Adaptive laboratory evolution accelerated glutarate production by Corynebacterium glutamicum

Abstract

Background: The demand for biobased polymers is increasing steadily worldwide. Microbial hosts for production of their monomeric precursors such as glutarate are developed. To meet the market demand, production hosts have to be improved constantly with respect to product titers and yields, but also shortening bioprocess duration is important.

Results: In this study, adaptive laboratory evolution was used to improve a C. glutamicum strain engineered for production of the C₅-dicarboxylic acid glutarate by flux enforcement. Deletion of the L-glutamic acid dehydrogenase gene gdh coupled growth to glutarate production since two transaminases in the glutarate pathway are crucial for nitrogen assimilation. The hypothesis that strains selected for faster glutarate-coupled growth by adaptive laboratory evolution show improved glutarate production was tested. A serial dilution growth experiment allowed isolating faster growing mutants with growth rates increasing from 0.10 h^-1 by the parental strain to 0.17 h^-1 by the fastest mutant. Indeed, the fastest growing mutant produced glutarate with a twofold higher volumetric productivity of 0.18 g L^-1 h^-1 than the parental strain. Genome sequencing of the evolved strain revealed candidate mutations for improved production. Reverse genetic engineering revealed that an amino acid exchange in the large subunit of L-glutamic acid-2-oxoglutarate aminotransferase was causal for accelerated glutarate production and its beneficial effect was dependent on flux enforcement due to deletion of gdh. Performance of the evolved mutant was stable at the 2 L bioreactor-scale operated in batch and fed-batch mode in a mineral salts medium and reached a titer of 22.7 g L^-1, a yield of 0.23 g g^-1 and a volumetric productivity of 0.35 g L^-1 h^-1. Reactive extraction of glutarate directly from the fermentation broth was optimized leading to yields of 58% and 99% in the reactive extraction and reactive re-extraction step, respectively. The fermentation medium was adapted according to the downstream processing results.

Conclusion: Flux enforcement to couple growth to operation of a product biosynthesis pathway provides a basis to select strains growing and producing faster by adaptive laboratory evolution. After identifying candidate mutations by genome sequencing causal mutations can be identified by reverse genetics. As exemplified here for glutarate production by C. glutamicum, this approach allowed deducing rational metabolic engineering strategies.

Keywords: Adaptive laboratory evolution; Corynebacterium glutamicum; Glutarate; Metabolic engineering; Reactive extraction; Reverse genetics; Volumetric productivity.

Fig. 1

Schematic representation of the metabolic pathway for glutarate production, flux enforcement by deletion of gdh (left panel) and ammonium assimilation by the GS/GOGAT system (right panel). Gene names are shown next to enzyme reactions (arrows), gene deletions are indicated by red crosses. Enzymes from P. stutzeri (dark grey), E. coli (light grey) and native enzymes (orange) are highlighted. gabT, GABA/5AVA amino transferase; gabD, succinate/glutarate-semialdehyde dehydrogenase; ldcC, l-lysine decarboxylase; patA, putrescine transaminase; patD, γ-aminobutyraldehyde dehydrogenase;glnA, glutamine synthetase (GS); gltBD, l-glutamic acid-2-oxoglutarate aminotransferase (GOGAT); gdh, l-glutamic acid dehydrogenase

Fig. 2

Influence of the amino acid exchange GabD^P134L on a growth and glutarate production and b the combined in vitro enzyme activities of GABA transaminase GabT and succinate semialdehyde dehydrogenase GabD. a Strain GluA and GluA T0 were cultivated in the BioLector microcultivation system with 40 g L⁻¹ glucose in CGXII minimal medium supplemented with 1 mM IPTG. Supernatant concentrations of glutarate were determined after 96 h and are given as means and standard deviations of three independent cultivations. b Crude extracts of DH5α (pEC-XT99A-gabTD) and DH5α (pEC-XT99A-gabTD^P134L) were assayed for combined in vitro enzyme activities of GABA transaminase GabT and succinate semialdehyde dehydrogenase GabD with 20 mM 5AVA as substrate and increasing glutarate concentrations. Statistical significance was assessed in Student’s unpaired t-test (*p < 0.05)

