Additional file 1 of A strategy to enhance and modify fatty acid synthesis in Corynebacterium glutamicum and Escherichia coli: overexpression of acyl-CoA thioesterases

Additional file 1: Fig. S1. Lipid metabolism and the predicted regulatory mechanisms in Corynebacterium glutamicum. There was a type I fatty acid synthase (FAS-I) system in C glutamicum, and the metabolites were CoA derivatives. As a TetR-type transcriptional regulator, FasR affected the transcriptional expression of genes including accD1, fasA, and fasB. Meanwhile, acyl-CoA could inhibit Acc, FasA, and FasB. This organism did not degrade fatty acid naturally due to the lack of the β-oxidation pathway. The red lines represented reference to previous studies (Ikeda et al. 2020), where double lines indicated inhibition and predicted inhibition and solid and dashed arrows indicated single and multiple enzymatic processes, respectively. The blue lines showed the predicted reverse β-oxidation pathway, a novel fatty acid synthesis pathway. Acetyl CoA carboxylase (Acc) was composed of AccBC, AccD1, and AccE. AccBC, acetyl-CoA carboxylase α subunit; AccD1, acetyl-CoA carboxylase β subunit; AccE, acyl carboxylase ε subunit; NCgl2309, acetyl-CoA acetyltransferase; NCgl0919, enoyl-CoA hydrolase; NCgl0973, acyl-CoA dehydrogenase; PD, pyruvate dehydrogenase; FasA, fatty acid synthase IA; FasB, fatty acid synthase IB; FasR, fatty acid synthesis inhibitory protein; Tes, acyl-CoA thioesterase; FadD, Acyl-CoA synthase. The dotted boxes in the figure were exogenous genes. FadE and Ter, acyl-CoA dehydrogenase; EchA, enoyl-CoA hydratase; FadB, multifunctional enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase; FadA, ketoacyl-CoA reductase. Fig S2. Agarose gel electrophoresis of the positive clones for heterologous expression in E. coli. A PCR validation of bacterial liquid. Lane M2, 2000 bp DNA marker; Lane bA, bacterial liquid PCR of BLtesA; Lane bB, bacterial liquid PCR of BLtesB; Lane b9, bacterial liquid PCR of BLte9; B double digestion validation of the recombinant plasmid. Lane M5, 5000 bp DNA marker; Lane dA, double digestion of plasmid pET_tesA extracted from BLtesA; Lane dB, double digestion of plasmid pET_tesB extracted from BLtesB; Lane d9, double digestion of plasmid pET_te9 extracted from BLte9. Fig S3. Agarose gel electrophoresis showed the PCR products of genomic DNA extracted from the knockouts. Lane M5, 5000 bp DNA marker; Lane −, negative control; Lane +, positive control; Lane aA and bA, PCR products of genomic DNA from CGΔtesA; Lane vA, PCR product of pK18_tesA without lacZ; Lane aB, bB and cB, PCR products of genomic DNA from CGΔtesB; Lane vB, PCR product of pK18_tesB without lacZ; Lane a9, b9 and c9, PCR products of genomic DNA from CGΔte9. Non-specific amplification in Lane a9 probably due to impure colonies, mixed with misassembled clones; Lane G, PCR product of genomic DNA from C. glutamicum. Fig. S4. Agarose gel electrophoresis of the positive complementations. A PCR validation of bacterial liquid. Lane M2, 2000 bp DNA marker; Lane hA, bacterial liquid PCR of CGtesA; Lane hB, bacterial liquid PCR of CGtesB; Lane h9, bacterial liquid PCR of CGte9; B double digestion validation of the recombinant plasmid. Lane M5, 5000 bp DNA marker; Lane qA, double digestion of plasmid pX_tesA extracted from CGtesA; Lane qB, double digestion of plasmid pX_tesB extracted from CGtesB; Lane q9, double digestion of plasmid pX_te9 extracted from CGte9. Fig. S5. Schematic diagram of gene knockout. A An efficient ‘‘blue spot selection’’ knockout method. LB, Luria-Bertani media; X-gal, 5-bromo-4-chloro-3-indolyl-D-galactopyranoside; Kan, kanamycin; Suc, sucrose. B Recombinant knockout vectors. Fig. S6 Colony morphology. A C glutamicum wild type grown on LB/X-gal plate. B C glutamicum electrotransformants grown on LB/X-gal/Kan plate, blue colonies might be positive clones carrying recombinant knockout vectors. Fig. S7. Standard curve of BSA. (OD: Optical Density; BSA: Bovine Serum Albumin). Fig. S8. Lineweaver-Burke plots of TesA A, TesB B, and TE9 C.