176 results on '"Shuangjun Lin"'
Search Results
2. A three-level regulatory mechanism of the aldo-keto reductase subfamily AKR12D
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Zhihong Xiao, Jinyin Zha, Xu Yang, Tingting Huang, Shuxin Huang, Qi Liu, Xiaozheng Wang, Jie Zhong, Jianting Zheng, Rubing Liang, Zixin Deng, Jian Zhang, Shuangjun Lin, and Shaobo Dai
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Science - Abstract
Abstract Modulation of protein function through allosteric regulation is central in biology, but biomacromolecular systems involving multiple subunits and ligands may exhibit complex regulatory mechanisms at different levels, which remain poorly understood. Here, we discover an aldo-keto reductase termed AKRtyl and present its three-level regulatory mechanism. Specifically, by combining steady-state and transient kinetics, X-ray crystallography and molecular dynamics simulation, we demonstrate that AKRtyl exhibits a positive synergy mediated by an unusual Monod-Wyman-Changeux (MWC) paradigm of allosteric regulation at low concentrations of the cofactor NADPH, but an inhibitory effect at high concentrations is observed. While the substrate tylosin binds at a remote allosteric site with positive cooperativity. We further reveal that these regulatory mechanisms are conserved in AKR12D subfamily, and that substrate cooperativity is common in AKRs across three kingdoms of life. This work provides an intriguing example for understanding complex allosteric regulatory networks.
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- 2024
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3. O-methyltransferase-like enzyme catalyzed diazo installation in polyketide biosynthesis
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Yuchun Zhao, Xiangyang Liu, Zhihong Xiao, Jie Zhou, Xingyu Song, Xiaozheng Wang, Lijun Hu, Ying Wang, Peng Sun, Wenning Wang, Xinyi He, Shuangjun Lin, Zixin Deng, Lifeng Pan, and Ming Jiang
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Science - Abstract
Abstract Diazo compounds are rare natural products possessing various biological activities. Kinamycin and lomaiviticin, two diazo natural products featured by the diazobenzofluorene core, exhibit exceptional potency as chemotherapeutic agents. Despite the extensive studies on their biosynthetic gene clusters and the assembly of their polyketide scaffolds, the formation of the characteristic diazo group remains elusive. l-Glutamylhydrazine was recently shown to be the hydrazine donor in kinamycin biosynthesis, however, the mechanism for the installation of the hydrazine group onto the kinamycin scaffold is still unclear. Here we describe an O-methyltransferase-like protein, AlpH, which is responsible for the hydrazine incorporation in kinamycin biosynthesis. AlpH catalyses a unique SAM-independent coupling of l-glutamylhydrazine and polyketide intermediate via a rare Mannich reaction in polyketide biosynthesis. Our discovery expands the catalytic diversity of O-methyltransferase-like enzymes and lays a strong foundation for the discovery and development of novel diazo natural products through genome mining and synthetic biology.
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- 2023
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4. Enhancing tylosin production by combinatorial overexpression of efflux, SAM biosynthesis, and regulatory genes in hyperproducing Streptomyces xinghaiensis strain
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Penghui Dai, Yuyao Qin, Luyuan Li, Haidi Li, Lihuo Lv, Danying Xu, Yuqing Song, Tingting Huang, Shuangjun Lin, Zixin Deng, and Meifeng Tao
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Streptomyces xinghaiensis ,Tylosin ,Combinational metabolic engineering ,Biotechnology ,TP248.13-248.65 ,Biology (General) ,QH301-705.5 - Abstract
Tylosin is a 16-membered macrolide antibiotic widely used in veterinary medicine to control infections caused by Gram-positive pathogens and mycoplasmas. To improve the fermentation titer of tylosin in the hyperproducing Streptomyces xinghaiensis strain TL01, we sequenced its whole genome and identified the biosynthetic gene cluster therein. Overexpression of the tylosin efflux gene tlrC, the cluster-situated S-adenosyl methionine (SAM) synthetase gene metKcs, the SAM biosynthetic genes adoKcs-metFcs, or the pathway-specific activator gene tylR enhanced tylosin production by 18%, 12%, 11%, and 11% in the respective engineered strains TLPH08-2, TLPH09, TLPH10, and TLPH12. Co-overexpression of metKcs and adoKcs-metFcs as two transcripts increased tylosin production by 22% in the resultant strain TLPH11 compared to that in TL01. Furthermore, combinational overexpression of tlrC, metKcs, adoKcs-metFcs, and tylR as four transcripts increased tylosin production by 23% (10.93g/L) in the resultant strain TLPH17 compared to that in TL01. However, a negligible additive effect was displayed upon combinational overexpression in TLPH17 as suggested by the limited increment of fermentation titer compared to that in TLPH08-2. Transcription analyses indicated that the expression of tlrC and three SAM biosynthetic genes in TLPH17 was considerably lower than that of TLPH08-2 and TLPH11. Based on this observation, the five genes were rearranged into one or two operons to coordinate their overexpression, yielding two engineered strains TLPH23 and TLPH24, and leading to further enhancement of tylosin production over TLPH17. In particular, the production of TLPH23 reached 11.35 g/L. These findings indicated that the combinatorial strategy is a promising approach for enhancing tylosin production in high-yielding industrial strains.
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- 2023
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5. Insertion sequence transposition inactivates CRISPR-Cas immunity
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Yong Sheng, Hengyu Wang, Yixin Ou, Yingying Wu, Wei Ding, Meifeng Tao, Shuangjun Lin, Zixin Deng, Linquan Bai, and Qianjin Kang
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Science - Abstract
Abstract CRISPR-Cas immunity systems safeguard prokaryotic genomes by inhibiting the invasion of mobile genetic elements. Here, we screened prokaryotic genomic sequences and identified multiple natural transpositions of insertion sequences (ISs) into cas genes, thus inactivating CRISPR-Cas defenses. We then generated an IS-trapping system, using Escherichia coli strains with various ISs and an inducible cas nuclease, to monitor IS insertions into cas genes following the induction of double-strand DNA breakage as a physiological host stress. We identified multiple events mediated by different ISs, especially IS1 and IS10, displaying substantial relaxed target specificity. IS transposition into cas was maintained in the presence of DNA repair machinery, and transposition into other host defense systems was also detected. Our findings highlight the potential of ISs to counter CRISPR activity, thus increasing bacterial susceptibility to foreign DNA invasion.
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- 2023
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6. Identification and characterization of a strong constitutive promoter stnYp for activating biosynthetic genes and producing natural products in streptomyces
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Wenli Guo, Zhihong Xiao, Tingting Huang, Kai Zhang, Hai-Xue Pan, Gong-Li Tang, Zixin Deng, Rubing Liang, and Shuangjun Lin
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Natural products ,Streptomyces ,Constitutive promoter ,stnYp ,Heterologous expression ,Biosynthetic gene cluster ,Microbiology ,QR1-502 - Abstract
Abstract Background Streptomyces are well known for their potential to produce various pharmaceutically active compounds, the commercial development of which is often limited by the low productivity and purity of the desired compounds expressed by natural producers. Well-characterized promoters are crucial for driving the expression of target genes and improving the production of metabolites of interest. Results A strong constitutive promoter, stnYp, was identified in Streptomyces flocculus CGMCC4.1223 and was characterized by its effective activation of silent biosynthetic genes and high efficiency of heterologous gene expression. The promoter stnYp showed the highest activity in model strains of four Streptomyces species compared with the three frequently used constitutive promoters ermEp*, kasOp*, and SP44. The promoter stnYp could efficiently activate the indigoidine biosynthetic gene cluster in S. albus J1074, which is thought to be silent under routine laboratory conditions. Moreover, stnYp was found suitable for heterologous gene expression in different Streptomyces hosts. Compared with the promoters ermEp*, kasOp*, and SP44, stnYp conferred the highest production level of diverse metabolites in various heterologous hosts, including the agricultural-bactericide aureonuclemycin and the antitumor compound YM-216391, with an approximately 1.4 − 11.6-fold enhancement of the yields. Furthermore, the purity of tylosin A was greatly improved by overexpressing rate-limiting genes through stnYp in the industrial strain. Further, the yield of tylosin A was significantly elevated to 10.30 ± 0.12 g/L, approximately 1.7-fold higher than that of the original strain. Conclusions The promoter stnYp is a reliable, well-defined promoter with strong activity and broad suitability. The findings of this study can expand promoter diversity, facilitate genetic manipulation, and promote metabolic engineering in multiple Streptomyces species.
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- 2023
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7. C–N bond formation by a polyketide synthase
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Jialiang Wang, Xiaojie Wang, Xixi Li, LiangLiang Kong, Zeqian Du, Dandan Li, Lixia Gou, Hao Wu, Wei Cao, Xiaozheng Wang, Shuangjun Lin, Ting Shi, Zixin Deng, Zhijun Wang, and Jingdan Liang
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Science - Abstract
Abstract Assembly-line polyketide synthases (PKSs) are molecular factories that produce diverse metabolites with wide-ranging biological activities. PKSs usually work by constructing and modifying the polyketide backbone successively. Here, we present the cryo-EM structure of CalA3, a chain release PKS module without an ACP domain, and its structures with amidation or hydrolysis products. The domain organization reveals a unique “∞”-shaped dimeric architecture with five connected domains. The catalytic region tightly contacts the structural region, resulting in two stabilized chambers with nearly perfect symmetry while the N-terminal docking domain is flexible. The structures of the ketosynthase (KS) domain illustrate how the conserved key residues that canonically catalyze C–C bond formation can be tweaked to mediate C–N bond formation, revealing the engineering adaptability of assembly-line polyketide synthases for the production of novel pharmaceutical agents.
