Schmollack Marc, Werner Felix, Huber Janine, Kiefer Dirk, Merkel Manuel, Hausmann Rudolf, Siebert Daniel, Blombach Bastian
Microbial Biotechnology, Campus Straubing for Biotechnology and Sustainability, Technical University of Munich, Uferstraße 53, 94315, Straubing, Germany.
Institute of Food Science and Biotechnology, Department of Bioprocess Engineering, University of Hohenheim, Stuttgart, Germany.
Biotechnol Biofuels Bioprod. 2022 Dec 14;15(1):139. doi: 10.1186/s13068-022-02238-3.
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.
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 IDH (pEKEx2-malEcad) produced 3.43 ± 0.59 g itaconic acid L with a product yield of 81 ± 9 mmol mol 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 and 116 ± 15 mmol mol, 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 IDH (pEKEx2-malEcad), produced 3.38 ± 0.28 g itaconic acid L at 25 °C from an acetate-containing aqueous side-stream of fast pyrolysis.
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.
衣康酸是生物基聚合物产业中一种很有前景的平台化学品。如今，衣康酸是通过工业规模的生物技术利用土曲霉从糖类生产的。从食品底物生产燃料和化学品是一个两难问题，因为未来的工艺应该依赖于不与食品或饲料竞争的碳源。因此，从乙酸盐等替代底物生产化学品对于在生物经济中开发新的价值链是可取的。
在本研究中，谷氨酸棒杆菌ATCC 13032被改造以从非食品底物乙酸盐高效生产衣康酸。因此，我们通过使转录调节因子RamB失活、降低异柠檬酸脱氢酶的活性、删除编码谷氨酸脱氢酶的gdh基因以及过表达为在谷氨酸棒杆菌中表达而优化的来自土曲霉的顺乌头酸脱羧酶（CAD），重新调整了中心碳和氮代谢。最终菌株谷氨酸棒杆菌ΔramBΔgdh IDH（pEKEx2-malEcad）在以乙酸盐作为唯一碳源和能源的氮限制基本培养基中进行小规模培养时，产生了3.43±0.59 g/L的衣康酸，产物得率为81±9 mmol/mol。将培养温度从30℃降至25℃提高了CAD活性，并进一步将产量和产物得率分别提高到5.01±0.67 g/L和116±15 mmol/mol。后者相当于理论最大值的35%，并且是迄今为止基于乙酸盐生产衣康酸的最高产物得率。此外，优化后的菌株谷氨酸棒杆菌ΔramBΔgdh IDH（pEKEx2-malEcad）在25℃下从快速热解含乙酸盐水相侧流中产生了3.38±0.28 g/L的衣康酸。
如本研究所示，乙酸盐是谷氨酸棒杆菌生产衣康酸的合适非食品碳源。调整中心碳和氮代谢能够从乙酸盐高效生产衣康酸，因此本研究为通过基因工程改造谷氨酸棒杆菌以利用乙酸盐生产其他产品提供了有用的设计原则。
