CRISPRi engineering E. coli for morphology diversification.
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. coli. Five sgRNAs associated with CRISPRi were designed and synthesized, respectively, to target five various locations on genes ftsZ or mreB encoded in the E. coli 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. coli fibers, and the larger the E. coli cells. Combined repressions on expressions of ftsZ and mreB generated long and larger E. coli 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.
Center of Synthetic and Systems Biology, School of Life Science, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China.
Dina
D
Elhadi
Center of Synthetic and Systems Biology, School of Life Science, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China.
Li
L
Lv
Center of Synthetic and Systems Biology, School of Life Science, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China.
Xiao-Ran
XR
Jiang
Center of Synthetic and Systems Biology, School of Life Science, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China.
Hong
H
Wu
Center of Synthetic and Systems Biology, School of Life Science, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China; Center for Nano and Micro Mechanics, Tsinghua University, Beijing 100084, China; MOE Key Lab for Industrial Biocatalysis, Tsinghua University, Beijing 100084, China. Electronic address: chengq@mail.tsinghua.edu.cn.
Guo-Qiang
GQ
Chen
D001426:Bacterial Proteins / Q000235:genetics*; D002473:Cell Wall / Q000378:metabolism; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D003598:Cytoskeletal Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000166:cytology* / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics*; D024861:Genetic Enhancement / Q000379:methods; D014644:Genetic Variation / Q000235:genetics; D006885:Hydroxybutyrates / Q000302:isolation & purification / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics
D001426:Bacterial Proteins; D003598:Cytoskeletal Proteins; D029968:Escherichia coli Proteins; C071525:FtsZ protein, Bacteria; D006885:Hydroxybutyrates; C078473:MreB protein, E coli
CRISPRi; Escherichia coli; Inclusion body; Morphology engineering; PHB; Polyhydroxyalkanoates; Synthetic biology; ftsZ; mreB