CRISPRi/dCpf1-mediated dynamic metabolic switch to enhance butenoic acid production in Escherichia coli.

Butenoic acid is a short-chain unsaturated fatty acid and important precursor for pharmaceutical and other applications. Heterologous thioesterases are able to convert a fatty acid biosynthesis intermediate in Escherichia coli to butenoic acid. In order to acquire high titer and yield of the product, dynamically switching the metabolic flux from fatty acid biosynthesis pathway to butenoic acid is critical after achieving enough cell mass of the host. A previous developed switch for butenoic acid fermentation is based on triclosan molecule as the FabI inhibitor in the fatty acid biosynthesis cycle. However, triclosan is toxic to human, which may limit its pharmaceutical application. Alternatively, we here purposed a nontoxic switch of carbon flux by harnessing recently developed CRISPR interference (CRISPRi) approach. In our work, we constructed a CRISPRi/dCpf1-mediated dynamic metabolic switch to separate the host growth and production phase via switching the expression of the fabI gene in fatty acid biosynthesis pathway. After optimizing the programmable targets, the CRISPRi-based switch boosted the titer of butenoic acid by 6-fold (1.41 g/L) in fed-batch fermentation. Our work supported that the CRISPRi/dCpf1 switch could replace triclosan-based switch as a nontoxic switch for butenoic acid production, and outcompeted the later switch in the biomass accumulation of the host cell. Moreover, the CRISPRi/dCpf1 system was integrated into the chromosome of the host to improve its genetic stability for long-term fermentation and other applications.Key Points• A programmable metabolic switch was developed to replace the toxic chemical switch to separate the growth phase and production phase of the butenoic acid.• The programmable CRISPRi/dCpf1 switch was efficiently and stably integrated into the host genome to increase their genetic stability during fermentation.• The optimized metabolic switch simultaneously increased the host biomass and butenoic acid titer, and solved the paradox of the competition between growth and production.

Engineering Escherichia coli for the synthesis of short- and medium-chain α,ß-unsaturated carboxylic acids.

Concerns over sustained availability of fossil resources along with environmental impact of their use have stimulated the development of alternative methods for fuel and chemical production from renewable resources. In this work, we present a new approach to produce α,ß-unsaturated carboxylic acids (α,ß-UCAs) using an engineered reversal of the ß-oxidation (r-BOX) cycle. To increase the availability of both acyl-CoAs and enoyl-CoAs for α,ß-UCA production, we use an engineered Escherichia coli strain devoid of mixed-acid fermentation pathways and known thioesterases. Core genes for r-BOX such as thiolase, hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and enoyl-CoA reductase were chromosomally overexpressed under the control of a cumate inducible phage promoter. Native E. coli thioesterase YdiI was used as the cycle-terminating enzyme, as it was found to have not only the ability to convert trans-enoyl-CoAs to the corresponding α,ß-UCAs, but also a very low catalytic efficiency on acetyl-CoA, the primer and extender unit for the r-BOX pathway. Coupling of r-BOX with YdiI led to crotonic acid production at titers reaching 1.5g/L in flask cultures and 3.2g/L in a controlled bioreactor. The engineered r-BOX pathway was also used to achieve for the first time the production of 2-hexenoic acid, 2-octenoic acid, and 2-decenoic acid at a final titer of 0.2g/L. The superior nature of the engineered pathway was further validated through the use of in silico metabolic flux analysis, which showed the ability of r-BOX to support growth-coupled production of α,ß-UCAs with a higher ATP efficiency than the widely used fatty acid biosynthesis pathway. Taken together, our findings suggest that r-BOX could be an ideal platform to implement the biological production of α,ß-UCAs.

The synthesis of (Z)-4-oxo-4-(arylamino)but-2-enoic acids derivatives and determination of their inhibition properties against human carbonic anhydrase I and II isoenzymes.

The synthesis of (Z)-4-oxo-4-(arylamino)but-2-enoic acid (4) derivatives containing structural characteristics that can be used for the synthesis of several active molecules, is presented. Some of the butenoic acid derivatives (4a, 4c, 4e, 4i, 4j, 4k) are synthesized following literature procedures and at the end of the reaction. In addition, structures of all synthesized derivatives (4a-4m) were determined by (1)H-NMR, (13)C-NMR and IR spectroscopy. Carbonic anhydrase is a metalloenzyme involved in many crucial physiologic processes as it catalyzes a simple but fundamental reaction, the reversible hydration of carbon dioxide to bicarbonate and protons. Significant results were obtained by evaluating the enzyme inhibitory activities of these derivatives against human carbonic anhydrase hCA I and II isoenzymes (hCA I and II). Butenoic acid derivatives (4a-4m) strongly inhibited hCA I and II with Kis in the low nanomolar range of 1.85 ± 0.58 to 5.04 ± 1.46 nM against hCA I and in the range of 2.01 ± 0.52 to 2.94 ± 1.31 nM against hCA II.

Ethnobotany, phytochemistry, pharmacology and quality control of Peucedanum decursivum (Miq.) Maxim: A critical review.

