MiR-196b-3p and miR-450b-3p are key regulators of adipogenesis in porcine intramuscular and subcutaneous adipocytes.

BACKGROUND: As components of white adipose tissue, porcine intramuscular (IM) and subcutaneous (SC) adipocytes undergo similar differentiation and adipogenesis processes. However, the adipogenic capacity of IM adipocytes is weaker than that of SC adipocytes. Identifying key regulators underlying this difference between IM and SC adipocytes will benefit pig breeding. RESULTS: In this study, we used BGISEQ-500 sequencing technology to analyze the expression of small RNAs in primary cultured IM and SC adipocytes on day 8 after adipogenic induction, and found 32-fold higher miR-196b-3p expression, as well as 8-fold lower miR-450b-3p expression in IM adipocytes than in SC adipocytes. Functional studies revealed that miR-196b-3p inhibits adipogenesis by targeting CD47 via the AMPK signaling pathway, and its effect was attenuated by the specific p-AMPKα activator AICAR. We also found that miR-450b-3p promotes adipogenesis by targeting SIRT1 via the Wnt/ß-catenin signaling pathway, and its effect was weakened by the Wnt/ß-catenin signaling activator LiCl. CONCLUSIONS: Our findings suggest that miR-196b-3p and miR-450b-3p are novel key regulatory factors that play opposite roles in porcine adipogenesis, helping us decipher the regulatory differences between porcine IM and SC fat deposition.

Observation of synergistic antibacterial properties of prodigiosin from Serratia marcescens jx-1 with metal ions in clinical isolates of Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are a major global health problem, and novel and effective antimicrobial drugs are urgently required to combat this life-threatening pathogen. Prodigiosin (PG) is a bacterial secondary metabolite with excellent anticancer and antibacterial properties. However, little is known about the antibacterial function of PG against MRSA. Therefore, the antibacterial efficacy of PG alone and PG in combination with different metal ions against clinic isolates of MRSA and methicillin-sensitive S. aureus (MSSA) strain was evaluated in the present study. The minimum inhibitory concentration of PG against both MRSA and MSSA was 0.25 µg/mL. However, 0.1 µg/mL PG showed a stronger inhibitory effect on MSSA cell growth (47.12%) than on MRSA cell growth (35.87%). Surprisingly, we observed a significant difference (p < 0.01) in membrane integrity between PG-treated MRSA and MSSA using the propidium iodide staining assay. Further, we found that in combination with PG, Zn2+, Al3+, and Cu2+ showed synergistic antibacterial effects against MRSA and MSSA. Our results could increase the current knowledge regarding the efficacy of PG in inhibiting the growth of different types of S. aureus clinical isolates and also offer a novel strategy for developing efficient antibacterial agents.

Insights into the anti-infective properties of prodiginines.

Prodiginines are a large family of tripyrrole alkaloids that contain natural members produced by various bacteria and non-natural members obtained from chemical synthesis, enzymatic synthesis, and mutasynthesis. These compounds have attracted a great deal of attention due to their wide range of fascinating properties including anti-infective, anticancer, and immunosuppressive activities. In consideration of the great need for novel and effective anti-infective agents, this review is mainly focused on the current status of research on the anti-infective properties of prodiginines, highlighting their antibacterial, antifungal, antiprotozoal, anti-larval, and antiviral activities. Additionally, the multiple mechanisms by which prodiginines exert their anti-infective effects will also be discussed.

Enhancement of prodigiosin synthetase (PigC) production from recombinant Escherichia coli through optimization of induction strategy and media.

PigC is a synthetase that catalyzes the condensation of 4-methoxy-2,2'-bipyrrole-5-carboxyaldehyde and 2-methyl-3-amylpyrrole to produce prodigiosin, which has a wide variety of impressive biological properties. In this study, we optimized PigC production from engineered Escherichia coli BL21(DE3). Investigation of different induction strategies revealed that autoinduction was the most appropriate method for PigC expression. As a result, PigC activity was elevated to 75.7 U/mL, nearly 2.1-fold higher than that with induction by isopropy-ß-d-thiogalactoside. To achieve maximum enzyme production, the automedium components were optimized. "Single-factor experiments" showed that PigC production was greatly affected by the concentrations of glucose, yeast extract, and lactose. The Box-Behnken design for response surface methodology was then used to determine the optimal concentrations of these three components. According to a statistical approach, the optimum values of the three most influential parameters were 0.73 g/L glucose, 13.17 g/L yeast extract, and 5.86 g/L lactose. In the optimized automedium, the best PigC activity was obtained at 179.3 U/mL, which was 2.4-fold higher than using the initial medium. This study maximized PigC production as a foundation for further study and future industrial application.