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- 2023
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8. The natural pyrazolotriazine pseudoiodinine from Pseudomonas mosselii 923 inhibits plant bacterial and fungal pathogens
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Ruihuan Yang, Qing Shi, Tingting Huang, Yichao Yan, Shengzhang Li, Yuan Fang, Ying Li, Linlin Liu, Longyu Liu, Xiaozheng Wang, Yongzheng Peng, Jiangbo Fan, Lifang Zou, Shuangjun Lin, and Gongyou Chen
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Science - Abstract
Natural antimicrobial metabolites produced by soil microorganisms can be used as green pesticides. Here, the authors isolated a Pseudomonas mosselii strain 923 from rice rhizosphere soils and identify the compound pyrazolotriazine pseudoiodinine inhibits the growth of plant bacterial and fungal pathogens.
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- 2023
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9. Catechol siderophores framed on 2,3-dihydroxybenzoyl-L-serine from Streptomyces varsoviensis
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Zhixiang Liu, Tingting Huang, Qing Shi, Zixin Deng, and Shuangjun Lin
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catecholate ,siderophore ,enterobactin ,streptomyces ,culture media ,NRPS ,Microbiology ,QR1-502 - Abstract
Enterobactin is an archetypical catecholate siderophore that plays a key role in the acquisition of ferric iron by microorganisms. Catechol moieties have been shown to be promising siderophore cores. Variants of the conserved 2,3-dihydroxybenzoate (DHB) moiety with structural modifications expand the bioactivity. Streptomyces are characterized by metabolites with diverse structures. The genomic sequence of Streptomyces varsoviensis indicated that it possessed a biosynthetic gene cluster for DHB containing siderophores and metabolic profiling revealed metabolites correlated with catechol-type natural products. Here, we report the discovery of a series of catecholate siderophores produced by S. varsoviensis and a scale-up fermentation was performed to purify these compounds for structural elucidation. A biosynthetic route for the catecholate siderophores is also proposed. These new structural features enrich the structural diversity of the enterobactin family compounds. One of the new linear enterobactin congeners shows moderate activity against a food-borne pathogen Listeria monocytogenes. This work demonstrated that changing culture conditions is still a promising approach to explore unexplored chemical diversity. The availability of the biosynthetic machinery will enrich the genetic toolbox of catechol siderophores and facilitate such engineering efforts.
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- 2023
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10. The Streptomyces viridochromogenes product template domain represents an evolutionary intermediate between dehydratase and aldol cyclase of type I polyketide synthases
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Yuanyuan Feng, Xu Yang, Huining Ji, Zixin Deng, Shuangjun Lin, and Jianting Zheng
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Biology (General) ,QH301-705.5 - Abstract
Structural analyses of a Streptomyces viridochromogenes product template (PT) domain suggests molecular and functional similarities with known fungal PTs involved in polyketide synthase activity.
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- 2022
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11. Intramolecular chaperone-mediated secretion of an Rhs effector toxin by a type VI secretion system
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Tong-Tong Pei, Hao Li, Xiaoye Liang, Zeng-Hang Wang, Guangfeng Liu, Li-Li Wu, Haeun Kim, Zhiping Xie, Ming Yu, Shuangjun Lin, Ping Xu, and Tao G. Dong
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Science - Abstract
Bacterial Rhs proteins with toxic domains are often secreted by type VI secretion systems. Here, the authors show that one of these proteins self-cleaves into three fragments, with the Rhs core and the N-terminal domain facilitating secretion and function of the C-terminal toxic domain.
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- 2020
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12. Biosynthesis of squalene-type triterpenoids in Saccharomyces cerevisiae by expression of CYP505D13 from Ganoderma lucidum
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Xin Song, Han Xiao, Shangwen Luo, Xiaozheng Wang, Wenfang Wang, and Shuangjun Lin
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Cytochrome P450s (CYPs) ,Squalene-type triterpenoids (STs) ,Saccharomyces cerevisiae ,Ganoderma lucidum ,Synthetic biology ,Technology ,Chemical technology ,TP1-1185 ,Biotechnology ,TP248.13-248.65 - Abstract
Abstract Background Squalene-type triterpenoids (STs) are a class of linearized triterpenoids with significant bioactivities, including anti-cancer, anti-oxidative, and anti-inflammatory activities. The efficient biosynthesis of STs has gained increasing attention. Results Using Saccharomyces cerevisiae as a heterologous host, we discovered that overexpression of CYP505D13 from Ganoderma lucidum, a famous medicinal mushroom capable of producing various triterpenoids as secondary metabolites, enables the engineered S. cerevisiae strain to produce two new STs, 4,8-dihydroxy-22,23-oxidosqualene (ST-1), 8-hydroxy-2,3;22,23-squalene dioxide (ST-2), and a known ST, 2,3; 22,23-squalene dioxide (ST-3), at the respective titers of 3.28 mg/L, 13.77 mg/L, and 12.23 mg/L after 59 h fermentation. Furthermore, our in vitro enzymatic assay implies that CYP505D13 is involved in the formation of ST-3. Conclusions This study provides a promising alternative to discover STs and facilitate their efficient bioproduction.
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- 2019
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13. Characterization of the Tellurite-Resistance Properties and Identification of the Core Function Genes for Tellurite Resistance in Pseudomonas citronellolis SJTE-3
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Wanli Peng, Yanqiu Wang, Yali Fu, Zixin Deng, Shuangjun Lin, and Rubing Liang
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tellurite resistance ,terZABCDE gene cluster ,core function gene ,terC gene ,terD gene ,terA gene ,Biology (General) ,QH301-705.5 - Abstract
Tellurite is highly toxic to bacteria and commonly used in the clinical screening for pathogens; it is speculated that there is a potential relationship between tellurite resistance and bacterial pathogenicity. Until now, the core function genes of tellurite resistance and their characteristics are still obscure. Pseudomonas citronellolis SJTE-3 was found able to resist high concentrations of tellurite (250 μg/mL) and formed vacuole-like tellurium nanostructures. The terZABCDE gene cluster located in the large plasmid pRBL16 endowed strain SJTE-3 with the tellurite resistance of high levels. Although the terC and terD genes were identified as the core function genes for tellurite reduction and resistance, the inhibition of cell growth was observed when they were used solely. Interestingly, co-expression of the terA gene or terZ gene could relieve the burden caused by the expression of the terCD genes and recover normal cell growth. TerC and TerD proteins commonly shared the conserved sequences and are widely distributed in many pathogenic bacteria, highly associated with the pathogenicity factors.
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- 2022
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14. Naphthoquinone-Based Meroterpenoids from Marine-Derived Streptomyces sp. B9173
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Xinqian Shen, Xiaozheng Wang, Tingting Huang, Zixin Deng, and Shuangjun Lin
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naphthoquinone ,meroterpenoid ,flaviogeranin family ,natural products ,Microbiology ,QR1-502 - Abstract
Naphthoquinone-based meroterpenoids are hybrid polyketide-terpenoid natural products with chemical diversity and a broad range of biological activities. Here, we report the isolation of a group of naphthoquinone-containing compounds from Streptomyces sp. B9173, and their structures were elucidated by using a combination of spectroscopic techniques, including 1D, 2D NMR, and high-resolution mass (HRMS) analysis. Seven flaviogeranin congeners or intermediates, three of which were new, have been derived from common naphthoquinone backbone and subsequent oxidation, methylation, prenylation, and amino group incorporation. Both flaviogeranin B1 (1) and B (2) contain an amino group which was incorporated into the C8 of 1,3,6,8-terhydroxynaphthalene (THN). Flaviogeranin D (3) contains an intact C-geranylgeranyl residue attached to the C2 of THN, while the O-geranylgeranyl group of 2 links with the hydroxyl on the C2 site of THN. Four compounds were selected and tested for antibacterial activity and cytotoxicity, with 3 and flaviogeranin C2 (5) displaying potent activity against selected bacteria and cancer cell lines. In light of the structure features of isolated compounds and the biosynthetic genes, a biosynthetic pathway of naphthoquinone-based flaviogeranins has been proposed. These isolated compounds not only extend the structural diversity but also represent new insights into the biosynthesis of naphthoquinone-based meroterpenoids.
