Improvement of gougerotin and nikkomycin production by engineering their biosynthetic gene clusters

Du, Deyao; Zhu, Yu; Wei, Junhong; Tian, Yun; Niu, Guoqing; Tan, Huarong

doi:10.1007/s00253-013-4836-7

Cited by 49 publications

(44 citation statements)

References 32 publications

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“…The RNA samples were treated using RQ1 RNase-free DNase (Promega) to remove genomic DNA. Synthesis of cDNA and subsequent quantitative real-time RT-PCR were essentially the same as described previously (25), except that transcription of the 16S rRNA gene was used as the housekeeping gene reference.…”

Section: Rna Extraction and Quantitative Real-time Rt-pcrmentioning

confidence: 99%

GouR, a TetR Family Transcriptional Regulator, Coordinates the Biosynthesis and Export of Gougerotin in Streptomyces graminearus

Wei

Tian

Niu

et al. 2014

Appl Environ Microbiol

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Gougerotin is a peptidyl nucleoside antibiotic. It functions as a specific inhibitor of protein synthesis by binding ribosomal peptidyl transferase and exhibits a broad spectrum of biological activities. gouR, situated in the gougerotin biosynthetic gene cluster, encodes a TetR family transcriptional regulatory protein. Gene disruption and genetic complementation revealed that gouR plays an important role in the biosynthesis of gougerotin. Transcriptional analysis suggested that GouR represses the transcription of the gouL-to-gouB operon consisting of 11 structural genes and activates the transcription of the major facilitator superfamily (MFS) transporter gene (gouM). Electrophoresis mobility shift assays (EMSAs) and DNase I footprinting experiments showed that GouR has specific DNA-binding activity for the promoter regions of gouL, gouM, and gouR. Our data suggested that GouR modulates gougerotin production by coordinating its biosynthesis and export in Streptomyces graminearus.

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Section: Rna Extraction and Quantitative Real-time Rt-pcrmentioning

confidence: 99%

GouR, a TetR Family Transcriptional Regulator, Coordinates the Biosynthesis and Export of Gougerotin in Streptomyces graminearus

Wei

Tian

Niu

et al. 2014

Appl Environ Microbiol

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“…Gene overexpression has been an effective way to enhance antibiotic production (Liu et al 2015;Du et al 2013;Yuan et al 2011;Malla et al 2010). In this study, the overexpression of some key enzymes involved in the 6-DCT biosynthetic pathway in the highyield strain A6-9 explained the enhanced production of 6-DCT.…”

Section: Discussionmentioning

confidence: 79%

Protein expression analysis of a high-demeclocycline producing strain of Streptomyces aureofaciens and the roles of CtcH and CtcJ in demeclocycline biosynthesis

Lin

et al. 2016

Bioresour. Bioprocess.

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Background: Streptomyces aureofaciens strain A6-9, obtained with traditional mutagenesis, produces elevated levels of 6-DCT. The increased formation of 6-DCT may be attributable to the changes in the expression of some proteins in the 6-DCT biosynthetic pathway. For this reason, we explored the differences in protein expression between A6-9 and wild-type (WT) strains of Streptomyces aureofaciens, and based on the differences (CtcH and CtcJ were overexpressed in A6-9), investigated the roles of CtcH and CtcJ in biosynthesis.Results: Two-dimensional gel electrophoresis and a Mascot search indicated that some enzymes (including CtcH and CtcJ) involved in the primary and secondary metabolism were more strongly expressed in the high-6-DCT-yielding strain A6-9 than in the WT strain DT1. To examine the roles of CtcH and CtcJ in 6-DCT biosynthesis, ctcH-deleted, ctcJdeleted, ctcH-overexpressing, and ctcJ-overexpressing mutants and a mutant overexpressing both ctcH and ctcJ were constructed. Compared with WT, 6-DCT production was 50 and 37 % higher in the ctcH-overexpressing and ctcJ-overexpressing strains, respectively, and increased by 60 % in the ctcH-ctcJ-overexpressing strain. The ctcH-deleted and ctcJ-deleted strains produced almost no 6-DCT. Analysis of the metabolic flux distribution indicated that ctcH encodes a hydroxyacyl-CoA dehydrogenase and ctcJ encodes a monooxygenase that are essential for 6-DCT biosynthesis. Conclusion:Protein expression differs between high-6-DCT-yielding and WT strains, and the enzymes increased in the high-6-DCT-yielding strain explain the increased 6-DCT production. ctcH encodes a hydroxyacyl-CoA dehydrogenase and ctcJ encodes a monooxygenase that are essential for 6-DCT biosynthesis.

