Metabolic engineering of Escherichia coli BL21 (DE3) for de novo production of l-DOPA from d-glucose

Abstract Background Production of l-tyrosine is gaining grounds as the market size of 3,4-dihydroxyphenyl-l-alanine (l-DOPA) is expected to increase due to increasing cases of Parkinson’s disease a neurodegenerative disease. Attempts to overproduce l-tyrosine for conversion to l-DOPA has stemmed on the overexpressing of critical pathway enzymes, an introduction of feedback-resistant enzymes, and deregulation of transcriptional regulators. Results An E. coli BL21 (DE3) was engineered by deleting tyrR, ptsG, crr, pheA and pykF while directing carbon flow through the overexpressing of galP and glk. TktA and PpsA were also overexpressed to enhance the accumulation of E4P and PEP. Directed evolution was then applied on HpaB to optimize its activity. Three mutants, G883R, G883A, L1231M, were identified to have improved activity as compared to the wild-type hpaB showing a 3.03-, 2.9- and 2.56-fold increase in l-DOPA production respectively. The use of strain LP-8 resulted in the production of 691.24 mg/L and 25.53 g/L of l-DOPA in shake flask and 5 L bioreactor, respectively. Conclusion Deletion of key enzymes to channel flux towards the shikimate pathway coupled with the overexpression of pathway enzymes enhanced the availability of l-tyrosine for L-DOPA production. Enhancing the activity of HpaB increased l-DOPA production from glucose and glycerol. This work demonstrates that increasing the availability of l-tyrosine and enhancing enzyme activity ensures maximum l-DOPA productivity

First-principles study on the electrical and thermal properties of the semiconducting Sc-3(CN)F-2 MXene

The two-dimensional materials MXenes have recently attracted interest for their excellent performance from diverse perspectives indicated by experiments and theoretical calculations. For the application of MXenes in electronic devices, the exploration of semiconducting MXenes arouses particular interest. In this work, despite the metallic properties of Sc3C2F2 and Sc3N2F2, we find that Sc-3(CN)F-2 is a semiconductor with an indirect band gap of 1.18 eV, which is an expansion of the semiconducting family members of MXene. Using first-principles calculations, the electrical and thermal properties of the semiconducting Sc-3(CN)F-2 MXene are studied. The electron mobilities are determined to possess strong anisotropy, while the hole mobilities show isotropy, i.e. 1.348 x 10(3) cm(2) V-1 s(-1) along x, 0.319 x 10(3) cm(2) V-1 s(-1) along the y directions for electron mobilities, and 0.517 x 10(3) cm(2) V-1 s(-1) along x, 0.540 x 10(3) cm(2) V-1 s(-1) along the y directions for hole mobilities. The room-temperature thermal conductivity along the M direction is determined to be 123-283 W m(-1) K-1 with a flake length of 1-100 m. Besides, Sc-3(CN)F-2 presents a relatively high specific heat of 547 J kg(-1) K-1 and a low thermal expansion coefficient of 8.703 x 10(-6) K-1. Our findings suggest that the Sc-3(CN)F-2 MXene might be a candidate material in the design and application of 2D nanoelectronic devices

Chemical Composition and Antioxidant Characteristic of Traditional and Industrial Zhenjiang Aromatic Vinegars during the Aging Process

Zhenjiang aromatic vinegar (ZAV) is one of the well-known fermented condiments in China, which is produced by solid-state fermentation. It can be classified into traditional Zhenjiang aromatic vinegar (TZAV) and industrial Zhenjiang aromatic vinegar (IZAV) because of different production methods. The purpose of the study was to evaluate the variations and differences on chemical compositions and antioxidant activities of TZAV and IZAV during the aging process. The proximate composition, organic acids content, total phenolic content (TPC), total flavonoid content (TFC), total antioxidant activity (TAA) and phenolic compounds composition of TZAV and IZAV were detected during the aging process. Organic acids contents, TPC, TFC, TAA and phenolic compounds contents in ZAV were increased during the aging process. Acetic acid, lactic acid and pyroglutamic acid in ZAV were major organic acids. With the extension of aging time, TZAV and IZAV had similar proximate compositions and organic acids content. The values of TPC, TFC and TAA were higher in TZAV than in IZAV when aging is more than 3 years. Rutin and p-coumaric acid were detected in TZAV but not in IZAV. In principal component analysis (PCA), TZAV and IZAV can be divided into two groups according to their phenolic compounds composition. These findings provide references for evaluating TZAV and IZAV on the basis of their characterizations

Obtaining a Panel of Cascade Promoter-5′-UTR Complexes in <i>Escherichia coli</i>

A promoter is one of the most important and basic tools used to achieve diverse synthetic biology goals. Escherichia coli is one of the most commonly used model organisms in synthetic biology to produce useful target products and establish complicated regulation networks. During the fine-tuning of metabolic or regulation networks, the limited number of well-characterized inducible promoters has made implementing complicated strategies difficult. In this study, 104 native promoter-5′-UTR complexes (PUTR) from <i>E. coli</i> were screened and characterized based on a series of RNA-seq data. The strength of the 104 PUTRs varied from 0.007% to 4630% of that of the P_BAD promoter in the transcriptional level and from 0.1% to 137% in the translational level. To further upregulate gene expression, a series of combinatorial PUTRs and cascade PUTRs were constructed by integrating strong transcriptional promoters with strong translational 5′-UTRs. Finally, two combinatorial PUTRs (P_ssrA-UTR_rpsT and P_dnaKJ-UTR_rpsT) and two cascade PUTRs (PUTR_ssrA-PUTR_infC‑rplT and PUTR_alsRBACE-PUTR_infC‑rplT) were identified as having the highest activity, with expression outputs of 170%, 137%, 409%, and 203% of that of the P_BAD promoter, respectively. These engineered PUTRs are stable for the expression of different genes, such as the red fluorescence protein gene and the β-galactosidase gene. These results show that the PUTRs characterized and constructed in this study may be useful as a plug-and-play synthetic biology toolbox to achieve complicated metabolic engineering goals in fine-tuning metabolic networks to produce target products

Outcomes in Newly Diagnosed Atrial Fibrillation and History of Acute Coronary Syndromes: Insights from GARFIELD-AF

BACKGROUND: Many patients with atrial fibrillation have concomitant coronary artery disease with or without acute coronary syndromes and are in need of additional antithrombotic therapy. There are few data on the long-term clinical outcome of atrial fibrillation patients with a history of acute coronary syndrome. This is a 2-year study of atrial fibrillation patients with or without a history of acute coronary syndromes

Analysis of Outcomes in Ischemic vs Nonischemic Cardiomyopathy in Patients With Atrial Fibrillation A Report From the GARFIELD-AF Registry

IMPORTANCE Congestive heart failure (CHF) is commonly associated with nonvalvular atrial fibrillation (AF), and their combination may affect treatment strategies and outcomes