Disruption of the Reductive 1,3-Propanediol Pathway Triggers Production of 1,2-Propanediol for Sustained Glycerol Fermentation by Clostridium pasteurianum

Abstract

Crude glycerol, the major by-product of biodiesel production, is an attractive bioprocessing feedstock owing to its abundance, low cost, and high degree of reduction. In line with the advent of the biodiesel industry, Clostridium pasteurianum has gained prominence as a result of its unique capacity to convert waste glycerol into n-butanol, a high-energy biofuel. However, no efforts have been directed at abolishing the production of 1,3-propanediol (1,3-PDO), the chief competing product of C. pasteurianum glycerol fermentation. Here, we report rational metabolic engineering of C. pasteurianum for enhanced n-butanol production through inactivation of the gene encoding 1,3-PDO dehydrogenase (dhaT). In spite of current models of anaerobic glycerol dissimilation, culture growth and glycerol utilization were unaffected in the dhaT disruption mutant (dhaT::Ll.LtrB). Metabolite characterization of the dhaT::Ll.LtrB mutant revealed an 83% decrease in 1,3-PDO production, encompassing the lowest C. pasteurianum 1,3-PDO titer reported to date (0.58 g liter ؊1 ). With 1,3-PDO formation nearly abolished, glycerol was converted almost exclusively to n-butanol (8.6 g liter ؊1 ), yielding a high n-butanol selectivity of 0.83 g n-butanol g ؊1 of solvents compared to 0.51 g n-butanol g ؊1 of solvents for the wild-type strain. Unexpectedly, high-performance liquid chromatography (HPLC) analysis of dhaT::Ll.LtrB mutant culture supernatants identified a metabolite peak consistent with 1,2-propanediol (1,2-PDO), which was confirmed by nuclear magnetic resonance (NMR). Based on these findings, we propose a new model for glycerol dissimilation by C. pasteurianum, whereby the production of 1,3-PDO by the wild-type strain and low levels of both 1,3-PDO and 1,2-PDO by the engineered mutant balance the reducing equivalents generated during cell mass synthesis from glycerol. IMPORTANCEOrganisms from the genus Clostridium are perhaps the most notable native cellular factories, owing to their vast substrate utilization range and equally diverse variety of metabolites produced. The ability of C. pasteurianum to sustain redox balance and glycerol fermentation despite inactivation of the 1,3-PDO pathway is a testament to the exceptional metabolic flexibility exhibited by clostridia. Moreover, identification of a previously unknown 1,2-PDO-formation pathway, as detailed herein, provides a deeper understanding of fermentative glycerol utilization in clostridia and will inform future metabolic engineering endeavors involving C. pasteurianum. To our knowledge, the C. pasteurianum dhaT disruption mutant derived in this study is the only organism that produces both 1,2-and 1,3-PDOs. Most importantly, the engineered strain provides an excellent platform for highly selective production of n-butanol from waste glycerol.A s a result of a 6-fold increase in global production between 2005 and 2012, biodiesel is presently the second most common biofuel in the world next to ethanol (1). This dramatic increase has generated an exceptional surplus of cru...

Towards improved butanol production through targeted genetic modification of Clostridium pasteurianum

“…Acetone formation was never observed in fermentations using the C. pasteurianum WT, in agreement with Biebl (2001), Sandoval et al (2015) and Pyne et al (2016). However, in the rex deletion mutant butyrate is visibly reassimilated which in the pathway proposed by Malaviya et al (2012) is coupled to acetone formation via the acetoacetyl- CoA: butyrate:CoA transferase in a similar fashion to C. acetobutylicum (Jones and Woods, 1986).…”

“…Reassimilation of butyrate without acetone production has been described in several C. acetobutylicum inactivational mutants, most notably mutants of ctfA (Millat et al, 2014), ptb (phosphotransbutrylase) and adc (acetoacetate decarboxylase) (Lehmann et al, 2012) and butyrate kinase ( buk ) gene (Desai et al, 1999). The fact that C. pasteurianum does not produce acetone under any condition tested here, or elsewhere (Biebl, 2001, Pyne et al, 2016, Sandoval et al, 2015), makes this organism an ideal target for investigations of the butyrate and acetate uptake mechanisms not reliant on CtfAB.…”

“…The conversion of glycerol to PDO involves a two-step transformation by glycerol dehydratase (DhaBCE), encoded by dhaBCE (CLPA_c22770-CLPA_c22760-CLPA_c22750) and PDO dehydrogenase (DhaT), encoded by dhaT (CLPA_c22740). Given the inability of Pyne et al (2016) to isolate a clean mutant of dhaT in the presence of the toxic intermediate 3-hydroxypropionaldehyde (3-HPA) (Barbirato et al, 1996, Maervoet et al, 2014), we chose to ablate PDO production by eliminating the synthesis of glycerol dehydratase through deletion of the entire dhaBCE operon. Accordingly, a KO cassette to achieve this was generated by SOE PCR, cloned into pMTL-KS15 and the resultant KO plasmid used to generate a dhaBCE mutant (CRG5532) by allelic exchange using our standard procedure (Table 1, Supplementary Fig.…”

“…This led to the search for, and apparent discovery of an additional ecotopic, sense strand, intron insertion in speA (encoding arginine decarboxylase) which was hypothesised as being essential. One consequence of these difficulties was that Pyne et al (2016) were unable to eliminate PDO production.…”

Genetically Engineered Strains: Application and Advances for 1,3-Propanediol Production from Glycerol

“…Besides, 1,3-PD has been applied widely in many other industrial fields, such as the production of composites, adhesives, laminates, coatings, mouldings, aliphatic polyesters, and antifreeze (12,13). 1,3-PD can be produced through two kinds of chemical pathways: the hydroformylation of ethylene oxide and the hydration of acrolein (11,14). However, many drawbacks like high consumption of energy, usage of expensive catalysts, and release of toxic intermediates increase its manufacturing cost by chemical methods.…”