Characterization of mutations induced by N-methyl-N′-nitro-N-nitrosoguanidine in an industrial Corynebacterium glutamicum strain

Trichoderma reesei (teleomorph Hypocrea jecorina) is the main industrial source of cellulases and hemicellulases harnessed for the hydrolysis of biomass to simple sugars, which can then be converted to biofuels such as ethanol and other chemicals. The highly productive strains in use today were generated by classical mutagenesis. To learn how cellulase production was improved by these techniques, we performed massively parallel sequencing to identify mutations in the genomes of two hyperproducing strains (NG14, and its direct improved descendant, RUT C30). We detected a surprisingly high number of mutagenic events: 223 single nucleotides variants, 15 small deletions or insertions, and 18 larger deletions, leading to the loss of more than 100 kb of genomic DNA. From these events, we report previously undocumented non-synonymous mutations in 43 genes that are mainly involved in nuclear transport, mRNA stability, transcription, secretion/vacuolar targeting, and metabolism. This homogeneity of functional categories suggests that multiple changes are necessary to improve cellulase production and not simply a few clear-cut mutagenic events. Phenotype microarrays show that some of these mutations result in strong changes in the carbon assimilation pattern of the two mutants with respect to the wild-type strain QM6a. Our analysis provides genome-wide insights into the changes induced by classical mutagenesis in a filamentous fungus and suggests areas for the generation of enhanced T. reesei strains for industrial applications such as biofuel production. biofuels ͉ biotechnology

show abstract

“…Interestingly, NTG mutagenesis is reported to lead mainly to A-T3G-C mutations (17), which is consistent with patterns observed here for NG14.…”

Section: Resultssupporting

confidence: 90%

Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using massively parallel DNA sequencing

Crom

Schackwitz

Pennacchio

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

175

170

View full text Add to dashboard Cite

show abstract

“…NTG induces a relatively wide spectrum of mutations by alkylating purines and pyrimidines. Most of the mutations induced by NTG are G ⅐ C 3 A ⅐ T transitions, but deletions and frameshifts occur to a lesser extent (24,38). The mutation frequencies were determined with the G-stretch reporter strain, and the mutated melA gene in this reporter strain can be reactivated only by a single-base frameshift mutation.…”

Section: Discussionmentioning

confidence: 99%

Indigenous and Environmental Modulation of Frequencies of Mutation in Lactobacillus plantarum

Machielsen

Alen-Boerrigter

Koole

et al. 2010

Appl Environ Microbiol

View full text Add to dashboard Cite

Reliability of microbial (starter) strains in terms of quality, functional properties, growth performance, and robustness is essential for industrial applications. In an industrial fermentation process, the bacterium should be able to successfully withstand various adverse conditions during processing, such as acid, osmotic, temperature, and oxidative stresses. Besides the evolved defense mechanisms, stress-induced mutations participate in adaptive evolution for survival under stress conditions. However, this may lead to accumulation of mutant strains, which may be accompanied by loss of desired functional properties. Defining the effects of specific fermentation or processing conditions on the mutation frequency is an important step toward preventing loss of genome integrity and maintaining the productivity of industrial strains. Therefore, a set of Lactobacillus plantarum mutator reporter strains suitable for qualitative and quantitative analysis of lowfrequency mutation events was developed. The mutation reporter system constructed was validated by using chemical mutagenesis (N-methyl-N-nitro-N-nitrosoguanidine) and by controlled expression of endogenous candidate mutator genes (e.g., a truncated derivative of the L. plantarum hexA gene). Growth at different temperatures, under low-pH conditions, at high salt concentrations, or under starvation conditions did not have a significant effect on the mutation frequency. However, incubation with sublethal levels of hydrogen peroxide resulted in a 100-fold increase in the mutation frequency compared to the background mutation frequency. Importantly, when cells of L. plantarum were adapted to 42°C prior to treatment with sublethal levels of hydrogen peroxide, there was a 10-fold increase in survival after peroxide treatment, and there was a concomitant 50-fold decrease in the mutation frequency. These results show that specific environmental conditions encountered by bacteria may significantly influence the genetic stability of strains, while protection against mutagenic conditions may be obtained by pretreatment of cultures with other, nonmutagenic stress conditions.

show abstract

“…Recently, there have been several reports on the use of omics analysis for the development of strains for amino acid production [19,20]. On the basis of more protein bands being shown in SYPA-9039 using SDS-PAGE, further metabolic engineering should be carried out.…”

Section: Discussionmentioning

confidence: 99%

Heterologous and homologous expression of the arginine biosynthetic argC~H cluster from Corynebacterium crenatum for improvement of l-arginine production

Rao

Yang

Xia

et al. 2012

Journal of Industrial Microbiology and Biotechnology

View full text Add to dashboard Cite

The genes involved in L: -arginine biosynthesis in Corynebacterium crenatum are organized as the argCJBDFRGH cluster like in Corynebacterium glutamicum. However, the argC~H cluster of the C. crenatum SYPA 5-5, which is an industrialized L: -arginine producer, had a lethal mutation occurring in the ArgR repressor encoding gene. The argC~H cluster with an inactive argR was overexpressed in E. coli and C. crenatum. In the recombinant E. coli JM109 enzyme activities were increased, and more L: -arginine was found in the supernatants from L: -glutamine. When the argC~H cluster was overexpressed in C. crenatum under its native promoter Parg, L: -arginine production was increased by 24.9%, but the presence of the recombinant plasmid pJC-9039 had a negative effect on cell growth. Surprisingly, the DO value of the recombinant strain dropped gently and stayed at a lower level from 24 h to the end of fermentation. The results demonstrated an increasing utilization of oxygen and the distinct enhancement of unit cell L: -arginine yields with the cluster argC~H-bearing in C. crenatum SYPA-9039. This study provides a kind of Corynebacteria with improved L: -arginine-producing ability and an efficient elevation for producing amino acid. Moreover, the promoter Parg would be used as a valid promoter to express objective genes for metabolic engineering in Corynebacteria.

show abstract

Characterization of mutations induced by N-methyl-N′-nitro-N-nitrosoguanidine in an industrial Corynebacterium glutamicum strain

Cited by 33 publications

References 26 publications

Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using massively parallel DNA sequencing

Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using massively parallel DNA sequencing

Indigenous and Environmental Modulation of Frequencies of Mutation in Lactobacillus plantarum

Heterologous and homologous expression of the arginine biosynthetic argC~H cluster from Corynebacterium crenatum for improvement of l-arginine production

Contact Info

Product

Resources

About