乙酰异戊酰泰乐菌素转化菌株可控式分化的RNAi调控系统的构建和分析

During the manufacturing process of acetylisovaleryltylosin (AIV), there is one problem on how to balance growth of manufacturing cells and conversion rate of AIV. In terms of maintaining the immobilized system concerning the Streptomyces thermotolerans, circumscribing the growth rate of cells will be a critical factor. In this project, we try to construct RNA interference system in Streptomyces thermotolerans, choosing spore-producing genes and temperature-induced promoter as the spacers and the leader of the CRISPR-Cas system, respectively. In this way, the differetiation of strain could be regulated accurately when the temperature rises to 40℃ since the differetiation genes of Streptomyces thermotolerans are disrupted by RNA interference system. And then the resting cells could be obtained which have a relatively slower growth rate and keep the AIV synthetic enzyme activity high enough to realize the manufacturing process at the same time. The utilization of this mutated strain avoids energy waste and equipment jamming resulting from excessive differetiation of cells. Additionally, the RNA interference system and the nutrients necessary for maintaining condition of the resting cell could also need fine adjustment from the transcriptomic and proteomics analysis. These analysis provide sufficient information for better understanding the mechanism of resting cells and optimization of fermentation process. As a consequence, an optimum industrial Streptomyces thermotolerans, which produces a higher amount of AIV, could be constructed. In this project, a novel method concerning improving the yield of the AIV is proposed. Furthermore, efforts will be done to unravel the mechanisms of resting cells.

耐热链霉菌固定化发酵转化生产乙酰异戊酰泰乐菌素的过程中，如何既保证菌体良好的细胞活力和转化能力，又能有效限制细胞分化是保证固定化体系高效稳定的瓶颈问题。本项目拟在菌体中构建CRISPR-Cas 介导的RNAi调控系统，利用温度敏感型启动子，设计并构建孢子分化生成关键步骤的基因开关，从而实现对菌体分化过程的准确调控；结合静息细胞不再生长但保持原有酶活的特性，降低菌体过度分化造成的能量浪费和设备堵塞；通过转录组学和蛋白质组学方法分析构建的RNAi调控系统和静息细胞，根据结果微调RNAi调控过程，并尝试阐释静息细胞内部机制，优化耐热链霉菌固定化发酵过程。本项目为从根本上解决链霉菌固定化发酵中的瓶颈问题，深入理解细胞活力与细胞分化的关系并构建适应固定化生产的高效菌株提供了新的思路，也为阐明静息细胞内部机制做出有益尝试。

耐热链霉菌转化生产的乙酰异戊酰泰乐菌素(AIV)是一种高效、不易产生耐药性的兽用抗生素。但在实际生产中AIV的产物浓度较低，因此有必要构建高产AIV生产菌株，并建立适合工业化生产的发酵工艺条件。本项目研究包括（1）克隆了原始菌株中活性较低的异戊酰基转移酶基因（acyB1-B2），构建了该基因的过表达菌株SP.11432，其发酵产物AIV浓度是原始菌株SP.11416的2倍。同时重组菌株SP.11432对乙酰异戊酰泰乐菌素、泰勒菌素和安普霉素具有很好的耐受性。（2）以固定菌体量为指标，在不同的固定化材料中选出最优的固定化材料聚氨酯泡沫（PUF），根据耐热链霉菌细胞膜的结构特性和菌体表面呈现电负性的特点，设计改造出最佳的定阳性-交联型聚氨酯泡沫（PCPUF）为耐热链霉菌SP.11432固定化材料。经80小时的单批发酵结果表明，以PCPUF为载体固定化重组SP.11432菌株进行发酵，得到AIV最高浓度为33.54mg/L，比游离发酵提高了1.6倍，且固定化发酵达到最高浓度的时间比游离菌株的缩短了12小时。（3）AIV等大环内酯类抗生素的生物合成基因簇进行了较为深入的系统进化分析，揭示了多烯大环内酯类抗生素生物合成基因簇的组成和进化规律。（4）为了精确控制链霉菌的生长发育过，本项目对天蓝色链霉菌sRNA scr4115及其靶标进行了系统性功能分析。对scr4115进行敲除分析表明，气生菌丝的形成和产孢受到阻遏，导致突变菌株呈现光秃表型。（5）通过生物信息学在线工具预测scr4115的靶标基因，结合eGFP融合蛋白、点突变以及qRT-PCR验证发现，scr4115的靶标蛋白为SCO4114和SCO6925。利用CRISPR-Cas9技术构建靶标基因SCO4114和SCO6925缺失突变株，发现其气生菌丝的形成和产孢同样受到抑制，呈现出光秃表型，与scr4115缺失菌株的表型相似。（6）以天蓝色链霉菌scr4115及其靶标SCO4114和SCO6925为研究对象，进行了对应缺失菌株和野生菌株的蛋白质组学分析。实验结果表明，与野生型菌株相比，scr4115敲除菌株出现了91个差异蛋白质，SCO4114敲除菌株出现了163个差异蛋白质，SCO6925敲除菌株出现了177个差异蛋白质。这些差异表达蛋白可能是天蓝色链霉菌气生菌丝的形成和孢子发育密切相关的蛋白，为天蓝色链霉菌形态发育机