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5. Additional file 1 of Identification and characterization of a strong constitutive promoter stnYp for activating biosynthetic genes and producing natural products in streptomyces

Author

Guo, Wenli, Xiao, Zhihong, Huang, Tingting, Zhang, Kai, Pan, Hai-Xue, Tang, Gong-Li, Deng, Zixin, Liang, Rubing, and Lin, Shuangjun

Abstract

Additional file 1: Figure S1. Transcription analyses of xylE in the control of different promoters; Figure S2. Nucleotide sequences of promoter stnYp339; Figure S3. The co-transcription analysis of genes in the ym cluster; Figure S4. Applications of promoter stnYp for aureonuclemycin overproduction in heterologous hosts; Figure S5. Applications of Promoter stnYp for YM-216391 overproduction in heterologous hosts; Figure S6. Transcription analyses of YM-216391 biosynthetic gene cluster; Figure S7. Applications of promoter stnYp for eliminating by-products of tylosin in industrial strains S. fradiae; Table S1. Bacterial strains used in this study; Table S2. Plasmids used in this study; Table S3. Primers used in this study.

Published
2023
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10. Newly Discovered Mechanisms of Antibiotic Self-Resistance with Multiple Enzymes Acting at Different Locations and Stages.

Author

Chen, Xiaorong, Pan, Hai-Xue, and Tang, Gong-Li

Subjects
DRUG discovery, NATURAL products, ANTIBIOTICS, ENZYMES, POISONS
Abstract

Self-resistance determinants are essential for the biosynthesis of bioactive natural products and are closely related to drug resistance in clinical settings. The study of self-resistance mechanisms has long moved forward on the discovery of new resistance genes and the characterization of enzymatic reactions catalyzed by these proteins. However, as more examples of self-resistance have been reported, it has been revealed that the enzymatic reactions contribute to self-protection are not confined to the cellular location where the final toxic compounds are present. In this review, we summarize representative examples of self-resistance mechanisms for bioactive natural products functional at different cell locations to explore the models of resistance strategies involved. Moreover, we also highlight those resistance determinants that are widespread in nature and describe the applications of self-resistance genes in natural product mining to interrogate the landscape of self-resistance genes in drug resistance-related new drug discovery. [ABSTRACT FROM AUTHOR]

Published
2023
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24. Characterization of a Membrane‐Bound O‐Acetyltransferase Involved in Trioxacarcin Biosynthesis Offers Insights into Its Catalytic Mechanism†.

Author

Yin, Yue, Shen, Yi, Meng, Song, Zhang, Mei, Pan, Hai‐Xue, and Tang, Gong‐Li

Subjects
BIOSYNTHESIS, AMINO acid residues, SITE-specific mutagenesis, NATURAL products, ACYLTRANSFERASES
Abstract

Summary of main observation and conclusion: Trioxacarcin A (TXN‐A) is a polycyclic aromatic natural product with remarkable biological activity. TxnB11, a membrane‐bound O‐acetyltransferase involved in TXN‐A biosynthesis that is difficult for protein manipulation, is biochemically characterized here for its acetylation function on C4‐sugar. A proposed catalytic mechanism is supported by transmembrane helix analysis and site‐directed mutagenesis, in which four conserved amino acid residues His35, Ser71, His100 and His317 are essential for enzyme activity. We tested the substrate tolerance of TxnB11 to acyl donors in vitro and found that TxnB11 can also utilize propionyl‐CoA and glycolyl‐CoA, resulting in two new TXN analogs with anti‐tumor activity. This work provides a first understanding of the catalytic mechanism of this unusual acyltransferase family and presents good candidates for creating structural diversity for natural products. [ABSTRACT FROM AUTHOR]

Published
2020
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32. Crystal Structure and Mutagenesis of an XYP Subfamily Cyclodipeptide Synthase Reveal Key Determinants of Enzyme Activity and Substrate Specificity

