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Polycyclic xanthones are characterized by highly oxygenated, angular hexacyclic frameworks and exhibit diverse biological activities. Although many of them have been isolated and chemically synthesized, the detailed biosynthetic machinery awaits discovery. Recently, xanthone construction in the xantholipin (1) pathway was shown to involve cryptic demethoxylation. This suggested a rationale for the existence of three O-methyltransferase (OMT) genes in the gene cluster, although there are only two O-methyl groups in the structure of 1. Here, in vivo and in vitro analysis have been used to show that the three paralogous OMTs, XanM1-M3, introduce individual methyl groups at specific points in the biosynthetic pathway. Each OMT can to some extent take over the role of the other OMTs, although they exhibit highly substrate-dependent regiospecificity. In addition, phylogenetic analysis suggests their evolution from a common ancestor. Four putative ancestral proteins were constructed, and one of them performed all the functions of XanM1-M3, while the others possessed more limited catalytic functions. The results suggest that a promiscuous common ancestor may have been able to catalyze all three reactions prior to gene duplication and functional divergence. The characterization of XanM1-M3 expands the enzyme inventory for polycyclic xanthone biosynthesis and suggests novel directed evolution approaches to diversifying natural product pathways.

This study aimed to investigate the role of sevoflurane on anti-oxidant effects in sepsis-induced multiple organ failure. Western blot and quantitative RT–PCR were used to measure the expression of...

Piericidin A1, a member of ɑ-pyridone antibiotic, exhibits various biological activities such as antimicrobial, antifungal, and antitumor properties and possesses potent respiration-inhibitory activity against insects due to its competitive binding capacity to mitochondrial complex I. The biosynthetic pathway of piericidin A1 has been reported in Streptomyces piomogeues var. Hangzhouwanensis, while the regulatory mechanism remains poorly understood. In this study, a Streptomyces antibiotic regulatory protein (SARP) family transcriptional regulator PieR was characterized. Genetic disruption and complementation manipulations revealed that PieR positively regulated the production of piericidin A1. Moreover, the overexpression of pieR contributed to the improvement of piericidin A1 productivity. The real-time quantitative PCR (RT-qPCR) was carried out and the data showed that pieR stimulated the transcription of all the biosynthesis-related genes for piericidin A1. In order to explore the regulatory mechanism, electrophoresis mobility shift assays (EMSA) and DNase I footprinting experiments have been conducted. A protected region covering 50 nucleotides within the upstream region of pieR was identified and two 5-nt direct repeat sequences (5′-CCGGA-3′) in the protected region were found. These findings, taken together, set stage for transcriptional control engineering in the view of optimizing piericidin A1 production and thus provide a viable potent route for the construction of strains with high productivity. Keywords: ɑ-pyridone antibiotic, Transcriptional regulation, SARP family regulator, Secondary metabolic, Streptomyces

The protein synthesis inhibitor anisomycin features a unique benzylpyrrolidine system and exhibits potent selective activity against pathogenic protozoa and fungi. It is one of the important effective components in Agricultural Antibiotic120, which has been widely used as naturally-originated agents for treatment of crop decay in China. The chemical synthesis of anisomycin has recently been reported, but the complex process with low productivity made the biosynthesis still to be a vital mainstay in efforts. The biosynthetic gene cluster (BGC) of anisomycin in Streptomyces hygrospinosus var. beijingensis has been identified in our previous work, while poor understanding of the regulatory mechanism limited the yield enhancement via regulation engineering of S. hygrospinosus var. beijingensis. In this study here, we characterized AniF as an indispensable LuxR family transcriptional regulator for the activation of anisomycin biosynthesis. The genetic manipulations of aniF and the real-time quantitative PCR (RT-qPCR) revealed that it positively regulated the transcription of the anisomycin BGC. Moreover, the overexpression of aniF contributed to the improvement of the production of anisomycin and its derivatives. Dissection of the mechanism underlying the function of AniF revealed that it directly activated the transcription of the genes aniR-G involved in anisomycin biosynthesis. Especially, one AniF-binding site in the promoter region of aniR was identified by DNase I footprinting assay and an inverted repeat sequence (5′-GGGC-3′) composed of two 4-nt half sites in the protected region was found. Taken together, our systematic study confirmed the positive regulatory role of AniF and might facilitate the future construction of engineering strains with high productivity of anisomycin and its derivatives. Keywords: Anisomycin, Agricultural Antibiotic120, Biosynthesis, AniF, LuxR regulator, Streptomyces

