A simple generic method for enhancing extracellular protein yields in engineered bacteria is still lacking. Here, we demonstrated that phage-encoded holin can be used to export proteins to the extracellular medium in both Gram-negative Escherichia coli and -positive Lactococcus lactis. When a putative holin gene LLNZ_RS10380 annotated in the genome of L. lactis NZ9000 (hol380) was recombinantly expressed in E. coli BL21(DE3), the Hol380 oligomerized up to hexamer in the cytoplasmic membrane, yielding membrane pore to allow the passage of cytosolic β-galatosidase (116 kDa), whose extracellular production reached 54.59 U/μl, accounting for 76.37% of the total activity. However, the overexpressed Hol380 could not release cytosolic proteins across the membrane in L. lactis NZ9000, but increased the secretory production of staphylococcal nuclease to 2.55-fold and fimbrial adhesin FaeG to 2.40-fold compared with those guided by signal peptide Usp45 alone. By using a combination of proteomics and transcriptional level analysis, we found that overexpression of the Hol380 raised the accumulation of Ffh and YidC involved in the signal recognition particle pathway in L. lactis, suggesting an alternative road participating in protein secretion. This study proposed a new approach by expressing holin in bacterial cell factories to export target proteins of economic or medical interest.

中文翻译：

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Holin 辅助细菌重组蛋白输出

仍然缺乏一种提高工程细菌细胞外蛋白质产量的简单通用方法。在这里，我们证明了噬菌体编码的 holin 可用于将蛋白质输出到革兰氏阴性大肠杆菌和阳性乳酸乳球菌的细胞外培养基中。当在L. lactis NZ9000 ( hol380 )基因组中注释的推定的 holin 基因LLNZ_RS10380在大肠杆菌中重组表达时BL21(DE3)，Hol380在细胞质膜上寡聚成六聚体，产生膜孔，允许胞质β-半乳糖苷酶（116 kDa）通过，其胞外产量达到54.59 U/μl，占总活性的76.37% . 然而，过表达的 Hol380 不能在乳酸乳球菌NZ9000 中跨膜释放胞质蛋白，但与单独由信号肽 Usp45 引导的相比，葡萄球菌核酸酶的分泌量增加了 2.55 倍，菌毛粘附素 FaeG 的分泌量增加了 2.40 倍。通过结合蛋白质组学和转录水平分析，我们发现 Hol380 的过表达提高了L. lactis信号识别粒子通路中 Ffh 和 YidC 的积累。，建议参与蛋白质分泌的替代途径。这项研究提出了一种新方法，通过在细菌细胞工厂中表达 holin 来输出具有经济或医学意义的目标蛋白质。