ETHNOPHARMACOLOGICAL RELEVANCE: Dried roots of Peucedanum decursivum, a traditional Chinese medicine (TCM), has historically respiratory diseases such as cough, thick phlegm, headache, fever, and gynecological diseases, rheumatoid arthritis, and nasopharyngeal carcinoma. AIM OF THE STUDY: Made an endeavor to evaluate the research trajectory of P. decursivum, comprehensively discern its developmental status, and offer a guideline for future investigations. MATERIALS AND METHODS: A meticulous search of literatures and books from 1955 to 2024 via databases like PubMed, Web of Science and CNKI was conducted, including topics and keywords of " P. decursivum" "Angelica decursivum" and "Zihua Qianhu". RESULTS: P. decursivum and its prescriptions have traditionally been used for treating phlegm-heat cough, wind-heat cough, gastrointestinal diseases, pain relief and so on. It contains 234 identified compounds, encompassing coumarins, terpenes, volatile oils, phenolic acids, fatty acids and derivatives. It exhibits diverse pharmacological activities, including anti-asthmatic, anti-inflammatory, antioxidant effects, anti-hypertensive, anti-diabetic, anti-Alzheimer, and anti-cancer properties, primarily attributed to coumarins. Microscopic identification, HPLC fingerprinting, and bioinformatics identification are the primary methods currently used for the quality control. CONCLUSION: P. decursivum demonstrates anti-asthmatic, anti-inflammatory, and antioxidant effects, aligning with its traditional use. However, experimental validation of its efficacy against phlegm and viruses is needed. Additionally, analgesic effects mentioned in historical texts lack modern pharmacological studies. Numerous isolated compounds exhibit highly valuable medicinal properties. Future research can delve into exploring these substances further. Rigorous of heavy metal contamination, particularly Cd and Pb, is necessary. Simultaneously, investigating its pharmacokinetics and toxicity in humans is crucial for the safety.

Development of a Transformation System for Nitratireductor sp.

Nitratireductor sp. OM-1 can accumulate butenoic acid, which is a short-chain unsaturated carboxylic acid utilized for chemical products. So far, we have predicted the thioesterase gene, te, as a candidate gene for butenoic acid biosynthesis, based on comparative transcriptome analysis. To confirm the function of te, the gene transfer system in Nitratireductor sp. OM-1 was required. Thus, in this study, we used electroporation as a transformation system and pRK415, a broad host range plasmid, and optimized the conditions. As a result, a maximum transformation efficiency of 7.9 × 104 colonies/µg DNA was obtained at 22.5 kV/cm. Moreover, an expression vector, pRK415-te, was constructed by insertion of te, which was successfully transferred into strain OM-1, using electroporation. The recombinant OM-1 strain produced butenoic acid at 26.7 mg/g of dried cell weight, which was a 254% increase compared to transformants harboring an empty vector. This is the first report of a gene transfer system for Nitratireductor sp., which showed that the te gene was responsible for butenoic acid production.

A high molar extinction coefficient bisterpyridyl homoleptic ru(II) complex with trans-2-methyl-2-butenoic acid functionality: potential dye for dye-sensitized solar cells.

In our continued efforts in the synthesis of ruthenium(II) polypyridine complexes as potential dyes for use in varied applications, such as the dye-sensitized solar cells (DSSCs), this work particularly describes the synthesis, absorption spectrum, redox behavior and luminescence properties of a new homoleptic ruthenium(II) complex bearing a simple trans-2-methyl-2-butenoic acid functionality as the anchoring ligand on terpyridine moiety. The functionalized terpyridine ligand: 4'-(trans-2-methyl-2-butenoic acid)-terpyridyl (L1) was synthesized by aryl bromide substitution on terpyridine in a basic reaction condition under palladium carbide catalysis. In particular, the photophysical and redox properties of the complex formulated as: bis-4'-(trans-2-methyl-2-butenoic acid)-terpyridyl ruthenium(II) bis-hexafluorophosphate [Ru(L1)(2)(PF(6))(2)] are significantly better compared to those of [Ru(tpy)(2)](2+) and compare well with those of the best emitters of Ru(II) polypyridine family containing tridentate ligands. Reasons for the improved photophysical and redox properties of the complex may be attributed partly to the presence of a substituted α,ß-unsaturated carboxylic acid moiety leading to increase in the length of π-conjugation bond thereby enhancing the MLCT-MC (Metal-to-ligand-charge transfer-metal centred) energy gap, and to the reduced difference between the minima of the excited and ground states potential energy surfaces.

4-(1-Benzyl-1H-benzo[d]imidazol-2-yl)-4-oxo-2-butenoic Acid Derivatives: Design, Synthesis and Anti-HIV-1 Activity.

Acquired immunodeficiency syndrome (AIDS) is still an incurable disease with increasing mortality rate. Despite the development of effective FDA-approved anti-HIV drugs, there are some problems due to the growing of resistant viral strands. Therefore, discovery of novel anti-HIV agents is so needed. Integrase, targeted in highly active antiretroviral therapy (HAART), is a crucial enzyme in viral replication. In this study, new benzimidazolyl diketo acid derivatives were designed according to required features for inhibitors of HIV-1 integrase. Designed compounds were synthesized and evaluated for anti-HIV-1 effects. According to the cell-based biological assay's results, most of the tested compounds demonstrated good anti-HIV-1 activity, ranging from 40-90 µM concentration with no severe cytotoxicity. The most potent compound was 13g with EC50 value of 40 µM and CC50 value of 550 µM. Docking analysis of compound 13g in integrase active site was in good agreement with well-known integrase inhibitors, proposing that anti-HIV-1 potency of compounds may be via integrase inhibition.