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“…4A). The second genetic element is a constitutive promoter, such as hrdBp, often used in Streptomyces genetic engineering (26). HrdB is a principal sigma factor that has been studied in S. griseus (40).…”

Section: Resultsmentioning

confidence: 99%

“…Iron-deficient medium was prepared by excluding ferrous sulfate from the production medium. The cell mass of the bacterial culture was measured by a modified method (26). A bacterial culture (50 ml) was centrifuged to sediment the cells.…”

Section: Methodsmentioning

confidence: 99%

A Branch Point of Streptomyces Sulfur Amino Acid Metabolism Controls the Production of Albomycin

Kulkarni

Zeng

Zhou

et al. 2016

Appl Environ Microbiol

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c Albomycin (ABM), also known as grisein, is a sulfur-containing metabolite produced by Streptomyces griseus ATCC 700974. Genes predicted to be involved in the biosynthesis of ABM and ABM-like molecules are found in the genomes of other actinomycetes. ABM has potent antibacterial activity, and as a result, many attempts have been made to develop ABM into a drug since the last century. Although the productivity of S. griseus can be increased with random mutagenesis methods, understanding of Streptomyces sulfur amino acid (SAA) metabolism, which supplies a precursor for ABM biosynthesis, could lead to improved and stable production. We previously characterized the gene cluster (abm) in the genome-sequenced S. griseus strain and proposed that the sulfur atom of ABM is derived from either cysteine (Cys) or homocysteine (Hcy). The gene product, AbmD, appears to be an important link between primary and secondary sulfur metabolic pathways. Here, we show that propargylglycine or iron supplementation in growth media increased ABM production by significantly changing the relative concentrations of intracellular Cys and Hcy. An SAA metabolic network of S. griseus was constructed. Pathways toward increasing Hcy were shown to positively impact ABM production. The abmD gene and five genes that increased the Hcy/Cys ratio were assembled downstream of hrdBp promoter sequences and integrated into the chromosome for overexpression. The ABM titer of one engineered strain, SCAK3, in a chemically defined medium was consistently improved to levels ϳ400% of the wild type. Finally, we analyzed the production and growth of SCAK3 in shake flasks for further process development. The Streptomyces genus was established at the beginning of the golden age of antibiotic discovery (1). Streptomyces griseus, a representative organism of the genus (2), was shown during this time to produce streptomycin (3), which has been clinically used to treat bacterial infections and other human diseases. It is well known that Streptomyces organisms in general have a large biosynthetic potential, being capable of producing many bioactive secondary metabolites-the S. griseus strains producing albomycin (ABM) are no exception. ABM was named by former Soviet Union scientists and underwent clinical investigations well before the information was released to the English-speaking scientific world (4). The biological activity and chemical constitution of ABM were later reported to be nearly identical to those of grisein, which was isolated from a distinct subtype of S. griseus by U.S. scientists in the 1940s (5-7). Despite its remarkable properties, continued studies of ABM were not pursued, partly because of the poor yields (8) and unpublicized research on the microorganism (9, 10). At present, the wealth of Streptomyces genomic information that is publically available provides the possibility of using a systems biology approach to uncover new aspects of Streptomyces metabolism and potentially overcome the production bottleneck.ABM and ABM-like secondary metabolites...

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Improvement of gougerotin and nikkomycin production by engineering their biosynthetic gene clusters

Cited by 49 publications

References 32 publications

GouR, a TetR Family Transcriptional Regulator, Coordinates the Biosynthesis and Export of Gougerotin in Streptomyces graminearus

GouR, a TetR Family Transcriptional Regulator, Coordinates the Biosynthesis and Export of Gougerotin in Streptomyces graminearus

Protein expression analysis of a high-demeclocycline producing strain of Streptomyces aureofaciens and the roles of CtcH and CtcJ in demeclocycline biosynthesis

A Branch Point of Streptomyces Sulfur Amino Acid Metabolism Controls the Production of Albomycin

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