Liu Kun, Liu Yan, Li Xiangfei, Zhang Xiushan, Xue Zhenglian, Zhao Ming
Anhui Engineering Laboratory for Industrial Microbiology Molecular Breeding, College of Biology and Food Engineering, Anhui Polytechnic University, Wuhu, 241000, China.
Appl Microbiol Biotechnol. 2023 Nov;107(21):6497-6506. doi: 10.1007/s00253-023-12757-0. Epub 2023 Sep 8.
The whole-cell catalysis strategy of alpha-ketoglutaric acid (α-KG) production from L-glutamic acid (L-Glu) using recombinant Escherichia coli, in which L-glutamate oxidase (LGox) was over-expressed, has replaced the traditional chemical synthesis strategy. However, large amounts of toxic by-product, HO, should be eliminated through co-expressing catalase (Cat), thus severely increasing burden in cells. To efficiently and economically produce α-KG, here, the genes SpLGox (from Streptomyces platensis NTU3304) and SlCat (from Streptomyces lividans TK24) were inserted into the low-dosage-IPTG (Isopropyl β-D-Thiogalactoside) inducible expression system, constructed in our previous work, in E. coli, respectively. Besides, a double-strain catalysis system was established and optimized to produce α-KG, and the productivity of α-KG was increased 97% compared with that through single strain catalysis. Finally, a double-strain cultivation strategy was designed and employed to simplify the scale-up fermentation. Using the optimized whole-cell biocatalyst conditions (pH 7.0, 35 °C), majority of the L-glutamic acid was transformed into α-KG and the titer reached 95.4 g/L after 6 h with the highest productivity at present. Therefore, this strategy may efficiently and cost-effectively produce α-KG, enhancing its potential for industrial applications. KEY POINTS: • SpLGox and SlCat were over-expressed to catalyze L-Glu to α-KG and eliminate by-product HO, respectively. • Double-strain cultivation and catalysis system can efficiently and cost-effectively produce α-KG from L-Glu.
利用重组大肠杆菌将L-谷氨酸（L-Glu）生产α-酮戊二酸（α-KG）的全细胞催化策略取代了传统的化学合成策略，该策略中L-谷氨酸氧化酶（LGox）过表达。然而，大量有毒副产物H₂O₂需通过共表达过氧化氢酶（Cat）来消除，这严重增加了细胞负担。为了高效且经济地生产α-KG，在此将来自天蓝色链霉菌NTU3304的基因SpLGox和来自变铅青链霉菌TK24的基因SlCat分别插入到我们之前构建的大肠杆菌低剂量异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达系统中。此外，建立并优化了双菌株催化系统以生产α-KG，与单菌株催化相比，α-KG的产量提高了97%。最后，设计并采用了双菌株培养策略以简化放大发酵过程。使用优化后的全细胞生物催化剂条件（pH 7.0，35℃），大部分L-谷氨酸转化为α-KG，6小时后效价达到95.4 g/L，是目前最高的生产率。因此，该策略可高效且经济地生产α-KG，增强其工业应用潜力。要点：• SpLGox和SlCat分别过表达以催化L-Glu生成α-KG并消除副产物H₂O₂。• 双菌株培养和催化系统可高效且经济地从L-Glu生产α-KG。
