Fig. 1

De novo production of branched-chain alcohols derived MBE. A Scheme of the branched-chain alcohols (BCA)-derived multi-methyl-branched esters (MBE) biosynthesis pathway in a recombinant E. coli strain. Overexpression of the native PrpE (propionyl-CoA ligase) and the heterologous propionyl-CoA carboxylase (PCC) complex (AccA, PccB and PccE subunits) from S. coelicolor (S. coel) leads to the production of methylmalonyl-CoA (MM-CoA) from exogenous propionate. The MBFA are produced by the MA system from M. tuberculosis (M. tb) (highlighted in light blue, plasmid pMB24); acyl-AMP ligase (FadD28), mycocerosic acid polyketide synthase (Mas), polyketide-associated protein A5 (PapA5). Sfp is a phosphopantetheinyl transferase of B. subtilis (B. sub). The BCA isobutanol and isopentanol are produced de novo by the conversion of 2-keto acids into the corresponding alcohols by the expression of L. lactis 2-ketoisovalerate decarboxylase Kivd and S. cerevisiae alcohol dehydrogenase Adh2 (dark blue, plasmid pIAA12), and the enzymes acetolactate synthase AlsS from B. subtilis, and E. coli ketol-acid reductoisomerase IlvC and dihydroxy-acid dehydratase IlvD (red, plasmid pIAA11). The genes encoding for the enzymes highlighted in brown are integrated into the chromosome and under the control of T7 promoters. B Representative TLC of three independent experiments showing total lipid pattern and MBE production of RQ5.1 and RQ5m (RQ5.1/pIAA11/pIAA12) strains 48 h post induction. (+), isobutanol supplemented to the culture medium at a final concentration of 20 mM. C Quantification of BCA production by strain RQ5m at 24 and 48 h post induction. Error bars represent the standard deviation of three independent experiments. PYR, pyruvate; AL, 2-acetolactate; DHIV, 2,3-dihydroxy-isovalerate; KIV, 2-ketoisovalerate; 2-IPPM, 2-isopropylmalate; 3-IPPM, 3-isopropylmalate; IPOS, 2-isopropyl-3-oxosuccinate; KIC, 2-ketoisocaproate; PP-CoA, propionyl-CoA; FAS, fatty acid synthase; FFA, free fatty acids. BCA-MBE, isobutanol and isopentanol-derived MBE; Et-MBE, ethanol derived MBE; Phe-MBE, 2-phenylethanol derived MBE; MAG, monoacylglycerides; DAG, diacylglycerides