Important role of a LAL regulator StaR in the staurosporine biosynthesis and high-production of Streptomyces fradiae CGMCC 4.576

Staurosporine, belonging to indolocarbazole compounds, is regarded as an excellent lead compound for synthesizing antitumor agents as a potent inhibitor against various protein kinases. In this study, two separate clusters(cluster A and cluster B),corresponding to biosyntheses of K-252 c(staurosporine aglycone) and sugar moiety, were identified in Streptomyces fradiae CGMCC 4.576 and heterologously expressed in Streptomyces coelicolor M1146 separately or together. Sta R, a cluster-situated LAL family regulator, activates staurosporine biosynthesis by binding to the promoter regions of sta O-sta C and sta G-sta N. The conserved sequences GGGGG and GCGCG were found through gradually truncating promoters of sta O and sta G, and further determined by mutational experiments. Overexpression of sta R with the supplementation of 0.01 g L–1 Fe SO4 increased staurosporine production to 5.2-fold compared with that of the parental strain Streptomyces fradiae CGMCC 4.576 in GYM medium. Our results provided an approach for improvement of staurosporine production mediated by a positive regulator and established the basis for dissecting the regulatory mechanisms of other indolocarbazole compounds with clinical application value.     

Tissue-Specific,Development-Dependent Phenolic Compounds Accumulation Profile and Gene Expression Pattern in Tea Plant [Camellia sinensis]

Phenolic compounds in tea plant [Camellia sinensis (L.)] play a crucial role in dominating tea flavor and possess a number of key pharmacological benefits on human health. The present research aimed to study the profile of tissue-specific, development-dependent accumulation pattern of phenolic compounds in tea plant. A total of 50 phenolic compounds were identified qualitatively using liquid chromatography in tandem mass spectrometry technology. Of which 29 phenolic compounds were quantified based on their fragmentation behaviors. Most of the phenolic compounds were higher in the younger leaves than that in the stem and root, whereas the total amount of proanthocyanidins were unexpectedly higher in the root. The expression patterns of 63 structural and regulator genes involved in the shikimic acid, phenylpropanoid, and flavonoid pathways were analyzed by quantitative real-time polymerase chain reaction and cluster analysis. Based on the similarity of their expression patterns, the genes were classified into two main groups: C1 and C2; and the genes in group C1 had high relative expression level in the root or low in the bud and leaves. The expression patterns of genes in C2-2-1 and C2-2-2-1 groups were probably responsible for the development-dependent accumulation of phenolic compounds in the leaves. Enzymatic analysis suggested that the accumulation of catechins was influenced simultaneously by catabolism and anabolism. Further research is recommended to know the expression patterns of various genes and the reason for the variation in contents of different compounds in different growth stages and also in different organs.     

链霉菌发育控制启动子—PTH4对孢子形成的影响

在原核生物发育分化的分子遗传学研究中,链霉菌因其复杂的分化生命周期,无与伦比的合成次级代谢产物的能力（目前世界上所知的数千种天然抗生素的７０％由链霉菌产生）,使之成为原核生物乃至微生物发育与分化研究的最好模式系统［１］,为研究基因时空表达和阐明分化调...     

Establishment of a novel virus-induced virulence effector assay for the identification of virulence effectors of plant pathogens using a PVX-based expression vector

Plant pathogens deliver virulence effectors into plant cells to modulate plant immunity and facilitate infection. Although species-specific virulence effector screening approaches have been developed for several pathogens, these assays do not apply to pathogens that cannot be cultured and/or transformed outside of their hosts. Here, we established a rapid and parallel screening assay, called the virus-induced virulence effector (VIVE) assay, to identify putative effectors in various plant pathogens, including unculturable pathogens, using a virus-based expression vector. The VIVE assay uses the potato virus X (PVX) vector to transiently express candidate effector genes of various bacterial and fungal pathogens into Nicotiana benthamiana leaves. Using the VIVE assay, we successfully identified Avh148 as a potential virulence effector of Phytophthora sojae. Plants infected with PVX carrying Avh148 showed strong viral symptoms and high-level Avh148 and viral RNA accumulation. Analysis of P. sojae Avh148 deletion mutants and soybean hairy roots overexpressing Avh148 revealed that Avh148 is required for full pathogen virulence. In addition, the VIVE assay was optimized in N. benthamiana plants at different developmental stages across a range of Agrobacterium cell densities. Overall, we identified six novel virulence effectors from seven pathogens, thus demonstrating the broad effectiveness of the VIVE assay in plant pathology research.     

