Strain construction for enhanced production of spinosad via intergeneric protoplast fusion

2009 ◽  
Vol 55 (9) ◽  
pp. 1070-1075 ◽  
Author(s):  
Chao Wang ◽  
XiaoLin Zhang ◽  
Zhi Chen ◽  
Ying Wen ◽  
Yuan Song
Keyword(s):  
Protoplast Fusion ◽  
Wild Type Strain ◽  
Molecular Genetic ◽  
Industrial Applications ◽  
Streptomyces Avermitilis ◽  
Wild Type ◽  
Saccharopolyspora Spinosa ◽  
New Class ◽  
Enhanced Production ◽  
Microbial Strains

Spinosad is a new class of insecticides produced by Saccharopolyspora spinosa . The aim of this study was to construct a starch-utilizing strain that overproduced spinosad by intergeneric fusion between S. spinosa and Streptomyces avermitilis . Protoplast fusion is an important technique for engineering microbial strains, especially for microorganisms with few available molecular genetic tools. Protoplast fusion was conducted with UV-irradiated protoplasts of S. spinosa and S. avermitilis. Among 76 recombinants screened by ESI-MS and HPLC, a starch-utilizing strain F17, identified as S. spinosa, was obtained. The yield of spinosad in F17 was increased by 447.22%, compared with the yield of the wild-type strain. This is the first report of intergeneric protoplast fusion between S. spinosa and S. avermilitis, which shows great potential for industrial applications.

scholarly journals Enhanced production of Clavulanic acid by improving glycerol utilization using reporter-guided mutagenesis of an industrial Streptomyces clavuligerus strain

2021 ◽  
Author(s):  
Chang-Hun Shin ◽  
Hang Soo Cho ◽  
Hyung-Jin Won ◽  
Ho Jeong Kwon ◽  
Chan-Wha Kim ◽  
...  
Keyword(s):  
Clavulanic Acid ◽  
Wild Type Strain ◽  
Random Mutagenesis ◽  
Streptomyces Clavuligerus ◽  
Wild Type ◽  
Mutant Selection ◽  
Enhanced Production ◽  
Industrial Strains ◽  
Glycerol Utilization

Abstract Clavulanic acid (CA) produced by Streptomyces clavuligerus is a clinically important β-lactamase inhibitor. It is known that glycerol utilization can significantly improve cell growth and CA production of S. clavuligerus. We found that the industrial CA-producing S. clavuligerus strain OR generated by random mutagenesis consumes less glycerol than the wild-type strain; we then developed a mutant strain in which the glycerol utilization operon is overexpressed, as compared to the parent OR strain, through iterative random mutagenesis and reporter-guided selection. The CA production of the resulting S. clavuligerus ORUN strain was increased by approximately 31.3 per cent (5.21 ± 0.26 g/L) in a flask culture and 17.4 per cent (6.11 ± 0.36 g/L) in a fermenter culture, as compared to that of the starting OR strain. These results confirmed the important role of glycerol utilization in CA production and demonstrated that reporter-guided mutant selection is an efficient method for further improvement of randomly mutagenized industrial strains.

Amylase hyper-producing haploid recombinant strains of Thermomyces lanuginosus obtained by intraspecific protoplast fusion

2000 ◽  
Vol 46 (7) ◽  
pp. 669-673 ◽  
Author(s):  
K Rubinder ◽  
B S Chadha ◽  
S Singh ◽  
H S Saini
Keyword(s):  
Protoplast Fusion ◽  
Culture Filtrate ◽  
Catabolite Repression ◽  
Type Strain ◽  
Wild Type Strain ◽  
Thermomyces Lanuginosus ◽  
Wild Type ◽  
Haploid Strain ◽  
Recombinant Strains ◽  
Specific Activities

Amylase hyper-producing, catabolite-repression-resistant, recombinant strains were produced by intraspecific protoplast fusion of thermophilic fungus Thermomyces lanuginosus strains, using well-characterized, morphological, and 2-deoxy-D-glucose resistant markers. The fusant heterokaryons exhibited enhanced amylase activities as compared to the amylase hyper-producing parental strain (T2). Diploids derived from heterokaryons segregated to stable haploid recombinant strains. In the haploid strain (Tlh 4q), approximately 5-fold higher specific activities of α-amylase and glucoamylase in the culture filtrate were observed as compared to the wild-type strain (W0).Key words: Thermomyces lanuginosus, protoplast fusion, amylase hyper-producing strain, catabolite repression.