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- 2020
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15. Characterization of the Phenanthrene-Degrading Sphingobium yanoikuyae SJTF8 in Heavy Metal Co-Existing Liquid Medium and Analysis of Its Metabolic Pathway
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Chong Yin, Weiliang Xiong, Hua Qiu, Wanli Peng, Zixin Deng, Shuangjun Lin, and Rubing Liang
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polycyclic aromatic hydrocarbons ,Sphingobium yanoikuyae SJTF8 ,phenanthrene ,heavy metal ,bioremediation ,tolerance ,Biology (General) ,QH301-705.5 - Abstract
Polycyclic aromatic hydrocarbons (PAHs) are common organic pollutants with great carcinogenic threaten, and metal/PAH-contaminated environments represent one of the most difficult remedial challenges. In this work, Sphingobium yanoikuyae SJTF8 was isolated and identified with great and stable PAH-degrading efficiency even under stress conditions. It could utilize typical PAHs (naphthalene, phenanthrene, and anthracene) and heterocyclic and halogenated aromatic compounds (dibenzothiophene and 9-bromophenanthrene) as the sole carbon source. It could degrade over 98% of 500 mg/L phenanthrene in 4 days, and the cis-3,4-dihydrophenanthrene-3,4-diol was the first-step intermediate. Notably, strain SJTF8 showed great tolerance to heavy metals and acidic pH. Supplements of 0.30 mM of Cu2+, 1.15 mM of Zn2+, and 0.01 mM of Cd2+ had little effect on its cell growth and phenanthrene degradation; phenanthrene of 250 mg/L could still be degraded completely in 48 h. Further, the whole genome sequence of S. yanoikuyae SJTF8 was obtained, and three plasmids were found. The potential genes participating in stress-tolerance and PAH-degradation were annotated and were found mostly distributed in plasmids 1 and 2. Elimination of plasmid 2 resulted in the loss of the PAH-degradation ability. On the basis of genome mining results, the possible degrading pathway and the metabolites of S. yanoikuyae SJTF8 to phenanthrene were predicted.
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- 2020
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16. Publisher Correction: Intramolecular chaperone-mediated secretion of an Rhs effector toxin by a type VI secretion system
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Tong-Tong Pei, Hao Li, Xiaoye Liang, Zeng-Hang Wang, Guangfeng Liu, Li-Li Wu, Haeun Kim, Zhiping Xie, Ming Yu, Shuangjun Lin, Ping Xu, and Tao G. Dong
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Science - Abstract
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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- 2020
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17. Structural insight into the tetramerization of an iterative ketoreductase siam through aromatic residues in the interfaces.
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Hua Wang, Huaidong Zhang, Yi Zou, Yanling Mi, Shuangjun Lin, Zhixiong Xie, Yunjun Yan, and Houjin Zhang
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Medicine ,Science - Abstract
In the biosynthesis of polyketides, ketoreductases (KRs) are an important group of enzymes that determine the chiralities of the carbon backbones. SiaM is a special member of this group that can recognize substrates with different lengths and can be used iteratively. Here we report the crystal structure of SiaM. Structural analysis indicates that the overall structure resembles those of other KRs. However, significant disparity can be found in the conserved LDD motif that is replaced with IRD motif in SiaM. The isoleucine and aspartic acid residues take similar orientations as leucine and aspartic acid in the conserved LDD motif, while the arginine residue points out towards the solvent. PISA analysis shows that SiaM forms a tetramer. Several aromatic residues are found in the interfaces, which have aromatic stacking interactions with the aromatic residues in the neighboring protomers. Mutagenesis studies performed on the aromatic residues show that these sites are important for maintaining the structural integrity of SiaM. However, the aromatic residues contribute differently to the enzymatic activity. In the N-terminal interface, the aromatic residues can be replaced with leucine without affecting the enzymatic activity while, in the other interface, such mutations abolish the enzymatic activity.
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- 2014
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18. Discovery and characterization of BlsE, a radical S-adenosyl-L-methionine decarboxylase involved in the blasticidin S biosynthetic pathway.
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Jun Feng, Jun Wu, Nan Dai, Shuangjun Lin, H Howard Xu, Zixin Deng, and Xinyi He
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Medicine ,Science - Abstract
BlsE, a predicted radical S-adenosyl-L-methionine (SAM) protein, was anaerobically purified and reconstituted in vitro to study its function in the blasticidin S biosynthetic pathway. The putative role of BlsE was elucidated based on bioinformatics analysis, genetic inactivation and biochemical characterization. Biochemical results showed that BlsE is a SAM-dependent radical enzyme that utilizes cytosylglucuronic acid, the accumulated intermediate metabolite in blsE mutant, as substrate and catalyzes decarboxylation at the C5 position of the glucoside residue to yield cytosylarabinopyranose. Additionally, we report the purification and reconstitution of BlsE, characterization of its [4Fe-4S] cluster using UV-vis and electron paramagnetic resonance (EPR) spectroscopic analysis, and investigation of the ability of flavodoxin (Fld), flavodoxin reductase (Fpr) and NADPH to reduce the [4Fe-4S](2+) cluster. Mutagenesis studies demonstrated that Cys31, Cys35, Cys38 in the C×××C×MC motif and Gly73, Gly74, Glu75, Pro76 in the GGEP motif were crucial amino acids for BlsE activity while mutation of Met37 had little effect on its function. Our results indicate that BlsE represents a typical [4Fe-4S]-containing radical SAM enzyme and it catalyzes decarboxylation in blasticidin S biosynthesis.
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- 2013
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19. A new β-carboline alkaloid from the Streptomyces flocculus CGMCC4.1223 mutant ΔstnK4
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Xiaozheng, Wang, Jing, Wo, Fei, Xu, Xinyue, Xie, Tingting, Huang, and Shuangjun, Lin
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- 2023
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20. Bifunctional NadC Homologue PyrZ Catalyzes Nicotinic Acid Formation in Pyridomycin Biosynthesis
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Zihua Zhou, Xu Yang, Tingting Huang, Jianting Zheng, Zixin Deng, Shaobo Dai, and Shuangjun Lin
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Molecular Medicine ,General Medicine ,Biochemistry - Abstract
Pyridomycin is a potent antimycobacterial natural product by specifically inhibiting InhA, a clinically validated antituberculosis drug discovery target. Pyridyl moieties of pyridomycin play an essential role in inhibiting InhA by occupying the reduced form of the nicotinamide adenine dinucleotide (NADH) cofactor binding site. Herein, we biochemically characterize PyrZ that is a multifunctional NadC homologue and catalyzes the successive formation, dephosphorylation, and ribose hydrolysis of nicotinic acid mononucleotide (NAMN) to generate nicotinic acid (NA), a biosynthetic precursor for the pyridyl moiety of pyridomycin. Crystal structures of PyrZ in complex with substrate quinolinic acid (QA) and the final product NA revealed a specific salt bridge formed between K184 and the C3-carboxyl group of QA. This interaction positions QA for accepting the phosphoribosyl group to generate NAMN, retains NAMN within the active site, and mediates its translocation to nucleophile D296 for dephosphorylation. Combining kinetic and thermodynamic analysis with site-directed mutagenesis, the catalytic mechanism of PyrZ dephosphorylation was proposed. Our study discovered an alternative and concise NA biosynthetic pathway involving a unique multifunctional enzyme.
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- 2022
21. Flavoprotein StnP2 Catalyzes the β-Carboline Formation during the Streptonigrin Biosynthesis
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Xiaozheng Wang, Dekun Kong, Tingting Huang, Fei Xu, Man-Cheng Tang, Zixin Deng, and Shuangjun Lin
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Molecular Medicine ,General Medicine ,Biochemistry - Abstract
β-Carboline (βC) alkaloids constitute a large family of indole alkaloids that exhibit diverse pharmacological properties, such as antitumor, antiviral, antiparasitic, and antimicrobial activities. Here, we report that a flavoprotein StnP2 catalyzes the dehydrogenation at C1-N2 of a tetrahydro-β-carboline (THβC) generating a 3,4-dihydro-β-carboline (DHβC), and the DHβC subsequently undergoes a spontaneous dehydrogenation to βC formation involved in the biosynthesis of the antitumor agent streptonigrin. Biochemical characterization showed that StnP2 catalyzed the highly regio- and stereo-selective dehydrogenation, and StnP2 exhibits promiscuity toward different THβCs. This study provides an alternative kind of enzyme catalyzing the biosynthesis of βC alkaloids and enhances the importance of flavoproteins.
- Published
- 2022
22. Structural basis of Streptomyces transcription activation by zinc uptake regulator
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Xu Yang, Yiqun Wang, Guiyang Liu, Zixin Deng, Shuangjun Lin, and Jianting Zheng
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DNA, Bacterial ,DNA-Binding Proteins ,Transcriptional Activation ,Zinc ,Bacterial Proteins ,Genetics ,Nucleic Acid Conformation ,Streptomyces coelicolor ,DNA-Directed RNA Polymerases ,Gene Expression Regulation, Bacterial ,Promoter Regions, Genetic - Abstract
Streptomyces coelicolor (Sc) is a model organism of actinobacteria to study morphological differentiation and production of bioactive metabolites. Sc zinc uptake regulator (Zur) affects both processes by controlling zinc homeostasis. It activates transcription by binding to palindromic Zur-box sequences upstream of −35 elements. Here we deciphered the molecular mechanism by which ScZur interacts with promoter DNA and Sc RNA polymerase (RNAP) by cryo-EM structures and biochemical assays. The ScZur-DNA structures reveal a sequential and cooperative binding of three ScZur dimers surrounding a Zur-box spaced 8 nt upstream from a −35 element. The ScRNAPσHrdB-Zur-DNA structures define protein-protein and protein-DNA interactions involved in the principal housekeeping σHrdB-dependent transcription initiation from a noncanonical promoter with a −10 element lacking the critical adenine residue at position −11 and a TTGCCC −35 element deviating from the canonical TTGACA motif. ScZur interacts with the C-terminal domain of ScRNAP α subunit (αCTD) in a complex structure trapped in an active conformation. Key ScZur-αCTD interfacial residues accounting for ScZur-dependent transcription activation were confirmed by mutational studies. Together, our structural and biochemical results provide a comprehensive model for transcription activation of Zur family regulators.