A widespread response of Gram-negative bacterial acyl-homoserine lactone receptors to Gram-positive Streptomyces γ-butyrolactone signaling molecules

Cell-cell communication is critical for bacterial survival in natural habitats, in which miscellaneous regulatory networks are encompassed. However, elucidating the interaction networks of a microbial community has been hindered by the population complexity. This study reveals that γ-butyrolactone(GBL) molecules from Streptomyces species, the major antibiotic producers,can directly bind to the acyl-homoserine lactone(AHL) receptor of Chromobacterium violaceum and influence violacein production controlled by the quorum sensing(QS) system. Subsequently, the widespread responses of more Gram-negative bacterial AHL receptors to Gram-positive Streptomyces signaling molecules are unveiled. Based on the cross-talk between GBL and AHL signaling systems, combinatorial regulatory circuits(CRC) are designed and proved to be workable in Escherichia coli(E. coli). It is significant that the QS systems of Gram-positive and Gram-negative bacteria can be bridged via native Streptomyces signaling molecules. These findings pave a new path for unlocking the comprehensive cell-cell communications in microbial communities and facilitate the exploitation of innovative regulatory elements for synthetic biology.     

Reconstitution of a mini-gene cluster combined with ribosome engineering led to effective enhancement of salinomycin production in Streptomyces albus

Salinomycin, an FDA-approved polyketide drug, was recently identified as a promising anti-tumour and anti-viral lead compound. It is produced by Streptomyces albus, and the biosynthetic gene cluster (sal) spans over 100 kb. The genetic manipulation of large polyketide gene clusters is challenging, and approaches delivering reliable efficiency and accuracy are desired. Herein, a delicate strategy to enhance salinomycin production was devised and evaluated. We reconstructed a minimized sal gene cluster (mini-cluster) on pSET152 including key genes responsible for tailoring modification, antibiotic resistance, positive regulation and precursor supply. These genes were overexpressed under the control of constitutive promoter P_kasO* or P_neo. The pks operon was not included in the mini-cluster, but it was upregulated by SalJ activation. After the plasmid pSET152::mini-cluster was introduced into the wild-type strain and a chassis host strain obtained by ribosome engineering, salinomycin production was increased to 2.3-fold and 5.1-fold compared with that of the wild-type strain respectively. Intriguingly, mini-cluster introduction resulted in much higher production than overexpression of the whole sal gene cluster. The findings demonstrated that reconstitution of sal mini-cluster combined with ribosome engineering is an efficient novel approach and may be extended to other large polyketide biosynthesis.     

燕麦盐胁迫响应基因的差异表达与生理响应的关系

以耐盐燕麦品种VAO-9为材料,通过Illumina测序与数字基因表达谱技术对300mmol/L NaCl处理前后的叶片cDNA文库进行RNA-Seq与DGE分析,同时测定0(CK)、100、200、250、300mmol/L NaCl胁迫下VAO-9幼苗叶片的相对电导率、丙二醛含量和脯氨酸含量,探讨燕麦盐胁迫响应基因的差异表达与生理响应的关系。结果表明:(1)RNA-Seq分析得到Unigenes 65 801条,其基因表达呈现高度的不均一性和冗余性;若差异基因表达谱鉴定分析以log2Ratio≥2且FDR值≤0.001为选择标准,则发现上调和下调表达基因在胁迫0.5h时分别有306和64个,在胁迫3h时分别有639和290个,胁迫24h时分别有1 488和882个。(2)KEGG代谢分析显示,有23 652条Unigenes比对到KEGG中的128条代谢途径,包括与逆境胁迫相关的植物激素信号转导途径、ABC转运蛋白途径、肌醇磷酸代谢途径、渗透调节途径等。(3)在300mmol/L NaCl处理下燕麦叶片的相对电导率、丙二醛含量、脯氨酸含量等生理指标的变化与相关差异表达基因的变化趋势基本一致,说明基因差异表达量与生理反应密切相关。研究认为,在相同的栽培及胁迫处理条件下,可根据植物盐响应生理指标的变化判断耐盐基因的表达情况。     

Hybrid antibiotics with the nikkomycin nucleoside and polyoxin peptidyl moieties

Acting as competitive inhibitors of chitin synthase, nikkomycins and polyoxins are potent antibiotics against pathogenic fungi. Taking advantage of the structural similarities between these two peptidyl nucleoside antibiotics, genes required for the biosynthesis of the dipeptidyl moiety of polyoxin from Streptomyces cacaoi were introduced into a Streptomyces ansochromogenes mutant producing the nucleoside moiety of nikkomycin X. Two hybrid antibiotics were generated. One of them was identified as polyoxin N, and the other, a novel compound, was named polynik A. The hybrid antibiotics exhibited merits from both parents: they had better inhibitory activity against phytopathogenic fungi than polyoxin B, and were more stable under different pH and temperature conditions than nikkomycin X. This study demonstrates the use of the combinatorial biosynthetic approach to produce valuable and novel hybrid antibiotics with improved properties.