Tuning and elucidation of the colony dimorphism in Rhodococcus ruber associated with cell flocculation in large scale fermentation.

Prevention of cell flocculation in large-scale fermentation is of great importance for most industrial microbes. Using Rhodococcus ruber TH3 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) in Rhodococcus showed 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.