CRISPR/dCas9干扰bcsD基因表达调控细菌纤维素结构

Regulating the structure of bacterial cellulose by altering the expression of bcsD using CRISPR/dCas9

摘要:

木葡糖酸醋杆菌（Gluconacetobacter xylinus）是细菌纤维素的主要生产菌株。在该菌中，BcsD是纤维素合酶的亚基之一，参与细菌纤维素的组装过程。利用CRISPR/dCas9系统调控bcsD基因的表达量，获得了一系列bcsD基因表达量不同的木葡糖酸醋杆菌。通过分析细菌纤维素的结构特征发现，细菌纤维素的结晶度和孔隙率随着木葡糖酸醋杆菌中bcsD表达量的变化而发生改变。其中孔隙率的变化范围在59.95%–84.05%之间，结晶度的变化范围在74.26%–93.75%之间，而细菌纤维素的产量并未因bcsD的表达量变化而发生显著下降。结果表明，bcsD的表达量低于55.34%后，细菌纤维素的孔隙率显著上升，并且细菌纤维素的结晶度与bcsD的表达量呈正相关。最终，通过干扰bcsD基因的表达，实现了一步发酵木葡糖酸醋杆菌获得了产量稳定且结构不同的细菌纤维素。

关键词:CRISPR/dCas9;木葡糖酸醋杆菌;细菌纤维素;bcsD

Abstract:

Gluconacetobacter xylinus is a primary strain producing bacterial cellulose (BC). In G. xylinus, BcsD is a subunit of cellulose synthase and is participated in the assembly process of BC. A series of G. xylinus with different expression levels of the bcsD gene were obtained by using the CRISPR/dCas9 technique. Analysis of the structural characteristics of BC showed that the crystallinity and porosity of BC changed with the expression of bcsD. The porosity varied from 59.95%-84.05%, and the crystallinity varied from 74.26%-93.75%, while the yield of BC did not decrease significantly upon changing the expression levels of bcsD. The results showed that the porosity of bacterial cellulose significantly increased, while the crystallinity was positively correlated with the expression of bcsD, when the expression level of bcsD was below 55.34%. By altering the expression level of the bcsD gene, obtaining BC with different structures but stable yield through a one-step fermentation of G. xylinus was achieved.

Key words:CRISPR/dCas9;Gluconacetobacter xylinus;bacterial cellulose;bcsD