Fig. 4: Optimizing in vivo production of cis-α-irone from a simple carbon source.
From: Total enzymatic synthesis of cis-α-irone from a simple carbon source
a Schematic representation of the plasmids used to produce cis-α-irone (Supplementary Table 3). Four plasmids were used. The first plasmid carried the SAR module, which contained the upper mevalonate enzymes: HMG-CoA synthase (HmgS), acetoacetyl-CoA thiolase (AtoB) and truncated HMG-CoA reductase (tHmgR), and engineered OfCCD1 fused with thioredoxin (TofCCD1m)10. The second plasmid carried the MPPI module, which contained the lower mevalonate enzymes: mevalonate kinase (MevK), phosphomevalonate kinase (PMK), mevalonate pyrophosphate decarboxylase (PMD) and isopentenyl pyrophosphate (IPP) isomerase (Idi). The third plasmid carried the lycopene synthesis EBIA module: geranylgeranyl pyrophosphate (GGPP) synthase (CrtE), phytoene synthase (CrtB), phytoene desaturase (CrtI) and farnesyl pyrophosphate (FPP) synthase (IspA). The fourth plasmid carried the pMT10 enzyme or pMT10 with the SAM cycle enzymes: S-adenosylmethionine synthase (MetK) and S-adenosylhomocysteine nucleosidase (Mtn).Tm1, Tm2, Tm3 are the mutated T7 promoters with different strengths (Tm1 > Tm2 > Tm3)21. b Schematic representation of the pathway to produce cis-α-irone from glucose or glycerol. SAM cycle enzymes (MetK and Mtn) and simplified regulation by metJ are shown. Dotted line represents the cell membrane. The bar charts represent the average amount of cis-α-irone produced in (c) the average amount of leftover psi-ionone in (d) the average specific titre of cis-α-irone produced in (e) the average specific titre of leftover psi-ionone in (f). c–f the average and the s.d. of three biologically independent experiments are shown. For c and e, significance (p-value) was evaluated by a two-sided Student’s t-test, n.s. represents p > 0.05. Refer to Supplementary Table 3 for strain-description. Three conditions were tested for in vivo α-irone production. Zero millimolar of L-Met: no methionine or dodecane was added. 10 mM L-Met: 10 mM methionine but no dodecane was added. 10 mM L-Met wDC: 10 mM methionine with 20 % by volume dodecane were added. Source data are provided as a Source Data file.