- Published
- 2022
23. Chirality-influenced antibacterial activity of methylthiazole- and thiadiazole-based supramolecular biocompatible hydrogels
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Sravan Baddi, Auphedeous Y. Dang-i, Tingting Huang, Chao Xing, Shuangjun Lin, and Chuan-Liang Feng
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Biomaterials ,Phenylalanine ,Thiadiazoles ,Nanofibers ,Biomedical Engineering ,Hydrogels ,General Medicine ,Molecular Biology ,Biochemistry ,Anti-Bacterial Agents ,Biotechnology - Abstract
Chiral stereochemistry is a unique and fundamental strategy that determines the interaction of bacteria cells with chiral biomolecules and stereochemical surfaces. The interaction between bacteria and material surface (molecular chirality or supramolecular chirality) plays a significant role in modulating antibacterial performance. Herein, we developed inherent chiral antibacterial hydrogels by modifying the carboxyl groups of our previously reported supramolecular gelator (LPF-left handed phenylalanine gelator and DPF- right handed phenylalanine gelator) with 2-amino-5-methylthiazole (MTZ) and 5-amino-1,3,4-thiadiazole-2- thiol (TDZ). The new L/D-gelator molecules initiate self-assembly to form hydrogels through non-covalent interactions (Hydrogen bonding and π-π interactions) verified by FTIR and CD spectroscopy. Morphological studies of the xerogels revealed left and right-handed chiral nanofibers for the gelators' L-form and D-form, respectively. The resulting hydrogels exhibited inherent antibacterial activity against Gram-positive (Bacillus subtilis, Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria, with TDZ hydrogels showing more significant antibacterial activity than MTZ hydrogels. Interestingly, the D-form (having right-handed nanofibers) of both hydrogels (MTZ and TDZ) exhibited higher antibacterial activities compared with the left-handed nanofibrous hydrogels (L-form) attributed to the stereoselective interaction of the chiral helical nanofiber. Moreover, the amplification of chirality moving from a molecular to a supramolecular level essentially improved the antibacterial action. Our results provide deep insight into the development of unique supramolecular chiral antimicrobial agents and hint at the potentiality of right-handed nanofibers (D-form) having enhanced antibacterial activity. STATEMENT OF SIGNIFICANCE: Chiral stereochemistry plays a significant role in many biological processes, which determines the interaction of bacteria cells with chiral biomolecules. The interaction between bacteria and material surface (molecular chirality or supramolecular chirality) plays a significant role in modulating antibacterial performance. Here, we deigned and synthesized unique inherent biocompatible supramolecular chiral hydrogel. From this study we concluded that the D-form (having right-handed nanofibers) of hydrogels exhibited higher antibacterial activities compared with the left-handed nanofibrous hydrogels (L-form) attributed to the stereoselective interaction of the chiral helical nanofiber. Additionally, this study also explored the amplification of chirality moving from a molecular to a supramolecular level essentially improved the antibacterial action.
- Published
- 2022
24. One-Pot Asymmetric Synthesis of an Aminodiol Intermediate of Florfenicol Using Engineered Transketolase and Transaminase
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Xinyue Xie, Qi Liu, Mancheng Tang, Shuangjun Lin, Zixin Deng, Wentao Tao, Yi-Lei Zhao, Yuanzhen Zhang, Tingting Huang, and Ting Shi
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Florfenicol ,chemistry.chemical_compound ,Stereochemistry ,Chemistry ,Enantioselective synthesis ,General Chemistry ,Transketolase ,Catalysis ,Transaminase - Published
- 2021
25. An N-N linked dimeric indole alkaloid from the marine sponge-associated rare actinomycetes
- Author
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Jingwei, Jia, Xiaozheng, Wang, Jin, Sang, Zhiyong, Li, Shuangjun, Lin, Zixin, Deng, and Tingting, Huang
- Abstract
Marine derived rare actinomycetes is emerging as one of the new sources for various natural products for further drug discovery. Dimeric indole alkaloids represent a group of structurally diverse natural products and N-N linkage is a special dimerization mode. Here, we report the isolation of 1,1'-([1,1'-biindole]-3,3'-diyl) bis (ethane-1,2-diol), a new tryptophan-derived indole alkaloid from the marine sponge-derived
- Published
- 2022
26. A natural pyrazolotriazine pseudoiodinine produced by Pseudomonas mosselii 923 inhibits bacterial and fungal rice pathogens
- Author
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Gongyou Chen, Ruihuan Yang, Qing Shi, Tingting Huang, Yichao Yan, Shengzhang Li, Yuan Fang, Ying Li, Linlin Liu, Longyu Liu, Xiaozheng Wang, Yongzheng Peng, Lifang Zou, and Shuangjun Lin
- Abstract
Natural products (NPs) are a consistent source of antimicrobial metabolites and pesticide leads and are largely produced by Pseudomonads-like soil-dwelling microorganisms. Herein we report the isolation of Pseudomonas mosselii strain 923 from rice rhizosphere soils of paddy fields; this strain specifically inhibited the growth of rice bacterial pathogens Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc) and the fungal pathogen Magnaporthe oryzae. The antimicrobial compound produced by P. mosselii 923 was purified and identified as pseudoiodinine using HRMS, NMR and single-crystal X-ray diffraction. Genome-wide random mutagenesis, transcriptome analysis and biochemical assays were used to define the pseudoiodinine biosynthetic cluster as a seven-gene operon, which was designated psdABCDEFG. Pseudoiodinine biosynthesis is proposed to initiate from GTP, which is converted into 2,5-diamino-6-(5-phospho-d-ribosylamino) pyrimidin-4(3H)-one by PsdD and converted to 5-amino-6-(5-phospho-d-ribitylamino) uracil by PsdG, followed by cyclization with glycine by PsdC/PsdE and an unknown ring contraction and two methylation reactions by PsdA and PsdF. Transposon mutagenesis indicated that pseudoiodinine biosynthesis is mediated by the global regulator GacA. Further regulation is mediated by two noncoding small RNAs, rsmY and rsmZ, that positively regulate pseudoiodinine transcription, and the carbon storage regulators CsrA2 and CsrA3, which negatively regulate expression. A 22.4-fold increase in pseudoiodinine production was achieved by optimizing the media used for fermentation, overexpressing the biosynthetic operon, and removing the CsrA binding sites. Use of P. mosselii strain 923 and purified pseudoiodinine in planta revealed that both of them inhibited X. oryzae without affecting the rice host, suggesting that pseudoiodinine can be used to control plant diseases.
- Published
- 2022
27. Bioaugmentation removal and microbiome analysis of the synthetic estrogen 17α-ethynylestradiol from hostile conditions and environmental samples by Pseudomonas citronellolis SJTE-3
- Author
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Wanli Peng, Shuangjun Lin, Zixin Deng, and Rubing Liang
- Subjects
Environmental Engineering ,Health, Toxicology and Mutagenesis ,Public Health, Environmental and Occupational Health ,Environmental Chemistry ,General Medicine ,General Chemistry ,Pollution - Published
- 2023
28. Oxidative Indole Dearomatization for Asymmetric Furoindoline Synthesis by a Flavin‐Dependent Monooxygenase Involved in the Biosynthesis of Bicyclic Thiopeptide Thiostrepton
- Author
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Wen Liu, Xiao-Wei Liang, Jian Wang, Shu-Li You, Jiang Tao, Shuangjun Lin, Yufeng Xue, and Zhi Lin
- Subjects
Indole test ,Indoles ,Molecular Structure ,Bicyclic molecule ,010405 organic chemistry ,Chemistry ,Stereochemistry ,Enantioselective synthesis ,Total synthesis ,General Chemistry ,Flavin group ,General Medicine ,010402 general chemistry ,Thiostrepton ,01 natural sciences ,Catalysis ,Mixed Function Oxygenases ,0104 chemical sciences ,Stereocenter ,chemistry.chemical_compound ,Flavins ,Stereoselectivity ,Oxidation-Reduction - Abstract
The interest in indole dearomatization, which serves as a useful tool in the total synthesis of related alkaloid natural products, has recently been renewed with the intention of developing new methods efficient in both yield and stereoselective control. Here, we report an enzymatic approach for the oxidative dearomatization of indoles in the asymmetric synthesis of a variety of furoindolines with a vicinal quaternary carbon stereogenic center. This approach depends on the activity of a flavin-dependent monooxygenase, TsrE, which is involved in the biosynthesis of bicyclic thiopeptide antibiotic thiostrepton. TsrE catalyzes 2,3-epoxidation and subsequent epoxide opening in a highly enantioselective manner during the conversion of 2-methyl-indole-3-acetic acid or 2-methyl-tryptophol to furoindoline , with up to > 99% conversion and > 99% ee under mild reaction conditions. Complementing current chemical methods for oxidative indole dearomatization, the TsrE activity-based approach enriches the toolbox in the asymmetric synthesis of products possessing a furoindoline skeleton.
