Microaerobic growth‐decoupled production of α‐ketoglutarate and succinate from xylose in a one‐pot process using Corynebacterium glutamicum

Abstract

Background Lignocellulosic biomass is the most abundant raw material on earth. Its efficient use for novel bio‐based materials is essential for an emerging bioeconomy. Possible building blocks for such materials are the key TCA‐cycle intermediates α‐ketoglutarate and succinate. These organic acids have a wide range of potential applications, particularly in use as monomers for established or novel biopolymers. Recently, Corynebacterium glutamicum was successfully engineered and evolved towards an improved utilization of d‐xylose via the Weimberg pathway, yielding the strain WMB2evo. The Weimberg pathway enables a carbon‐efficient C5‐to‐C5 conversion of d‐xylose to α‐ketoglutarate and a shortcut route to succinate as co‐product in a one‐pot process. Methods and Results C. glutamicum WMB2evo was grown under dynamic microaerobic conditions on d‐xylose, leading to the formation of comparably high amounts of succinate and only small amounts of α‐ketoglutarate. Subsequent carbon isotope labeling experiments verified the targeted production route for both products in C. glutamicum WMB2evo. Fed‐batch process development was initiated and the effect of oxygen supply and feeding strategy for a growth‐decoupled co‐production of α‐ketoglutarate and succinate were studied in detail. The finally established fed‐batch production process resulted in the formation of 78.4 mmol L−1 (11.45 g L−1) α‐ketoglutarate and 96.2 mmol L−1 (11.36 g L−1) succinate. Conclusion The developed one‐pot process represents a promising approach for the combined supply of bio‐based α‐ketoglutarate and succinate. Future work will focus on tailor‐made down‐stream processing of both organic acids from the fermentation broth to enable their application as building blocks in chemical syntheses. Alternatively, direct conversion of one or both acids via whole‐cell or cell‐free enzymatic approaches can be envisioned; thus, extending the network of value chains starting from cheap and renewable d‐xylose.

Nitrogen-controlled Valorization of Xylose-derived Compounds by Metabolically Engineered Corynebacterium glutamicum

“…is overexpressed to enable utilization of xylose for growth. As way out, either native xylulokinase was be replaced by a D-xylulokinase variant from Pichia stipitis [85], which does not accept xylitol as substrate, or introduction of the oxidative Weimberg pathway for xylose utilization may help [86]. The inhibition by xylitol 5-phosphate formed during xylose utilization in our strains also occurs during xylonate production.…”

Perspectives in growth production trade-off in microbial bioproduction

“…This avoids the metabolic burden of the production pathway during the growth phase and helps in the allocation of all metabolic resources to the synthesis of the product during the production phase. These approaches for microbial conversion have been widely used for the utilization of non-canonical carbon sources such as glycerol, 19 xylose, 20 protocatechuate, 21 hydrocinnamic acids, 22 and toxic products such as 1,2-indandiol production in Rhodococcus sp. 23 The transition from one phase to another is induced by several different strategies.…”

“…23 Examples of co-production include a growth-decoupled fed-batch production process, which resulted in the formation of about 11 g L −1 α-ketoglutarate and succinate from xylose in C. glutamicum . 20 Advancement in computational implementations for identifying a range of operating points within a feasible solution space has also been reported. 34…”

“…23 Examples of co-production include a growth-decoupled fed-batch production process, which resulted in the formation of about 11 g L −1 a-ketoglutarate and succinate from xylose in C. glutamicum. 20 Advancement in computational implementations for identifying a range of operating points within a feasible solution space has also been reported. 34 A major challenge of decoupled microbial conversions that use constitutive production pathways is that during the growth phase, there may be a phenotypic dri due to suboptimal growth or toxicity and low-producing variants with tness advantages may overtake the population in the bioconversion process.…”

Metabolic engineering of Corynebacterium glutamicum for the production of anthranilate from glucose and xylose

“…Since C. glutamicum cannot utilize xylose naturally, several different pathways such as the isomerase pathway or the Weimberg pathway were implemented into the metabolism of this bacterium (Kawaguchi et al., 2006 ; Meiswinkel et al., 2013 ; Radek et al., 2014 ). In this context, C. glutamicum was engineered to produce several compounds such as protocatechuate, succinate or α‐ketoglutarate from xylose or glucose/xylose mixtures (Brüsseler et al., 2019 ; Labib et al., 2021 ; Tenhaef et al., 2021 ). Noteworthy, xylose utilization via the isomerase pathway has the advantage of providing the SA pathway precursor E4P (Kogure et al., 2016 ).…”