RNA interference reveals chloride channel 7 gene helps short-term hypersalinity stress resistance in Hong Kong oyster Crassostrea hongkongensis

The chloride channel 7 gene(CLC 7)of the Hong Kong oyster Crassostrea hongkongensis was cloned and named ChCLC 7.The cDNA was 2572 bp in length,with a 5′non-coding region containing 25 bp,a 3′non-coding region containing 327 bp,and an open reading frame of 2298 bp.ChCLC 7 has 96.8%and 92.1%homology with CLC 7 of Crassostrea gigas and Crassostrea virginica,respectively,and it was clustered with CLC 7 of C.gigas and C.virginica.QRT-PCR showed that ChCLC 7 was expressed in all eight tissues,with the highest in adductor muscle and second in gill.The ChCLC 7 expression pattern in gill was altered significantly under high salinity stress with an overall upward and then downward trend.After RNA interference,the expression of ChCLC 7 and survival rate of oyster under high salinity stress was reduced significantly,and so did the concentration of hemolymph chloride ion in 48-96 h after RNA interference.We believed that ChCLC 7 could play an important role in osmoregulation of C.hongkongensis by regulating Cl^(-)transport.This study provided data for the analysis of molecular mechanism against oyster salinity stress.

O-methyltransferase CbzMT catalyzes iterative 3,4-dimethylations for carbazomycin biosynthesis

Carbazomycins(1-8)are a subgroup of carbazole derivatives that contain oxygen at the C3 and C4 positions and an unusual asymmetric substitution pattern.Several of these compounds exhibit antifungal and antioxidant activities.To date,no systematic biosynthetic studies have been conducted on carbazomycins.In this study,carbazomycins A and B(1 and 2)were isolated from Streptomyces luteosporeus NRRL 2401 using a one-strain-many-compound(OSMAC)-guided natural product mining screen.A biosynthetic gene cluster(BGC)was iden-tified,and possible biosynthetic pathways for 1 and 2 were proposed.The in vivo genetic manipulation of the O-methyltransferase-encoding gene cbzMT proved indispensable for 1 and 2 biosynthesis.Size exclusion chro-matography indicated that CbzMT was active as a dimer.In vitro biochemical assays confirmed that CbzMT could repeatedly act on the hydroxyl groups at C3 and C4,producing monomethylated 2 and dimethylated 1.Monomethylated carbazomycin B(2)is not easily methylated;however,CbzMT seemingly prefers the dimethy-lation of the dihydroxyl substrate(12)to 1,even with a low conversion efficiency.These findings not only improve the understanding of carbazomycin biosynthesis but also expand the inventory of OMT-catalyzing it-erative methylations on different acceptor sites,paving the way for engineering biocatalysts to synthesize new active carbazomycin derivatives.

基于基因组挖掘的农抗120活性成分制霉菌素和丰加霉素的发现和鉴定

【目的】分析刺孢吸水链霉菌北京变种(农抗120产生菌)基因组和次级代谢产物组分,研究并鉴定农抗120产生菌中未被发现的活性组分。【方法】利用antiSMASH在线分析农抗120产生菌Streptomyces hygrospinosusvar.beijingensis基因组信息,锁定可能的制霉菌素和丰加霉素生物合成基因簇。利用HPLC和LC-MS等分析方法对农抗120产生菌发酵产物进行分析,同时利用制霉菌素和丰加霉素标准品作为对照,以鉴定该菌株代谢组分中的次级代谢产物。此外,通过构建目标基因簇大片段缺失突变株,并对所得突变株发酵产物进行检测,以确定生物合成基因簇与目的代谢产物的对应关系。【结果】本研究综合利用基因组序列分析、基因缺失突变株构建以及代谢产物检测方法,鉴定了农抗120产生菌中制霉菌素和丰加霉素两种活性成分,并确定了负责这些化合物合成的基因簇。【结论】本研究所构建的多重基因簇失活突变株为挖掘刺孢吸水链霉菌北京变种更多的天然次级代谢产物奠定了基础。

