Figure captions
FIGURE 1 Construction and verification of IUP in E. colifor lycopene production. (A) Two steps catalysis from isoprenol and prenol to two building blocks IPP and DMAPP, followed by conversion to lycopene by CrtEBI . ADP: adenosine diphosphate; ATP: adenosine triphosphate; IP: isopentenyl phosphate; IPP: isopentenyl diphosphate; DMAP: dimethylallyl phosphate; DMAPP: dimethylallyl diphosphate; GPP: geranyl diphosphate; FPP: farnesyl diphosphate; GGPP: geranylgeranyl diphosphate. ThiM , E. coli kinase: hydroxyethylthiazole kinase; IPK isopentenol kinase; idi : IPP Δ-isomerase;IspA : FPP synthase; CrtE : GGPP synthase; CrtB : phytoene synthase; CrtI phytoene desaturase (B) YZ0: BL21(DE3) harboring pEBI; YZ1: BL21(DE3) harboring pEBI and pC19IUP. (C) Lycopene titer of strain YZ0 with or without prenol and YZ1 with prenol.
FIGURE 2 The optimization of ThiMEc andIPKMTH by promoter engineering and IDI selection. (A) IPKMTH and ThiMEc are under control of constitutive promotors of three level: strong (***): PJ23119, medium (**): PJ23106 and low (*): PJ23109, respectively. (B) Lycopene titer of 9 strains in combination of constitutive promotors. (C) Both IPKMTH and ThiMEcare under control of four kinds of inducible promotor: L-rhamnose monohydrate inducible promotor (PRha), Anhydrotetracycline hydrochloride inducible promotor (Ptet), L-Arabinose inducible promotor (PBAD) and IPTG inducible promotor (Ptac). Type I IDI: idi from E.coli ; Type II IDI: idi from Bacillus subtilis and idi fromSaccharomyces cerevisiae (D) Lycopene titer of strain YZ4~7 referring to BL21(DE3), respectively harboring pEBI and IUP pathway controlled by Ptac, Ptet, PBAD and PRha. Lycopene titer of strain YZ7 mixed with 1g/L prenol and 1g/L of combined prenol and isoprenol (1p/1i means the ratio of prenol and isoprenol is 1:1). (F) Lycopene titer of strains YZ7, YZ71 and YZ72, separately employing IDIEc, IDIBc and IDISc.
FIGURE 3 Screening and verification of RBS of ThiMEc to increase lycopene production. (A) The IUP expression frame after preliminary optimization. IPKMTH ,ThiMEc and idiSc are under control inducible promoter PRha. The streamline of best RBS screen and verification for ThiMEc . a) Circular PCR utilizing degenerate primer containing R(A/G) for construction of RBS library of ThiMEc . b) The RBS library of ThiMEc . was obtained by transferring PCR product to DH5α and extraction of mixed plasmids. c) The mixed library combined with pEBI was transferred to BL21(DE3), obtaining a series of single colonies. d) Randomly selection and fermentation of transformants e) Selection of cells with crimson followed by sequencing g) The ligation of ThiMEc . controlled by original RBS(RBS0) and selected RBS with GFP and fermented the according strains at 2 hours after induction) The test of cell density and expression level of ThiMEc .with RBS0 and RBS5 through fluorescence by microplate reader. (B) The lycopene titer of 11 strains (YZ72-1~11) randomly selected with crimson. Control strain YZ72 harbors IUP, of which the ThiMEc .is controlled by RBS0. (C) The fluorescence value per unit cell ofThiMEc . with RBS0 and RBS5 (calculation: total fluorescence/OD600).
FIGURE 4 The optimization of fermentation condition for lycopene production through IUP pathway. (A) Effects on transcription and cell growth of IPTG concentration. (B) Lycopene titer of strain YZ72-5 mixed with a series of concentration of IPTG (0.5, 0.1, 0.05, 0.01 and 0.005 mMol/L). (C) Lycopene titer of strain YZ72-5 provided with 0.005 mMol/L IPTG and 1g/L substrates of different ratio (p: 100% prenol; prenol: isoprenol=5:1, 3:1; 1:1; 1:3 and 1:5; i: 100% isoprenol). (D) Lycopene titer of strain YZ72-5 supplied with 0.005 mMol/L IPTG and different concentration (1g/L; 2g/L and 3g/L) of substrates, of which prenol: isoprenol is 1:3.
FIGURE 5 Rapid construction of the multicopy IUP genome-harboring chassis through PtrCAST for lycopene production (A) Transposition targeting 8 loci/IS1 loci on BL21(DE3); Verification strain library with different copies of IUP expression cassette; Transformation of pEBI into strain library and random selection of transformants for fermentation, followed by picking strains with crimson for detection and sequencing. (B) Lycopene titer of strain YZ72-5 with IUP in plasmids, YZJ3 with 7 copies IUP expression cassette in genome and YZJ3-4 with 13 copies IUP expression cassette in genome both cultivated in flask and fermenter. (C) Fed-batch fermentation profile of the strain YZJ3-4.