scholarly journals Identification and Gene Disruption of Small Noncoding RNAs in Streptomyces griseus

2009 ◽  
Vol 191 (15) ◽  
pp. 4896-4904 ◽  
Author(s):  
Takeaki Tezuka ◽  
Hirofumi Hara ◽  
Yasuo Ohnishi ◽  
Sueharu Horinouchi
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Noncoding Rnas ◽  
Streptomyces Griseus ◽  
Streptomyces Avermitilis ◽  
Dependent Manner ◽  
Wild Type ◽  
Srna Candidate ◽  
Small Noncoding Rnas ◽  
Nuclease Mapping

ABSTRACT Small noncoding RNAs (sRNAs) have been shown to control diverse cellular processes in prokaryotes. To identify and characterize novel bacterial sRNAs, a gram-positive, soil-inhabiting, filamentous bacterium, Streptomyces griseus, was examined, on the assumption that Streptomyces should express sRNAs as important regulators of morphological and physiological differentiation. By bioinformatics investigation, 54 sRNA candidates, which were encoded on intergenic regions of the S. griseus chromosome and were highly conserved in those of both Streptomyces coelicolor A3(2) and Streptomyces avermitilis, were selected. Of these 54 sRNA candidates, 17 transcripts were detected by Northern blot analysis of the total RNAs isolated from cells grown on solid medium. Then, the direction of transcription of each sRNA candidate gene was determined by S1 nuclease mapping, followed by exclusion of four sRNA candidates that were considered riboswitches of their downstream open reading frames (ORFs). Finally, a further sRNA candidate was excluded because it was cotranscribed with the upstream ORF determined by reverse transcription-PCR. Thus, 12 sRNAs ranging in size from 40 to 300 nucleotides were identified in S. griseus. Seven of them were apparently transcribed in a growth phase-dependent manner. Furthermore, of the 12 sRNAs, the expression profiles of 7 were significantly influenced by a mutation of adpA, which encodes the central transcriptional regulator of the A-factor regulatory cascade involved in both morphological differentiation and secondary metabolism in S. griseus. However, disruption of all 12 sRNA genes showed no detectable phenotypic changes; all the disruptants grew and formed aerial mycelium and spores with the same time course as the wild-type strain on various media and produced streptomycin similarly to the wild-type strain.

Effects of Acuc on the Growth Development and Spinosad Biosynthesis of Saccharopolyspora Spinosa

2021 ◽  
Author(s):  
Zhudong Liu ◽  
Jie Xiao ◽  
Jianli Tang ◽  
Yang Liu ◽  
Ling Shuai ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Type Strain ◽  
Wild Type Strain ◽  
Shuttle Vector ◽  
Type I ◽  
Wild Type ◽  
Saccharopolyspora Spinosa ◽  
Fermentation Products ◽  
Overexpression Strain ◽  
Mutant Strains

Abstract Background: The interaction between acuC and spinosad biosynthesis is complex. In this study, acetoin utilization protein (acuC) was characterized. It is a type I histone deacetylase that is highly conserved in bacteria. This study first explored the effect of acuC on the growth and development of secondary metabolites of S. spinosa. Results: The knockout strain and overexpression strain were constructed separately with the shuttle vector pOJ260. The overexpression of the acuC gene affects the growth and phenotype of S. spinosa. Moreover, the spore production ability of the S. spinosa-acuC strain on solid medium was weaker than that of the wild-type strain. HPLC analysis of the fermentation products for the wild-type and mutant strains demonstrated that the yield of the overexpression strain was 87% higher than that of the wild-type strain. Conclusions: We concluded that the overexpression of acuC positively regulated the biosynthesis of spinosad and affected the acetylation pathway and the growth of S. spinosa. A comparative proteomic analysis between the wild-type and overexpression strains revealed related genes in different metabolic pathways that were affected. We envision that these results can be extended to other actinomycetes for secondary metabolite improvement.