Chemical and enzymatic approaches to the synthesis of optically pure ethyl (R)-4-cyano-3-hydroxybutanoate.

Ethyl (R)-4-cyano-3-hydroxybutanoate (HN) is an important chiral synthon for side chain of the cholesterol-lowering drug atorvastatin (Lipitor), which is the hydroxymethylglutaryl CoA reductase inhibitor. HN is also used as a synthon in the production of L-carnitine and (R)-4-amino-3-hydroxybutanoic acid. It is necessary to have a clear understanding of the synthesis process of HN for its extensive use. This review gives an overview of different synthetic strategies of optically active HN, including chemical and enzymatic approaches. The emphasis is focused mainly on the synthetic routes using biocatalysts, such as halohydrin dehalogenase, nitrilase, carbonyl reductase, and lipase.

Characterization of a newly synthesized carbonyl reductase and construction of a biocatalytic process for the synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate with high space-time yield.

A carbonyl reductase (SCR2) gene was synthesized and expressed in Escherichia coli after codon optimization to investigate its biochemical properties and application in biosynthesis of ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), which is an important chiral synthon for the side chain of cholesterol-lowering drug. The recombinant SCR2 was purified and characterized using ethyl 4-chloro-3-oxobutanoate (COBE) as substrate. The specific activity of purified enzyme was 11.9 U mg(-1). The optimum temperature and pH for enzyme activity were 45 °C and pH 6.0, respectively. The half-lives of recombinant SCR2 were 16.5, 7.7, 2.2, 0.41, and 0.05 h at 30 °C, 35 °C, 40 °C, 45 °C, and 50 °C, respectively, and it was highly stable in acidic environment. This SCR2 displayed a relatively narrow substrate specificity. The apparent K m and V max values of purified enzyme for COBE are 6.4 mM and 63.3 µmol min(-1) mg(-1), respectively. The biocatalytic process for the synthesis of (S)-CHBE was constructed by this SCR2 in an aqueous-organic solvent system with a substrate fed-batch strategy. At the final COBE concentration of 1 M, (S)-CHBE with yield of 95.3% and e.e. of 99% was obtained after 6-h reaction. In this process, the space-time yield per gram of biomass (dry cell weight, DCW) and turnover number of NADP(+) to (S)-CHBE were 26.5 mmol L(-1) h(-1) g(-1) DCW and 40,000 mol/mol, respectively, which were the highest values as compared with other works.

[Advances in mechanisms of biofilm formation and drug resistance of Staphylococcus aureus].

Staphylococcus aureus is a common pathogenic bacterium. However, due to the abuse of antibiotics, multiple drug-resistant S. aureus (DR S. aureus) has emerged in a large number, which seriously threatens human health. DR S. aureus usually forms biofilms by attaching on contact surfaces and secreting macromolecules including polysaccharides, proteins, and lipids, thus encasing themselves in a self-generated polymeric matrix. A biofilm provides an efficacious barrier that protects bacteria from detrimental environmental factors. Simultaneously, it protects DR S. aureus from the host immune system and attenuates the penetration and killing effects of drugs, serving as a key structure for the development of drug resistance. Therefore, gaining an in-depth understanding of the DR S. aureus biofilm is crucial for treating related infectious diseases. In this paper, we summarize recent research progress in the biofilm formation mechanism, drug resistance mechanism, and measures for inhibition and clearance of DR S. aureus and provide an outlook on the future research directions.