- Published
- 2021
29. Identification of a 17β-estradiol-degrading Microbacterium hominis SJTG1 with high adaptability and characterization of the genes for estrogen degradation
- Author
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Weiliang Xiong, Wanli Peng, Yali Fu, Zixin Deng, Shuangjun Lin, and Rubing Liang
- Subjects
Biodegradation, Environmental ,Environmental Engineering ,Estradiol ,Microbacterium ,Health, Toxicology and Mutagenesis ,Humans ,Environmental Chemistry ,Estrogens ,Pollution ,Waste Management and Disposal - Abstract
Environmental estrogen contamination poses severe threat to wildlife and human. Biodegradation is an efficient strategy to remove the wide-spread natural estrogen, while strains suitable for hostile environments and fit for practical application are rare. In this work, Microbacterium hominis SJTG1 was isolated and identified with high degrading efficiency for 17β-estradiol (E2) and great environment fitness. It could degrade nearly 100% of 10 mg/L E2 in minimal medium in 6 days, and remove 93% of 1 mg/L E2 and 74% of 10 mg/L E2 in the simulated E2-polluted solid soil in 10 days. It maintained stable E2-degrading efficiency in various harsh conditions like non-neutral pH, high salinity, stress of heavy metals and surfactants. Genome mining and comparative genome analysis revealed that there are multiple genes potentially associated with steroid degradation in strain SJTG1. One 3β/17β-hydroxysteroid dehydrogenase HSD-G129 induced by E2 catalyzed the 3β/17β-dehydrogenation of E2 and other steroids efficiently. The transcription of hsd-G129 gene was negatively regulated by the adjacent LysR-type transcriptional regulator LysR-G128, through specific binding to the conserved site. E2 can release this binding and initiate the degradation process. This work provides an efficient and adaptive E2-degrading strain and promotes the biodegrading mechanism study and actual remediation application.
- Published
- 2023
30. Two-enzyme cascade catalyzed trideuteromethylative modification of natural products
- Author
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Zi-Ruo Zou, Kai Zhang, Ting-Yan Han, Qiang Zhou, Shuangjun Lin, Xian-Feng Hou, and Gong-Li Tang
- Subjects
Organic Chemistry ,Drug Discovery ,Biochemistry - Published
- 2022
31. Characterization of Pyridomycin B Reveals the Formation of Functional Groups in Antimycobacterial Pyridomycin
- Author
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Zixing Deng, Shuangjun Lin, Tingting Huang, Maolong Wei, Lin Chen, Zihua Zhou, and Zhihong Xiao
- Subjects
Alanine ,chemistry.chemical_classification ,Ecology ,biology ,Chemistry ,medicine.drug_class ,Stereochemistry ,INHA ,Active site ,Mycobacterium tuberculosis ,Antimycobacterial ,Applied Microbiology and Biotechnology ,Streptomyces ,Nonribosomal peptide ,Polyketide synthase ,Catalytic triad ,biology.protein ,medicine ,Moiety ,Peptide Synthases ,Oxidoreductases ,Oligopeptides ,Polyketide Synthases ,Food Science ,Biotechnology - Abstract
Pyridomycin, a cyclodepsipeptide with potent antimycobacterial activity, specifically inhibits the InhA enoyl reductase of Mycobacterium tuberculosis. Structure-activity relationship studies indicated that the enolic acid moiety in the pyridomycin core system is an important pharmacophoric group, and the natural configuration of the C-10 hydroxyl contributes to the bioactivity of pyridomycin. The ring structure of pyridomycin was generated by the nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) hybrid system (PyrE-PyrF-PyrG). Bioinformatics analysis reveals that short-chain dehydrogenase/reductase (SDR) family protein Pyr2 functions as a 3-oxoacyl acyl carrier protein (ACP) reductase in the pyridomycin pathway. Inactivation of pyr2 resulted in accumulation of pyridomycin B, a new pyridomycin analogue featured with enol moiety in pyridyl alanine moiety and a saturated 3-methylvaleric acid group. The elucidated structure of pyridomycin B suggests that rather than functioning as a post-tailoring enzyme, Pyr2 catalyzes ketoreduction to form the C-10 hydroxyl group in pyridyl alanine moiety and the double bond formation of the enolic acid moiety derived from isoleucine when the intermediate assembled by PKS-NRPS machinery is still tethered to the last NRPS module in a special energy-saving manner. Ser-His-Lys residues constitute the active site of Pyr2, which is different from the typically conserved Tyr-based catalytic triad in the majority of SDRs. Site-directed mutation identified that His154 in the active site is a critical residue for pyridomycin B production. These findings will improve our understanding of pyridomycin biosynthetic logic, identify the missing link for the double bound formation of enol ester in pyridomycin, and enable the creation of chemical diversity of pyridomycin derivatives. IMPORTANCE Tuberculosis (TB) is one of the world’s leading causes of death by infection. Recently, pyridomycin, the antituberculous natural product from Streptomyces has garnered considerable attention for being determined as a target inhibitor of InhA enoyl reductase of Mycobacterium tuberculosis. In this study, we report a new pyridomycin analogue from mutant HTT12, demonstrate the essential role of a previously ignored gene pyr2 in pyridomycin biosynthetic pathway, and imply that Pyr2 functions as a trans ketoreductase (KR) contributing to the formation of functional groups of pyridomycin utilizing a distinct catalytic mechanism. As enol moiety are important for pharmaceutical activities of pyridomycin, our work would expand our understanding of the mechanism of SDR family proteins and set the stage for future bioengineering of new pyridomycin derivatives.
- Published
- 2022
32. An N-N linked dimeric indole alkaloid from the marine sponge-associated rare actinomycetes Kocuria sp. S42
- Author
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Jingwei Jia, Xiaozheng Wang, Jin Sang, Zhiyong Li, Shuangjun Lin, Zixin Deng, and Tingting Huang
- Subjects
Organic Chemistry ,Plant Science ,Biochemistry ,Analytical Chemistry - Abstract
Marine derived rare actinomycetes is emerging as one of the new sources for various natural products for further drug discovery. Dimeric indole alkaloids represent a group of structurally diverse natural products and N-N linkage is a special dimerization mode. Here, we report the isolation of 1,1'-([1,1'-biindole]-3,3'-diyl) bis (ethane-1,2-diol), a new tryptophan-derived indole alkaloid from the marine sponge-derived Kocuria sp. S42. The structure was established based on extensive spectroscopic analyses, including nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass (HR-ESI-MS) spectrometry. The new dimeric indole alkaloid via N-N linkage exhibits moderate antimicrobial activity.
- Published
- 2022
- Full Text
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33. Efficient and Stable Removal of the Synthetic Estrogen 17α-Ethynylestradiol by Pseudomonas Citronellolis Sjte-3 from Various Environments
- Author
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Wanli Peng, Shuangjun Lin, Zixin Deng, and liang rubing
- Subjects
History ,Polymers and Plastics ,Business and International Management ,Industrial and Manufacturing Engineering - Published
- 2022
34. Bacterial Product Template Domain: An Evolutionary Intermediate between Dehydratase and Aldol Cyclase of Type I Polyketide Synthases
- Author
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yuanyuan Feng, Xu Yang, Huining Ji, Zixin Deng, Shuangjun Lin, and Jianting Zheng
- Abstract
The product template (PT) domains act as an aldol cyclase to control the regiospecific aldol cyclization of the extremely reactive poly-β-ketone intermediate assembled by an iterative type I polyketide synthases (PKSs). Up to now, only the structure of fungal PksA PT that mediates the first-ring cyclization via C4-C9 aldol cyclization is available. We describe here the structural and computational characterization of a bacteria PT domain that controls C2-C7 cyclization in orsellinic acid (OSA) synthesis. Mutating the catalytic His949 of the PT abolishes production of OSA and results in a tetraacetic acid lactone (TTL) generated by spontaneous O-C cyclization of the acyl carrier protein (ACP)-bound tetraketide intermediate. Crystal structure of the bacterial PT domain closely resembles dehydrase (DH) domains of modular type I PKSs in the overall fold, dimerization interface and catalytic “His-Asp” dyad organization, but is significantly different from PTs of fungal iterative type I PKSs. QM/MM calculation reveals that the catalytic His949 abstracts a proton from C2 and transfers it to C7 carbonyl to mediate the cyclization reaction. According to the structural similarity to DHs and the functional similarity to fungal PTs, we propose that the bacterial PT represents an evolutionary intermediate between the two tailoring domains of type I PKSs.
