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Guoping Zhao – 1st expert on this subject based on the ideXlab platform
crispr cas12a assisted genome editing in Amycolatopsis MediterraneiFrontiers in Bioengineering and Biotechnology, 2020Co-Authors: Yajuan Zhou, Guoping Zhao, Jiacheng Wu, Jin WangAbstract:
Amycolatopsis Mediterranei U32 is an industrial producer of rifamycin SV, whose derivatives have long been the first-line antimycobacterial drugs. In order to perform genetic modification in this important industrial strain, a lot of efforts have been made in the past decades and a homologous recombination-based method was successfully developed in our laboratory, which, however, requires the employment of an antibiotic resistance gene for positive selection and did not support convenient markerless gene deletion. Here in this study, the clustered regularly interspaced short palindromic repeat (CRISPR) system was employed to establish a genome editing system in A. Mediterranei U32. Specifically, the Francisella tularensis subsp. novicida Cas12a (FnCas12a) gene was first integrated into the U32 genome to generate target-specific double-stranded DNA (dsDNA) breaks (DSBs) under the guidance of CRISPR RNAs (crRNAs). Then, the DSBs could be repaired by either the non-homologous DNA end-joining (NHEJ) system or the homology-directed repair (HDR) pathway, generating inaccurate or accurate mutations in target genes, respectively. Besides of A. Mediterranei, the present work may also shed light on the development of CRISPR-assisted genome editing systems in other species of the Amycolatopsis genus.
glnr dominates rifamycin biosynthesis by activating the rif cluster genes transcription both directly and indirectly in Amycolatopsis MediterraneiFrontiers in Microbiology, 2020Co-Authors: Guoping Zhao, Jin WangAbstract:
: Because of the remarkable efficacy in treating mycobacterial infections, rifamycin and its derivatives are still first-line antimycobacterial drugs. It has been intensely studied to increase rifamycin yield from Amycolatopsis Mediterranei, and nitrate is found to provide a stable and remarkable stimulating effect on the rifamycin production, a phenomenon known as “nitrate-stimulating effect (NSE)”. Although the NSE has been widely used for the industrial production of rifamycin, its detailed molecular mechanism remains ill-defined. And our previous study has established that the global nitrogen regulator GlnR may participate in the NSE, but the underlying mechanism is still enigmatic. Here, we demonstrate that GlnR directly controls rifamycin biosynthesis in A. Mediterranei and thus plays an essential role in the NSE. Firstly, GlnR specifically binds to the upstream region of rifZ, which leads us to uncover that rifZ has its own promoter. As RifZ is a pathway-specific activator for the whole rif cluster, GlnR indirectly upregulates the whole rif cluster transcription by directly activating the rifZ expression. Secondly, GlnR specifically binds to the upstream region of rifK, which is also characterized to have its own promoter. It is well-known that RifK is a 3-amino-5-hydroxybenzoic acid (AHBA, the starter unit of rifamycin) synthase, thus GlnR can promote the supply of the rifamycin precursor by directly activating the rifK transcription. Notably, GlnR and RifZ independently activate the rifK transcription through binding to different sites in rifK promoter region, which suggests that the cells have a sophisticated regulatory mechanism to control the AHBA biosynthesis. Collectively, this study reveals that GlnR activates the rif cluster transcription in both direct (for rifZ and rifK) and indirect (for the whole rif cluster) manners, which well interprets the phenomenon that the NSE doesn’t occur in the glnR null mutant. Furthermore, this study deepens our understanding about the molecular mechanism of the NSE.
glnr positive transcriptional regulation of the phosphate specific transport system pstscab in Amycolatopsis Mediterranei u32Acta Biochimica et Biophysica Sinica, 2018Co-Authors: Yuhui Zhang, Guoping Zhao, Ying Wang, Zhi Hui Shao, Yixuan Zhang, Peng Li, Jing WangAbstract:
: Amycolatopsis Mediterranei U32 is an important industrial strain for the production of rifamycin SV. Rifampicin, a derivative of rifamycin SV, is commonly used to treat mycobacterial infections. Although phosphate has long been known to affect rifamycin biosynthesis, phosphate transport, metabolism, and regulation are poorly understood in A. Mediterranei. In this study, the functional phosphate transport system pstSCAB was isolated by RNA sequencing and inactivated by insertion mutation in A. Mediterranei U32. The mycelium morphology changed from a filamentous shape in the wild-type and pstS1+ strains to irregular swollen shape at the end of filamentous in the ΔpstS1 strain. RT-PCR assay revealed that pstSCAB genes are co-transcribed as a polycistronic messenger. The pstSCAB transcription was significantly activated by nitrate supplementation and positively regulated by GlnR which is a global regulator of nitrogen metabolism in actinomycetes. At the same time, the yield of rifamycin SV decreased after mutation (ΔpstS1) compared with wild-type U32, which indicated a strong connection among phosphate metabolism, nitrogen metabolism, and rifamycin production in actinomycetes.
