Methanol-Essential Growth of Corynebacterium glutamicum: Adaptive Laboratory Evolution Overcomes Limitation due to Methanethiol Assimilation Pathway

Abstract

Methanol is a sustainable substrate for biotechnology. In addition to natural methylotrophs, metabolic engineering has gained attention for transfer of methylotrophy. Here, we engineered Corynebacterium glutamicum for methanol-dependent growth with a sugar co-substrate. Heterologous expression of genes for methanol dehydrogenase from Bacillus methanolicus and of ribulose monophosphate pathway genes for hexulose phosphate synthase and isomerase from Bacillus subtilis enabled methanol-dependent growth of mutants carrying one of two independent metabolic cut-offs, i.e., either lacking ribose-5-phosphate isomerase or ribulose-5-phosphate epimerase. Whole genome sequencing of strains selected by adaptive laboratory evolution (ALE) for faster methanol-dependent growth was performed. Subsequently, three mutations were identified that caused improved methanol-dependent growth by (1) increased plasmid copy numbers, (2) enhanced riboflavin supply and (3) reduced formation of the methionine-analogue O-methyl-homoserine in the methanethiol pathway. Our findings serve as a foundation for the engineering of C. glutamicum to unleash the full potential of methanol as a carbon source in biotechnological processes.

Keywords: adaptive laboratory evolution; isotopic labeling; metabolic engineering; methanol; ribulose monophosphate pathway; synthetic methylotrophy.

Figure 1

The ribulose monophosphate pathway (RuMP) implemented in C. glutamicum. (A) Δrpe and (B) Δrpi concepts for methanol-dependent complementation of two metabolic cut-offs of the pentose phosphate pathway in C. glutamicum, respectively. Substrates in grey boxes: MeOH, methanol; metabolites in black boxes: E4P, erythrose 4-phosphate; F6P, fructose 6-phosphate; FA, formaldehyde; GAP, glyceraldehyde 3-phosphate; Hu6P, hexulose 6-phosphate; R5P, ribose 5-phosphate; Ru5P, ribulose 5-phosphate; S7P, sedoheptulose 7-phosphate; Xul, xylulose; Xu5P, xylulose 5-phosphate; interconnected pathways, violet boxes: PPP, pentose phosphate pathway; native or homologous overexpression of genes in orange circles: rpe, ribulose 5-phosphate epimerase; rpi, ribose 5-phosphate isomerase; tal, transaldolase; tkt, transketolase; xylA, xylose isomerase; xylB, xylulokinase; heterologous overexpression of xylA gene (xylose isomerase) from X. campestris in green circle; heterologous overexpression of RuMP pathway genes from B. subtilis in blue circles: hxlA, 3-hexulose 6-phosphate synthase; hxlB, 6-phospho 3-hexulose isomerase; heterologous overexpression of mdh gene (methanol dehydrogenase) from B. methanolicus in pink circle; red arrows, knocked out reactions; green arrows, complementing reactions.

Figure 2

Loss and gain of Rpi Function. C. glutamicum strains cultivated in BioLector with 1 mL CGXII medium including carbon sources and 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) at 30 °C. (A) Mutants Δald ΔfadH (MDS0) (circles) and Δald ΔfadH Δrpi (pEKEx3-xylAB) (MDS1) (triangles) comprising plasmid pEKEx3-xylAB were supplemented with 20 mM ribose (red), xylose (blue) and both (brown). (B) MDS0 (circles), MDS1(pVWEx1) (rectangles) and MDS1(pVWEx1-rpi) (diamonds) were supplemented with 20 mM ribose (red). (C) MDS1(pVWEx1-mdh-hxlAB) cultivated in 100 mL shakings flasks with 10 mL CGXII medium including carbon sources, 0.1 g L⁻¹ yeast extract and 1 mM IPTG at 30 °C. Supplementation with either 20 mM xylose (blue) or gluconate (black) with and without 500 mM methanol (full, empty). Error bars indicate standard deviations of biological triplicates.

Figure 3

Labeling of cadaverine from ¹³C-methanol by C. glutamicum strains MDS1 and MDS2T8. (A) Possible ¹³C-isotopologue distributions in the central carbon pathway. (B) Cadaverine was produced by the methanol-independent strain C. glutamicum MDS0 (pEKEx3-mdh-hxlAB) (pVWEx1-lysC^fbr-ldcC) [20] (C) MDS1 (pVWEx1-mdh-hxlAB) (pEC-XT99A-lysC^fbr-ldcC) and (D) MDS2T8 (pEC-XT99A-lysC^fbr-ldcC), respectively in shake flasks containing M9 minimal medium with 20 mM gluconate, 0.5 g/L yeast extract and 500 mM ¹³C-methanol for 72 h. The pie charts show mean ¹³C enrichment in cadaverine determined at carbon positions C1/C5 and C2/C4 for assimilation of carbon via RuMP pathway reactions (green) and C3 for assimilation of ¹³CO₂ that has arisen from dissimilation of ¹³C-methanol (blue).

Figure 4

Biomass formation with methanol as co-substrate (A) and relative copy numbers of plasmid pEKEx3-mdh-hxlAB (B) and HxlAB activities (C) in strain C. glutamicum MDS2 and its derivatives. (A) C. glutamicum wildtype (WT) and MDS2 were cultivated in CGXII medium with 20 mM ribose for 48 h and the maximal ΔOD was determined. C. glutamicum Δald ΔfadH Δrpe (pEKEx3-mdh-hxlAB) (= MDS2), and derivatives were cultivated in CGXII medium containing 20 mM ribose and 500 mM methanol for 48 h to determine the maximum ΔOD and investigate the influence of mutations found in strain MDS2T14, obtained by ALE. Blue bars indicate addition of 20 µM riboflavin. (B) Quantitative PCR (qPCR) was performed on isolated genomic and plasmid DNA from overnight cultures of MDS2, MDS2T14 and MDS2 Δcg3104. Relative plasmid copy numbers (PCN) were calculated using serial dilutions from 100 to 0.1 ng/μL as standard curve for the chromosomal (gntK) and plasmid (oriV_Cg) targets. Error bars depict standard deviations of technical triplicates. (C) Coupled HxlAB enzyme assays were performed with crude extracts of MDS2, MDS2T14 and MDS2 Δcg3104.

Figure 5

The influence of ALE mutations on methanol-dependent growth. (A) The insertion at the rpe locus in MDS2T14 is depicted. (B) C. glutamicum MDS2 (circles), MDS2T8 (triangles), MDS2T14 (squares) were cultivated using the BioLector with 1 mL filling volume of CGXII minimal medium with 20 mM glucose as sole carbon source (open symbols) or supplemented with 20 μM riboflavin (filled symbols). Backscatter was detected in BioLector with a gain of 20. Error bars depict standard deviations from biological triplicates. (C) The effect of ΔmetY and metK^S288N on methanol-essential growth and S-adenosylmethionine (SAM) synthesis is shown. (D) C. glutamicum strain MDS2 Δcg3104 ΔmetY was cultivated using the BioLector with 1 mL filling volume of CGXII minimal medium with 20 mM ribose and 20 μM riboflavin (empty symbols) and additional 500 mM methanol (filled symbols). Backscatter was detected in BioLector with a gain of 20. Error bars depict standard deviations from biological triplicates.