- Published
- 2021
35. Functional Genome Mining Reveals a Class V Lanthipeptide Containing a<scp>d</scp>‐Amino Acid Introduced by an F420H2‐Dependent Reductase
- Author
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Zhuo Cheng, Fei Zhang, Ghader Bashiri, Jing Wang, Jiali Hong, Sheng-Xiong Huang, Zixin Deng, Yemin Wang, Shuangjun Lin, Min Xu, Lijun Xu, Meifeng Tao, and Xuefei Chen
- Subjects
chemistry.chemical_classification ,010405 organic chemistry ,Stereochemistry ,General Chemistry ,General Medicine ,Lantibiotics ,Reductase ,010402 general chemistry ,01 natural sciences ,Genome ,Catalysis ,0104 chemical sciences ,chemistry.chemical_compound ,Enzyme ,chemistry ,Biosynthesis ,Dehydroalanine ,Gene cluster ,Lanthionine - Abstract
Lantibiotics are a type of ribosomally synthesized and post-translationally modified peptides (termed lanthipeptides) with often potent antimicrobial activity. Herein, we report the discovery of a new lantibiotic, lexapeptide, using the library expression analysis system (LEXAS) approach. Lexapeptide has rare structural modifications, including N-terminal (N,N)-dimethyl phenylalanine, C-terminal (2-aminovinyl)-3-methyl-cysteine, and d-Ala. The characteristic lanthionine moiety in lexapeptide is formed by three proteins (LxmK, LxmX, and LxmY), which are distinct from enzymes known to be involved in lanthipeptide biosynthesis. Furthermore, a novel F420 H2 -dependent reductase (LxmJ) from the lexapeptide biosynthetic gene cluster (BGC) is identified to catalyze the reduction of dehydroalanine to install d-Ala. Our findings suggest that lexapeptide is the founding member of a new class of lanthipeptides that we designate as class V. We also identified further class V lanthipeptide BGCs in actinomycetes and cyanobacteria genomes, implying that other class V lantibiotics await discovery.
- Published
- 2020
36. Bioconversion of a ganoderic acid 3-hydroxy-lanosta-8,24-dien-26-oic acid by a crude enzyme from Ganoderma lucidum
- Author
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Jian-Jiang Zhong, Han Xiao, Shuangjun Lin, Siqin Cai, and Xiaozheng Wang
- Subjects
chemistry.chemical_classification ,Chromatography ,Bioconversion ,Lanosterol ,Ganoderic acid ,Substrate (chemistry) ,Bioengineering ,Dehydrogenase ,Applied Microbiology and Biotechnology ,Biochemistry ,chemistry.chemical_compound ,Enzyme ,chemistry ,Biocatalysis ,Ganoderma lucidum - Abstract
Ganoderic acids (GAs) are oxygenated lanostane-type triterpenoids from the traditional medicinal mushroom Ganoderma lucidum and of significant biological activities. Although a ganoderic acid 3-hydroxy-lanosta-8,24-dien-26-oic acid (HLDOA) was found to be biosynthesized from lanosterol, further post-modification of HLDOA is yet unclear. In this work, by using HLDOA as the substrate and a crude enzyme from G. lucidum as the biocatalyst, we observed a new peak in liquid chromatography from the reaction system. The product was purified and identified to be 3-oxo-lanosta-8,24-dien-26-oic acid (OLDOA), which may be converted from HLDOA by a putative dehydrogenase of G. lucidum. The work is useful to future manufacture of GAs as well as their biosynthetic pathway elucidation.
- Published
- 2020
37. Antimicrobial Activity with Enhanced Mechanical Properties in Phenylalanine-Based Chiral Coassembled Hydrogels: The Influence of Pyridine Hydrazide Derivatives
- Author
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Chuanliang Feng, Chao Xing, Vincent Mukwaya, Li Yang, Shuangjun Lin, Auphedeous Yinme Dang-i, Tingting Huang, Nabila Mehwish, and Xiaoqiu Dou
- Subjects
Biochemistry (medical) ,Biomedical Engineering ,Supramolecular chemistry ,Phenylalanine ,General Chemistry ,Hydrazide ,Antimicrobial ,Combinatorial chemistry ,Biomaterials ,Bipyridine ,chemistry.chemical_compound ,chemistry ,Pyridine ,Self-healing hydrogels ,Chirality (chemistry) - Abstract
Hydrazide derivatives are known to display a wide range of biological properties including antimicrobial activities, hence making them desirable candidates for soft biomaterials. Herein, we report chiral supramolecular coassembled hydrogels obtained from two phenylalanine gelators (L/DPF and B2L/D) and two dicarbohydrazide molecules (pyridine-2,6-dicarbohydrazide (PDH) and (2,2'-bipyridine)-5,5'-dicarbohydrazide (BDH)) that exhibited enhanced mechanical properties, chirality modulation, and antimicrobial activity. Four lines of coassembled hydrogels were obtained (
- Published
- 2020
38. Biosynthetic access to the rare antiarose sugar via an unusual reductase-epimerase
- Author
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Jing Yang, Sheng-Xiong Huang, Meifeng Tao, Geoff P. Horsman, Li Wang, Yijun Yan, Xiaowei Guo, Shuangjun Lin, Dongdong Xu, and Zhiyin Yu
- Subjects
chemistry.chemical_classification ,010405 organic chemistry ,Biological activity ,General Chemistry ,Reductase ,010402 general chemistry ,01 natural sciences ,Tropolone ,0104 chemical sciences ,chemistry.chemical_compound ,Enzyme ,Aglycone ,chemistry ,Biosynthesis ,Biochemistry ,Epimer ,Gene - Abstract
Rubrolones, isatropolones, and rubterolones are recently isolated glycosylated tropolonids with notable biological activity. They share similar aglycone skeletons but differ in their sugar moieties, and rubterolones in particular have a rare deoxysugar antiarose of unknown biosynthetic provenance. During our previously reported biosynthetic elucidation of the tropolone ring and pyridine moiety, gene inactivation experiments revealed that RubS3 is involved in sugar moiety biosynthesis. Here we report the in vitro characterization of RubS3 as a bifunctional reductase/epimerase catalyzing the formation of TDP-d-antiarose by epimerization at C3 and reduction at C4 of the key intermediate TDP-4-keto-6-deoxy-d-glucose. These new findings not only explain the biosynthetic pathway of deoxysugars in rubrolone-like natural products, but also introduce RubS3 as a new family of reductase/epimerase enzymes with potential to supply the rare antiarose unit for expanding the chemical space of glycosylated natural products.
- Published
- 2020
39. The Streptomyces viridochromogenes product template domain represents an evolutionary intermediate between dehydratase and aldol cyclase of type I polyketide synthases
- Author
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Yuanyuan Feng, Xu Yang, Huining Ji, Zixin Deng, Shuangjun Lin, and Jianting Zheng
- Subjects
Aldehydes ,Sleep Apnea, Obstructive ,Medicine (miscellaneous) ,Humans ,General Agricultural and Biological Sciences ,Polyketide Synthases ,General Biochemistry, Genetics and Molecular Biology ,Hydro-Lyases ,Streptomyces - Abstract
The product template (PT) domains act as an aldol cyclase to control the regiospecific aldol cyclization of the extremely reactive poly-β-ketone intermediate assembled by an iterative type I polyketide synthases (PKSs). Up to now, only the structure of fungal PksA PT that mediates the first-ring cyclization via C4–C9 aldol cyclization is available. We describe here the structural and computational characterization of a bacteria PT domain that controls C2–C7 cyclization in orsellinic acid (OSA) synthesis. Mutating the catalytic H949 of the PT abolishes production of OSA and results in a tetraacetic acid lactone (TTL) generated by spontaneous O-C cyclization of the acyl carrier protein (ACP)-bound tetraketide intermediate. Crystal structure of the bacterial PT domain closely resembles dehydrase (DH) domains of modular type I PKSs in the overall fold, dimerization interface and His-Asp catalytic dyad organization, but is significantly different from PTs of fungal iterative type I PKSs. QM/MM calculation suggests that the catalytic H949 abstracts a proton from C2 and transfers it to C7 carbonyl to mediate the cyclization reaction. According to structural similarity to DHs and functional similarity to fungal PTs, we propose that the bacterial PT represents an evolutionary intermediate between the two tailoring domains of type I PKSs.
- Published
- 2021
40. [Advances in the bioaugmentation-assisted remediation of petroleum contaminated soil]
- Author
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Jin, Zheng, Yali, Fu, Quanwei, Song, Jiacai, Xie, Shuangjun, Lin, and Rubing, Liang
- Subjects
Soil ,Biodegradation, Environmental ,Petroleum ,Soil Pollutants ,Soil Microbiology - Abstract
Bioremediation is considered as a cost-effective, efficient and free-of-secondary-pollution technology for petroleum pollution remediation. Due to the limitation of soil environmental conditions and the nature of petroleum pollutants, the insufficient number and the low growth rate of indigenous petroleum-degrading microorganisms in soil lead to long remediation cycle and poor remediation efficiency. Bioaugmentation can effectively improve the biodegradation efficiency. By supplying functional microbes or microbial consortia, immobilized microbes, surfactants and growth substrates, the remediation effect of indigenous microorganisms on petroleum pollutants in soil can be boosted. This article summarizes the reported petroleum-degrading microbes and the main factors influencing microbial remediation of petroleum contaminated soil. Moreover, this article discusses a variety of effective strategies to enhance the bioremediation efficiency, as well as future directions of bioaugmentation strategies.
