Genetic improvement of processes yielding microbial products

Adrio, José L.; Demain, Arnold L.

doi:10.1111/j.1574-6976.2005.00009.x

Cited by 201 publications

(113 citation statements)

References 309 publications

(276 reference statements)

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“…Although it has been more than 60 years since the first penicillinoverproducing mutant was obtained by X-ray treatment of P. chrysogenum X-1612, 190 conventional breeding (random mutagenesis and massive tracing of strains) remains the preferred method when molecular genetic tools have not been developed for the producer microorganism. 191 Nutritional repression can be decreased by mutation to antimetabolite resistance.…”

Section: Strain Improvement By Avoiding or Removing Ccrmentioning

confidence: 99%

Carbon source regulation of antibiotic production

et al. 2010

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Section: Strain Improvement By Avoiding or Removing Ccrmentioning

confidence: 99%

Carbon source regulation of antibiotic production

et al. 2010

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“…Genetic markers are an indispensable component of modern molecular cloning strategies, because they facilitate the high-efficiency screening of target-trans formants. The two most widely used genetic markers are antibiotic resistance marker and biosynthetic marker (27). Among these, antibiotic resistance markers are widely applied in the genetic manipulation of yeast (28).…”

Section: Discussionmentioning

confidence: 99%

Amplification, Sequencing and Cloning of Iranian Native Bacillus subtilis Alpha-amylase Gene in Saccharomyces cerevisiae

Afzaljavan

Mobini-Dehkordi

2013

Jundishapur J Microbiol

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Background: Alpha-amylases are digestive enzymes which hydrolyze starch glycosidic bonds to glucose, maltose, maltotriose and dextrin which have diverse applications in a wide range of industries such as food, textile, paper, detergents representing approximately 30% of the world enzyme production. Objectives: In this study, the gene encoding the alpha-amylase enzyme of native isolated Bacillus subtilis was amplified with specific primers containing of NotI and AscI restriction sites by PCR and then sequenced. Purified PCR product and shuttle episomal vector p316TDH3 were cut by restriction enzymes and cloned into Escherichia coli and yeast hosts. Materials and Methods:The haploid auxotroph (ura3-) strain of Saccharomyces cerevisiae and p316TDH3 were used as the host and vector for cloning and expression of the alpha amylase gene, respectively. In native Bacillus sp. the amyE gene without signal sequence was amplified with specific primers that introduced AscI and NotI restriction sites. After constructing the recombinant plasmid, it was transformed into E. coli competent cells. Then, colonies selection and confirmation were performed and the extracted plasmid was introduced to competent yeast cells using carrier sperm DNA. Recombinant yeast cells could grow on minimal media and produce extracellular enzyme. Results:The presence of alpha-amylase gene in recombinant bacteria was certificated by colony-PCR method. After extraction of recombinant vector from E. coli, the competent S. cerevisiae cells were transformed using polyethylene glycol and carrier sperm DNA. The recombinant yeast strains were screened by URA3 auxotrophic marker and analyzed for alpha-amylase gene existence. In the other hand, the amylase gene length of native B. subtilis was 1887 base pairs (bp) with an approximately 93.65% similarity with standard bacterial strain. Conclusions: Based on this similarity and our bioinformatics evaluations, this mentioned alpha-amylase gene can be expressed in S. cerevisiae as extracellular enzyme.

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“…The process of mutagenesis and rational selection has been utilized for improvement of industrially important strains for overproduction of desired metabolites [19,20,37]. In our previous study, an ethyl methane sulfonate (EMS) induced (exposure at 20 ll ml -1 for 30 min with 92 % reduction in viability), atorvastatin (20 lg ml -1 ) tolerating mutant EA22 was generated from S. johnsonii ATCC-20490 with 2-fold improvement in CoQ 10 titer (HPTLC method) [17].…”

Section: Mutagenesis Selection Of Mutants and Screening In Shake Flaskmentioning

confidence: 99%

Morphological and Molecular Differentiation of Sporidiobolus johnsonii ATCC 20490 and Its Coenzyme Q10 Overproducing Mutant Strain UF16

et al. 2014

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Coenzyme Q 10 (CoQ 10 ) is an industrially important molecule having nutraceutical and cosmeceutical applications. CoQ 10 is mainly produced by microbial fermentation and the process demands the use of strains with high productivity and yields of CoQ 10 . During strain improvement program consisting of sequential induced mutagenesis, rational selection and screening process, a mutant strain UF16 was generated from Sporidiobolus johnsonii ATCC 20490 with 2.3-fold improvements in CoQ 10 content. EMS and UV rays were used as mutagenic agents for generating UF16 and it was rationally selected based on atorvastatin resistance as well as survival at free radicals exposure. We investigated the genotypic and phenotypic changes in UF16 in order to differentiate it from wild type strain. Morphologically it was distinct due to reduced pigmentation of colony, reduced cell size and significant reduction in mycelial growth forms with abundance of yeast forms. At molecular level, UF16 was differentiated based on PCR fingerprinting method of RAPD as well as large and small-subunit rRNA gene sequences.Rapid molecular technique of RAPD analysis using six primers showed 34 % polymorphic fragments with mean genetic distance of 0.235. The partial sequences of rRNAgene revealed few mutation sites on nucleotide base pairs. However, the mutations detected on rRNA gene of UF16 were less than 1 % of total base pairs and its sequence showed 99 % homology with the wild type strain. These mutations in UF16 could not be linked to phenotypic or genotypic changes on CoQ 10 biosynthetic pathway that resulted in improved yield. Hence, investigating the mutations responsible for deregulation of CoQ 10 pathway is essential to understand the cause of overproduction in UF16. Phylogenetic analysis based on RAPD bands and rRNA gene sequences coupled with morphological variations, exhibited the novelty of mutant UF16 having potential for improved CoQ 10 production.

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Genetic improvement of processes yielding microbial products

Cited by 201 publications

References 309 publications

Carbon source regulation of antibiotic production

Carbon source regulation of antibiotic production

Amplification, Sequencing and Cloning of Iranian Native Bacillus subtilis Alpha-amylase Gene in Saccharomyces cerevisiae

Morphological and Molecular Differentiation of Sporidiobolus johnsonii ATCC 20490 and Its Coenzyme Q10 Overproducing Mutant Strain UF16

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