Co-reporter:Xiangxi Meng;Wenzhao Wang;Zhoujie Xie;Pengwei Li;Yue Li
Science China Life Sciences 2017 Volume 60( Issue 9) pp:980-991
Publication Date(Web):14 August 2017
DOI:10.1007/s11427-017-9120-8
Neomycins are a group of aminoglycoside antibiotics with both clinical and agricultural applications. To elucidate the regulatory mechanism of neomycin biosynthesis, we completed draft genome sequencing of a neomycin producer Streptomyces fradiae CGMCC 4.7387 from marine sediments, and the neomycin biosynthesis gene cluster was identified. Inactivation of the afsA-g gene encoding a γ-butyrolactone (GBL) synthase in S. fradiae CGMCC 4.7387 resulted in a significant decrease of neomycin production. Quantitative RT-PCR analysis revealed that the transcriptional level of neoR and the aphA-neoGH operon were reduced in the afsA-g::aac(3)IV mutant. Interestingly, a conserved binding site of AdpA, a key activator in the GBL regulatory cascade, was discovered upstream of neoR, a putative regulatory gene encoding a protein with an ATPase domain and a tetratricopeptide repeat domain. When neoR was inactivated, the neomycin production was reduced about 40% in comparison with the WT strain. Quantitative RT-PCR analysis revealed that the transcriptional levels of genes in the aphA-neoGH operon were reduced clearly in the neoR::aac(3)IV mutant. Finally, the titers of neomycin were improved considerably by overexpression of afsA-g and neoR in S. fradiae CGMCC 4.7387.
Co-reporter:Zhoujie Xie;Zhao Zhang;Zhenju Cao;Meng Chen
Applied Microbiology and Biotechnology 2017 Volume 101( Issue 9) pp:3811-3820
Publication Date(Web):28 March 2017
DOI:10.1007/s00253-017-8252-2
Since the lacZα-based blue/white screening system was introduced to molecular biology, several different visual reporter systems were developed and used for various purposes in Escherichia coli. A common limit to the existent visual reporter systems is that an extracellular chromogenic substrate has to be added for the visible pigment production. In this study, we developed a new blue/white screening system based on a non-ribosomal peptide synthetase encoded by idgS from Streptomyces and a phosphopantetheinyl transferase encoded by sfp from Bacillus. When IdgS is activated from an apo-form to a holo-form via a posttranslational modification catalyzed by Sfp, it can synthesize a blue pigment indigoidine using L-glutamine, the amino acid abundant in cells, as a substrate. The new blue/white screening system contains a recipient E. coli strain with an optimized idgS gene cassette and a cloning vector harboring an sfp gene with an in-frame insertion of a multiple cloning site close to its N-terminal. We demonstrated that the IdgS/Sfp-based blue/white screening system is a powerful alternative to the lacZα-based screening system, which does not require any external substrate addition.
Co-reporter:Dr. Zhengyan Guo;Dr. Jine Li;Dr. Hua Qin;Dr. Min Wang;Dr. Xun Lv; Xuebing Li; Yihua Chen
Angewandte Chemie International Edition 2015 Volume 54( Issue 17) pp:
Publication Date(Web):
DOI:10.1002/anie.201412190
Abstract
Streptothricins (STNs) are atypical aminoglycosides containing a rare carbamoylated D-gulosamine (D-GulN) moiety, and the antimicrobial activity of STNs has been exploited for crop protection. Herein, the biosynthetic pathway of the carbamoylated D-GulN moiety was delineated. An N-acetyl-D-galactosamine is first attached to the streptolidine lactam by the glycosyltransferse StnG and then epimerized to N-acetyl-D-gulosamine by the putative epimerase StnJ. After carbamoylation by the carbamoyltransferase StnQ, N-acetyl-D-GulN is deacetylated by StnI to furnish the carbamoylated D-GulN moiety. In vitro studies characterized two novel enzymes: StnG is an unprecedented GT-A fold N-glycosyltransferase that glycosylates the imine nitrogen atom of guanidine, and StnI is the first reported N-acetyl-D-GulN deacetylase.
Co-reporter:Dr. Zhengyan Guo;Dr. Jine Li;Dr. Hua Qin;Dr. Min Wang;Dr. Xun Lv; Xuebing Li; Yihua Chen
Angewandte Chemie 2015 Volume 127( Issue 17) pp:
Publication Date(Web):
DOI:10.1002/ange.201412190
Abstract
Streptothricins (STNs) are atypical aminoglycosides containing a rare carbamoylated D-gulosamine (D-GulN) moiety, and the antimicrobial activity of STNs has been exploited for crop protection. Herein, the biosynthetic pathway of the carbamoylated D-GulN moiety was delineated. An N-acetyl-D-galactosamine is first attached to the streptolidine lactam by the glycosyltransferse StnG and then epimerized to N-acetyl-D-gulosamine by the putative epimerase StnJ. After carbamoylation by the carbamoyltransferase StnQ, N-acetyl-D-GulN is deacetylated by StnI to furnish the carbamoylated D-GulN moiety. In vitro studies characterized two novel enzymes: StnG is an unprecedented GT-A fold N-glycosyltransferase that glycosylates the imine nitrogen atom of guanidine, and StnI is the first reported N-acetyl-D-GulN deacetylase.
