Journal
METABOLIC ENGINEERING
Volume 38, Issue -, Pages 358-369Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymben.2016.09.001
Keywords
Morphology engineering; CRISPRi; PHB; Polyhydroxyalkanoates; ftsZ; mreB; Inclusion body; Escherichia coli; Synthetic biology
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Funding
- State Basic Science Foundation 973 [2012CB725201]
- National Natural Science Foundation of China [31430003, 31270146]
- special Tsinghua President Grant [2015THZ10]
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Microbial morphology engineering has recently become interesting for biotechnology. Genes ftsZ and mreB encoding proteins of bacterial fission ring and skeletons, respectively, are essential for cell growth, they both are the most important genes keeping the bacterial shapes including the cell length and width, respectively. Clustered regularly interspaced short palindromic repeats interference, abbreviated as CRISPRi, was for the first time used in this study to regulate expression intensities of ftsZ or/and mreB in E. coll. Five sgRNAs associated with CRISPRi were designed and synthesized, respectively, to target five various locations on genes ftsZ or mreB encoded in the E. coil chromosome, resulting in various reduced expression levels of ftsZ or/and mreB, respectively, forming elongated or/and fatter cells. Repressions on gene expressions of ftsZ or/and mreB could be further intensified by combining various sgRNAs together. It was found that the stronger the repression on genes ftsZ or/and mreB, the longer the E. coil fibers, and the larger the E. coil cells. Combined repressions on expressions of ftsZ and mreB generated long and larger E. coil with diverse morphologies including various sizes of gourds, bars, coccus, spindles, multi-angles and ellipsoids. In all cases, accumulations of intracellular biopolyester polyhydroxybutyrate (PHB) were in direct proportional to the intracellular volumes, ranging from 40% to 80% PHB in bacterial cell dry weights, depending on the cell volumes increases by the above CRISPRi applications.
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