Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid (UCM), Avenida Puerta de Hierro, s/n, 28040, Madrid, Spain.
J Ind Microbiol Biotechnol. 2013 Sep;40(9):977-93. doi: 10.1007/s10295-013-1302-6. Epub 2013 Jun 22.
Mature sakacin A (SakA, encoded by sapA) and its cognate immunity protein (SakI, encoded by sapiA), and two SakA-derived chimeras mimicking the N-terminal end of mature enterocin P (EntP/SakA) and mature enterocin A (EntA/SakA) together with SakI, were fused to different signal peptides (SP) and cloned into the protein expression vectors pNZ8048 and pMG36c for evaluation of their production and functional expression by different lactic acid bacteria. The amount, antimicrobial activity, and specific antimicrobial activity of SakA and its chimeras produced by Lactococcus lactis subsp. cremoris NZ9000 depended on the SP and the expression vector. Only L. lactis NZ9000 (pNUPS), producing EntP/SakA, showed higher bacteriocin production and antimicrobial activity than the natural SakA-producer Lactobacillus sakei Lb706. The lower antimicrobial activity of the SakA-producer L. lactis NZ9000 (pNUS) and that of the EntA/SakA-producer L. lactis NZ9000 (pNUAS) could be ascribed to secretion of truncated bacteriocins. On the other hand, of the Lb. sakei Lb706 cultures transformed with the pMG36c-derived vectors only Lb. sakei Lb706 (pGUS) overproducing SakA showed a higher antimicrobial activity than Lb. sakei Lb706. Finally, cloning of SakA and EntP/SakA into pPICZαA and pKLAC2 permitted the production of SakA and EntP/SakA by recombinant Pichia pastoris X-33 and Kluyveromyces lactis GG799 derivatives although their antimicrobial activity was lower than expected from their production.
成熟的 sakacin A（SakA，由 sapA 编码）及其同源免疫蛋白（SakI，由 sapiA 编码），以及两个模拟成熟肠球菌素 P（EntP/SakA）和成熟肠球菌素 A（EntA/SakA）N 端的 SakA 衍生嵌合体，与 SakI 一起融合到不同的信号肽（SP）中，并克隆到蛋白表达载体 pNZ8048 和 pMG36c 中，以评估它们在不同乳酸菌中的产生和功能表达。乳球菌乳亚种 NZ9000 产生的 SakA 和其嵌合体的数量、抗菌活性和特异性抗菌活性取决于 SP 和表达载体。只有生产 EntP/SakA 的乳球菌 NZ9000（pNUPS）比天然 SakA 生产者德氏乳杆菌 Lb706 显示出更高的细菌素产生和抗菌活性。SakA 生产者乳球菌 NZ9000（pNUS）和 EntA/SakA 生产者乳球菌 NZ9000（pNUAS）的抗菌活性较低，可以归因于截短的细菌素分泌。另一方面，在转化了 pMG36c 衍生载体的 Lb. sakei Lb706 培养物中，只有过度生产 SakA 的 Lb. sakei Lb706（pGUS）显示出比 Lb. sakei Lb706 更高的抗菌活性。最后，将 SakA 和 EntP/SakA 克隆到 pPICZαA 和 pKLAC2 中，使得重组毕赤酵母 X-33 和乳酸克鲁维酵母 GG799 衍生物能够产生 SakA 和 EntP/SakA，尽管它们的抗菌活性低于预期。