Weihong Jiang – 2nd expert on this subject based on the ideXlab platform
nitrate stimulating effect in Amycolatopsis Mediterranei from discovery to mechanistic studiesChinese Journal of Biotechnology, 2015Co-Authors: Zhi Hui Shao, Weihong Jiang, Xiaoming Ding, Ying Wang, Jin Wang, Guoping ZhaoAbstract:
: Nitrate not only remarkably stimulates the rifamycinbiosynthesis in Amycolatopsis Mediterranei, but also influences the primary metabolisms, including the inhibition of fatty acids biosynthesis in the bacterial. This phenomenon has been designated as “Nitrate Stimulating Effect” by the late Prof. J.S. Chiaosince its discovery in the 1970’s, and has been found in many other antibiotics-producing actinomycetes subsequently. Based on the research in his laboratory, we have revealed that the nitrate stimulation effect mainly manifests in two aspects over the last two decades. First, nitrate promotes the supply of rifamycin precursors, e.g., UDP-glucose, AHBA, malonyl-CoA and methylmalonyl-CoA. Specifically, the biosynthesis of fatty acids is inhibited by nitrate consequently the acetyl-CoA is shunted into malonyl-CoA. Second, nitrate facilitates the expression of genes in the rifclulsterthat encodes rifamycin biosynthetic enzymes. Following our current understanding, the future research will focus on the signals, the signal transduction pathway and the molecular mechanisms that dictate nitrate-mediated transcriptional and post-translational regulations.
three of four glnr binding sites are essential for glnr mediated activation of transcription of the Amycolatopsis Mediterranei nas operonJournal of Bacteriology, 2013Co-Authors: Ying Wang, Weihong Jiang, Guoping Zhao, Zhi Hui Shao, Yinhua Lu, Jingzhi Wang, Hua Yuan, Jing WangAbstract:
In Amycolatopsis Mediterranei U32, genes responsible for nitrate assimilation formed one operon, nasACKBDEF, whose transcription is induced by the addition of nitrate. Here, we characterized GlnR as a direct transcriptional activator for the nas operon. The GlnR-protected DNA sequences in the promoter region of the nas operon were characterized by DNase I footprinting assay, the previously deduced Streptomyces coelicolor double 22-bp GlnR binding consensus sequences comprising a1, b1, a2, and b2 sites were identified, and the sites were then mutated individually to test their roles in both the binding of GlnR in vitro and the GlnR-mediated transcriptional activation in vivo. The results clearly showed that only three GlnR binding sites (a1, b1, and b2 sites) were required by GlnR for its specific binding to the nas promoter region and efficient activation of the transcription of the nas operon in U32, while the a2 site seemed unnecessary.
a complex role of Amycolatopsis Mediterranei glnr in nitrogen metabolism and related antibiotics productionArchives of Microbiology, 2007Co-Authors: Hao Yu, Weihong Jiang, Ruishen Jiao, Guoping ZhaoAbstract:
Amycolatopsis, genus of a rare actinomycete, produces many clinically important antibiotics, such as rifamycin and vancomycin. Although GlnR of Amycolatopsis Mediterranei is a direct activator of the glnA gene expression, the production of GlnR does not linearly correlate with the expression of glnA under different nitrogen conditions. Moreover, A. Mediterranei GlnR apparently inhibits rifamycin biosynthesis in the absence of nitrate but is indispensable for the nitrate-stimulating effect for its production, which leads to the hyper-production of rifamycin. When glnR of A. Mediterranei was introduced into its phylogenetically related organism, Streptomyces coelicolor, we found that GlnR widely participated in the host strain’s secondary metabolism, resemblance to the phenotypes of a unique S. coelicolorglnR mutant, FS2. In contrast, absence or increment in copy number of the native S. coelicolor glnR did not result in a detectable pleiotrophic effect. We thus suggest that GlnR is a global regulator with a dual functional impact upon nitrogen metabolism and related antibiotics production.
