Random or Targeted Genetic Approaches for the Production of Isopropanol By Clostridium Beijerinckii DSM6423

Isopropanol (isopropyl alcohol) is a chemical compound widely used in cosmetics and for household use. It is mainly obtained by hydration of propylene but it can also be produced by microorganisms and used in a dehydrogenation process for biosourced propylene production. Despite their natural capacity to produce isopropanol, natural isopropanol producing strains such as Clostridium beijerinckiiDSM6423 (NRRL B-593 ; George et al 1983) have been poorly studied. Recently, few publications have shown that this strain was able to produce relatively high levels of a mixture  of isopropanol, butanol and ethanol either in continuous culture (Survase & Jurgens, 2011) or in an extractive fermentation system (Lopez-Contreras et al. 2013).

In order to improve the isopropanol production capacity of C. beijerinckii, two complementary approaches were investigated aiming at redirecting carbon fluxes toward this solvent. The first strategy consisted in combining random mutagenesis and genome shuffling. This technique is based on the possibility to mix entire genomes using protoplasts fusion of interesting mutated strains. It has already been successfully applied to C. acetobutylicum CICC 8012 (Gao et al., 2012) to improve yield and solvent titers of acetone-butanol fermentation. In our study (Mate de Gerando et al, 2016), we have been able to obtain 36 NTG mutants of C. beijerinckii DSM6423 on selective plates containing either high concentrations of isopropanol (up to 35 g/L), analogous substrates (ethyl-or methyl- bromobutyrate) or allyl alcohol, a well-known inhibitor of alcohol dehydrogenases. Considering stability and fermentation performances, the best NTG mutants were selected for the genome shuffling step. Three highly isopropanol tolerant strains able to withstand up to 50 g/L isopropanol were finally obtained. Moreover, the best strain produced up to 22% more isopropanol than the wild type. Genome analysis of the NTG and shuffled mutants revealed SNP’s mutations located in several genes involved in the central metabolism or the transcriptional regulation network of the strain.

In the second approach, we used the genetic tools developed for engineering Clostridia. Targeted modifications of C. beijerinckii DSM6423 were tested but multiple attempts to transform this strain failed. To understand the recalcitrance of the strain to genetic modifications, its genome was sequenced and the physical map detailed. Genome reconstruction showed the presence of two extrachromosmic elements, a double strand linear DNA bacteriophage and a natural plasmid which could explain the difficulties to introduce another mobile genetic element for genetic engineering.This work on C. beijerinckii DSM6423 lead us to better understand the genetic content of the strain and its IBE metabolic pathway, paving the way to future targeted engineering approaches.

References

George et al, 1983. Appl Environ Microbiol.45:1160-3

Survase et al, 2011. Appl Microbiol Biotechnol. 91:1305-13

Gao et al, 2012. Curr Microbiol. 2012 65:128-32

de Vrije et al, 2013. Bioresour Technol. 137:153-9.

Mate de Gerando et al 2016 Appl Microbiol Biotechnol DOI 10.1007/s00253-016-7302-5