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.