BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Tuning and elucidation of the colony dimorphism in Rhodococcus
ruber associated with cell flocculation in large scale fermentation
Received: 18 December 2016 /Revised: 29 March 2017 / Accepted: 29 April 2017 / Published online: 27 May 2017
Springer-Verlag Berlin Heidelberg 2017
Abstract Prevention of cell flocculation in large-scale fer-
mentation 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 associ-
ated 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 hydro-
phobicity 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.
Keywords Cell flocculation
Stearic acid bound EPS
Rhodococcus is a genus of aerobic and nonsporulating gram-
positive bacteria. Rhodococcus species are of great chemical,
pharmaceutical, and environmental importance due to their
powerful ability to produce valuable chemicals such as acryl-
amide and steroids (Yamada and Kobayashi 1996), to degrade
diverse compounds in polluted environments, and their
involvement in the biodesulfurization of fossil fuel
(Larkin et al. 2005; van der Geize and Dijkhuizen 2004).
Over the years, advances in Rhodococcus were empha-
sized, and many studies on its cellular and enzymatic modifi-
cations were reported (i.e., cell tolerance enhancement
(Ma and Yu 2012), genome gene-knockout (Ma et al.
2010), and in vivo nitrile hydratase evolution (Liu et al. 2014;
Yu et al. 2006)). An interesting phenomenon was observed in
the applications of Rhodococcus in bioremediation, in which
many strains showed spontaneous changes in their colony
morphotypes (e.g., from rough to smooth). Hydrophobic
Electronic supplementary material The online version of this article
(doi:10.1007/s00253-017-8319-0) contains supplementary material,
which is available to authorized users.
* Huimin Yu
Department of Chemical Engineering, Tsinghua University,
Beijing 100084, China
Key Laboratory of Industrial Biocatalysis (Tsinghua University), the
Ministry of Education, Beijing 100084, China
Center for Synthetic and Systems Biology, Tsinghua University,
Beijing 100084, China
Appl Microbiol Biotechnol (2017) 101:6321–6332