多拷贝整合血红蛋白基因对嗜盐细菌生长及PHA合成的影响

Polyhydroxyalkanoates (PHA) are natural polyesters produced by bacteria and have attracted attentions due to the application as tissue engineering materials. Halophiles are considered as a potential resource for PHA production due to their ability of producing PHA under high salt growth conditions. In previous studies, we have isolated a halophilic strain, Halomonas sp., from Chaerhan salt lake in Qinghai province, which can use a cheap carbon source such as starch to grow and produce PHA. We also found that introduction of Vitreoscilla hemoglobin gene (vgb) into Halomonas sp., significantly improved its cell growth and PHA production. In order to understand the role of VHb better and to improve the PHA production in Halomonas sp, in the present work we planned to insert 1-5 copies of vgb gene into Halomonas sp. genome through a modified Mu-transposon. Later we will investigate the effect of VHb on cell growth, PHA production, PHA related enzymes activities and the concentration of key co-enzymes and growth factors in different Halomonas sp. recombinants which express different levels of vgb gene. Finally, we will analyze the level of transcriptome and metabolic flux of carbon source in these strains with different copies of vgb gene. The main goal of this study is to investigate the regulatory mechanism of VHb and enhance the PHA production in Halomonas sp., thus providing a new efficient approach of metabolic engineering in halophiles to increase their potential of PHA production.

聚羟基脂肪酸酯(PHA)是微生物合成的生物聚酯，在医学组织工程领域应用前景广阔。利用嗜盐菌生产PHA有诸多优势，但相关的分子生物学改造却罕见报道。前期研究发现细菌血红蛋白（Vitreoscilla Hemoglobin, VHb）基因（vgb）的导入能够明显提高嗜盐细菌生产PHA的能力。为研究VHb蛋白促进嗜盐细菌生长以及提高PHA合成能力的具体机制，本项目拟采用改造后的人工Mu转座技术在该菌株中构建DNA片段的多次染色体整合系统，将1-5个拷贝的vgb基因整合到中度嗜盐细菌Halomonas sp.基因组上，获得遗传稳定的重组体。在此基础上，重点研究VHb的不同表达水平对嗜盐细菌生长速度、PHA产量、PHA相关酶活性、重要辅酶及生长因子浓度、菌体转录组以及碳代谢终产物分布的影响，以期揭示VHb调控嗜盐细菌生长及PHA合成的内在机制，为嗜盐细菌代谢工程改造提供新的思路与手段。

聚羟基脂肪酸酯（PHA）是一种可降解的生物高分子材料，广泛存在于微生物体内。PHA是性质优良的生物塑料但工业化进程却进展缓慢，阻碍PHA进一步大规模应用的是其较高的成本。本课题研究利用嗜盐菌有效降低PHA成本的可行性，并在此基础上尝试建立不灭菌连续发酵工艺。取得以下研究结果：.从青海察尔汗盐湖采集的土样、水样中分离纯化出一株生长快速、高效积累PHA中度嗜盐菌，经鉴定为盐单胞菌属，其最适生长温度为30度，最适pH为9.0，最适盐浓度为60 g/L。在此基础上进行培养和生产条件优化，其细胞干重（CDW）和PHA含量分别提高了2倍和5倍，且细菌生长和PHA的积累是同步进行的，表明可利用该嗜盐菌进行连续发酵。.嗜盐菌的生长生产需要高盐浓度维持，因此不灭菌培养成为可能。当NaCl浓度达到60 g/L或更高，pH为9.0的条件下，不灭菌发酵是可行的，尤其是当发酵通气、接种之前将pH调节至约11.0并维持约0.5小时。.利用嗜盐菌开发不灭菌连续发酵工艺，该过程中利用双发酵罐进行尝试，第一个发酵罐用于生长，而第二个发酵罐主要用于提高PHA含量，同时上清液可回收利用。该工艺连续进行14天而没有染菌等问题。该过程的成功开发可显著降低PHA生产过程的能源、底物等成本，使生产过程更加简单，为低成本PHA的大规模工业化生产打下了良好的基础，将成为PHA工业化过程的一个重大突破。