Improved electrocompetence and metabolic engineering of Clostridium pasteurianum reveals a new regulation pattern of glycerol fermentation

Abstract

Clostridium pasteurianum produces industrially valuable chemicals such as n-butanol and 1,3-propanediol from fermentations of glycerol and glucose. Metabolic engineering for increased yields of selective compounds is not well established in this microorganism. In order to study carbon fluxes and to selectively increase butanol yields, we integrated the latest advances in genome editing to obtain an electrocompetent Clostridium pasteurianum strain for further engineering. Deletion of the glycerol dehydratase large subunit (dhaB) using an adapted S. pyogenes Type II CRISPR/Cas9 nickase system resulted in a 1,3-propanediol-deficient mutant producing butanol as the main product. Surprisingly, the mutant was able to grow on glycerol as the sole carbon source. In spite of reduced growth, butanol yields were highly increased. Metabolic flux analysis revealed an important role of the newly identified electron bifurcation pathway for crotonyl-CoA to butyryl-CoA conversion in the regulation of redox balance. Compared to the parental strain, the electron bifurcation pathway flux of the dhaB mutant increased from 8 to 46% of the overall flux from crotonyl-CoA to butyryl-CoA and butanol, indicating a new, 1,3-propanediol-independent pattern of glycerol fermentation in Clostridium pasteurianum.

Keywords: 1,3‐propanediol, n‐butanol; CRISPR/Cas9 nickase; Clostridium pasteurianum; electron bifurcation pathway; glycerol metabolism.

Figure 1

Transformation efficiency of C. pasteurianum R525. Error bars indicate standard deviations from three experiments of transformation with plasmid pMTL85141. CFU = colony‐forming units.

Figure 2

Generation of a glycerol dehydratase (dhaB) deletion mutant of C. pasteurianum. (A) Genomic DNA region of C. pasteurianum to be modified. A 20 nt target sequence (N20) was chosen at the 5´end of dhaB for Cas9 nickase single‐strand break. Homology regions H1 and H2 flank the dhaB region to be deleted. (B) Glycerol dehydratase deletion vector pMTL85141‐Cas9n‐dhaB, containing Cas9 nickase, guide RNA targeting dhaB as well as an editing template, consisting of H1 + H2. (C) Screening for dhaB deletion. Genomic DNA from transformants was used as a template for PCR using primers as depicted in Fig. 5A. A 700 bp band indicates successful deletion of dhaB. Wt = R525 genomic DNA as template; H₂O = no template control.

Figure 3

Biomass and product formation by the dhaB mutant and its complementation strain compared to the parental strain R525. Strains were cultivated in Biebl medium with 5 g/L yeast extract. dhaB = dhaB deletion mutant, BuOH = butanol, AcAc = acetic acid, BuAc = butyric acid

Figure 4

Growth and product formation of dhaB deletion mutant on glycerol. BuOH = butanol, BuAc = butyric acid, AcAc = acetic acid, LaAc = lactic acid, EtOH = ethanol

Figure 5

Growth and product formation of dhaB deletion mutant on glycerol/glucose co‐substrate. BuOH = butanol, BuAc = butyric acid, AcAc = acetic acid, LaAc = lactic acid, EtOH = ethanol

Figure 6

Metabolic network and flux distribution from MFA of C. pasteurianum (R525/dhaB mutant) grown on glycerol. Solid lines with arrows indicate reactions with calculated rates, dashed lines with arrows indicate measured rates. Displayed fluxes are given relative to the glycerol uptake rate. Box A states the calculated percentage of r11 on the overall flux of Crotonyl‐CoA to Butyryl‐CoA (r11 + r12) for both strains. Box B contains the corresponding values for r12. 3HP = 3 Hydroxypropanal