Department of Food Sciences and Technology, University of Natural Resources and Life Sciences, Institute of Food Technology, Muthgasse 18, Vienna, Vienna, 1190, Austria.
Department of Biotechnology, Institute of Microbiology and Microbial Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, Vienna, Vienna, 1190, Austria.
Microb Cell Fact. 2023 Apr 17;22(1):72. doi: 10.1186/s12934-023-02075-5.
Laccases are multicopper enzymes that oxidize a wide range of aromatic and non-aromatic compounds in the presence of oxygen. The majority of industrially relevant laccases are derived from fungi and are produced in eukaryotic expression systems such as Pichia pastoris and Saccharomyces cerevisiae. Bacterial laccases for research purposes are mostly produced intracellularly in Escherichia coli, but secretory expression systems are needed for future applications. Bacterial laccases from Streptomyces spp. are of interest for potential industrial applications because of their lignin degrading activities.
In this study, we expressed small laccases genes from Streptomyces coelicolor, Streptomyces viridosporus and Amycolatopsis 75iv2 with their native signal sequences in Gram-positive Bacillus subtilis and Streptomyces lividans host organisms. The extracellular activities of ScLac, SvLac and AmLac expressed in S. lividans reached 1950 ± 99 U/l, 812 ± 57 U/l and 12 ± 1 U/l in the presence of copper supplementation. The secretion of the small laccases was irrespective of the copper supplementation; however, activities upon reconstitution with copper after expression were significantly lower, indicating the importance of copper during laccase production. The production of small laccases in B. subtilis resulted in extracellular activity that was significantly lower than in S. lividans. Unexpectedly, AmLac and ScLac were secreted without their native signal sequences in B. subtilis, indicating that B. subtilis secretes some heterologous proteins via an unknown pathway.
Small laccases from S. coelicolor, S. viridosporus and Amycolatopsis 75iv2 were secreted in both Gram-positive expression hosts B. subtilis and S. lividans, but the extracellular activities were significantly higher in the latter.
漆酶是一类多铜酶，能够在氧气存在的条件下氧化广泛的芳香族和非芳香族化合物。大多数具有工业应用价值的漆酶来自真菌，并且在毕赤酵母和酿酒酵母等真核表达系统中产生。出于研究目的，细菌漆酶主要在大肠杆菌中进行细胞内生产，但未来的应用需要分泌表达系统。由于具有木质素降解活性，链霉菌属的细菌漆酶引起了潜在工业应用的关注。
在这项研究中，我们使用其天然信号序列，在革兰氏阳性的枯草芽孢杆菌和链霉菌属宿主生物中表达了来自变铅青链霉菌、深绿链霉菌和衣康酸放线菌 75iv2 的小漆酶基因。在补充铜的情况下，在链霉菌属中表达的 ScLac、SvLac 和 AmLac 的细胞外活性分别达到 1950±99 U/l、812±57 U/l 和 12±1 U/l。小漆酶的分泌与铜的补充无关；然而，在表达后用铜再构成时的活性显著降低，这表明铜在漆酶生产过程中的重要性。在枯草芽孢杆菌中生产小漆酶导致细胞外活性显著低于链霉菌属。出乎意料的是，AmLac 和 ScLac 在枯草芽孢杆菌中没有其天然信号序列就被分泌出来，这表明枯草芽孢杆菌通过未知途径分泌一些异源蛋白。
来自变铅青链霉菌、深绿链霉菌和衣康酸放线菌 75iv2 的小漆酶在革兰氏阳性表达宿主枯草芽孢杆菌和链霉菌属中都被分泌，但在后者中的细胞外活性显著更高。