- Published
- 2021
41. A Novel Mice Model of Catecholaminergic Polymorphic Ventricular Tachycardia Generated by CRISPR/Cas9
- Author
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Changliang Hou, Xue Jiang, Q. Qiu, Jiancheng Zheng, Sun Chen, Yunting Zhang, M. Xu, Shuangjun Lin, Lijian Xie, and x. tingting
- Subjects
medicine.medical_specialty ,business.industry ,chemistry.chemical_element ,Calcium ,medicine.disease ,Ventricular tachycardia ,Catecholaminergic polymorphic ventricular tachycardia ,Ryanodine receptor 2 ,Calcium in biology ,Sudden cardiac death ,Contractility ,Endocrinology ,chemistry ,Internal medicine ,Ca2+/calmodulin-dependent protein kinase ,cardiovascular system ,medicine ,business - Abstract
Catecholaminergic polymorphic ventricular tachycardia (CPVT) has been considered as one of the most important causes of children’s sudden cardiac death. Mutations in the genes for RyR2 and CASQ2, two mainly subtypes of CPVT, have been identified. However, the mutation in the gene of TECRL was rarely reported, which could be another genetic cause of CPVT. We evaluated myocardial contractility, electrophysiology, calcium handling in Tecrl knockout (Tecrl KO) mice and human induced pluripotent stem cell-derived cardiomyocytes. Immediately after epinephrine plus caffeine injection, Tecrl KO mice showed much more multiple premature ventricular beats and ventricular tachycardia. The Tecrl KO mice demonstrate CPVT phenotypes. Mechanistically, intracellular calcium amplitude was reduced, while time to baseline of 50 was increased in acute isolated cardiomyocytes. RyR2 protein levels decreased significantly upon cycloheximide treatment in TECRL deficiency cardiomyocytes. Overexpression of TECRL and KN93 can partially reverse cardiomyocytes calcium dysfunction, and this is p-CaMKII/CaMKII dependent. Therefore, a new CPVT mouse model was constructed. We propose a previously unrecognized mechanism of TECRL and provide support for the therapeutic targeting of TECRL in treating CPVT.
- Published
- 2021
42. The transcriptome analysis of Escherichia coli responding to tellurite
- Author
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Rubing Liang, Shuangjun Lin, Wanli Peng, Sushu Hu, and Zixin Deng
- Subjects
Transcriptome ,medicine ,Biology ,medicine.disease_cause ,Escherichia coli ,Microbiology - Abstract
Tellurite is a strong antimicrobial agent highly toxic to many microorganisms, while its toxicity mechanism is still unclear. In this study, the comparative transcriptome analysis of E. coli MG1655 responding to the stress of tellurite was performed and the differentially transcribed genes were analyzed, to understand toxicity mechanisms of tellurite preliminaryly and uncover metabolism processes changes resulted from tellurite globally. After treated with 10 µg/mL tellurite for 1 h, high concentration and long time, the cells exhibited an obvious adaptive reaction and many metabolic processes were influenced. The transcription of the genes involved in the ribosome metabolism and the flagella assembly were changed significantly, implying they might be the major pathway affected by tellurite. The transcription of the genes encoding the transcriptional factors and small RNAs, and the genes functioned in the cell motility, metal ion metabolism and membrane function were also varied, which may participate in the metabolism adjustment and damage repair to resist the toxicity of tellurite. This work can facilitate the study of the toxicity mechanism of tellurite and promote the clinical application of this chemical.
- Published
- 2021
43. The 3-Oxoacyl-(Acyl-Carrier-Protein) Reductase HSD-X1 of Pseudomonas Citronellolis SJTE-3 Catalyzes the Conversion of 17β-estradiol to Estrone
- Author
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Rubing Liang, Yali Fu, Wanli Peng, Shuangjun Lin, and Zixin Deng
- Subjects
endocrine system ,Estradiol ,Structural Biology ,Estrone ,Pseudomonas ,Acyl Carrier Protein ,3-Oxoacyl-(Acyl-Carrier-Protein) Reductase ,General Medicine ,Oxidoreductases ,Biochemistry - Abstract
Background: Pseudomonas citronellolis SJTE-3 can efficiently degrade 17β-estradiol (E2) and other estrogenic chemicals. However, the enzyme responsible for E2 metabolism within strain SJTE-3 has remained unidentified. Objective: Here, a novel 3-oxoacyl-(acyl-carrier protein) (ACP) reductase, HSD-X1 (WP_ 009617962.1), was identified in SJTE-3 and its enzymatic characteristics for the transformation of E2 were investigated. Methods: Multiple sequence alignment and homology modelling were used to predict the protein structure of HSD-X1. The concentrations of different steroids in the culture of recombinant strains expressing HSD-X1 were determined by high performance liquid chromatography. Additionally, the transcription of hsd-x1 gene was investigated using reverse transcription and quantitative PCR analysis. Heterologous expression and affinity purification were used to obtain recombinant HSD- X1. Results: The transcription of hsd-x1 gene in P. citronellolis SJTE-3 was induced by E2. Multiple sequence alignment (MSA) indicated that HSD-X1 contained the two consensus regions and conserved residues of short-chain dehydrogenase/reductases (SDRs) and 17β-hydroxysteroid dehydrogenases (17β-HSDs). Over-expression of hsd-x1 gene allowed the recombinant strain to degrade E2. Recombinant HSD-X1 was purified with a yield of 22.15 mg/L and used NAD+ as its cofactor to catalyze the oxidization of E2 into estrone (E1) while exhibiting a Km value of 0.025 ± 0.044 mM and a Vmax value of 4.92 ± 0.31 mM/min/mg. HSD-X1 could tolerate a wide range of temperature and pH, while the presence of divalent ions exerted little influence on its activity. Further, the transformation efficiency of E2 into E1 was over 98.03% across 15 min. Conclusion: Protein HSD-X1 efficiently catalyzed the oxidization of E2 and participated in estrogen degradation by P. citronellolis SJTE-3.
- Published
- 2021
44. Biosynthetic access to the rare antiarose sugar
- Author
-
Yijun, Yan, Jing, Yang, Li, Wang, Dongdong, Xu, Zhiyin, Yu, Xiaowei, Guo, Geoff P, Horsman, Shuangjun, Lin, Meifeng, Tao, and Sheng-Xiong, Huang
- Subjects
Chemistry - Abstract
Rubrolones, isatropolones, and rubterolones are recently isolated glycosylated tropolonids with notable biological activity. They share similar aglycone skeletons but differ in their sugar moieties, and rubterolones in particular have a rare deoxysugar antiarose of unknown biosynthetic provenance. During our previously reported biosynthetic elucidation of the tropolone ring and pyridine moiety, gene inactivation experiments revealed that RubS3 is involved in sugar moiety biosynthesis. Here we report the in vitro characterization of RubS3 as a bifunctional reductase/epimerase catalyzing the formation of TDP-d-antiarose by epimerization at C3 and reduction at C4 of the key intermediate TDP-4-keto-6-deoxy-d-glucose. These new findings not only explain the biosynthetic pathway of deoxysugars in rubrolone-like natural products, but also introduce RubS3 as a new family of reductase/epimerase enzymes with potential to supply the rare antiarose unit for expanding the chemical space of glycosylated natural products., Rubrolones, isarubrolones, and rubterolones are recently isolated glycosylated tropolonids with notable biological activity.
- Published
- 2021
45. Characterization of Lysozyme-Like Effector TseP Reveals the Dependence of Type VI Secretion System (T6SS) Secretion on Effectors in Aeromonas dhakensis Strain SSU
- Author
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Tong-Tong Pei, Tao G. Dong, Weiliang Xiong, Shuangjun Lin, Zeng-Hang Wang, Li-Li Wu, Ping Xu, and Xiaoye Liang
- Subjects
Mutant ,Cell ,Virulence ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Dictyostelium discoideum ,03 medical and health sciences ,Bacterial Proteins ,Phagocytosis ,Cell Wall ,medicine ,Escherichia coli ,Environmental Microbiology ,Secretion ,Dictyostelium ,030304 developmental biology ,Type VI secretion system ,0303 health sciences ,Mutation ,Ecology ,biology ,030306 microbiology ,Chemistry ,Effector ,Type VI Secretion Systems ,biology.organism_classification ,Cell biology ,medicine.anatomical_structure ,Muramidase ,Aeromonas ,Food Science ,Biotechnology - Abstract
The type VI secretion system (T6SS) is a widespread weapon employed by Gram-negative bacteria for interspecies interaction in complex communities. Analogous to a contractile phage tail, the double-tubular T6SS injects toxic effectors into prokaryotic and eukaryotic neighboring cells. Although effectors dictate T6SS functions, their identities remain elusive in many pathogens. Here, we report the lysozyme-like effector TseP in Aeromonas dhakensis, a waterborne pathogen that can cause severe gastroenteritis and systemic infection. Using secretion, competition, and enzymatic assays, we demonstrate that TseP is a T6SS-dependent effector with cell wall-lysing activities, and TsiP is its cognate immunity protein. Triple deletion of tseP and two known effector genes, tseI and tseC, abolished T6SS-mediated secretion, while complementation with any single effector gene partially restored bacterial killing and Hcp secretion. In contrast to whole-gene deletions, the triple-effector inactivation in the 3eff(c) mutant abolished antibacterial killing but not T6SS secretion. We further demonstrate that the 3eff(c) mutation abolished T6SS-mediated toxicity of SSU to Dictyostelium discoideum amoebae, suggesting that the T6SS physical puncture is nontoxic to eukaryotic cells. These data highlight not only the necessity of possessing functionally diverse effectors for survival in multispecies communities but also that effector inactivation would be an efficient strategy to detoxify the T6SS while preserving its delivery efficiency, converting the T6SS to a platform for protein delivery to a variety of recipient cells. IMPORTANCE Delivery of cargo proteins via protein secretion systems has been shown to be a promising tool in various applications. However, secretion systems are often used by pathogens to cause disease. Thus, strategies are needed to detoxify secretion systems while preserving their efficiency. The T6SS can translocate proteins through physical puncture of target cells without specific surface receptors and can target a broad range of recipients. In this study, we identified a cell wall-lysing effector, and by inactivating it and the other two known effectors, we have built a detoxified T6SS-active strain that may be used for protein delivery to prokaryotic and eukaryotic recipient cells.
