基因组岛上的删除在假单胞菌 KT2440 基因组可以创建一个最佳底盘的合成生物学应用。
Deletion of genomic islands in the Pseudomonas putida KT2440 genome can create an optimal chassis for synthetic biology applications.
机构信息
Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin, 300071, China.
State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300071, China.
出版信息
Microb Cell Fact. 2020 Mar 18;19(1):70. doi: 10.1186/s12934-020-01329-w.
BACKGROUND
Genome streamlining is a feasible strategy for constructing an optimum microbial chassis for synthetic biology applications. Genomic islands (GIs) are usually regarded as foreign DNA sequences, which can be obtained by horizontal gene transfer among microorganisms. A model strain Pseudomonas putida KT2440 has broad applications in biocatalysis, biotransformation and biodegradation.
RESULTS
In this study, the identified GIs in P. putida KT2440 accounting for 4.12% of the total genome size were deleted to generate a series of genome-reduced strains. The mutant KTU-U13 with the largest deletion was advantageous over the original strain KTU in several physiological characteristics evaluated. The mutant KTU-U13 showed high plasmid transformation efficiency and heterologous protein expression capacity compared with the original strain KTU. The metabolic phenotype analysis showed that the types of carbon sources utilized by the mutant KTU-U13 and the utilization capabilities for certain carbon sources were increased greatly. The polyhydroxyalkanoate (PHA) yield and cell dry weight of the mutant KTU-U13 were improved significantly compared with the original strain KTU. The chromosomal integration efficiencies for the γ-hexachlorocyclohexane (γ-HCH) and 1,2,3-trichloropropane (TCP) biodegradation pathways were improved greatly when using the mutant KTU-U13 as the recipient cell and enhanced degradation of γ-HCH and TCP by the mutant KTU-U13 was also observed. The mutant KTU-U13 was able to stably express a plasmid-borne zeaxanthin biosynthetic pathway, suggesting the excellent genetic stability of the mutant.
CONCLUSIONS
These desirable traits make the GIs-deleted mutant KTU-U13 an optimum chassis for synthetic biology applications. The present study suggests that the systematic deletion of GIs in bacteria may be a useful approach for generating an optimal chassis for the construction of microbial cell factories.
背景
基因组精简是构建合成生物学应用的最佳微生物底盘的可行策略。基因组岛（GI）通常被视为外来 DNA 序列，可以通过微生物之间的水平基因转移获得。模式菌株恶臭假单胞菌 KT2440 在生物催化、生物转化和生物降解方面有广泛的应用。
结果
本研究中，鉴定出的恶臭假单胞菌 KT2440 中的 GI 占总基因组大小的 4.12%，通过删除这些 GI 生成了一系列基因组缩减菌株。与原始菌株 KTU 相比，具有最大缺失的突变株 KTU-U13 在几个评估的生理特性方面具有优势。与原始菌株 KTU 相比，突变株 KTU-U13 具有更高的质粒转化效率和异源蛋白表达能力。代谢表型分析表明，突变株 KTU-U13 利用的碳源类型和某些碳源的利用能力大大增加。与原始菌株 KTU 相比，突变株 KTU-U13 的聚羟基烷酸（PHA）产量和细胞干重显著提高。当使用突变株 KTU-U13 作为受体细胞时，γ-六氯环己烷（γ-HCH）和 1,2,3-三氯丙烷（TCP）生物降解途径的染色体整合效率大大提高，并且观察到突变株 KTU-U13 对 γ-HCH 和 TCP 的增强降解。突变株 KTU-U13 能够稳定表达质粒携带的玉米黄质生物合成途径，表明突变株具有优异的遗传稳定性。
结论
这些理想的特性使 GI 缺失的突变株 KTU-U13 成为合成生物学应用的最佳底盘。本研究表明，系统删除细菌中的 GI 可能是构建微生物细胞工厂的最佳底盘的有用方法。
Zotero 插件