Juishen Chiao – 3rd expert on this subject based on the ideXlab platform
identification and functional analysis of a nitrate assimilation operon nasackbdef from Amycolatopsis Mediterranei u32Archives of Microbiology, 2011Co-Authors: Zhi Hui Shao, Juishen Chiao, Xiaoming Ding, Jin Wang, Guoping ZhaoAbstract:
Nitrate assimilation has been well studied for Gram-negative bacteria but not so much in the Gram-positive actinomycetes up to date. In a rifamycin SV-producing actinomycete, Amycolatopsis Mediterranei strain U32, nitrate not only can be used as a sole nitrogen source but also remarkably stimulates the antibiotic production along with regulating the related metabolic enzymes. A gene cluster of nasACKBDEF was cloned from a U32 genomic library by in situ hybridization screening with a heterogeneous nasB probe and confirmed later by whole genome sequence, corresponding to the protein coding genes of AMED_1121 to AMED_1127. These genes were co-transcribed as an operon, concomitantly repressed by ammonium while activated with supplement of either nitrate or nitrite. Genetic and biochemical analyses identified the essential nitrate/nitrite assimilation functions of the encoded proteins, orderly, the assimilatory nitrate reductase catalytic subunit (NasA), nitrate reductase electron transfer subunit (NasC), nitrate/nitrite transporter (NasK), assimilatory nitrite reductase large subunit (NasB) and small subunit (NasD), bifunctional uroporphyrinogen-III synthase (NasE), and an unknown function protein (NasF). Comparing rifamycin SV production and the level of transcription of nasB and rifE from U32 and its individual nas mutants in Bennet medium with or without nitrate indicated that nitrate assimilation function encoded by the nas operon played an essential role in the “nitrate stimulated” rifamycin production but had no effect upon the transcription regulation of the primary and secondary metabolic genes related to rifamycin biosynthesis.
bacterial type i glutamine synthetase of the rifamycin sv producing actinomycete Amycolatopsis Mediterranei u32 is the only enzyme responsible for glutamine synthesis under physiological conditionsActa Biochimica et Biophysica Sinica, 2006Co-Authors: Wentao Peng, Guoping Zhao, Juishen Chiao, Jin Wang, Ting Wu, Jianqiang HuangAbstract:
The structural gene for glutamine synthetase, glnA, from Amycolatopsis Mediterranei U32 was cloned via screening a genomic library using the analog gene from Streptomyces coelicolor. The clone was functionally verified by complementing for glutamine requirement of an Escherichia coli glnA null mutant under the control of a lac promoter. Sequence analysis showed an open reading frame encoding a protein of 466 amino acid residues. The deduced amino acid sequence bears significant homologies to other bacterial type I glutamine synthetases, specifically, 71% and 72% identical to the enzymes of S. coelicolor and Myco- bacterium tuberculosis, respectively. Disruption of this glnA gene in A. Mediterranei U32 led to glutamine auxotrophy with no detectable glutamine synthetase activity in vivo. In contrast, the cloned glnA + gene can complement for both phenotypes in trans. It thus suggested that in A. Mediterranei U32, the glnA gene encoding glutamine synthetase is uniquely responsible for in vivo glutamine synthesis under our laboratory defined physiological conditions.
a novel two component system amrb amkb involved in the regulation of central carbohydrate metabolism in rifamycin sv producing Amycolatopsis Mediterranei u32Current Microbiology, 2004Co-Authors: Weiwu Wang, Juishen Chiao, Guoping Zhao, Weihong JiangAbstract:
A novel two-component signal transduction system amrB-amkB was cloned from rifamycin SV-producing Amycolatopsis Mediterranei U32, and their biochemical functions as a response regulator and a histidine protein kinase, respectively, were proven. The amrB disruption mutant was generated by insertional inactivation with the aparmycin resistance gene. The metabolic response to the absence of amrB gene was determined by a biochemical profiling technique in which the concentration changes of metabolic intermediates were measured by gas chromatography with time-of-flight mass spectrometry (GC/TOF-MS). Although the phenotype analyses of the amrB gene disruption mutant showed no significant change with respect to rifamycin SV production and morphological differentiation, the global metabolomic analyses found the concentration levels of some key intermediates in the TCA cycle and glycolysis pathway were affected by an amrB gene disruption event. The primary results suggested that amrB-amkB genes might be involved in the regulation of central carbohydrate metabolism in A. Mediterranei U32.