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Exploring the Potential of Corynebacterium glutamicum to Produce the Compatible Solute Mannosylglycerate
Frontiers in Bioengineering and Biotechnology ( IF 4.3 ) Pub Date : 2021-09-21 , DOI: 10.3389/fbioe.2021.748155
Andreas Schwentner 1 , Heiko Neugebauer 1 , Serin Weinmann 1 , Helena Santos 2 , Bernhard J Eikmanns 1
Affiliation  

The compatible solute mannosylglycerate (MG) has exceptional properties in terms of protein stabilization and protection under salt, heat, and freeze-drying stresses as well as against protein aggregation. Due to these characteristics, MG possesses large potential for clinical and biotechnological applications. To achieve efficient MG production, Corynebacterium glutamicum was equipped with a bifunctional MG synthase (encoded by mgsD and catalyzing the condensation of 3-phosphoglycerate and GDP-mannose to MG) from Dehalococcoides mccartyi. The resulting strain C. glutamicum (pEKEx3 mgsD) intracellularly accumulated about 111 mM MG (60 ± 9 mg gCDW−1) with 2% glucose as a carbon source. To enable efficient mannose metabolization, the native manA gene, encoding mannose 6-phosphate isomerase, was overexpressed. Combined overexpression of manA and mgsD from two plasmids in C. glutamicum resulted in intracellular MG accumulation of up to ca. 329 mM [corresponding to 177 mg g cell dry weight (CDW)−1] with glucose, 314 mM (168 mg gCDW−1) with glucose plus mannose, and 328 mM (176 mg gCDW−1) with mannose as carbon source(s), respectively. The product was successfully extracted from cells by using a cold water shock, resulting in up to 5.5 mM MG (1.48 g L−1) in supernatants. The two-plasmid system was improved by integrating the mgsD gene into the manA-bearing plasmid and the resulting strain showed comparable production but faster growth. Repeated cycles of growth/production and extraction of MG in a bacterial milking-like experiment showed that cells could be recycled, which led to a cumulative MG production of 19.9 mM (5.34 g L−1). The results show that the newly constructed C. glutamicum strain produces MG from glucose and mannose and that a cold water shock enables extraction of MG from the cytosol into the medium.



中文翻译:


探索谷氨酸棒杆菌生产相容性溶质甘露糖基甘油酸的潜力



相容的溶质甘露糖甘油酸酯 (MG) 在盐、热和冷冻干燥胁迫下的蛋白质稳定和保护以及防止蛋白质聚集方面具有卓越的特性。由于这些特性,MG在临床和生物技术应用方面具有巨大的潜力。为了实现高效的MG生产,谷氨酸棒杆菌配备有双功能 MG 合酶(编码为毫克数并催化3-磷酸甘油酸和GDP-甘露糖缩合成MG)麦卡氏脱盐球菌。由此产生的应变谷氨酸棒杆菌(pEKEx3毫克数)以2%葡萄糖作为碳源,细胞内积累约111 mM MG (60 ± 9 mg g CDW -1 )。为了实现有效的甘露糖代谢,天然曼A编码甘露糖6-磷酸异构酶的基因过度表达。联合过度表达曼A和毫克数来自两个质粒谷氨酸棒杆菌导致细胞内 MG 积累高达约。葡萄糖为 329 mM [相当于 177 mg g细胞干重 (CDW) -1 ],葡萄糖加甘露糖为 314 mM (168 mg g CDW -1 ),甘露糖作为碳为 328 mM (176 mg g CDW -1 )分别来源。通过冷水冲击成功从细胞中提取产物,上清液中的 MG 含量高达 5.5 mM (1.48 g L -1 )。 通过整合双质粒系统进行了改进毫克数基因进入曼A携带质粒和所得菌株显示出可比的产量但生长更快。在类似细菌挤奶的实验中,重复生长/产生和提取 MG 的循环表明,细胞可以循环利用,从而导致累积 MG 产量为 19.9 mM (5.34 g L -1 )。结果表明,新建的谷氨酸棒杆菌该菌株从葡萄糖和甘露糖中产生 MG,并且冷水冲击能够将 MG 从细胞质提取到培养基中。

更新日期:2021-09-21
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