Fig. 3

Characterization of the evolved strains after eight transfers (T0–T8) regarding a growth and b production of glutarate and 5AVA. Cells were grown in the BioLector microcultivation system using 40 g L⁻¹ glucose minimal medium supplemented with 1 mM IPTG and harvested after 56 h. Values and error bars represent mean and standard deviation values (n = 3 cultivations)

Fig. 4

Influence of the SNP gltB^E686Q and the plasmid pEC-XT99A-tetA(Z)^Δ21bp-gabTD^P134L on maximal growth rate, and glutarate and 5AVA production by stepwise reverse genetics. Cells were grown in the BioLector microcultivation system using 40 g L⁻¹ glucose minimal medium supplemented with 1 mM IPTG and harvested after 96 h. Values and error bars represent mean and standard deviation values (n = 3 cultivations)

Fig. 5

Influence of the SNP GltB^E686Q on l-lysine production in dependency of the gdh deletion. Cells were grown in the BioLector microcultivation system using 40 g L⁻¹ glucose minimal medium and harvested after 48 h. Values and error bars represent mean and standard deviation values (n = 3 cultivations). Statistical significance was assessed in Student’s unpaired t-test (***p < 0.001, *p < 0.05, n.s. not significant)

Fig. 6

Glutarate production by C. glutamicum GluA T0 (a) and GluA T7 (b) in bioreactors operated in batch mode. Both strains were cultivated in CGXII minimal medium in batch mode over 48 h, containing 40 g L⁻¹ glucose. Glutarate production is indicated in green squares (g L⁻¹), biomass concentration (CDW) is shown in black diamonds (g L⁻¹), glucose concentration (g L⁻¹) is plotted as pink hollow triangles, and 5AVA concentration (g L⁻¹) in red diamonds and the relative dissolved oxygen saturation (rDOS) is indicated in light blue (%). Cultivation was performed at 30 °C and pH 7.0 regulated with 10% (v/v) H₃PO₄ and 4 M KOH. 0.6 mL L⁻¹ of antifoam agent AF204 (Sigma Aldrich, Taufkirchen, Germany) was added to the medium manually before inoculation

Fig. 7

Glutarate production by C. glutamicum GluA T7 in fed-batch mode. GluA T7 was cultivated in CGXII minimal medium in fed-batch mode over 64 h, containing 40 g L⁻¹ glucose and 150 g L⁻¹ glucose from the feeding solution. Glutarate production is indicated in green squares (g L⁻¹), biomass concentration (CDW) is shown in black diamonds (g L⁻¹), glucose concentration (g L⁻¹) is plotted as pink hollow triangles, and 5AVA concentration (g L⁻¹) in red diamonds, feed solution (mL) is plotted as pink line and the relative dissolved oxygen saturation (rDOS) is indicated in light blue (%). Cultivation was performed at 30 °C and pH 7.0 regulated with 10% (v/v) H₃PO₄ and 4 M KOH. An overpressure of 0.4 bar was applied. 0.6 mL L⁻¹ of antifoam agent AF204 (Sigma Aldrich, Taufkirchen, Germany) was added to the medium manually before inoculation

Fig. 8

Reactive extraction yields of glutarate from fermentation broth at T = 25 °C and p = 1 bar using either T-C6 (a) or T-C8 (b) as amine extractant. The pH of the fermentation broth was adjusted to 2.5 using highly concentrated sulfuric acid before reactive extraction. a T-C6 was used as amine extractant in the organic solvent ethyl oleate at molar ratios of T-C6/glutarate ranging from 1/1 to 10/1. 10 wt% of 1-dodecanol were added to the organic phase as modifier. b T-C8 was used as amine extractant in the organic solvent ethyl oleate at molar ratios of T-C8/glutarate ranging from 1/1 to 10/1. Systems with a molar ratio of T-C8/glutarate = 6/1 or higher contained 10 wt% 1-dodecanol as modifier in the organic solvent (+Mod.)

Fig. 9

Re-extraction yields of glutarate from the organic phase after reactive extraction using T-C6 (T-C6/glutarate = 9/1) in ethyl oleate (containing 10 wt% of 1-dodecanol) at T = 25 °C and p = 1 bar. The water-soluble amines a M-C3 and b M-C4 were used for re-extraction at molar ratios of WSA/glutarate (organic) ranging from 1/1 to 5/1