Tuning and elucidation of the colony dimorphism in Rhodococcus ruber associated with cell flocculation in large scale fermentation
Tuning and elucidation of the colony dimorphism in Rhodococcus ruber associated with cell flocculation in large scale fermentation
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DOI：
10.1007/s00253-017-8319-0
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发表时间：
2017-05
影响因子：
5
通讯作者：
Song Jiao;Jie Chen;Huimin Yu;Zhongyao Shen
中科院分区：
文献类型：
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作者：
Song Jiao;Jie Chen;Huimin Yu;Zhongyao Shen
对大多数工业微生物来说，防止大规模发酵过程中的细胞絮凝是非常重要的。以橡胶红球菌（grhodococcus ruberTH3）为模型菌株，我们发现发酵罐中不期望的细胞絮凝与菌落二态现象有关，并且只发生在粗糙型细胞（R-TH3）中，而不发生在光滑型细胞（S-TH3）中。通过分析R-TH3和S-TH3的转录组差异，选择了6个在S-TH3中转录显著上调的代表性基因，并在R-TH3中过表达。6个工程菌株的菌落形态发生了不同程度的变化，其中三种脂质代谢相关蛋白LM1、LM2和LM3的过表达使菌落形态从粗糙变为几乎与S-TH3一样光滑。扫描电镜观察证实了R-TH3工程菌株的细胞表面差异。它们的细胞表面疏水性也随之降低，细胞沉降行为也随之改变。以R-TH3/LM1为工程菌代表，测定了细胞包膜的脂肪酸含量。S-TH3、R-TH3/LM1和R-TH3脂肪酸含量分别为27.21%、24.10%和22.24%。在所有脂肪酸中，硬脂酸与红球菌亲水性胞外多糖（EPS）的结合在细胞间存在显著差异。S-TH3、R-TH3/LM1和R-TH3细胞EPS含量分别为191、163和137 mg/g。因此，S-TH3细胞的亲水性主要是由于细胞最外层的EPS。脂肪酸尤其是硬脂酸的增加导致结合EPS的增加，最终导致亲水性增强。
Prevention of cell flocculation in large-scale fermentation is of great importance for most industrial microbes. UsingRhodococcus ruberTH3 as a model strain, we revealed that the undesired cell flocculation in a fermenter was associated with the colony dimorphism phenomenon, and it only occurred in the rough-type of cells (R-TH3) instead of the smooth-type of cells (S-TH3). By analyzing the transcriptome differences of R-TH3 and S-TH3, six representative genes with significantly upregulated transcription in S-TH3 were selected and overexpressed in R-TH3. The colony morphotypes of the six engineered strains changed to different extents, in which overexpressions of three lipid metabolism-related proteins LM1, LM2, and LM3 tuned the colony morphotype from rough to almost as smooth as in S-TH3. SEM observation confirmed the cell surface difference of the engineered strains from R-TH3. Their cell surface hydrophobicity also reduced, and the cell sedimentation behaviors were consequently changed as expected. Using R-TH3/LM1 as the representative of the engineered bacteria, fatty acids of the cell envelopes were measured. Fatty acid contents of S-TH3, R-TH3/LM1, and R-TH3 were 27.21, 24.10, and 22.24%, respectively. Among all the fatty acids, stearic acid binding to hydrophilic extracellular polysaccharides (EPS) inRhodococcusshowed significant differences among the cells. The EPS contents of S-TH3, R-TH3/LM1, and R-TH3 were 191, 163, and 137 mg/g cells. Hence, the hydrophilicity of the S-TH3 cells was mainly due to the EPS in the outermost layer of the cells. Increase of fatty acids especially stearic acid results in the increase of the bound EPS, finally bringing about the hydrophilicity enhancement.