Metabolic engineering of Corynebacterium glutamicum for efficient production of succinate from lignocellulosic hydrolysate

Abstract

Background: Succinate has been recognized as one of the most important bio-based building block chemicals due to its numerous potential applications. However, efficient methods for the production of succinate from lignocellulosic feedstock were rarely reported. Nevertheless, Corynebacterium glutamicum was engineered to efficiently produce succinate from glucose in our previous study.

Results: In this work, C. glutamicum was engineered for efficient succinate production from lignocellulosic hydrolysate. First, xylose utilization of C. glutamicum was optimized by heterologous expression of xylA and xylB genes from different sources. Next, xylA and xylB from Xanthomonas campestris were selected among four candidates to accelerate xylose consumption and cell growth. Subsequently, the optimal xylA and xylB were co-expressed in C. glutamicum strain SAZ3 (ΔldhAΔptaΔpqoΔcatP_sod-ppcP_sod-pyc) along with genes encoding pyruvate carboxylase, citrate synthase, and a succinate exporter to achieve succinate production from xylose in a two-stage fermentation process. Xylose utilization and succinate production were further improved by overexpressing the endogenous tkt and tal genes and introducing araE from Bacillus subtilis. The final strain C. glutamicum CGS5 showed an excellent ability to produce succinate in two-stage fermentations by co-utilizing a glucose-xylose mixture under anaerobic conditions. A succinate titer of 98.6 g L^-1 was produced from corn stalk hydrolysate with a yield of 0.87 g/g total substrates and a productivity of 4.29 g L^-1 h^-1 during the anaerobic stage.

Conclusion: This work introduces an efficient process for the bioconversion of biomass into succinate using a thoroughly engineered strain of C. glutamicum. To the best of our knowledge, this is the highest titer of succinate produced from non-food lignocellulosic feedstock, which highlights that the biosafety level 1 microorganism C. glutamicum is a promising platform for the envisioned lignocellulosic biorefinery.

Keywords: Corynebacterium glutamicum; Lignocellulosic hydrolysate; Succinate; Xylose.

Fig. 1

Succinate biosynthesis pathway of C. glutamicum. The bold black arrows indicate metabolic fluxes increased by overexpression or introduction of the corresponding genes. The gray arrows indicate the reactions leading to a byproduct or presumably irrelevant reactions. Deleted genes are indicated with crosses. Metabolites: G6P glucose-6-phosphate, 6PGL 6-phosphoglucono-1,5-lactone, 6PG 6-phosphogluconate, Ru5P ribulose-5-phosphate, Xu5P xylulose-5-phosphate, R5P ribose-5-phosphate, G3P glyceraldehyde-3-phosphate, S7P sedoheptulose-7-phosphate, F6P fructose-6-phosphate, FBP fructose-1,6-bisphosphate, E4P erythrose-4-phosphate, DHAP dihydroxyacetone, DPG glycerate-1,3-diphosphate, 3PG glycerate-3-phosphate, 2PG glycerate-2-phosphate, PEP phosphoenolpyruvate, PYR pyruvate, AcP acetyl phosphate, AcCoA acetyl-CoA. Genes and their encoded enzymes: iolT encoding myo-inositol permease, glk encoding glucokinase, ptsG encoding glucose-EII of phosphoenolpyruvate phosphotransferase system (PTS), pgi encoding glucose-6-phosphate isomerase, araE encoding a H⁺ symporter protein, xylA encoding xylose isomerase, xylB encoding xylulokinase, zwf and opcA encoding glucose-6-phosphate dehydrogenase, devB encoding 6-phosphogluconolactonase, gnd encoding 6-phosphogluconate dehydrogenase, tkt encoding transketolase, tal encoding transaldolase, pqo encoding pyruvate: quinone oxidoreductase, pta encoding phosphotransacetylase, ackA encoding acetate kinase, cat encoding acetyl-CoA:CoA transferase, aceE encoding pyruvate complex dehydrogenase E1 component, ppc encoding phosphoenolpyruvate carboxylase, pyc encoding pyruvate carboxylase, mdh encoding malate dehydrogenase, gltA encoding citrate synthase, sucE encoding succinate exporter

Fig. 2

Batch cultivation of C. glutamicum I-pXMJ19 (squares), I-eco (diamonds), I-ppm (circles), I-sco (upward triangles), and I-xcb (downward triangles). a Profiles of cell growth (filled symbols) and xylose consumption (open symbols) under aerobic conditions. The strains were cultured in CGXIIA medium containing 20 g L⁻¹ xylose in 500-mL flasks at 30 °C and 220 rpm with an initial OD₆₀₀ of 0.8, and were induced with 0.5 mM IPTG. b SDS–PAGE analysis of intracellular proteins extracted from I-eco, I-ppm, I-sco and I-xcb from cultures with an OD₆₀₀ of 5. Proteins were separated on a 12% SDS-PAGE gel. c Profiles of xylose consumption (open symbols) under anaerobic conditions with an initial OD₆₀₀ of 30. 30 g L⁻¹ 4MgCO₃·Mg(OH)₂·5H₂O, 200 mM sodium bicarbonate and 1 mM IPTG were added into the CGXIIB medium

Fig. 3

Batch fermentation of C. glutamicum CGS1 (squares), CGS3 (circles) and CGS5 (upward triangles). a Profiles of cell growth (filled symbols) and xylose consumption (open symbols) in CGXIIA medium with 30 g L⁻¹ xylose. b Profiles of succinate production (filled symbols) and xylose consumption (open symbols) under anaerobic conditions with an initial OD₆₀₀ of 30. 30 g L⁻¹ 4MgCO₃·Mg(OH)₂·5H₂O, 200 mM sodium bicarbonate, and 1 mM IPTG were added into the CGXIIB medium. c Profiles of succinate yield (black bars), xylose consumption rate (white bars, calculated at 8 h), and succinate productivity (grey bars, calculated at 8 h) under anaerobic conditions

Fig. 4

Succinate production by CGS5 at high cell density (OD₆₀₀ = 150) under anaerobic conditions. 100 g L⁻¹ 4MgCO₃·Mg(OH)₂·5H₂O, 300 mM sodium bicarbonate, and 2 mM IPTG were added into the CGXIIB medium. The consumption of glucose (open squares), xylose (open circles), and concentration of succinate (filled upward triangles), ketoglutarate (filled stars), pyruvate (filled downward triangles), fumarate (filled leftward triangles), and acetate (filled rightward triangles) are shown. a Profiles of the production of organic acids and consumption of sugars from mixtures of glucose and xylose. b Profiles of the production of organic acids and consumption of sugars from lignocellulosic hydrolysate