Co-reporter:Haiyan Chen, Ning Liu, Ying Huang, Yihua Chen
Tetrahedron: Asymmetry 2014 Volume 25(Issue 2) pp:113-116
Publication Date(Web):31 January 2014
DOI:10.1016/j.tetasy.2013.11.013
The anthrabenzoxocinones (ABXs) are hexacyclic aromatic compounds with various bioactivities. Some ABXs with two stereogenic carbon centers have been isolated from different actinomycetes. However, none of them have an absolute configuration assigned, which precludes further studies on the structure–activity relationship and biosynthetic mechanisms of the ABXs. An ABX compound 1.264-C was isolated from Streptomyces sp. FXJ1.264. The absolute configuration of 1.264-C was characterized by X-ray crystallographic, electronic, and experimental circular dichroism (CD) analyses. Based on the reported CD and specific rotation data, the absolute configurations of several other ABXs were also assigned.(6S,16S)-3,11,13,15-Tetrahydroxy-1,6,9,9-tetramethyl-9,16-dihydro-6H-6,16-epoxyanthra[2,3-e]benzo[b]oxocin-14(7H)-oneC27H24O7[α]D23=+375.4 (c 0.013, CH2Cl2)Source of chirality: natural product from Streptomyces sp. FXJ1.264Absolute configuration: (6S,16S)
Co-reporter:JinE Li;ZhengYan Guo;Wei Huang;XiangXi Meng;GuoMin Ai
Science China Life Sciences 2013 Volume 56( Issue 7) pp:619-627
Publication Date(Web):2013 July
DOI:10.1007/s11427-013-4504-2
Streptothricins (STs) are used commercially to treat bacterial and fungal diseases in agriculture. Mining of the sequenced microbial genomes uncovered two cryptic ST clusters from Streptomyces sp. C and Streptomyces sp. TP-A0356. The ST cluster from S. sp. TP-A0356 was verified by successful heterologous expression in Streptomyces coelicolor M145. Two new ST analogs were produced together with streptothricin F and streptothricin D in the heterologous host. The ST cluster was further confirmed by inactivation of gene stnO, which was proposed encoding an aminomutase supplying β-lysines for the poly-β-Lys chain formation. A putative biosynthetic pathway for STs is proposed based on bioinformatics analyses of the ST genes and experimental evidence.
![1-Cyclohexene-1-carboxylicacid,5-[6-O-acetyl-3-O-[2,6-dideoxy-3-O-methyl-4-C-[1-(2-methyl-1-oxobutoxy)ethyl]-a-L-lyxo-hexopyranosyl]-4-O-[(5-methyl-2-thioxo-4-thiazolidinyl)carbonyl]-b-D-allopyranosyl]-2-amino-5-hydroxy-3,6-dioxo-](http://img.cochemist.com/ccimg/118700/118625-41-5.png)
![1-Cyclohexene-1-carboxylicacid,5-[6-O-acetyl-3-O-[2,6-dideoxy-3-O-methyl-4-C-[1-(2-methyl-1-oxobutoxy)ethyl]-a-L-lyxo-hexopyranosyl]-4-O-[(5-methyl-2-thioxo-4-thiazolidinyl)carbonyl]-b-D-allopyranosyl]-2-amino-5-hydroxy-3,6-dioxo-](http://img.cochemist.com/ccimg/118700/118625-41-5_b.png)
![1-Cyclohexene-1-carboxylicacid,5-[6-O-acetyl-3-O-[2,6-dideoxy-3-O-methyl-4-C-[(1S)-1-(1-oxopropoxy)ethyl]-a-L-lyxo-hexopyranosyl]-4-O-[(2Z)-2-isothiocyanato-1-oxo-2-buten-1-yl]-b-D-allopyranosyl]-2-amino-5-hydroxy-3,6-dioxo-,(5S)-](http://img.cochemist.com/ccimg/113700/113603-73-9.png)