Transcriptomic analysis of cell envelope inhibition by prodigiosin in methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading threat to public health as it is resistant to most currently available antibiotics. Prodigiosin is a secondary metabolite of microorganisms with broad-spectrum antibacterial activity. This study identified a significant antibacterial effect of prodigiosin against MRSA with a minimum inhibitory concentration as low as 2.5 mg/L. The results of scanning electron microscopy, crystal violet staining, and confocal laser scanning microscopy indicated that prodigiosin inhibited biofilm formation in S. aureus USA300, while also destroying the structure of the cell wall and cell membrane, which was confirmed by transmission electron microscopy. At a prodigiosin concentration of 1.25 mg/L, biofilm formation was inhibited by 76.24%, while 2.5 mg/L prodigiosin significantly reduced the vitality of MRSA cells in the biofilm. Furthermore, the transcriptomic results obtained at 1/8 MIC of prodigiosin indicated that 235and 387 genes of S. aureus USA300 were significantly up- and downregulated, respectively. The downregulated genes were related to two-component systems, including the transcriptional regulator LytS, quorum sensing histidine kinases SrrB, NreA and NreB, peptidoglycan biosynthesis enzymes (MurQ and GlmU), iron-sulfur cluster repair protein ScdA, microbial surface components recognizing adaptive matrix molecules, as well as the key arginine synthesis enzymes ArcC and ArgF. The upregulated genes were mainly related to cell wall biosynthesis, as well as two-component systems including vancomycin resistance-associated regulator, lipoteichoic acid biosynthesis related proteins DltD and DltB, as well as the 9 capsular polysaccharide biosynthesis proteins. This study elucidated the molecular mechanisms through which prodigiosin affects the cell envelope of MRSA from the perspectives of cell wall synthesis, cell membrane and biofilm formation, providing new potential targets for the development of antimicrobials for the treatment of MRSA.

Properties and biotechnological applications of halohydrin dehalogenases: current state and future perspectives.

Halohydrin dehalogenases (HHDHs) are lyases that catalyze the cleavage of carbon-halogen bond of halohydrins. They also can catalyze the reverse reaction in the presence of nucleophiles such as cyanide, azide, and nitrite ions. HHDHs have been recognized as the ideal tools for the degradation of various halogenated environmental pollutants. Moreover, they can be used as biocatalysts for the kinetic resolution of halohydrins and epoxides, and for the preparation of various substituted alcohols. This review is mainly focused on the current status of research on HHDHs, highlighting the production, characterization, structures and mechanism, protein engineering, and biotechnological applications of HHDHs.

Characterization and application of a newly synthesized 2-deoxyribose-5-phosphate aldolase.

A codon-optimized 2-deoxyribose-5-phosphate aldolase (DERA) gene was newly synthesized and expressed in Escherichia coli to investigate its biochemical properties and applications in synthesis of statin intermediates. The expressed DERA was purified and characterized using 2-deoxyribose-5-phosphate as the substrate. The specific activity of recombinant DERA was 1.8 U/mg. The optimum pH and temperature for DERA activity were pH 7.0 and 35 °C, respectively. The recombinant DERA was stable at pH 4.0-7.0 and at temperatures below 50 °C. The enzyme activity was inhibited by 1 mM of Ni(2+), Ba(2+) and Fe(2+). The apparent K (m) and V (max) values of purified enzyme for 2-deoxyribose-5-phosphate were 0.038 mM and 2.9 µmol min(-1) mg(-1), for 2-deoxyribose were 0.033 mM and 2.59 µmol min(-1) mg(-1), respectively, which revealed that the enzyme had similar catalytic efficiency towards phosphorylated and non-phosphorylated substrates. To synthesize statin intermediates, the bioconversion process for production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose from chloroacetaldehyde and acetaldehyde by the recombinant DERA was developed and a conversion of 94.4 % was achieved. This recombinant DERA could be a potential candidate for application in production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose.

[Progress in microbial production of prodigiosin].

Prodigiosin is an important natural red pigment that is produced as a secondary metabolite by microorganisms, and has great potential applications in the field of pharmaceutical development, environmental management and dye preparation. This paper reviews recent research progress in the production of prodigiosin by microbial fermentation, including discovery and modification of the prodigiosin-producing microorganisms, regulation and optimization of prodigiosin fermentation and extraction process, and resolution of biosynthetic pathway of prodigiosin and related transcriptional regulation. Finally, we discussed the future research directions in microbial production of prodigiosin.

A novel synthesis of iminodiacetic acid: biocatalysis by whole Alcaligenes faecalis ZJB-09133 cells from iminodiacetonitrile.

Iminodiacetic acid (IDA) has been widely used as an important intermediate in the fine chemical industry. In this study, a novel synthesis route of IDA from iminodiacetonitrile by whole microorganisms was investigated. A strain with the capability of producing nitrilase, ZJB-09133, was isolated and identified, and later named Alcaligenes faecalis ZJB-09133. In addition, the detailed biocatalysis of iminodiacetonitrile to produce IDA using ZJB-09133 was investigated. The results showed that the conversion reached 65.3% in Na(2)HPO(4)-NaH(2)PO(4) buffer of pH 8.0 under the following conditions: cells in the amount of 0.075-g DCW/L, 1.5% substrate, conversion time of 8 h, and a reaction temperature of 35°C. To the best of our knowledge, this is the first time that the production of IDA using a biocatalysis method has been reported.