Unravelling the roadblocks to 1,2-propanediol biosynthesis in select solventogenicClostridiumspecies

Abstract

BackgroundThe compound 1,2-propanediol is an important industrial bulk chemical that has proven particularly recalcitrant to bio-production. Solvent-producingClostridiumspecies represent promising candidates for engineering 1,2-propaediol production. Co-production of 1,2-popanediol and butanol has the potential to improve the economics of the acetone-butanol-ethanol (ABE) fermentation.ResultsIn this study, the methylglyoxal synthase gene (mgsA) fromClostridium beijerinckiiNCIMB 8052 was homologously expressed in this organism. Additionally, a separate strain ofClostridium beijerinckiiNCIMB 8052 was engineered by cloning and expressingmgsAand methylglyoxal/glyoxal reductase (mgR) fromClostridium pasteurianumATCC 6013 as a fused protein linked by polyglycine linker in the former. Both strains ofC. beijerinckiiNCIMB 8052 failed to produce 1,2-propaneol. Instead, traces of acetol—the precursor of 1,2-propanediol—were detected in cultures of both strains. When the recombinant strains were exposed to acetol, both strains exhibited ∼100% acetol-to-1,2-propanediol conversion efficiency. Conversely, methylglyoxal supplementation led to the production of traces of acetol but not lactaldehyde or 1,2-propanediol. When wildtypeC. beijerinckiiNCIMB 8052,C. pasteurianumATCC 6013 andClostridium tyrobutyricumATCC 25755 were challenged with methylglyoxal,C. beijerinckiiproduced ∼0.1 g/L (S)-(+)-1,2-Propanediol, whileC. tyrobutyricumproduced traces of lactate.C. pasteurianumproduced neither 1,2-propanediol nor lactate. The wild types of all three species above exhibited ∼100% acetol-to-1,2-propanediol conversion efficiency. The recombinant strain ofC. beijerinckiiexpressing fused MgsA and MgR fromC. pasteurianumATCC 6013 showed enhanced growth and solvent production, producing as high as 88% more butanol on both glucose and lactose than the control strain and the recombinant strain of the same organism expressing the native MgsA.ConclusionsRecombinant and native strains ofC. beijerinckii,C. pasteurianumandC. tyrobutyricumstudied in this work exhibit extremely poor capacity to catalyze the conversion of the intermediates of the methylglyoxal bypass to 1,2-propanediol. This is indicative of lack of appropriate enzymes to catalyze the reactions from methylglyoxal to acetol or lactaldehyde. Inability to detect methylglyoxal in the recombinant strains harboringmgsA(both homologous and heterologous)— whereas the strain expressing bothmgsAandmgRfromC. pasteurianum, under the same promoter (Padc) produced higher concentrations of butanol—suggests thatC. beijerinckiimight possess a regulatory mechanism that limits the activity of methylglyoxal-producing MgsA. The protein product ofmgRfromC. pasteurianumrepresents a promising metabolic engineering candidate towards increasing butanol production.

Transcriptomic characterization of recombinantClostridium beijerinckiiNCIMB 8052 expressing methylglyoxal synthase and glyoxal reductase fromClostridium pasteurianumATCC 6013

“…However, it is not clear what engendered this trend in C. beijerinckii _mgsA+mgR in comparison to C. beijerinckii _p459. Nonetheless, it is conceivable that greater reliance on organic nitrogen sources (peptides) by C. beijerinckii _mgsA+mgR, coupled with significantly improved lactose uptake and catabolism in this strain, led to improved growth and plausibly, enhanced solventogenesis when compared to C. beijerinckii _p459 (16). Furthermore, biosynthesis of purines and pyrimidines is ‘resource- and energy-intensive’, hence, salvaging these molecules from the immediate environment reduces cellular metabolic burden, which promotes growth.…”

“…Conversely, lower concentrations of iron favor sugar utilization, growth and ABE production (49). Notably, C. beijerinckii _mgsA+mgR consumed 183.33% more lactose than C. beijerinckii _p459 (16). It is important to highlight that both C. beijerinckii _mgsA+mgR and C. beijerinckii _p459 were grown in the same medium containing 10 mg/L FeSO 4 .7H 2 O.…”

“…Possible formation of iron binding MG-protein complexes may account at least in part, for inability to detect MG in cultures of C. beijerinckii _mgsA+mgR. Additionally, co-expression of mgsA and mgR (16) likely resulted in enhanced detoxification of MG by the aldehyde reductase enzyme product of mgR . Previously, we observed that a strain of C. beijerinckii expressing mgsA alone (without the tethered aldehyde reductase in C. beijerinckii _mgsA+mgR exhibited diminished growth, and the presence of MG in the culture (16).…”

“…Additionally, co-expression of mgsA and mgR (16) likely resulted in enhanced detoxification of MG by the aldehyde reductase enzyme product of mgR . Previously, we observed that a strain of C. beijerinckii expressing mgsA alone (without the tethered aldehyde reductase in C. beijerinckii _mgsA+mgR exhibited diminished growth, and the presence of MG in the culture (16). This assertion is supported by the collective expression profiles of csd (cysteine desulfurase) cysteine and methionine biosynthesis and metabolic enzymes, and upregulation of iron import permeases ( feoA and feoB ), hemerythrin, ideR , flavodoxin and other flavoproteins.…”

“…We previously reported the engineering of the strains of C. beijerinckii used in this study (16). To isolate total RNA, C. beijerinckii _p459 and C. beijerinckii _mgsA+mgR were grown in P2 medium as previously described (11), with a slight modification.…”