Discovery of a new nosiheptide-producing strain and its fermentation optimization for nosiheptide production / 中国药科大学学报

@#Nosiheptide is a typical thiopeptide antibiotic displaying potent activity toward various drug-resistant strains of Gram-positive pathogens.Although nosiheptide lacks in vivo activity, and good water-solubility with a series of uncontrollable analogues, which may limit its clinical application, glycosylated analogues may overcome problem of low activity and may improve its druggability.In search of novel glycosylated nosiheptide producers, we applied a genome mining strategy that identified Actinoalloteichus sp.AHMU CJ021 that contains all genes required.However, despite the presence of a predicted glycosyltransferase, glycosylated derivatives of nosiheptide were not detected, after following one strain many compounds (OSMAC) strategy and heterologous expression of a regulatory protein NocP.Nevertheless, nosiheptide produced by this strain was remarkably pure, and further experiments were conducted to improve its production by optimization of the culture medium.Under optimal conditions, 58.73 mg/L nosiheptide was produced, representing an almost 6-fold improvement compared to the original fermentation medium.Therefore, we consider Actinoalloteichus sp.AHMU CJ021 a suitable potential candidate for industrial production of nosiheptide, which provides the basis for solving the problem of nosiheptide structural analogues.

Advances in strategies for activating silent biosynthetic gene clusters in Streptomyces / 中国药科大学学报

@#Microbial secondary metabolites have always been one of the important sources of discovery and development of new drugs due to their remarkable biological activities. The explosion of genome sequences has revealed that Streptomyces harbor an immensely untapped biosynthetic potential. However, the number of active secondary metabolites with new skeletons or structural units found from Streptomyces is much lower than that of biosynthetic gene clusters(BGCs), mainly due to the fact that many BGCs are either expressed weakly or transcriptionally silent under conventional laboratory conditions. Beginning with the bioinformatics tools for BGCs prediction, this review focuses on the classical approaches to activate silent BGCs of Streptomyces in native and heterologous hosts. Moreover, several new strategies including transcriptional factors decoy, reporter-guided high-throughput selection and muliplexed CRISPR-TAR were detailed, which provide methodological references for mining new secondary metabolites from Streptomyces.

Regio-selective glycosylation of mogroside IIIE by glycosyltransferase / 中国药科大学学报

@#Mogrosides, the main sweet components isolated from Siraitia grosvenorii, are a family of cucurbitane-type tetracyclic triterpenoid saponins. Given that the high sweetness, low calorie and excellent taste, mogrosides have become the important resource for the development of natural non-nutritive sweeteners. As reported, 11α-hydroxyl group in the structural skeleton of mogrosides was closely related to sweetness and taste, but it had not been confirmed experimentally. In this work, we used mogroside IIIE as a model compound, which was 300 times sweeter than 5% sucrose and tasted better, and modified its 11α-hydroxyl group through glycosyltransferase to elucidate the relations between structure and sweetness of mogroside compounds. The glycosyltransferase HXSW-GT-2 was obtained to regio-selectively glycosylate the 11α-hydroxyl group of mogroside IIIE through the screening of glycosyltransferase library. And then, the soluble expression of HXSW-GT-02 in Escherichia coli was efficiently achieved by optimizing the induction conditions. Subsequently, the yield of glycosylated mogroside IIIE(MG-IIIE-Glu)was increased to > 85% through optimizing reaction pH, temperature, UDP-G dosage and biocatalyst loading. The product MG-IIIE-Glu was bio-prepared at a 0. 5 L scale and the final purity was 97. 8%. A “mouth feel” test showed that MG-IIIE-Glu had no sweetness and displayed obvious bitterness through the comparison with mogroside IIIE and 5% sucrose. In conclusion, the function of the 11α-hydroxyl group of mogrosides in sweetness and taste was preliminarily elucidated which would be beneficial for the structural modification and development of mogroside sweeteners.

Rapid and sensitive high-throughput determination method for alanine-dependent transaminase / 中国药科大学学报

@#A new method for determining transaminase activity based on the color change of the reaction solution was established, by using alanine-dependent transaminase VfTA from Vibrio fluvialis JS17 as the research object coupled with pyruvate oxidase and horseradish peroxidase. After the optimization of the conditions, the linear relationship between VfTA activity units and the absorbance at 400 nm was investigated. This method was also applied to determine the activity of commercial transaminase ATA117. The results showed that the detection limit of transaminase VfTA activity was up to 0. 45 U/mL and the detection limit of ATA117 activity was up to 0. 5 U/mL. The transaminase activity could be quickly judged according to the color depth of the reaction solution.