- Published
- 2021
46. Enzymatic Pyran Formation Involved in Xiamenmycin Biosynthesis
- Author
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Xu-Liang Bu, Jun Xu, Jianting Zheng, Min-Juan Xu, Ting Zhou, Jing-Yi Weng, Shuangjun Lin, Bei-Bei He, and Yi-Lei Zhao
- Subjects
chemistry.chemical_compound ,chemistry ,Biosynthesis ,Pyran ,Stereochemistry ,Furan ,Epoxide ,General Chemistry ,Monooxygenase ,Ring (chemistry) ,Cyclase ,Catalysis ,Benzopyran - Abstract
The pyran ring is a very common structural unit of many natural, bioactive molecules that are widely found in plants, bacteria, and fungi. However, the enzymatic processes by which many of these pyran-containing molecules are formed are unclear. Herein, we report the construction of the pyran ring catalyzed by the cooperation of a flavin-dependent monooxygenase, XimD, and a SnoaL-like cyclase, XimE, in the biosynthesis of xiamenmycins. XimD catalyzes the formation of an epoxide intermediate that spontaneously transforms to furan and pyran products (43:1) in vitro. XimE then catalyzes the formation of the pyran ring in a 6-endo configuration from the epoxide to yield a benzopyran, xiamenmycin B. Further, we obtained the crystallographic structure of XimE, with and without product, which suggests a synergistic mechanism in which a group of four residues (Y46–Y90–H102–E136) acts cooperatively as the general acid and base. Subsequent structure-based analysis of possible viable substrates indicates that both X...
- Published
- 2019
47. The molecular basis for the intramolecular migration (NIH shift) of the carboxyl group duringpara-hydroxybenzoate catabolism
- Author
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Jim C. Spain, Ying Xu, Huan Zhao, Ning-Yi Zhou, and Shuangjun Lin
- Subjects
0301 basic medicine ,Stereochemistry ,Gentisates ,030106 microbiology ,Substituent ,Parabens ,Biology ,Hydroxylation ,Thioester ,Microbiology ,03 medical and health sciences ,chemistry.chemical_compound ,Thioesterase ,Molecular Biology ,Biotransformation ,chemistry.chemical_classification ,DNA ligase ,Brevibacillus ,Acetyl-CoA ,Enzymes ,030104 developmental biology ,chemistry ,Hydroxybenzoate ,NIH shift ,lipids (amino acids, peptides, and proteins) ,Metabolic Networks and Pathways - Abstract
The NIH shift is a chemical rearrangement in which a substituent on an aromatic ring undergoes an intramolecular migration, primarily during an enzymatic hydroxylation reaction. The molecular mechanism for the NIH shift of a carboxyl group has remained a mystery for 40 years. Here, we elucidate the molecular mechanism of the reaction in the conversion of para-hydroxybenzoate (PHB) to gentisate (GA, 2, 5-dihydroxybenzoate). Three genes (phgABC) from the PHB utilizer Brevibacillus laterosporus PHB-7a encode enzymes (p-hydroxybenzoyl-CoA ligase, p-hydroxybenzoyl-CoA hydroxylase and gentisyl-CoA thioesterase, respectively) catalyzing the conversion of PHB to GA via a route involving CoA thioester formation, hydroxylation concomitant with a 1, 2-shift of the acetyl CoA moiety and thioester hydrolysis. The shift of the carboxyl group was established rigorously by stable isotopic experiments with heterologously expressed phgABC, converting 2, 3, 5, 6-tetradeutero-PHB and [carboxyl-13 C]-PHB to 3, 4, 6-trideutero-GA and [carboxyl-13 C]-GA respectively. This is distinct from the NIH shifts of hydrogen and aceto substituents, where a single oxygenase catalyzes the reaction without the involvement of a thioester. The discovery of this three-step strategy for carboxyl group migration reveals a novel role of the CoA thioester in biochemistry and also illustrates the diversity and complexity of microbial catabolism in the carbon cycle.
- Published
- 2018
48. A novel streptonigrin type alkaloid from the
- Author
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Xiaozheng, Wang, Fei, Xu, Tingting, Huang, Zixin, Deng, and Shuangjun, Lin
- Subjects
Alkaloids ,Antineoplastic Agents ,Streptonigrin ,Streptomyces - Abstract
Streptonigrin (STN) is a highly functionalized aminoquinone alkaloid with broad and potent antitumor activities. Previously, the biosynthetic gene cluster of STN was identified in
- Published
- 2020
49. Metabolism analysis of 17α-ethynylestradiol by Pseudomonas citronellolis SJTE-3 and identification of the functional genes
- Author
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Yali Fu, Yanqiu Wang, Wanli Peng, Zixin Deng, Xin Sun, Rubing Liang, Shuangjun Lin, and Ben Jia
- Subjects
Environmental Engineering ,Ethanol ,biology ,Health, Toxicology and Mutagenesis ,Cytochrome P450 ,Estrogens ,Dehydrogenase ,Metabolism ,Ethinyl Estradiol ,biology.organism_classification ,Pollution ,chemistry.chemical_compound ,Biodegradation, Environmental ,chemistry ,Biochemistry ,Pseudomonas ,biology.protein ,Environmental Chemistry ,Yeast extract ,Pseudomonas citronellolis ,Energy source ,Waste Management and Disposal ,Gene - Abstract
Synthetic estrogens are the most hazardous and persistent environmental estrogenic contaminants, with few reports on their biodegradation. Pseudomonas citronellolis SJTE-3 degraded natural steroids efficiently and metabolized 17α-ethynylestradiol (EE2) with the addition of different easily used energy sources (glucose, peptone, ethanol, yeast extract, fulvic acid and ammonia). Over 92% of EE2 (1 mg/L) and 55% of EE2 (10 mg/L) in culture were removed in seven days with the addition of 0.1% ethanol, and the EE2-biotransforming efficiency increased with the increasing ethanol concentrations. Two novel intermediate metabolites of EE2 (C22H22O and C18H34O2) were identified with high-performance liquid chromatography (HPLC) and GC-Orbitrap/MS. Comparative analysis and genome mining revealed strain SJTE-3 contained a unique genetic basis for EE2 metabolism, and the putative EE2-degrading genes exhibited dispersed distribution. The EE2 metabolism of strain SJTE-3 was inducible and the transcription of eight genes were significantly induced by EE2. Three genes (sdr3, yjcH and cyp2) encoding a short-chain dehydrogenase, a membrane transporter and a cytochrome P450 hydroxylase, respectively, were vital for EE2 metabolism in strain SJTE-3; their over-expression accelerated EE2 metabolic processes and advanced the generation of intermediate metabolites. This work could promote the study of bacterial EE2 metabolism mechanisms and facilitate efficient bioremediation for EE2 pollution.
- Published
- 2022
50. Spot 42 RNA regulates putrescine catabolism in Escherichia coli by controlling the expression of puuE at the post-transcription level
- Author
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Wanli Peng, Guochen Wan, Zixin Deng, Xin Sun, Shuangjun Lin, Ruyan Li, and Rubing Liang
- Subjects
Spot 42 RNA ,0303 health sciences ,Binding Sites ,Transcription, Genetic ,030306 microbiology ,Chemistry ,Catabolism ,Catabolite repression ,Repressor ,General Medicine ,Gene Expression Regulation, Bacterial ,Applied Microbiology and Biotechnology ,Microbiology ,03 medical and health sciences ,chemistry.chemical_compound ,RNA, Bacterial ,Biochemistry ,Transcription (biology) ,Putrescine ,Escherichia coli ,Animals ,Energy source ,Gene ,030304 developmental biology - Abstract
Putrescine, a typical polyamine compound important for cell growth and stress resistance, can be utilized as an energy source. However, the regulation of its catabolism is unclear. Here the small RNA (sRNA) Spot 42, an essential regulator of carbon catabolite repression (CCR), was confirmed to participate in the post-transcriptional regulation of putrescine catabolism in Escherichia coli. Its encoding gene spf exclusively exists in the γ-proteobacteria and contains specific binding sites to the 5'-untranslated regions of the puuE gene, which encodes transaminase in the glutamylated putrescine pathway of putrescine catabolism converting γ-aminobutyrate (GABA) into succinate semialdehyde (SSA). The transcription of the spf gene was induced by glucose, inhibited by putrescine, and unaffected by PuuR, the repressor of puu genes. Excess Spot 42 repressed the expression of PuuE significantly in an antisense mechanism through the direct and specific base-pairing between the 51`-57 nt of Spot 42 and the 5'-UTR of puuE. Interestingly, Spot 42 mainly influenced the stability of the puuCBE transcript. This work revealed the regulatory role of Spot 42 in putrescine catabolism, in the switch between favorable and non-favorable carbon source utilization, and in the balance of metabolism of carbon and nitrogen sources.
- Published
- 2020
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