![1-Cyclohexene-1-carboxylicacid,5-[6-O-acetyl-3-O-[2,6-dideoxy-3-O-methyl-4-C-[(1S)-1-(1-oxopropoxy)ethyl]-a-L-lyxo-hexopyranosyl]-4-O-[(2Z)-2-isothiocyanato-1-oxo-2-buten-1-yl]-b-D-allopyranosyl]-2-amino-5-hydroxy-3,6-dioxo-,(5S)-](http://img.cochemist.com/ccimg/113700/113603-73-9_b.png)
![L-Cysteine, N-acetyl-,[2-[[2-(acetylamino)-2-carboxyethyl]thio]-1-carboxypropyl]carbamodithioate(ester), 4'-ester with 5-[6-O-acetyl-3-O-[2,6-dideoxy-3-O-methyl-4-C-[1-(2-methyl-1-oxobutoxy)ethyl]-a-L-lyxo-hexopyranosyl]-b-D-allopyranosyl]-2-amino-5-hydroxy-3,6-dioxo-1-cyclohexene-1-carboxylicacid (9CI)](http://img.cochemist.com/ccimg/101500/101411-70-5.png)
![L-Cysteine, N-acetyl-,[2-[[2-(acetylamino)-2-carboxyethyl]thio]-1-carboxypropyl]carbamodithioate(ester), 4'-ester with 5-[6-O-acetyl-3-O-[2,6-dideoxy-3-O-methyl-4-C-[1-(2-methyl-1-oxobutoxy)ethyl]-a-L-lyxo-hexopyranosyl]-b-D-allopyranosyl]-2-amino-5-hydroxy-3,6-dioxo-1-cyclohexene-1-carboxylicacid (9CI)](http://img.cochemist.com/ccimg/101500/101411-70-5_b.png)
![1-Cyclohexene-1-carboxylicacid,5-[6-O-acetyl-3-O-[2,6-dideoxy-3-O-methyl-4-C-[(S)-1-(3-methyl-1-oxobutoxy)ethyl]-a-L-lyxo-hexopyranosyl]-4-O-[(Z)-2-isothiocyanato-1-oxo-2-buten-1-yl]-b-D-allopyranosyl]-2-amino-5-hydroxy-3,6-dioxo-,(5S)-](http://img.cochemist.com/ccimg/101500/101411-68-1.png)
![1-Cyclohexene-1-carboxylicacid,5-[6-O-acetyl-3-O-[2,6-dideoxy-3-O-methyl-4-C-[(S)-1-(3-methyl-1-oxobutoxy)ethyl]-a-L-lyxo-hexopyranosyl]-4-O-[(Z)-2-isothiocyanato-1-oxo-2-buten-1-yl]-b-D-allopyranosyl]-2-amino-5-hydroxy-3,6-dioxo-,(5S)-](http://img.cochemist.com/ccimg/101500/101411-68-1_b.png)
![1-Cyclohexene-1-carboxylicacid, 5-[6-O-acetyl-3-O-[2,6-dideoxy-4-C-(1-hydroxyethyl)-3-O-methyl-a-L-lyxo-hexopyranosyl]-4-O-[(2Z)-2-isothiocyanato-1-oxo-2-buten-1-yl]-b-D-allopyranosyl]-2-amino-5-hydroxy-3,6-dioxo-,(5S)-](http://img.cochemist.com/ccimg/114500/114413-27-3.png)
![1-Cyclohexene-1-carboxylicacid, 5-[6-O-acetyl-3-O-[2,6-dideoxy-4-C-(1-hydroxyethyl)-3-O-methyl-a-L-lyxo-hexopyranosyl]-4-O-[(2Z)-2-isothiocyanato-1-oxo-2-buten-1-yl]-b-D-allopyranosyl]-2-amino-5-hydroxy-3,6-dioxo-,(5S)-](http://img.cochemist.com/ccimg/114500/114413-27-3_b.png)
![1-Cyclohexene-1-carboxylicacid, 5-[6-O-acetyl-3-O-(4-C-acetyl-2,6-dideoxy-3-O-methyl-a-L-lyxo-hexopyranosyl)-4-O-[(Z)-2-isothiocyanato-1-oxo-2-buten-1-yl]-b-D-allopyranosyl]-2-amino-5-hydroxy-3,6-dioxo-,(5S)-](http://img.cochemist.com/ccimg/101500/101411-69-2.png)
![1-Cyclohexene-1-carboxylicacid, 5-[6-O-acetyl-3-O-(4-C-acetyl-2,6-dideoxy-3-O-methyl-a-L-lyxo-hexopyranosyl)-4-O-[(Z)-2-isothiocyanato-1-oxo-2-buten-1-yl]-b-D-allopyranosyl]-2-amino-5-hydroxy-3,6-dioxo-,(5S)-](http://img.cochemist.com/ccimg/101500/101411-69-2_b.png)
