RESUMO
ω-Hydroxyundec-9-enoic acid (ω-HUA) was reported as a valuable medium-chain fatty acid with industrial potentials. For bioconversion of ricinoleic acid to ω-HUA, in this study, an alcohol dehydrogenase (Adh) from Micrococcus luteus, a Baeyer-Villiger monooxygenase (BVMO) from Pseudomonas putida KT2440 and an esterase (Pfe1) from Pseudomonas fluorescens SIK WI were overexpressed in Escherichia coli BL21(DE3). In order to enhance accessibility of Pfe1 to the (E)-11-(heptanoyloxy) undec-9-enoic acid (11-HOUA) substrate, a truncated PelB signal sequence without the recognition site of signal peptidase (tPelB) was attached to the N-terminus of Pfe1, resulting in the construction of E. coli AB-tPE strain expressing Adh, BVMO and the tPelB-Pfe1 fusion protein. A batch-type biotransformation of ricinoleic acid by E. coli AB-tPE resulted in 1.8- and 2.2-fold increases in ω-HUA conversion yield and productivity, respectively, relative to those for the control strain without the PelB sequence (AB-E). By fed-batch-type biotransformation with glycerol feeding, the AB-tPE strain produced 23.7 mM (equivalent to 4.7 g/L) of ω-HUA with 60.8%(mol/mol) of conversion yield and 1.2 mM/h of productivity, which were 13.2, 2.9, and 2.6 times higher than those in a batch-type biotransformation using the AB-E strain. In conclusion, combination of the truncated PelB-Pfe1 fusion and fed-batch process with glycerol feeding provided the highest efficiency of ω-HUA biotransformation, of which strategies might be applicable for biotransformation of hydrophobic substances.
Assuntos
Escherichia coli/metabolismo , Esterases/genética , Microbiologia Industrial , Polissacarídeo-Liases/química , Sinais Direcionadores de Proteínas , Ácidos Undecilênicos/metabolismo , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biotransformação , Escherichia coli/genética , Esterases/metabolismo , Expressão Gênica , Glicerol/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Polissacarídeo-Liases/genética , Proteínas Recombinantes/metabolismo , Ácidos Ricinoleicos/metabolismoRESUMO
This study aimed at the development of biotransformation strategies with feeding of energy sources for bioconversion of ricinoleic acid to (E)-11-(heptanoyloxy) undec-9-enoic acid (11-HOUA), a key intermediate of brassylic acid, by recombinant Escherichia coli overexpressing an alcohol dehydrogenase from Micrococcus luteus and a Baeyer-Villiger monooxygenase from Pseudomonas putida KT2440. Feeding of glucose or glycerol facilitated both the preparation of high-density cell biocatalyst and supply of the NAD+ and NADPH cofactors. By the glucose feeding strategy, 30.8g/L of the engineered E. coli cells produced 29.7mM of 11-HOUA with 1.9mM/h of productivity, which were 1.8 and 1.6 times higher than the same biotransformation without the glucose feeding, respectively. Intermittent addition of glycerol increased 11-HOUA productivity by 16% compared to that by the glucose feeding. Finally, 34.5mM of 11-HOUA concentration, 77% conversion and 2.2mM/h productivity were obtained using 31.6g/L of cell biocatalyst along with the glycerol addition. It was concluded that supplementation of additional carbon sources in biotransformation process would be a potent strategy to increase the performance of fatty acid conversion.
Assuntos
Escherichia coli/efeitos dos fármacos , Glucose/farmacologia , Glicerol/farmacologia , Ácidos Ricinoleicos/metabolismo , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Reatores Biológicos , Biotransformação , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Undecilênicos/metabolismoRESUMO
For the analysis of multiple-reaction intermediates for long-chain dicarboxylic acid biotransformation, simple and reproducible methods of extraction and derivatization were developed on the basis of gas chromatography with flame ionization detector (GC-FID) instead of mass spectrometry. In the derivatization step, change of the ratio of pyridine to MSTFA from 1:3 to 9:1 resulted in higher peak intensity (p = 0.021) and reproducibility (0.6%CV) when analyzing 32 g/l ricinoleic acid (RA). Extraction of RA and ω-hydroxyundec- 9-enoic acid with water containing 100 mM Tween 80 showed 90.4-99.9% relative extraction efficiency and 2-7%CV compared with those with hydrophobic ethyl acetate. In conclusion, reduction of the pyridine content and change of the extraction solvent to water with Tween 80 provided compatible derivatization and extraction methods to GC-FID-based analysis of longchain carboxylic acids.