Isolation of thermotolerant mutants by using proofreading-deficient DNA polymerase .DELTA. as an effective mutator in Saccharomyces cerevisiae

Shimoda, Chikashi; Itadani, Akiko; Sugino, Akio; Furusawa, Mitsuru

doi:10.1266/ggs.81.391

Cited by 40 publications

(34 citation statements)

References 15 publications

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“…However, the temperatures suitable for conventional strains of Saccharomyces cerevisiae are relatively low (25 to 30°C). While screens for S. cerevisiae mutants able to produce ethanol efficiently at high temperature have been performed, only a modest increase in temperature has been obtained, 40°C maximum (35,40). Alternatively, attention has also focused on thermotolerant yeast species capable of producing ethanol at elevated temperatures.…”

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confidence: 99%

High-Temperature Ethanol Fermentation and Transformation with Linear DNA in the Thermotolerant Yeast Kluyveromyces marxianus DMKU3-1042

Nonklang

Abdel-Banat

Cha-aim

et al. 2008

Appl Environ Microbiol

197

136

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We demonstrate herein the ability of Kluyveromyces marxianus to be an efficient ethanol producer and host for expressing heterologous proteins as an alternative to Saccharomyces cerevisiae. Growth and ethanol production by strains of K. marxianus and S. cerevisiae were compared under the same conditions. K. marxianus DMKU3-1042 was found to be the most suitable strain for high-temperature growth and ethanol production at 45°C. This strain, but not S. cerevisiae, utilized cellobiose, xylose, xylitol, arabinose, glycerol, and lactose. To develop a K. marxianus DMKU3-1042 derivative strain suitable for genetic engineering, a uracil auxotroph was isolated and transformed with a linear DNA of the S. cerevisiae ScURA3 gene. Surprisingly, Ura ؉ transformants were easily obtained. By Southern blot hybridization, the linear ScURA3 DNA was found to have inserted randomly into the K. marxianus genome. Sequencing of one Lys ؊ transformant confirmed the disruption of the KmLYS1 gene by the ScURA3 insertion. A PCR-amplified linear DNA lacking K. marxianus sequences but containing an Aspergillus ␣-amylase gene under the control of the ScTDH3 promoter together with an ScURA3 marker was subsequently used to transform K. marxianus DMKU3-1042 in order to obtain transformants expressing Aspergillus ␣-amylase. Our results demonstrate that K. marxianus DMKU3-1042 can be an alternative cost-effective bioethanol producer and a host for transformation with linear DNA by use of S. cerevisiaebased molecular genetic tools.Ethanol production at high temperature has received much attention because fermentation processes conducted at elevated temperatures will significantly reduce cooling costs (14). Other advantages of elevated temperatures include more-efficient simultaneous saccharification and fermentation, a continuous shift from fermentation to distillation, reduced risk of contamination, and suitability for use in tropical countries (3,5,27). However, the temperatures suitable for conventional strains of Saccharomyces cerevisiae are relatively low (25 to 30°C). While screens for S. cerevisiae mutants able to produce ethanol efficiently at high temperature have been performed, only a modest increase in temperature has been obtained, 40°C maximum (35,40). Alternatively, attention has also focused on thermotolerant yeast species capable of producing ethanol at elevated temperatures. Isolates of Kluyveromyces marxianus appear to be particularly promising (3,5,20,27). This species has been reported to grow at 47°C (3), 49°C (20), and even 52°C (6) and to produce ethanol at temperatures above 40°C (14). Moreover, K. marxianus offers additional benefits (36) including a high growth rate (34) and the ability to utilize a wide variety of industrially relevant substrates such as sugar cane, corn silage juice, molasses, and whey powder (17,27,32,42,49). Because of these advantages, K. marxianus is currently being promoted as a viable alternative to S. cerevisiae as an ethanol producer. However, systematic comparison of the ethanol productivity of...