假单胞菌Pseudomonas fluorescens Pf0-1中转录调控因子SpfB负调控DNA磷硫酰化修饰（英文）

【目的】DNA磷硫酰化修饰是DNA骨架上非桥接的氧原子以序列选择性和R-构型被硫取代的一种新型DNA修饰。目前,磷硫酰化修饰在多种细菌、古生菌以及人类致病菌中多有发现,但其分子调控机制尚不清楚。为了全面解析磷硫酰化修饰的调控机制,本文选择荧光假单胞菌Pf0-1为研究对象,开展了其DNA磷硫酰化修饰的调控机制研究。【方法】首先,构建了spfB基因缺失和回补菌株,使用碘能特异性断裂磷硫酰化修饰DNA的方法,研究了该基因缺失对修饰表型的影响。利用cDNA在相邻同方向的基因间隔区进行PCR,确定了磷硫酰化修饰基因簇spf BCDE内的共转录单元。通过荧光定量RT-PCR,分析了spfB基因缺失突变株中磷硫酰化修饰基因的转录量。利用异源表达并纯化得到的重组蛋白SpfB进行了体外功能研究。通过EMSA实验,验证了SpfB蛋白具有与spfB启动子序列结合活性。通过DNase I footprinting实验,精确定位了Spf B蛋白与DNA结合序列。【结果】spf B基因的缺失加剧了磷硫酰化修饰DNA断裂所致电泳条带弥散的表型,spf B基因的回补能够恢复该表型,证明spf B基因负调控磷硫酰化修饰。鉴定了spf基因簇中只含有1个共转录单元,且该共转录单元在?spfB突变株中转录水平明显上升。通过EMSA和DNase I footprint实验,检测了SpfB蛋白与磷硫酰化修饰基因spf BCDE的启动子区域5′-TGTTTGT-3′相结合。【结论】SpfB作为转录调控因子负调控磷硫酰化修饰基因spf BCDE的表达,为解析磷硫酰化修饰的调控机制和全面理解基因组上的部分修饰特征奠定了基础。

A LuxR family transcriptional regulator AniF promotes the production of anisomycin and its derivatives in Streptomyces hygrospinosus var.beijingensis

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.

Characterization of the positive SARP family regulator PieR for improving piericidin A1 production in Streptomyces piomogeues var.Hangzhouwanensis

Piericidin A1,a member ofɑ-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.

Enhanced synergy between Cu^(0) and Cu^(+) on nickel doped copper catalyst for gaseous acetic acid hydrogenation

As the substitution of common noble catalysts in the hydrogenation of carboxylic acid,a highly effective Cu-Ni/SiO_(2) catalyst was prepared by a novel stepwise ammonia evaporation method.Its performance in the gasphase hydrogenation of acetic acid was further examined.With the introduction of Ni dopant,more stable Cu^(δ+) sites,which can adsorb more acetic acid,were formed due to the electron transfer from Cu to Ni.This makes more Cu^(0) sites available for hydrogen adsorption,which was suggested as the rate-determining step in acetic acid hydrogenation.A conversion of 99.6% was successfully achieved on this new Cu/SiO_(2)-0.5Ni catalyst,accompanied by the ethanol selectivity of 90%.The incorporation of nickel between copper nanoparticles enhances the synergistic effect between Cu^(0) and Cu^(+).It also helps mitigate the aggregation of copper nanoparticles due to the Ostwald ripening effect induced by acetic acid and enhance the stability of copper catalyst in the conversion of carboxylic acid.

Metabolic engineering of a methyltransferase for production of drug precursors demecycline and demeclocycline in Streptomyces aureofaciens

Demecycline(DMTC)and demeclocycline(DMCTC)are C6-demethylated derivatives of tetracycline(TC)and chlortetracycline(CTC),respectively.They are precursors of minocycline and tigecycline,which showed remarkable bioactivity against TC-resistant bacteria and have been used clinically for decades.In order to biosynthesize drug precursors DMTC and DMCTC,the function of a possible C-methyltransferase encoding gene ctcK was studied systematically in the CTC high-yielding industrial strain Streptomyces aureofaciens F3.TheΔctcK mutant accumulated two new products,which were turned out to be DMTC and DMCTC.Meanwhile,timecourse analysis of the fermentation products detected the epimers of DMTC and DMCTC transformed spontaneously.Finally,an engineering strain with higher productivity of DMCTC was constructed by deleting ctcK and overexpressing ctcP of three extra copies simultaneously.Construction of these two engineering strains not only served as a successful example of synthesizing required products through metabolic engineering,but also provided original strains for following elaborate engineering to synthesize more effective tetracycline derivatives.