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UVC-mutagenesis in acetogens: resistance to methanol, ethanol, acetone, or n-butanol in recombinants with tailored genomes as the step in engineering of commercial biocatalysts for continuous CO2/H2 blend fermentations

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Abstract

Time- and cost-efficient six-step UVC-mutagenesis was developed and validated to generate acetogen mutants with preliminary reduced genomes to prevent product inhibition in the to-be-engineered commercial biocatalysts. Genome reduction was performed via elimination of pta, ack, spo0A, spo0J and some pro-phage genes. UVC-mutants such as Clostridium sp. MT1784RG, Clostridium sp. MT653RG, Clostridium sp. MT896RG, and Clostridium sp. MT1962RG (all 4 share 97 % DNA homology with Clostridium ljungdahlii ATCC 55383) were selected based on resistance to methanol (3 M), ethanol (3.6 M), acetone (2.5 M), or n-butanol (0.688 M), respectively. As a part of the biocatalyst engineering algorithm, genome reduction step was associated with integration of attTn7 recognition sequence to the chromosomes of each of the above strains to prepare the defined integration sites for future integration of multi-copy synthetic operons encoding biosynthesis of methanol, ethanol, acetone or n-butanol. Reduced genome mutants had cell duplication times decreased compared to the same for the respective parental strains. All groups of mutants had decreased share of palmitic (C16:0) and increased share of oleic (C18:1) acids along with detection of isopropylstearate (C20) compared to the parental strains. Mutants resistant to acetone and n-butanol also had monounsaturated fatty acid (C20:1) not found in parental strains. Cyclopropane fatty acid (C21) was identified only in n-butanol resistant mutants.

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Acknowledgments

The research was supported by the angel funds of MT family friends. Biocatalyst Gurus, Inc,. is the sole distributor of the electroporation and electrofusion equipment, custom multi-fermentor modules and MT254UVnator: www.syngasbiofuelsenergy.com.

Conflict of interest

The authors declare that they have no conflict of interest.

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Author notes

  1. Michael Kiriukhin & Eugene Gak

    Present address: Ajinomoto-Genetika Research Institute, 1st Dorozhny Pr. 1-1, 117545, Moscow, Russia

Authors and Affiliations

  1. Syngas Biofuels Energy, Inc., P.O. Box 300819, Houston, TX, 77230, USA

    Michael Tyurin

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  1. Michael Kiriukhin
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Correspondence to Michael Tyurin.

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Kiriukhin, M., Tyurin, M. & Gak, E. UVC-mutagenesis in acetogens: resistance to methanol, ethanol, acetone, or n-butanol in recombinants with tailored genomes as the step in engineering of commercial biocatalysts for continuous CO2/H2 blend fermentations. World J Microbiol Biotechnol 30, 1559–1574 (2014). https://doi.org/10.1007/s11274-013-1579-7

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  • DOI: https://doi.org/10.1007/s11274-013-1579-7

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