Application of TAR cloning in microbial secondary metabolites / 中国药科大学学报

@#Microbial secondary metabolites have been a major source for drug discovery and development due to their structural novelty and diversity. Microbial secondary metabolites, typically encoded by specific biosynthetic gene cluster(BGC), are non-essential for the growth and propagation of the microbes. Despite the abundant existence of microbes, the majority of them are unculturable under laboratory conditions. Moreover, given that most of the BGCs from culturable microbes are silent, the discovery of novel microbial secondary metabolites has been hampered. Recently, the heterologous expression of BGCs has become an attractive approach to discover various microbial secondary metabolites, among which TAR-based heterologous expression is one of the important tools. This review summarized the principle of TAR, the applications and the advanced strategies of TAR-based methods for heterologous expression of secondary metabolites, which may help the advancements of drug discovery and development from microbial sources.

Enzymatic synthesis of mogroside ⅢE / 中国药科大学学报

The aim was to develop the simple preparation method of mogroside ⅢE,and to lay the foundation for the development of the mogroside sweeteners. In the present study,the glycosidase CPU-GH17,which can regio-selectively biosynthesize mogroside ⅢE from mogroside V,was screened from the established library of glycosi-dases. Then,the soluble expression condition of CPU-GH17 in E. coli was exploited by investigating isopropyl β-D-thiogalactoside (IPTG)concentration,culture temperature and induction time,and 0. 4 mmol/L IPTG,15 °C and 12 h was used as optimal condition. The result showed that mogroside V could be completely converted into mogroside ⅢE under the conditions of pH 6. 0,45 °C,3 U/mL enzyme loading,5 mg/mL substrate concentration for 20 h. In conclusion,a biosynthetic system for the regio-selective preparation of mogroside ⅢE by recombinant CPU-GH17 was successfully established and verified at a preparative scale.

Applications of glycosyltransferases in the improvement of druggability of natural products / 中国药科大学学报

Natural products are one of the most important sources for druggable lead compounds given their diverse structures and activities.Nevertheless,very few of them can be directly developed to drugs.The undesirable druggability of natural products result from their weaker activity,lower aqueous solubility and poorer stability.Since improving druggability by chemical modifications is restricted by the limited access to the complex structures of natural products,enzymatic modification has gradually become one of important tools for the structural modification of natural products.Meanwhile,since enzymatic glycosylation by glycosyltransferases has shown certain advantages such as excellent regio-and stereo-selectivity,high catalytic efficiency and mild conditions.It has been a promising route for the improvement of druggability for natural products.In the present review,we summarize different glycosyltransferases and their application for structural modification of natural products as well as the challenges issues involved in the glycosylation,which provides a general perspective on the enzymatic modification of natural products for the improvement of their draggability.

Current progress in quantitative detection of microRNA / 中国药科大学学报

@#With the recent development of investigating biological functions of microRNA(miRNA), the importance of miRNA in the biological processes including developmental biology, metabolic regulation, disease progression and treatment has been well recognized. Due to its the instability, low level of expression and minor difference in sequence, more sophisticated analytical methods for rapid and easy quantitative detection of miRNA with strong specificity and high sensitivity play an important role in the study of the biological functions of miRNA. This review analyzes and compares recent quantitative methods to detect miRNA, with an attempt to provide a foundation for choosing an appropriate method to quantitatively analyze miRNA.

Strategy to solve cofactor issues in oxidoreductase catalyzed biocatalytic applications / 生物工程学报

NAD(P)(H)-dependent oxidoreductase catalyzes the reduction of ketones or aldehydes to prepare a wide variety of valuable chiral alcohols or amines. However, expensive cofactors are absolutely required for the biocatalytic processes with oxidoreductases, which severely hinder their industrial applications. Consequently, the issue on reducing cofactor costs has become one of the major focuses in the field of biocatalysis. With the substantial development in recent years, a number of strategies have been proposed and implemented to solve the cofactor issues in the oxidoreductase catalyzed biocatalysis, including the establishment of cofactor regeneration system, the improvement of endogenous cofactor availability via metabolic engineering and the development of biomimetic agents to replace cofactors. In this review, recent trends and advances on these strategies are presented, and respective advantages and shortcomings are also discussed with a number of examples.

Two step process for diketo-reduction catalyzed by diketoreductase / 中国药科大学学报

Diketoreductase can catalyze a double reduction of β,δ-diketo ester to corresponding dihydroxv product with enantiomeric excess(ee)and diastereomeric excess(de)both greater than 99.5%.In order to explore the catalytic mechanism of this unique enzyme,the present study investigated the diketone reduction process and reaction characteristics by diketoreductase.We found that two mono-hydroxy intermediates were produced during the diketone reduction and its reverse reaction.The two intermediates were further separated by C18 column chromatography and structurally confirmed by chiral HPLC with authentic standards.Two mono-hydroxy intermediates could be served as the substrates for the reduction by diketoreductase with different reaction velocities.The formation and disappearance of intermediates were largely affected by temperature and substrate concentration.In addition,steady state kinetics with the two intermediates showed different reaction rates in their disappearance.Collectively,the results indicate that the diketone reduction undergoes a two-step process with the formation of both intermediates,but the disappearance rates for the two intermediates are slightly different.