Microbial catalysis for the production of hydroxy- and amino-fatty acids

This study focused on the microbial synthesis of 3-hydroxybutyric acid (3HBA), which is industrially relevant but can also be converted into non-coded amino fatty acids. Synthetic operons combining the acetoacetyl-CoA thiolase gene (phaA) from C. necator, either the (R)- (phaB) or (S)-3-hydroxybutyryl-CoA dehydrogenase gene (paaH1) from C. necator, and thioesterase (tesB) from E. coli or 3-hydroxyisobutyryl-CoA hydrolase (bch) from B. cereus were previously developed in our laboratory to synthesize 3HBA from glucose. While successful, 3HBA synthesis produced acetate as a by-product of overflow metabolism. To address acetate co-production, genes encoding phosphotransacetylases Pta and EutD were targeted for deletion using modified CRISPR-Cas9 methods. Testing in flasks revealed that the Δpta mutant showed no 3HBA synthesis, while the double deletionΔeutDΔpta produced 1.2-fold less acetate but also decreased product yield by 2.3-fold. The ΔeutD mutant increased product yield by 1.3-fold, but acetate levels rose as well. To optimize 3HBA synthesis further, a bioprocess was developed. Aerobic fermentation with BL21_tesB_RHBD yielded 6.89 g/L of 3HBA when protein expression was induced at higher densities, representing a 4.6-fold increase from previous flask conditions. Although flask cultivation showed poor results, the ΔeutD_tesB_RHBD strain in the bioprocess achieved a 1.6-fold increase compared to BL21, resulting in 11.2 g/L (R)3HBA while maintaining acetate at about 1 g/L. Additionally, a cascade reaction was designed to convert 3HBA to 3-oxobutyric acid (3OBA) and then to the non-coded amino acid 3-amino butyric acid (3ABA). Since 3OBA is an intermediate in 3HBA synthesis, it was hypothesized that replacing 3-hydroxybutyryl-CoA with an ω-transaminase (CV_TA) known for 3OBA-methyl ester conversion would yield 3ABA from glucose. However, the new pathway did not produce 3ABA, as CV_TA only acted on the methyl ester form of 3OBA. Screening of three commercially available transaminases identified ATA-117 as a promising candidate, with molecular docking suggesting its potential interaction with 3OBA but not 3OBA-CoA due to unfavorable interactions in the active site. A one-pot cascade reaction was also tested to produce α-amino fatty acids and 3ABA from hydroxy fatty acids. Five enzymes were evaluated for hydroxy fatty acid oxidation to provide substrates for transaminase reactions. The two alcohol dehydrogenases (ADHs) did not exhibit activity towards the target substrates. While glycolate oxidase (GO) exhibited activity with all tested 2-hydroxy fatty acids, both GO and CV_TA showed mutual inhibition in one-pot assays. Two lactate dehydrogenases were cloned, but their activity remained untested due to time constraints. The potential applications of non-coded amino acids (ncAAs) are numerous. Here their application was tested regarding the enhancement of antimicrobial peptide activity. Based on previous research, a nonapeptide derived from bovine lactorerricin was modified by adding ncAAs with acyl chains ranging from C4 to C10 at the C- or N-terminus. The modified peptides were tested against E. coli, B. subtilis, P. aeruginosa, S. typhimurium, and S. aureus using minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) assays, along with electron microscopy to assess peptide-bacteria interactions. Results indicated a correlation between increased antimicrobial peptide potency and fatty amino acid chain length. While modifications at N- and C-terminals exhibited varying effects i.e. in some instances the C-terminal modification had a more profound effect, while in other the N-terminal modification appeared more powerful, no consistent pattern emerged. SEM and TEM images showed membrane damage in E. coli and B. subtilis, suggesting membrane disruption as the primary action mechanism. Circular dichroism analysis revealed that ncAA incorporation did not alter the secondary structure.