MUTANTS OF METARHIZIUM ANISOPLIAE WITH INCREASE VIRULENCE TOWARD MOSQUITO LARVAE

1978 ◽  
Vol 20 (2) ◽  
pp. 211-219 ◽  
Author(s):  
Karen Al-Aidroos ◽  
Donald W. Roberts
Keyword(s):  
Metarhizium Anisopliae ◽  
Culex Pipiens ◽  
Type Strain ◽  
Wild Type Strain ◽  
Spontaneous Mutant ◽  
Wild Type ◽  
Culex Pipiens Pipiens ◽  
Enhanced Production ◽  
Imperfect Fungus

A wild-type strain of Metarhizium anisopliae (Metsch.) Sorokin, an imperfect fungus, killed 76% of Culex pipiens pipiens larvae within five days after spore administration. A spontaneous mutant strain was selected whose LD50 was less than half that of the wild type, and whose LT50 was one day faster. This mutant also showed early, dense sporulation, rapid in vitro spore germination, and enhanced production of destruxins. It is probable that at least one of these additional characteristics is related to the mutation to hypervirulence.

scholarly journals Effects of acuC on the growth development and spinosad biosynthesis of Saccharopolyspora spinosa

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Zhudong Liu ◽  
Jie Xiao ◽  
Jianli Tang ◽  
Yang Liu ◽  
Ling Shuai ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Posttranslational Modification ◽  
Wild Type Strain ◽  
Shuttle Vector ◽  
Underlying Mechanism ◽  
Type I ◽  
Wild Type ◽  
Saccharopolyspora Spinosa ◽  
Proteomics Analysis ◽  
Overexpression Strain

Abstract Background Acetoin utilization protein (acuC) is a type I histone deacetylase which is highly conserved in bacteria. The acuC gene is related to the acetylation/deacetylation posttranslational modification (PTM) system in S. spinosa. Spinosyns, the secondary metabolites produced by Saccharopolyspora spinosa, are the active ingredients in a family of insect control agents. However, the specific functions and influences of acuC protein in S. spinosa are yet to be characterized. Results The knockout strain and overexpression strain were constructed separately with the shuttle vector pOJ260. The production of spinosyns A and D from S. spinosa-acuC were 105.02 mg/L and 20.63 mg/L, which were 1.82-fold and 1.63-fold higher than those of the wild-type strain (57.76 mg/L and 12.64 mg/L), respectively. The production of spinosyns A and D from S. spinosa-ΔacuC were 32.78 mg/L and 10.89 mg/L, respectively. The qRT-PCR results of three selected genes (bldD, ssgA and whiA) confirmed that the overexpression of acuC affected the capacities of mycelial differentiation and sporulation. Comparative proteomics analysis was performed on these strains to investigate the underlying mechanism leading to the enhancement of spinosad yield. Conclusions This study first systematically analysed the effects of overexpression acuC on the growth of S. spinosa and the production of spinosad. The results identify the differentially expressed proteins and provide evidences to understand the acetylation metabolic mechanisms which can lead to the increase of secondary metabolites.

The G243D mutation (afsB mutation) in the principal sigma factor σ HrdB alters intracellular ppGpp level and antibiotic production in Streptomyces coelicolor A3(2)

Microbiology ◽  
2010 ◽  
Vol 156 (8) ◽  
pp. 2384-2392 ◽  
Author(s):  
Guojun Wang ◽  
Yukinori Tanaka ◽  
Kozo Ochi
Keyword(s):  
Sigma Factor ◽  
Parent Strain ◽  
Wild Type Strain ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Fine Tuning ◽  
Wild Type ◽  
New Class ◽  
In The Wild ◽  
Amino Acid Depletion

Deficient antibiotic production in an afsB mutant, BH5, of Streptomyces coelicolor A3(2) was recently shown to be due to a mutation (G243D) in region 1.2 of the primary sigma factor σ HrdB. Here we show that intracellular ppGpp levels during growth, as well as after amino acid depletion, in the mutant BH5 are lower than those of the afsB+ parent strain. The introduction of certain rifampicin resistance (rif) mutations, which bypassed the requirement of ppGpp for transcription of pathway-specific regulatory genes, actII-ORF4 and redD, for actinorhodin and undecylprodigiosin, respectively, completely restored antibiotic production by BH5. Antibiotic production was restored also by introduction of a new class of thiostrepton-resistance (tsp) mutations, which provoked aberrant accumulation of intracellular ppGpp. Abolition of ppGpp synthesis in the afsB tsp mutant Tsp33 again abolished antibiotic production. These results indicate that intracellular ppGpp level is finely tuned for successful triggering of antibiotic production in the wild-type strain, and that this fine tuning was absent from the afsB mutant BH5, resulting in a failure to initiate antibiotic production in this strain.