Anisomycin (compound 1), a pyrrolidine antibiotic, exhibits diverse biological and pharmacologic activities. The biosynthetic gene cluster of compound 1 has been identified previously, and the multistep assembly of the core benzylpyrrolidine scaffold was characterized. However, enzymatic modifications, such as acylation, involved in compound 1 biosynthesis are unknown. In this study, the genetic manipulation of aniI proved that it encoded an indispensable acetyltransferase for compound 1 biosynthesis. Bioinformatics analysis suggested AniI as a member of maltose (MAT) and galactoside O-acetyltransferases (GAT) with C-terminal left-handed parallel beta-helix (LbH) subdomain, which were referred to as LbH-MAT-GAT sugar O-acetyltransferases. However, the biochemical assay identified that its target site was the hydroxyl group of the pyrrolidine ring. AniI was found to be tolerant of acyl donors with different chain lengths for the biosynthesis of compound 1 and derivatives 12 and 13 with butyryl and isovaleryl groups, respectively. Meanwhile, it showed comparable activity toward biosynthetic intermediates and synthesized analogues, suggesting promiscuity to the pyrrolidine ring structure of compound 1. These data may inspire new viable synthetic routes for the construction of more complex pyrrolidine ring scaffolds in compound 1. Finally, the overexpression of aniI under the control of strong promoters contributed to the higher productivities of compound 1 and its analogues. These findings reported here not only improve the understanding of anisomycin biosynthesis but also expand the substrate scope of O-acetyltransferase working on the pyrrolidine ring and pave the way for future metabolic engineering construction of high-yield strains. IMPORTANCE Acylation is an important tailoring reaction during natural product biosynthesis. Acylation could increase the structural diversity and affect the chemical stability, volatility, biological activity, and even the cellular localization of specialized compounds. Many acetyltransferases have been reported in natural product biosynthesis. The typical example of the LbH-MAT-GAT sugar O-acetyltransferase subfamily was reported to catalyze the coenzyme A (CoA)-dependent acetylation of the 6-hydroxyl group of sugars. However, no protein of this family has been characterized to acetylate a nonsugar secondary metabolic product. Here, AniI was found to catalyze the acylation of the hydroxyl group of the pyrrolidine ring and be tolerant of diverse acyl donors and acceptors, which made the biosynthesis more efficient and exclusive for biosynthesis of compound 1 and its derivatives. Moreover, the overexpression of aniI serves as a successful example of genetic manipulation of a modification gene for the high production of final products and might set the stage for future metabolic engineering.

The potential of using hydrolyzed egg white (hEW) as a food additive to decrease shrinkage, cooking loss of pork meat slices was investigated. Functional characteristics including solubility, Water-Holding Capacity (WHC) and Oil-Binding Capacity (OBC) of three hEWs by using different kinds of enzymes, a commercial small size peptide S and native egg white (NEw) were evaluated. Pork meat slices (8 cm×6 cm) with thickness of 4 mm were soaked in the same weight of solution contained hEW, small peptide S or NEw at a protein concentration of 10% (w/v) at 4°C for 24 h respectively. Results showed that, cooking loss (%) and shrinkage rate (%) decreased when raw meat was immersed in the solution which contained hEWs or NEw. Meat treated by hEW obtained by using enzyme-Thermoase PC10F® showed an excellent WHC and resulted in the least shrinkage rate (3.2%) compared with the meat soaked in the same portion of water (shrinkage rate of 22.7%). Moreover, by using a scanning electron microscopy, relative larger holes and a grainy appearance of endomysium sheaths were also observed in the meat treated by this hEW. Treated by hEWs and NEw contributed to the decrease of elasticity and toughness of cooked meat. Thus, treated by hEWs may permit reduction in cooking loss as and shrinkage rate of pork meat slices.

In this study, we assessed the antioxidant efficacy of four different size distributions of Egg White (EW) hydrolysates and evaluated their potential as natural antioxidants for meat preservation. Water solubility and Oxygen Radical Absorbance Capacity (ORAC) of EW hydrolysates were measured. Hydrolysis contributed to the increase of ORAC values of EW hydrolysates in comparison with native egg white. Based on this result, we investigated the effects of EW hydrolysates on color stability of pork slices. Pork slices were individually soaked in four EW hydrolysates solutions at 10% (w/w) protein concentration. After storage at 4°C for 24 h, objective measurements of the color of pork slices were performed using a colorimeter, the content of metmyoblobin before and after soak was also determined. Small size EW hydrolysate S showed a good ORAC value (389 µmol TE/g) and an excellent water solubility, inhibited color change (ΔE = 2.8) in treated pork slices, the lowest metmyoglobin proportion was also found in the meat treated by this small size EW hydrolysate S. Moreover, it was demonstrated that EW hydrolysates were generally better than the Control (only with water) in improving color stability during storage time. These results indicate that EW hydrolysates are promising sources of biological antioxidants and can potentially be used as preservatives in meat products.

The importance of user satisfaction is increasing in the competitive market. However, with the development of the Internet, user satisfaction models that based on time-consuming survey become unsuitable for software products, which evolve faster than ever. To support software evolution, this paper presents a new user satisfaction evaluation approach by analyzing user comments, the results of which are instructive for the next-step of evolution. We give an evaluation framework and calculate methods for user satisfaction analysis. Based on a case, we show the feasibility and maneuverability of this approach.