生物合成ε-聚赖氨酸聚合度调控的分子机制解析

ε-polylysine (ε-PL) is a kind of biopolymer with great applied values and its application area is closely related to its molecular weight. For ε-PL, the difference on molecular weight is determined by the production strain. Based on the difference on polymerization degree of ε-PL and the results of whole-genome sequencing, in this study, we will use bioinformatics and molecular simulation to analyze the differences on gene sequences and subunits structures of the two ε-PL synthetases, from Steptomyces albulus PD-1 and Kitasatospora sp. PL6-3 respectively. Next, gene shuffling will be applied to construct mutants in which the domains of ε-PL synthetases are exchanged, which will help to screen the key domain affecting polymerization degree of ε-PL. Then, on basis of simulative mutations by computational biology, site-specific mutagenesis and saturated mutagenesis will be applied to the possible sites which regulate polymerization degree in the key domain. Finally, a series of mutants was obtained to synthesize ε-PL efficiently and precisely control the molecular weight of ε-PL. The expected results of this study will help to elucidate the regulatory mechanism of polymerization degree of biosynthesized ε-PL, furthermore, this study will enhance the exploration of ε-PL downstream application. Therefore, this study is of great academic and applied values.

ε-聚赖氨酸是一类具有重要应用价值的生物高分子，其应用领域与分子量密切相关，而该类生物高分子的分子量差异因菌种差异稳定存在。本研究针对生物合成ε-PL聚合度的差异，以来源于Steptomyces albulus PD-1和Kitasatospora sp. PL6-3的ε-PL聚合酶为研究对象，基于全基因组测序结果，采用生物信息学和分子模拟的方法分析两种Pls基因序列及亚基结构差异；通过基因重排技术构建Pls结构域替换的重组菌株，筛选影响ε-PL聚合度的关键结构域；在计算生物学模拟突变的基础上，对关键结构域中调控聚合度的可能位点进行饱和突变，阐明特定位点氨基酸残基种类对ε-PL聚合度的影响规律；基于此构建系列Pls突变株以实现ε-PL的高效合成与分子量的精准控制。本研究预期结果将有助于阐明生物合成ε-PL聚合度的调控机理，并对ε-PL下游应用开发起到积极的作用，学术意义和应用价值重大。

本项目以两种不同来源的聚赖氨酸合成酶（Pls）为研究对象，采用生物信息学和分子模拟的方法分析两种合成酶氨基酸序列及亚基结构差异。通过构建Pls结构域替换的重组菌株，筛选影响聚赖氨酸（ε-PL）聚合度的关键结构域。在计算生物学模拟的基础上，对关键结构域中的氨基酸位点进行定点突变分析，阐明特定位点氨基酸残基对ε-PL聚合度和肽键聚合的的影响。主要研究结果包括：(1) 根据已发现Pls蛋白序列中的保守结构域，利用生物信息学方法从基因组数据库中挖掘1843个潜在聚氨基酸合成酶，进一步，对这些蛋白序列进行进化树和保守模序识别分析，为研究ε-PL聚合度调控和肽键催化机理提供基础。 (2)通过构建Pls结构域替换的重组菌株，筛选出了影响ε-PL聚合度的关键结构域：Linker 1-跨膜结构域和 Linker 2-跨膜结构域。(2) 通过多序列比对分析在C3-domain发现了QTHLFxDR保守模序，并提出以组氨酸为基础的肽键聚合机制。该研究将丰富对非核糖体肽合成酶合成机制的认识，对研究其他聚氨基酸合成酶催化机理具有重要借鉴意义。本项目共发表SCI论文9篇，申请和授权专利共3项。