compared. Multivariate logistic regression analysis was used to determine the independent risk factors for END. Results:A total of 1 060 patients with SSI were included, of which 268 (25.28%) developed END. There were statistically significant differences in the infarct involving internal capsule on diffusion-weighted imaging, SSI classification, maximum diameter and classification of the infarcts, and the number of infarct layers between the END group and the non-END group (all P<0.05). Multivariate logistic regression analysis showed that the infarct involved the internal capsule (odds ratio [ OR] 1.52, 95% confidence interval [ CI] 1.27-1.96; P=0.015), the maximum diameter of the infarct ( OR 1.33, 95% CI 1.12-1.42; P=0.023), proximal SSI ( OR 1.49, 95% CI 1.28-1.92; P=0.018), and the number of infarct layers (compared to 1 layer, 3 layers: OR 15.01, 95% CI 4.12-12.45, P=0.013; ≥ 4 layers: OR 15.42, 95% CI 5.67-18.43, P=0.004) were independent risk factors for END. Conclusion:Infarct involved internal capsule on diffusion-weighted imaging, larger diameter of the infarct, proximal SSI, and ≥3 layers of infarct may be predictive factors of the occurrence of END in patients with SSI.

Objective:To investigate the efficacy and safety of tirofiban in patients with acute ischemic stroke (AIS) with early neurological deterioration (END).Methods:Consecutive patients with AIS who had END and did not receive reperfusion treatment admitted to Xuzhou Municipal Hospital Affiliated to Xuzhou Medical University within 24 h of onset from January 2017 to December 2022 were retrospectively included. END was defined as an increase of ≥2 in the National Institutes of Health Stroke Scale score within 72 h after onset compared at admission, or an increase of 1 in motor function. The tirofiban group was given tirofiban within 2 h after the occurrence of END, while the control group was given aspirin+clopidogrel. A score of ≤2 on the modified Rankin Scale at 90 d after onset was considered as a good outcome.Results:A total of 502 patients with AIS who had END were enrolled; including 322 males (64.14%) aged 65 ± 9 years. There was no statistically significant difference in baseline characteristics between the tirofiban group ( n=252) and the control group ( n=250). The good outcome rate of the tirofiban group at 90 d was significantly higher than that of the control group (60.32% vs. 42.00%; P<0.05), while there were no statistically significant differences in the incidence of symptomatic hemorrhagic transformation, asymptomatic hemorrhagic transformation, death and serious adverse events within 90 d. There were statistically significant differences in diabetes, atrial fibrillation, use of tirofiban, and the classification of stroke etiology between the good outcome group and the poor outcome group ( P<0.05). Multivariate logistic regression analysis showed that the use of tirofiban was an independent correlated factor for good outcome (odds ratio 0.33, 95% confidence interval 0.22-0.48; P<0.001). Conclusion:Tirofiban has good efficacy and safety for END in patients with AIS who did not received reperfusion therapy.

Objective:This study aimed to evaluate the efficacy of decitabine (DAC) and identify factors influencing treatment responses in patients with primary immune thrombocytopenia (ITP) who had failed glucocorticoid therapy.Methods:Clinical data of 61 patients with glucocorticoid-resistant ITP who received DAC therapy (5 mg·m -2·d -1×3 d via intravenous infusion) for at least three cycles with 3-4-week intervals at the Department of Hematology, Qilu Hospital of Shandong University, from November 2015 to June 2021 were analyzed retrospectively. Results:The 61 patients comprised 20 males and 41 females, with a median age of 45 years (range: 15-81 years). Among them, 43 patients were glucocorticoid-dependent (glucocorticoid-dependent group), while 18 patients were glucocorticoid-resistant (glucocorticoid-resistant group). Following DAC treatment, 12 patients (19.67% ) achieved complete response (CR), and 16 patients (26.23% ) exhibited response (R), resulting in an overall response (OR) rate of 45.90% (28/61). Comparison between the OR group ( n=28) and the non-response (NR) group ( n=33) revealed significant differences in responses to glucocorticoids (dependent or resistant) and platelet counts before treatment ( χ2=8.789, P=0.003; z=-2.416, P=0.016). The glucocorticoid-dependent group showed higher platelet counts than the glucocorticoid-resistant group after the second and third cycles of DAC treatment ( P=0.032, 0.024). Moreover, the OR rates after the first, second, and third cycles of DAC treatment in the glucocorticoid-dependent group were all higher than those in the glucocorticoid-resistant group ( P=0.042, P=0.012, P=0.029). A significant correlation was observed between glucocorticoid dependence and responses to DAC treatment ( OR=9.213, 95% CI 1.937-43.820, P=0.005) . Conclusion:DAC demonstrates definitive efficacy with mild adverse effects in a subset of patients with glucocorticoid-resistant primary ITP. Glucocorticoid dependence and higher platelet counts before treatment are associated with a favorable response to DAC therapy.

As one of the first institutions to carry out integrated education in China, the Shanghai Jiao Tong University School of Medicine has carried out a pilot reform of the organ system centered vertical single cycle integrated curriculum in the undergraduate medical education (five-year English stream) based on the Sino-foreign cooperative education program. The curriculum has been formed with multi-disciplinary single cycle integrated courses, longitudinal integrated courses, and three-dimensional specialized expansion electives. The curriculum focuses on the competency training of medical student. The aim is to highlight the teaching concept of early contact with clinic, attach importance to the cultivation and shaping of medical personality, achieve effective connection with post-graduation education, and realize the intersection and deep integration of social, humanities and medical professional courses through the construction of a series of course modules.

Objective:To investigate the correlation between fibrinogen and lipoprotein (a) and early neurological deterioration (END) in acute ischemic stroke patients with diabetes.Methods:From January 2017 to December 2020, patients with acute ischemic stroke admitted to the Department of Neurology, Xuzhou Municipal Hospital Affiliated to Xuzhou Medical University within 48 h of onset were enrolled retrospectively. END was defined as the National Institutes of Health Stroke Scale (NIHSS) score within 7 d after onset increased by ≥2 or motor function score increased by ≥1 compared with the baseline. Demographic and baseline clinical data were collected. Multivariate logistic regression analysis was used to identify the independent risk factors for END in general and diabetic patients with acute ischemic stroke. Results:A total of 1 504 patients with acute ischemic stroke were enrolled. Two hundred and fifty-two (16.76%) patients had END. The age, baseline NIHSS score, random blood glucose, fibrinogen, lipoprotein (a) levels, and the proportion of patients with diabetes in the END group were higher than those in the non-END group. There were also significant differences in various stroke etiologic subtypes between the END group and the non-END group (all P<0.05). Multivariate logistic regression analysis showed that fibrinogen and lipoprotein (a) levels were not the independent risk factors for END in patients with acute ischemic stroke. Three hundred and thirty-seven patients also had diabetes mellitus, of which 85 had END (25.22%). The levels of fibrinogen and lipoprotein (a) in the END group were significantly higher than those in the non-END group ( P<0.05). Multivariate logistic regression analysis showed that fibrinogen (odds ratio 2.23, 95% confidence interval 1.75-4.54; P=0.002) and lipoprotein (a) (odds ratio 1.98, 95% confidence interval 1.57-3.65; P=0.003) were the independent risk factors for END in acute ischemic stroke patients with diabetes. Conclusion:Higher fibrinogen and lipoprotein (a) levels are associated with END in acute ischemic stroke patients with diabetes.

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*