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mentioning

confidence: 99%

High-Temperature Ethanol Fermentation and Transformation with Linear DNA in the Thermotolerant Yeast Kluyveromyces marxianus DMKU3-1042

Nonklang

Abdel-Banat

Cha-aim

et al. 2008

Appl Environ Microbiol

197

136

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“…The GREACE method implants a pool of genetically modified proofreading elements into host cells to act as "evolution accelerator", thus provides multiple mutators with diverse characteristics which might guarantee accelerated evolution of host cells under diverse conditions. Accelerating microbial evolution by mutators with elevated mutation rates during genome replication have been reported previously [47][48][49]. All these studies used a single specific mutator with a certain mutation rate.…”

Section: Discussionmentioning

confidence: 97%

Genome Replication Engineering Assisted Continuous Evolution (GREACE) to Improve Microbial Tolerance for Biofuels Production

Luan¹,

Zhang²,

Li³

et al. 2015

New Microbial Technologies for Advanced Biofuels

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Background: Microbial production of biofuels requires robust cell growth and metabolism under tough conditions. Conventionally, such tolerance phenotypes were engineered through evolutionary engineering using the principle of "Mutagenesis followed-by Selection". The iterative rounds of mutagenesis-selection and frequent manual interventions resulted in discontinuous and inefficient strain improvement processes. This work aimed to develop a more continuous and efficient evolutionary engineering method termed as "Genome Replication Engineering Assisted Continuous Evolution" (GREACE) using "Mutagenesis coupled-with Selection" as its core principle. Results: The core design of GREACE is to introduce an in vivo continuous mutagenesis mechanism into microbial cells by introducing a group of genetically modified proofreading elements of the DNA polymerase complex to accelerate the evolution process under stressful conditions. The genotype stability and phenotype heritability can be stably maintained once the genetically modified proofreading element is removed, thus scarless mutants with desired phenotypes can be obtained. Kanamycin resistance of E. coli was rapidly improved to confirm the concept and feasibility of GREACE. Intrinsic mechanism analysis revealed that during the continuous evolution process, the accumulation of genetically modified proofreading elements with mutator activities endowed the host cells with enhanced adaptation advantages. We further showed that GREACE can also be applied to engineer n-butanol and acetate tolerances. In less than a month, an E. coli strain capable of growing under an n-butanol concentration of 1.25% was isolated. As for acetate tolerance, cell growth of the evolved E. coli strain increased by 8-fold under 0.1% of acetate. In addition, we discovered that adaptation to specific stresses prefers accumulation of genetically modified elements with specific mutator strengths. Conclusions: We developed a novel GREACE method using "Mutagenesis coupled-with Selection" as core principle. Successful isolation of E. coli strains with improved n-butanol and acetate tolerances demonstrated the potential of GREACE as a promising method for strain improvement in biofuels production.

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“…Genetic analysis showed that at least two stepwise mutations were necessary for acquiring this phenotype. Namely, the first mutation at hot1 locus provided 38.5℃-resistant properties, and by the addition of the second mutation (not identified) the mutant was able to make colonies at 40℃ [28] .…”

Section: Mutator Of Budding Yeastmentioning

confidence: 99%

“…In our previous simulation studies [6,7,15] and adaptive evolution experiments with living organisms [24,28] , we have never used another disparity-mutator which performed in biased-mutagenesis in the leading strand. There is a decent reason.…”

Section: Closing Remarksmentioning

confidence: 99%

The role of disparity-mutagenesis model on tumor development with special reference to increased mutation rate

Furusawa¹

2013

JST

Self Cite

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Human solid tumors are believed to have very high mutation rates at least in the early stage of extension. Irrespective of whether the increased mutation rate is a necessary condition for the tumor development or not, extremely high mutation rates such as in excess of the so-called "threshold" would before long result in the natural death of tumor cells. In reality, however, we are dying by cancer. Thus, it has been hypothesized that tumor cells should make a quick transition from the higher mutation state to the lower one. According to our "disparity-mutagenesis model", however, carcinogenesis could continue without any incident even under a prolonged period of high mutation rates. Namely, if lagging-strand-biased mutations far beyond the threshold of mutation rate are introduced in tumor cells, the tumor could progress to malignant extension without extinction. The results of evolution experiments using mutator microorganisms are discussed in terms of carcinogenesis.

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Isolation of thermotolerant mutants by using proofreading-deficient DNA polymerase .DELTA. as an effective mutator in Saccharomyces cerevisiae

Cited by 40 publications

References 15 publications

High-Temperature Ethanol Fermentation and Transformation with Linear DNA in the Thermotolerant Yeast Kluyveromyces marxianus DMKU3-1042

High-Temperature Ethanol Fermentation and Transformation with Linear DNA in the Thermotolerant Yeast Kluyveromyces marxianus DMKU3-1042

Genome Replication Engineering Assisted Continuous Evolution (GREACE) to Improve Microbial Tolerance for Biofuels Production

The role of disparity-mutagenesis model on tumor development with special reference to increased mutation rate

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