Characterization of recombinant strains of the Clostridium acetobutylicum butyrate kinase inactivation mutant: Need for new phenomenological models for solventogenesis and butanol inhibition?

Abstract

Two metabolic engineering tools, namely gene inactivation and gene overexpression, were employed to examine the effects of two genetic modifications on the fermentation characteristics of Clostridium acetobutylicum. Inactivation of the butyrate kinase gene (buk) was examined using strain PJC4BK, while the combined effect of buk inactivation and overexpression of the aad gene-encoding the alcohol aldehyde dehydrogense (AAD) used in butanol formation-was examined using strain PJC4BK(pTAAD). The two strains were characterized in controlled pH > or = 5.0 fermentations, and by a recently enhanced method of metabolic flux analysis. Strain PJC4BK was previously genetically characterized, and fermentation experiments at pH > or = 5.5 demonstrated good, but not exceptional, solvent-production capabilities. Here, we show that this strain is a solvent superproducer in pH > or = 5.0 fermentations producing 225 mM (16.7 g/L) of butanol, 76 mM of acetone (4.4 g/L), and 57 mM (2.6 g/L) of ethanol. Strain PJC4BK(pTAAD) produced similar amounts of butanol and acetone but 98 mM (4.5 g/L) of ethanol. Both strains overcame the 180 mM (13 g/L) butanol toxicity limit, without any selection for butanol tolerance. Work with strain PJC4BK(pTAAD) is the first reported use of dual antibiotic selection in C. acetobutylicum. One antibiotic was used for selection of strain PJC4BK while the second antibiotic selected for the pTAAD presence. Overexpression of aad from pTAAD resulted in increased ethanol production but did not increase butanol titers, thus indicating that AAD did not limit butanol production under these fermentation conditions. Metabolic flux analysis showed a decrease in butyrate formation fluxes by up to 75% and an increase in acetate formation fluxes of up to 100% during early growth. The mean specific butanol and ethanol formation fluxes increased significantly in these recombinant strains, up to 300% and 400%, respectively. Onset of solvent production occurred during the exponential-growth phase when the culture optical density was very low and when total and undissociated butyric acid levels were <1 mM. Butyrate levels were low throughout all fermentations, never exceeding 20 mM. Thus, threshold butyrate concentrations are not necessary for solvent production in these stains, suggesting the need for a new phenomenological model to explain solvent formation.

Targeted gene disruption by use of a group II intron (targetron) vector in Clostridium acetobutylicum

“…As indicated in Figure 1C, buk mutant and solR mutant produced 44% and 37% more butanol than wild type, respectively. These results were consistent with the previously published data [7,8].…”

“…The gene buk, encoding the butyrate kinase, catalyzes the production of butyrate, and the gene solR located on the megaplasmid of the strain, encodes a putative repressor of solvent formation genes [7,8]. pSY6-buk and pSY6-solR vectors, constructed based on pSY6 ( Figure 1A and Supplementary information, Figure S1), were electroporated into C. acetobutylicum ATCC 824, respectively.…”

“…Without a proper replicon and/or promoter, the targetron plasmid pJIR750ai for C. perfringens from Sigma Aldrich was not applicable for gene disruption in the C. acetobutylicum directly (data not shown). Therefore, a modified targetron plasmid pSY6 was created by cloning the L1.LtrB group II intron fragment into the pIMP1-ptb, which was an E. coli-C. acetobutylicum shuttle vector containing a ptb (phosphotransbutyrylase) promoter [6].The gene buk, encoding the butyrate kinase, catalyzes the production of butyrate, and the gene solR located on the megaplasmid of the strain, encodes a putative repressor of solvent formation genes [7,8]. pSY6-buk and pSY6-solR vectors, constructed based on pSY6 ( Figure 1A and Supplementary information, Figure S1), were electroporated into C. acetobutylicum ATCC 824, respectively.…”

Comparative phenotypic analysis and genome sequence of Clostridium beijerinckii SA-1, an offspring of NCIMB 8052

“…SA-1 displays a metabolic carbon flow that is redirected mainly to the production of increased amounts of butanol and acetone. Redirecting the metabolic flow of carbon towards solvents to reach higher final butanol titres and yields has been shown previously by genetically modified C. acetobutylicum strains with disrupted genes responsible for butyric acid production (Harris et al, 2000;Jang et al, 2012b). Moreover, the overall butanol accumulation, ABE yields and solvent productivity of SA-1 are comparable to values previously reported for C. beijerinckii BA101, another butanol hyperproducing offspring of NCIMB 8052 (Formanek et al, 1997;Ezeji et al, 2007a, b;Qureshi & Blaschek, 2000;Lienhardt et al, 2002).…”

Lignocellulosic Biomass Utilization Toward Biorefinery Using Meshophilic Clostridial Species

“…Significant advances for C. acetobutylicum have been made to methods for gene integration [24]. Superior performance has also been demonstrated from genetically engineered derivatives of C. acetobutylicum ATCC 824 [25,26]. Methods based on a group II intron system for gene knockout have been described [27,28].…”