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Fig. 1.

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Fig. 1. Overall experimental design of this study. (A) Schematic diagram of carotenoid biosynthesis in E. coli. The native E. coli MEP pathway (right) and exogenous MVA pathway (left) are shown. The biosynthesis of lycopene and β-carotene from the precursors IPP and DMAPP are described (bottom). The genes we introduced using the plasmid system are shown in blue and orange. All genes and their corresponding enzymes are the following; mvaE: acetoacetyl-CoA thiolase/HMG-CoA reductase, mvaS: HMG-CoA synthase, mvaK1/erg12: mevalonate kinase, mvaK2/erg8: phosphomevalonate kinase, mvaD/erg19: mevalonate 5-diphosphate decarboxylase, Idi/ipiHP1: IPP isomerase, ispA: FPP synthase, crtE: GGPP synthase, crtB: phytoene synthase, crtI: phytoene desaturase, crtY: lycopene cyclase, dxs: DXP synthase, dxr: DXP isomerase reductase. Pathway intermediates G3P: glyceraldehyde 3 phosphate, DXP: 1-deoxy-D-xylose 5 phosphate, MEP: 2-C-methyl-D-erythriol 4-phosphate, HMBPP: 1-hydroxy-2-methyl-2(E) butenyl 4-pyrophosphate, IPP: isopentenyl diphosphate, DMAPP dimethylallyl diphosphate, GPP: geranyl pyrophosphate, FPP: farnesyl diphosphate, GGPP geranylgeranyl diphosphate. (B) Design of MVA pathway constructs we used in this study. Both the pSNA and pSCS constructs were divided into 3 parts: bottom, top, and IPP isomerase. The pSNA construct consisted of the top portion (mvaE and mvaS from E. faecalis), the bottom portion (mvaK1, mvaD, and mvaK2 from S. pneumoniae), and E. coli idi. In the case of the pSCS constructs, the top portion was from E. saccharolyticus, and the bottom portion was from S. cerevisiae. IPP isomerase was prepared in 3 different versions: native E. coli idi for pSCS1, E. coli codon-optimized E. coli idi for pSCS2, and B. subtilis fni for pSCS3. C. Plasmid constructs employed in this study to facilitate carotenoid biosynthesis. pT-LYCm4 and pT-HB are introduced in E. coli for lycopene and β-carotene biosynthesis. The pT-LYCm4 contains crtE, crtB, and crtI derived from P. agglomerans and ipiHP1 of H. pluvialis. The pT-HB was constructed by introducing crtY from P. ananatis right next to the ipiHP1 into the pT-LYCm4 plasmid construct.
J. Microbiol. Biotechnol. 2024;34:2338~2346 https://doi.org/10.4014/jmb.2408.08053
© J. Microbiol. Biotechnol.