Additional file 1 of Metabolic engineering of Rhodococcus jostii RHA1 for production of pyridine-dicarboxylic acids from lignin

Additional file 1: Figure S1. Vector for construction of the ΔpcaHG markerless deletion R. jostii strain; Figure S2. PCR confirmation of the ΔpcaHG markerless deletion; Figure S3. Growth characteristics of ΔpcaHG markerless deletion and wild-type R. jostii strains; Figure S4. HPLC analysis of ΔpcaHG markerless deletion and wild-type R. jostii strains grown in M9 minimal media containing 0.1% 4-hydroxybenzoic acid; Figure S5. Production of 2,4-PDCA observed by HPLC analysis in R. jostii ΔpcaHG containing pTipQC2ligAB; Figures S6. HPLC traces for production of 2,4-PDCA by R. jostii ΔpcaHG containing pTipQC2ligAB, and for production of 2,5-PDCA by R. jostii ΔpcaHG containing pTipQC2praA; Figure S7. Agarose gels showing loss of plasmid DNA from R. jostii ΔpcaHG containing pTipQC2ligAB; Figures S8,S9. Chromosomal integration vector (S8) for insertion of ligAB or praA genes onto R. jostii chromosome, and relevant PCR primers (S9); Figure S10. Gene expression for praA and ligAB genes for chromosomal gene insertion constructs (without promoter) by RT-PCR; Figure S11. Nucleotide sequence of constitutive promoter Ptpc5; Figures S12,S13. LigAB activity (S12) and protein production (S13) from pcaHG:ligAB(Picl) construct, induced with 1-8% methanol; Figure S14. LigAB activity observed using R. jostii ppcaHG:ligAB constructs containing four different promoters, grown in LB media; Figure S15. Whole cell biotransformation of protocatechuic acid (PCA) to 2,4-PDCA using R. jostii pcaHG:ligAB(Picl) and R. jostii pcaHG:ligAB(Ptpc5); Figure S16. Production of 2,4-PDCA using R. jostii pcaHG:ligAB(Ptpc5) grown on M9 minimal media containing 0.1% 4-hydroxybenzoic acid and 0.1% yeast extract, analysed by HPLC; Figure S17. Production of 2,4-PDCA from minimal media containing Green Value Protobind lignin by constructs containing chromosomal expression of ligAB genes, analysed by HPLC; Figure S18. SDS-PAGE gel of cell extracts of R. jostii pcaHG:ligAB(Ptpc5) with or without pTipQC2-dyp2, showing expression of recombinant Dyp2 peroxidase; Figure S19. Production of 2,4-PDCA by R. jostii pcaHG:ligAB(Ptpc5) with or without pTipQC2-dyp2, grown on M9 minimal media containing 1% wheat straw lignocellulose; Figure S20. Small scale (4 mL) testing of the production of 2,4-PDCA and protocatchuic acid (PCA) by R. jostii pcaHG:ligAB(Ptpc5) with or without expression of Amycolatopsis dyp2 gene; Figure S21. Titre of 2,4-PDCA vs time from 2.5L bioreactor for R. jostii pcaHG:ligAB(Ptpc5) grown on M9 minimal media containing 1% Green Value Protobind lignin at 30 °C; Figures S22–S24. Isolation of 2,4-PDCA product from fermentation broth: S22, Extraction of 2,4-PDCA into isopropanol; S23, HPLC analysis of extracted product; S24, 1H NMR analysis of isolated product.