A genome-reduced Corynebacterium glutamicum derivative discloses a hidden pathway relevant for 1,2-propanediol production

Abstract

Background: 1,2-propanediol (1,2-PDO) is widely used in the cosmetic, food, and drug industries with a worldwide consumption of over 1.5 million metric tons per year. Although efforts have been made to engineer microbial hosts such as Corynebacterium glutamicum to produce 1,2-PDO from renewable resources, the performance of such strains is still improvable to be competitive with existing petrochemical production routes.

Results: In this study, we enabled 1,2-PDO production in the genome-reduced strain C. glutamicum PC2 by introducing previously described modifications. The resulting strain showed reduced product formation but secreted 50 ± 1 mM D-lactate as byproduct. C. glutamicum PC2 lacks the D-lactate dehydrogenase which pointed to a yet unknown pathway relevant for 1,2-PDO production. Further analysis indicated that in C. glutamicum methylglyoxal, the precursor for 1,2-PDO synthesis, is detoxified with the antioxidant native mycothiol (MSH) by a glyoxalase-like system to lactoylmycothiol and converted to D-lactate which is rerouted into the central carbon metabolism at the level of pyruvate. Metabolomics of cell extracts of the empty vector-carrying wildtype, a 1,2-PDO producer and its derivative with inactive D-lactate dehydrogenase identified major mass peaks characteristic for lactoylmycothiol and its precursors MSH and glucosaminyl-myo-inositol, whereas the respective mass peaks were absent in a production strain with inactivated MSH synthesis. Deletion of mshA, encoding MSH synthase, in the 1,2-PDO producing strain C. glutamicum ΔhdpAΔldh(pEKEx3-mgsA-yqhD-gldA) improved the product yield by 56% to 0.53 ± 0.01 mM_1,2-PDO mM_glucose^-1 which is the highest value for C. glutamicum reported so far.

Conclusions: Genome reduced-strains are a useful basis to unravel metabolic constraints for strain engineering and disclosed in this study the pathway to detoxify methylglyoxal which represents a precursor for 1,2-PDO production. Subsequent inactivation of the competing pathway significantly improved the 1,2-PDO yield.

Keywords: 1,2-propanediol; Chassis organism; Corynebacterium glutamicum; Genome reduction; Lactoylmycothiol; Methylglyoxal; Mycothiol.

Fig. 1

Overview of the 1,2-PDO pathway introduced into C. glutamicum [22], including the proposed bypass from methylglyoxal to d-lactate and pyruvate via lactoylmycothiol in blue. Black arrows represent native pathways; dotted arrows indicate more than one reaction; green and disrupted arrows represent heterologously expressed proteins and deletions of gene sequences of mentioned proteins, respectively. Abbreviations: TCA, tricarboxylic acid cycle; DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde 3-phosphate; UDP-GlcNAc, uridine diphosphate-N-acetylglucosamine; 1-l-Ins-1-P, 1-l-myo-inositol 1-phosphate; GldA, glycerol dehydrogenase (from E. coli); HdpA, dihydroxyacetone phosphate phosphatase; MgsA, methylglyoxal synthase (from E. coli); Dld, quinone-dependent d-lactate dehydrogenase; LldD, quinone-dependent l-lactate dehydrogenase; Ldh, NAD-dependent l-lactate dehydrogenase; YqhD, aldehyde reductase (from E. coli); MshA, glycosyltransferase; “GloA”, proposed lactoylglutathione lyase homolog in C. glutamicum; “GloB”, proposed hydroxyacylglutathione hydrolase homolog in C. glutamicum

Fig. 2

(A) Growth (black) and glucose consumption (green) and (B) 1,2-PDO (blue) and lactate (grey) accumulation of the strains C. glutamicum GRSΔhdpAΔldh(pEKEx3-mgsA-yqhD-gldA) (GRS) and PC2ΔhdpAΔldh(pEKEx3-mgsA-yqhD-gldA) (PC2) in shaking flasks with modified CGXII minimal medium. Error bars represent the standard deviation of the mean values of three biological replicates

Fig. 3

(A) Growth (black), (B) glucose consumption (green), (C) 1,2-PDO (blue) and (D) lactate (grey) accumulation of the strains C. glutamicum ΔhdpAΔldh(pEKEx3-mgsA-yqhD-gldA) (-) and C. glutamicum ΔhdpAΔldhΔdld(pEKEx3-mgsA-yqhD-gldA) (Δdld) in shaking flasks with modified CGXII minimal medium. Error bars represent the standard deviation of the mean values of three biological replicates

Fig. 4

Glucose-specific 1,2-PDO yields of C. glutamicum ΔhdpAΔldh(pEKEx3-mgsA-yqhD-gldA) (-), C. glutamicum ΔhdpAΔldhΔmshA(pEKEx3-mgsA-yqhD-gldA) (ΔmshA), C. glutamicum ΔhdpAΔldhΔcg1426(pEKEx3-mgsA-yqhD-gldA) (Δcg1426), C. glutamicum ΔhdpAΔldhΔcg1073(pEKEx3-mgsA-yqhD-gldA) (Δcg1073), C. glutamicum ΔhdpAΔldhΔcg0071(pEKEx3-mgsA-yqhD-gldA) (Δcg0071), C. glutamicum ΔhdpAΔldhΔcg1482(pEKEx3-mgsA-yqhD-gldA) (Δcg1482) and C. glutamicum ΔhdpAΔldhΔcg1856(pEKEx3-mgsA-yqhD-gldA) (Δcg1856) cultivated in shaking flasks with modified CGXII minimal medium and glucose as sole carbon and energy source. Error bars represent the standard deviation of the mean values of three biological replicates