Fermentative Production of l-2-Hydroxyglutarate by Engineered Corynebacterium glutamicum via Pathway Extension of l-Lysine Biosynthesis

Abstract

l-2-hydroxyglutarate (l-2HG) is a trifunctional building block and highly attractive for the chemical and pharmaceutical industries. The natural l-lysine biosynthesis pathway of the amino acid producer Corynebacterium glutamicum was extended for the fermentative production of l-2HG. Since l-2HG is not native to the metabolism of C. glutamicum metabolic engineering of a genome-streamlined l-lysine overproducing strain was required to enable the conversion of l-lysine to l-2HG in a six-step synthetic pathway. To this end, l-lysine decarboxylase was cascaded with two transamination reactions, two NAD(P)-dependent oxidation reactions and the terminal 2-oxoglutarate-dependent glutarate hydroxylase. Of three sources for glutarate hydroxylase the metalloenzyme CsiD from Pseudomonas putida supported l-2HG production to the highest titers. Genetic experiments suggested a role of succinate exporter SucE for export of l-2HG and improving expression of its gene by chromosomal exchange of its native promoter improved l-2HG production. The availability of Fe²⁺ as cofactor of CsiD was identified as a major bottleneck in the conversion of glutarate to l-2HG. As consequence of strain engineering and media adaptation product titers of 34 ± 0 mM were obtained in a microcultivation system. The glucose-based process was stable in 2 L bioreactor cultivations and a l-2HG titer of 3.5 g L^-1 was obtained at the higher of two tested aeration levels. Production of l-2HG from a sidestream of the starch industry as renewable substrate was demonstrated. To the best of our knowledge, this study is the first description of fermentative production of l-2HG, a monomeric precursor used in electrochromic polyamides, to cross-link polyamides or to increase their biodegradability.

Keywords: C. glutamicum; L-2-hydroxyglutarate; bioreactor; glutarate hydroxylase; metabolic engineering; wheat sidestream concentrate.

Figure 1

Schematic overview of metabolically engineered C. glutamicum overproducing l-2HG. Enzyme names are shown next to the reaction represented by the arrows. Dashed arrows represent several reaction steps. Heterologous enzymes are boxed, while gene deletions for enzymes are indicated by red crosses. Dark gray boxes depict enzymes encoded in P. stutzeri (gabT, GABA/5AVA amino transferase; gabD, succinate/glutarate-semialdehyde dehydrogenase) and light gray boxes those from E. coli (ldcC, l-lysine decarboxylase; patA, putrescine transaminase; patD, γ-aminobutyraldehyde dehydrogenase). In the case of glutarate hydroxylase (EC 1.14.1.64) CsiD enzymes (violet box) were sourced from either E. coli (HEGluA), P. putida (HPGluA), or Halobacillus sp. (HBGluA).

Figure 2

Product titers of l-2HG and its precursor glutarate obtained with strains overproducing different glutarate hydroxylases. C. glutamicum strains HEGluA, HPGluA, and HBGluA expressing csiD from E. coli MG1655, P. putida KT2440, and Halobacillus sp. BA-2008, respectively, were grown in 40 g L⁻¹ glucose CGXII minimal medium supplemented with 1 mM IPTG in the microcultivation device BioLector. Values and error bars represent means and standard deviations from 3 replicate cultivations with supernatants analyzed after 96 h. Statistical significance was assessed by Student's paired t-testing (*p < 0.05, n.s, not significant).

Figure 3

Influence of overexpression and deletion of sucE on production of l-2HG, glutarate and 5AVA. (A) Strains GluA2 and HPGluA2 differed from strains GluA and HPGluA by overexpression of sucE. (B) Strains GluAΔsucE and HPGluAΔsucE were derived from strains GluA and HPGluA, respectively, by deletion of sucE. Strains were grown in the BioLector using 40 g L⁻¹ glucose minimal medium supplemented with 1 mM IPTG and supernatants were analyzed after 120 h. Values and error bars represent mean and standard deviation values (n = 3 cultivations). Statistical significance was assessed in Student's paired t-test (***p < 0.001, *p < 0.05, n.s. not significant).

Figure 4

Influence of the iron concentration on the maximal growth rate and production of l-2HG and glutarate. C. glutamicum HPGluA2 was grown in the BioLector with 40 g L⁻¹ glucose minimal medium supplemented with 1 mM IPTG and the indicated iron concentrations. Supernatants were analyzed after 96 h. Values and error bars represent means and standard deviations (n = 3 cultivations).

Figure 5

Influence of l-2HG on the combined in vitro enzyme activities of GABA transaminase GabT and succinate semialdehyde dehydrogenase GabD (A) and influence of extracellularly added glutarate on production of l-2HG (B). (A) Crude extracts of GluA were assayed for combined in vitro enzyme activities of GABA transaminase GabT and succinate semialdehyde dehydrogenase GabD in the presence of increasing concentrations of l-2HG. (B) Strain HPGluA2 was cultivated in the BioLector with 40 g L⁻¹ glucose minimal medium supplemented with 1 mM IPTG, 2 mM iron (II)-sulfate and increasing concentrations of glutarate (0, 20, 40 mM). Supernatant concentrations of l-2HG (filled violet triangles), glutarate (open green squares) as well as the net glutarate concentrations produced in addition to the added glutarate concentration (closed green squares) were determined after 96 h and are given as means and standard deviations of three independent cultivations.

Figure 6

l-2HG production by C. glutamicum HPGluA2 in fed-batch fermentation with (A) 0.5 vvm and (B) 1 vvm aeration rate. HPGluA2 was cultivated in CGXII minimal medium in fed-batch mode over 240 h, containing 40 g L⁻¹ glucose and feeding 600 g L⁻¹ glucose solution. l-2HG concentration is indicated in violet stars (mM), biomass concentration (CDW) is shown in black diamands (g L⁻¹), glucose concentration (g L⁻¹) is plotted as pink hollow triangles, and glutarate concentration (mM) in green squares, 600 g L⁻¹ glucose feed (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 0.2 bar was applied. 0.6 mL L⁻¹ of antifoam agent AF204 (Sigma Aldrich, Taufkirchen, Germany) was added to the medium manually before inoculation.

Figure 7

Comparison of glutarate and l-2HG production based on glucose (A) or wheat sidestream concentrate (B). C. glutamicum glutarate producer GluA and l-2HG producer HPGluA2 were grown in the Duetz microcultivation plates with low or high oxygen supply using covers of different air permeability and different volumes (3 and 2 mL). Strains were cultivated in CGXII minimal medium with 40 g L⁻¹ glucose or with a mixture containing 246 g L⁻¹ WSC, 20 g L⁻¹ ammonium sulfate and 42 g L⁻¹ MOPS. Both media were supplemented with 1 mM IPTG and 2 mM FeSO₄. Supernatants were analyzed after 96 h. Values and error bars represent means and standard deviations of 3 cultivations.