Enhanced heterologous protein productivity by genome reduction in Lactococcus lactis NZ9000.
Enhanced heterologous protein productivity by genome reduction in Lactococcus lactis NZ9000.
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通过乳酸乳球菌 NZ9000 基因组减少提高异源蛋白质生产力
DOI：
10.1186/s12934-016-0616-2
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发表时间：
2017-01-03
影响因子：
6.4
通讯作者：
Qiao M
中科院分区：
文献类型：
--
作者：
Zhu D;Fu Y;Liu F;Xu H;Saris PE;Qiao M
背景：在工业应用中将新型底盘生物体用作微生物细胞工厂是一个深入的研究领域。乳酸乳球菌是研究最广泛的模式生物之一，具有用作发酵所需产品的工程宿主的卓越能力。然而，很少有研究报道乳酸乳球菌基因组还原作为功能基因组研究的干净背景和理想产品发酵的模型底盘。结果：使用 Cre-loxP 删除系统删除了乳酸乳球菌 NZ9000 (L.lactis 9 k) 基因组 (2,530,294 bp) 中占 2.83% 的四个大的非必需 DNA 区域，这是实现乳酸乳球菌基因组还原的第一步。 本研究中基因组最小化。将突变体与亲本菌株的几个生理特征进行比较，并以红色荧光蛋白 (RFP) 和细菌素白素 C (LecC) 作为报告基因，评估其作为异源蛋白生产（细胞内和分泌表达）的微生物细胞工厂的能力。与测试的两种培养基中的野生菌株相比，这四种突变体生长更快，产生的生物量增加，三磷酸腺苷含量增加，并且维护需求减少。特别是，具有最大缺失的乳酸乳球菌 9 k-4 被确定为重组蛋白生产的最佳候选宿主。通过乳链菌肽诱导，与野生菌株相比，不仅转录效率而且表达报告基因的生产水平提高了大约三到四倍。强组成型启动子P5和P8控制的lecC基因在L.lactis 9 k-4中的表达也显着提高。结论：基因组精简后的L.lactis 9 k-4在多项生理性状评估中均优于亲本菌株。此外，乳酸乳球菌 9 k-4 作为蛋白质生产的平台生物表现出良好的特性。在未来的工作中，通过使用我们的特定设计，将最大限度地减少乳酸乳球菌的基因组，为功能基因组学研究提供比本研究中构建的乳酸乳球菌 9 k-4 更干净的背景。此外，改进的背景将有可能用于生物技术。
Background:The implementation of novel chassis organisms to be used as microbial cell factories in industrial applications is an intensive research field. Lactococcus lactis, which is one of the most extensively studied model organisms, exhibits superior ability to be used as engineered host for fermentation of desirable products. However, few studies have reported about genome reduction of L. lactis as a clean background for functional genomic studies and a model chassis for desirable product fermentation.Results:Four large nonessential DNA regions accounting for 2.83% in L. lactis NZ9000 (L. lactis 9 k) genome (2,530,294 bp) were deleted using the Cre-loxP deletion system as the first steps toward a minimized genome in this study. The mutants were compared with the parental strain in several physiological traits and evaluated as microbial cell factories for heterologous protein production (intracellular and secretory expression) with the red fluorescent protein (RFP) and the bacteriocin leucocin C (LecC) as reporters. The four mutants grew faster, yielded enhanced biomass, achieved increased adenosine triphosphate content, and diminished maintenance demands compared with the wild strain in the two media tested. In particular, L. lactis 9 k-4 with the largest deletion was identified as the optimum candidate host for recombinant protein production. With nisin induction, not only the transcriptional efficiency but also the production levels of the expressed reporters were approximately three- to fourfold improved compared with the wild strain. The expression of lecC gene controlled with strong constitutive promoters P5 and P8 in L. lactis 9 k-4 was also improved significantly.Conclusions:The genome-streamlined L. lactis 9 k-4 outcompeted the parental strain in several physiological traits assessed. Moreover, L. lactis 9 k-4 exhibited good properties as platform organism for protein production. In future works, the genome of L. lactis will be maximally reduced by using our specific design to provide an even more clean background for functional genomics studies than L. lactis 9 k-4 constructed in this study. Furthermore, an improved background will be potentially available for use in biotechology.
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影响因子：
6.4
作者：
Bahey-El-Din, Mohammed;Griffin, Brendan T.;Gahan, Cormac G. M.
通讯作者：
Gahan, Cormac G. M.
影响因子：
4.4
作者：
Lambert, Jolanda M.;Bongers, Roger S.;Kjeerebezem, Michiel
通讯作者：
Kjeerebezem, Michiel
影响因子：
3.6
作者：
Hashimoto, M;Ichimura, T;Kato, J
通讯作者：
Kato, J
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影响因子：
2.4
作者：
Gao, G.;Qiao, J. -J.;Qiao, M. -Q.
通讯作者：
Qiao, M. -Q.
影响因子：
4.4
作者：
Hernandez, Igor;Molenaar, Douwe;van Hylckama Vlieg, Johan E. T.
通讯作者：
van Hylckama Vlieg, Johan E. T.