Author

He, Jun-Bin, Ren, Yichen, Li, Peifeng, Liu, Yi-Pei, Pan, Hai-Xue, Huang, Lin-Juan, Wang, Jiayuan, Fang, Pengfei, and Tang, Gong-Li

Abstract

Cyclodipeptide synthases (CDPSs) catalyze the synthesis of diverse cyclodipeptides (CDPs) by utilizing two aminoacyl-tRNA (aa-tRNA) substrates in a sequential ping-pong reaction mechanism. Numerous CDPSs have been characterized to provide precursors for diketopiperazines (DKPs) with diverse structural characteristics and biological activities. BcmA, belonging to the XYP subfamily, is a cyclo(l-Ile-l-Leu)-synthesizing CDPS involved in the biosynthesis of the antibiotic bicyclomycin. The structural basis and determinants influencing BcmA enzyme activity and substrate selectivity are not well understood. Here, we report the crystal structure of SsBcmA from Streptomyces sapporonensis. Through structural comparison and systematic site-directed mutagenesis, we highlight the significance of key residues located in the aminoacyl-binding pocket for enzyme activity and substrate specificity. In particular, the nonconserved residues D161 and K165 in pocket P2 are essential for the activity of SsBcmA without significant alteration of the substrate specificity, while the conserved residues F158 as well as F210 and S211 in P2 are responsible for determining substrate selectivity. These findings facilitate the understanding of how CDPSs selectively accept hydrophobic substrates and provide additional clues for the engineering of these enzymes for synthetic biology applications.

Published
2024
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44. Refolding and purification of recombinant human interferon-γ expressed as inclusion bodies inEscherichia coliusing size exclusion chromatography

Author

Guan, Yi-Xin, Pan, Hai-Xue, Gao, Yong-Gui, Yao, Shan-Jing, and Cho, Man-Gi

Abstract

A size exclusion chromatography (SEC) process, in the presence of denaturant in the refolding buffer was developed to refold recombinant human interferon-γ (rhIFN-γ) at a high concentration. The rhIFN-γ was overexpressed inE. coli, resulting in the formation of inactive inclusion bodies (IBs). The IBs were first solubilized in 8 M urea as the denaturant, and then the refolding process performed by decreasing the urea concentration on the SEC column to suppress protein aggregation. The effects of the urea concentration, protein loading mode and column height during the refolding step were investigated. The combination of the bufferexchange effect of SEC and a moderate urea concentration in the refolding buffer resulted in an efficient route for producing correctly folded rhIFN-γ, with protein recovery of 67.1% and specific activity up to 1.2×107IU/mg.

Published
2005
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45. Characterization of a Membrane‐Bound O‐Acetyltransferase Involved in Trioxacarcin Biosynthesis Offers Insights into Its Catalytic Mechanism†.

Author

Yin, Yue, Shen, Yi, Meng, Song, Zhang, Mei, Pan, Hai‐Xue, and Tang, Gong‐Li

Subjects
*BIOSYNTHESIS, *AMINO acid residues, *SITE-specific mutagenesis, *NATURAL products, *ACYLTRANSFERASES
Abstract

Summary of main observation and conclusion: Trioxacarcin A (TXN‐A) is a polycyclic aromatic natural product with remarkable biological activity. TxnB11, a membrane‐bound O‐acetyltransferase involved in TXN‐A biosynthesis that is difficult for protein manipulation, is biochemically characterized here for its acetylation function on C4‐sugar. A proposed catalytic mechanism is supported by transmembrane helix analysis and site‐directed mutagenesis, in which four conserved amino acid residues His35, Ser71, His100 and His317 are essential for enzyme activity. We tested the substrate tolerance of TxnB11 to acyl donors in vitro and found that TxnB11 can also utilize propionyl‐CoA and glycolyl‐CoA, resulting in two new TXN analogs with anti‐tumor activity. This work provides a first understanding of the catalytic mechanism of this unusual acyltransferase family and presents good candidates for creating structural diversity for natural products. [ABSTRACT FROM AUTHOR]

Published
2020
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