Isolation and differential expression of β-1,3-glucanase messenger RNAs, SrGLU3 and SrGLU4, following inoculation of Sesbania rostrata

2006 ◽  
Vol 33 (11) ◽  
pp. 983 ◽  
Author(s):  
Chi-Te Liu ◽  
Toshihiro Aono ◽  
Misako Kinoshita ◽  
Hiroki Miwa ◽  
Taichiro Iki ◽  
...  
Keyword(s):  
Differential Expression ◽  
Type Strain ◽  
Wild Type Strain ◽  
Sesbania Rostrata ◽  
Messenger Rnas ◽  
Azorhizobium Caulinodans ◽  
Wild Type ◽  
Transcript Accumulation ◽  
Ethylene Treatment ◽  
New Class

We report here the isolation and characterisation of two new β-1,3-glucanase cDNAs, SrGLU3 and SrGLU4, from a tropical legume Sesbania rostrata Bremek. & Oberm., which form N2-fixing nodules on the stem after infection by Azorhizobium caulinodans. SrGLU3 was characterised as being grouped in a branch with tobacco class I β-1,3-glucanases, where the isoforms were reported to be induced by either pathogen infection or ethylene treatment. SrGLU4 was characterised as separate from other classes, and we propose this new branch as a new class (Class VI). The SrGLU3 gene was constitutively expressed in normal stem nodules induced by the wild type strain of A. caulinodans (ORS571), and also even in immature stem nodules induced by a mutant (ORS571-C1), which could not form mature stem-nodules. In contrast, the transcript accumulation of SrGLU4 was hardly detectable in immature nodules inoculated by the ORS571-C1 mutant. We suggest that S. rostrata makes use of SrGLU4 to discriminate between symbionts and non-symbionts (mutants) in developing nodules. We propose the SrGLU4 gene as a new nodulin during nodulation.

scholarly journals Identification of ε-Poly-L-lysine as an Antimicrobial Product from an Epichloë Endophyte and Isolation of Fungal ε-PL Synthetase Gene

Molecules ◽  
2020 ◽  
Vol 25 (5) ◽  
pp. 1032 ◽  
Author(s):  
Enkhee Purev ◽  
Tatsuhiko Kondo ◽  
Daigo Takemoto ◽  
Jennifer T. Niones ◽  
Makoto Ojika
Keyword(s):  
Type Strain ◽  
Plant Pathogens ◽  
Wild Type Strain ◽  
Bioactive Metabolites ◽  
Wild Type ◽  
Enhanced Production ◽  
Enhanced Expression ◽  
In The Wild ◽  
Eukaryotic Genomes ◽  
Epichloë Endophyte

The endophytic fungus Epichloë festucae is known to produce bioactive metabolites, which consequently protect the host plants from biotic and abiotic stresses. We previously found that the overexpression of vibA (a gene for transcription factor) in E. festucae strain E437 resulted in the secretion of an unknown fungicide. In the present study, the active substance was purified and chemically identified as ε-poly-L-lysine (ε-PL), which consisted of 28–34 lysine units. The productivity was 3.7-fold compared with that of the wild type strain E437. The isolated ε-PL showed inhibitory activity against the spore germination of the plant pathogens Drechslera erythrospila, Botrytis cinerea, and Phytophthora infestans at 1–10 μg/mL. We also isolated the fungal gene “epls” encoding ε-PL synthetase Epls. Overexpression of epls in the wild type strain E437 resulted in the enhanced production of ε-PL by 6.7-fold. Interestingly, overexpression of epls in the different strain E. festucae Fl1 resulted in the production of shorter ε-PL with 8–20 lysine, which exhibited a comparable antifungal activity to the longer one. The results demonstrate the first example of ε-PL synthetase gene from the eukaryotic genomes and suggest the potential of enhanced expression of vibA or/and epls genes in the Epichloë endophyte for constructing pest-tolerant plants.

scholarly journals Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 383-391 ◽  
Author(s):  
Yasumasa Tsukamoto ◽  
Jun-ichi Kato ◽  
Hideo Ikeda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quantitative Analysis ◽  
Structural Analysis ◽  
Recombination Rate ◽  
Type Strain ◽  
Negative Selection ◽  
Wild Type Strain ◽  
Illegitimate Recombination ◽  
Wild Type ◽  
In The Wild

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

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