Metabolic engineering of Corynebacterium glutamicum for acetate-based itaconic acid production

Abstract

Background Itaconic acid is a promising platform chemical for a bio-based polymer industry. Today, itaconic acid is biotechnologically produced with Aspergillus terreus at industrial scale from sugars. The production of fuels but also of chemicals from food substrates is a dilemma since future processes should rely on carbon sources which do not compete for food or feed. Therefore, the production of chemicals from alternative substrates such as acetate is desirable to develop novel value chains in the bioeconomy. Results In this study, Corynebacterium glutamicum ATCC 13032 was engineered to efficiently produce itaconic acid from the non-food substrate acetate. Therefore, we rewired the central carbon and nitrogen metabolism by inactivating the transcriptional regulator RamB, reducing the activity of isocitrate dehydrogenase, deletion of the gdh gene encoding glutamate dehydrogenase and overexpression of cis-aconitate decarboxylase (CAD) from A. terreus optimized for expression in C. glutamicum. The final strain C. glutamicum ΔramB Δgdh IDHR453C (pEKEx2-malEcadopt) produced 3.43 ± 0.59 g itaconic acid L−1 with a product yield of 81 ± 9 mmol mol−1 during small-scale cultivations in nitrogen-limited minimal medium containing acetate as sole carbon and energy source. Lowering the cultivation temperature from 30 °C to 25 °C improved CAD activity and further increased the titer and product yield to 5.01 ± 0.67 g L−1 and 116 ± 15 mmol mol−1, respectively. The latter corresponds to 35% of the theoretical maximum and so far represents the highest product yield for acetate-based itaconic acid production. Further, the optimized strain C. glutamicum ΔramB Δgdh IDHR453C (pEKEx2-malEcadopt), produced 3.38 ± 0.28 g itaconic acid L−1 at 25 °C from an acetate-containing aqueous side-stream of fast pyrolysis. Conclusion As shown in this study, acetate represents a suitable non-food carbon source for itaconic acid production with C. glutamicum. Tailoring the central carbon and nitrogen metabolism enabled the efficient production of itaconic acid from acetate and therefore this study offers useful design principles to genetically engineer C. glutamicum for other products from acetate.

Improving growth properties of Corynebacterium glutamicum by implementing an iron‐responsive protocatechuate biosynthesis

“…acting as an electron shuttle, Perron & Brumaghim, 2009 ) or whether the enzymatic stress‐response benefits from the increased Fe 2+ availability of the strain. Given the fact, that acetate gains increasing importance as an alternative carbon and energy source for microbial production processes (Kiefer et al, 2020 ; Merkel et al, 2022 ; Schmollack et al, 2023 ), future research needs to address this effect as well as the performance of C. glutamicum IRON+ when exposed to different environmental stresses.…”

High-quality physiology of Alcanivorax borkumensis SK2 producing glycolipids enables efficient stirred-tank bioreactor cultivation

“…A prolonged lag phase and reduced biomass yield with increasing acetate concentrations, but not higher than 15 g L -1 , was also observed in this study ( Figure 5B ; Figures 6A,B ). Several other bacteria, including P. putida , E. coli , and Corynebacterium glutamicum , have demonstrated biotechnological production of different products using acetate, with the ability to grow on acetate concentrations of at least 10 g L -1 ( Noh et al, 2018 ; Arnold et al, 2019 ; Wolf et al, 2020 ; Schmollack et al, 2022 ). Acetate serves as a crucial precursor for various biotechnological products, such as itaconic acid production in E. coli and C. glutamicum , as well as rhamnolipid production in P. putida ( Noh et al, 2018 ; Arnold et al, 2019 ; Merkel et al, 2022 ).…”

Metabolic engineering of Corynebacterium glutamicum for fatty alcohol production from glucose and wheat straw hydrolysate

“…Growth during shaking flask and bioreactor cultivations was monitored by measuring the optical density at 600 nm (OD 600 ) with an Ultrospec 10 cell density meter (Harvard Biochrom, Holliston, MA, USA). A previously determined correlation factor of 0.23 [ 63 ] was used to convert OD 600 to a biomass concentration in g cell dry weight (CDW) L −1 . The growth rate µ, biomass yield Y X/S , product yield Y P/S and biomass-specific glucose consumption rate q S were determined as described elsewhere [ 31 ].…”

“…Corynebacterium glutamicum shows a comparable high tolerance to inhibitors such as aromatic compounds typically found in lignocellulosic hydrolysates [ 6 , 20 , 69 , 70 ]. Therefore, in several studies, this bacterium was utilized for the production of chemicals and fuels such as lactic, succinic, cis , cis -muconic, itaconic, 5-aminovaleric acid or 1,2-propanediol and isobutanol from non-food biomass hydrolysates [ 11 , 39 , 40 , 46 , 48 , 62 , 63 ]. The non-oleaginous C. glutamicum lacks essential genes for the β-oxidation of fatty acids [ 4 ] and has already been engineered for the production of lipids and fatty acids (FA) [ 33 , 54 , 72 , 74 ].…”