Courtade Gaston, Le Simone Balzer, Sætrom Gerd Inger, Brautaset Trygve, Aachmann Finn L
NOBIPOL, Department of Biotechnology and Food Science, NTNU Norwegian University of Science and Technology, N-7491, Trondheim, Norway.
Department of Biotechnology and Nanomedicine, SINTEF Materials and Chemistry, N-7034, Trondheim, Norway.
Carbohydr Res. 2017 Aug 7;448:212-219. doi: 10.1016/j.carres.2017.02.003. Epub 2017 Feb 14.
Lytic polysaccharide monooxygenases (LPMOs) are key enzymatic players of lignocellulosic biomass degradation processes. As such, they have been introduced in cellulolytic cocktails for more efficient and less expensive lignocellulose saccharification. The recombinant production of LPMOs in bacteria for scientific investigations using vectors typically based on the T7 and lacUV5 promoters has been hampered by low yields. Reasons for this have been catabolite repression when producing the proteins in defined media with glucose as the sole carbon source, as well as the lack of an inducible expression system that allows controlled production of LPMOs that are correctly processed during translocation to the periplasmic space. A cassette vector design containing the XylS/Pm system was constructed and evaluated, showing that the expression cassette could easily be used for exchanging LPMO coding genes with or without signal sequences. The cassette was shown to reliably produce mature (translocated) LPMOs under controlled conditions that were achieved by using a low dosage (0.1 mM) of the Pm inducer m-toluic acid and a low (16 °C) cultivation temperature after induction. Furthermore, the signal sequences of five bacterial LPMOs were tested, and the signal sequence of LPMO10A from Serratia marcescens was found to give highest levels of recombinant protein production and translocation. The LPMO expression cassette was also evaluated in cultivations using defined media with glucose as the sole carbon source with a product yield of 7-22 mg per L of culture in shaking flasks. The integrity of the recombinant proteins were analyzed using NMR spectroscopy, showing that the system produced correctly processed and folded LPMOs. Finally, high cell-density cultivations of the recombinant strains were carried out, demonstrating stable protein production levels at similar relative yields (42-1298 mg per L of culture; 3.8-11.6 mg per OD unit) as in shaking flasks, and showing the scale-up potential of the system.
裂解多糖单加氧酶（LPMOs）是木质纤维素生物质降解过程中的关键酶。因此，它们已被引入纤维素分解酶混合物中，以实现更高效、更廉价的木质纤维素糖化。使用通常基于T7和lacUV5启动子的载体在细菌中重组生产LPMOs用于科学研究时，产量一直很低。原因包括在以葡萄糖为唯一碳源的确定培养基中生产蛋白质时的分解代谢物阻遏，以及缺乏一种可诱导表达系统，该系统能够控制LPMOs的生产，使其在转运到周质空间的过程中得到正确加工。构建并评估了一种包含XylS/Pm系统的盒式载体设计，结果表明该表达盒可轻松用于交换有无信号序列的LPMO编码基因。该盒式载体在通过使用低剂量（0.1 mM）的Pm诱导剂间甲苯甲酸和诱导后较低的培养温度（16°C）实现的可控条件下，能够可靠地产生成熟（转运的）LPMOs。此外，测试了五种细菌LPMOs的信号序列，发现粘质沙雷氏菌的LPMO10A信号序列产生的重组蛋白产量和转运水平最高。LPMO表达盒还在以葡萄糖为唯一碳源的确定培养基的培养中进行了评估，在摇瓶中每升培养物的产物产量为7 - 22 mg。使用核磁共振光谱分析了重组蛋白的完整性，结果表明该系统产生了正确加工和折叠的LPMOs。最后，对重组菌株进行了高细胞密度培养，结果表明在与摇瓶中相似的相对产量（每升培养物42 - 1298 mg；每OD单位3.8 - 11.6 mg）下，蛋白质生产水平稳定，显示了该系统的放大潜力。
