Department of Biotechnology, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Paterna, Valencia, Spain.
Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB. Edifici CRAG, Bellaterra, Barcelona, Spain.
Sci Rep. 2017 Nov 7;7(1):14663. doi: 10.1038/s41598-017-15277-w.
Filamentous fungi encode distinct antifungal proteins (AFPs) that offer great potential to develop new antifungals. Fungi are considered immune to their own AFPs as occurs in Penicillium chrysogenum, the producer of the well-known PAF. The Penicillium digitatum genome encodes only one afp gene (afpB), and the corresponding protein (AfpB) belongs to the class B phylogenetic cluster. Previous attempts to detect AfpB were not successful. In this work, immunodetection confirmed the absence of AfpB accumulation in wild type and previous recombinant constitutive P. digitatum strains. Biotechnological production and secretion of AfpB were achieved in P. digitatum with the use of a P. chrysogenum-based expression cassette and in the yeast Pichia pastoris with the α-factor signal peptide. Both strategies allowed proper protein folding, efficient production and single-step purification of AfpB from culture supernatants. AfpB showed antifungal activity higher than the P. chrysogenum PAF against the majority of the fungi tested, especially against Penicillium species and including P. digitatum, which was highly sensitive to the self-AfpB. Spectroscopic data suggest that native folding is not required for activity. AfpB also showed notable ability to withstand protease and thermal degradation and no haemolytic activity, making AfpB a promising candidate for the control of pathogenic fungi.
丝状真菌编码独特的抗真菌蛋白（AFPs），为开发新的抗真菌药物提供了巨大的潜力。真菌被认为对自身的 AFP 具有免疫力，就像青霉素生产菌——产黄青霉那样，它是著名的 PAF 的产生菌。橘青霉基因组仅编码一个 afp 基因（afpB），相应的蛋白（AfpB）属于 B 类系统发育簇。以前尝试检测 AfpB 并没有成功。在这项工作中，免疫检测证实野生型和以前重组的组成型橘青霉菌株中没有 AfpB 积累。使用基于产黄青霉的表达盒和酵母毕赤酵母的α因子信号肽，在橘青霉中实现了 AfpB 的生物技术生产和分泌。这两种策略都允许 AfpB 正确折叠，从培养上清液中高效生产和进行一步纯化。AfpB 对大多数测试的真菌表现出比产黄青霉 PAF 更高的抗真菌活性，特别是对青霉属真菌，包括对自身 AfpB 高度敏感的橘青霉。光谱数据表明，活性不需要天然折叠。AfpB 还表现出显著的抵抗蛋白酶和热降解的能力，并且没有溶血活性，这使得 AfpB 成为控制病原真菌的有前途的候选物。
