Towards renewable commodity chemicals: Biosynthesis of phloroglucinol and chemoenzymatic synthesis of caprolactam.

摘要

In rise of the uncertainty associated with the chemical industries reliance on non-renewable resources for virtually all commodity chemical manufacture, the research for sources with renewable feedstocks are of importance. The following research comprises of chemistry which progresses towards the deviation from a petroleum-based chemical economy, to one that thrives on starting materials from renewable sources through utilization of rapid and efficient syntheses dependent upon microbial biocatalysts. Efforts aimed to biocatalytically produce 1,3,5-trihydroxybenzene and an epsilon-caprolactam precursor from renewable resources at a level with commercial importance. To enhance the viability of commercial manufacture, following a successful biocatalytic production, efforts focused on delivering chemicals with the same level and purity to compete with nonrenewable routes.;epsilon-Caprolactam, the monomer for nylon 6, was synthesized from L-beta-lysine. As a renewable feedstock, D-glucose derived L-lysine had been shown to cyclize and deaminate to form epsilon-caprolactam in previous works. Alternatively, L-beta-lysine was researched for applicability in an analogous synthesis of epsilon-caprolactam. Under anaerobic conditions, Clostridium subterminale SB4 degrades L-lysine as a carbon source with two initial metabolic, amino group isomerizations, to L-beta-lysine and then 3,5-diaminohexanoate. Successful, selective inhibition of the second aminomutase enzyme was achieved through intense irradiation with light, providing an in vivo microbial synthesis of L-beta-lysine. The resulting biosynthetic L-beta-lysine product, ultimately from renewable D-glucose, was cyclized to beta-aminocaprolactam in near quantitative yields by reflux in high temperature alcohols. Hydrodenitrogenation of beta-aminocaprolactam to epsilon-caprolactam was achieved only in trace amounts.;Phloroglucinol's vast synthetic utility is overshadowed by the problematic synthesis, which precludes its use in commodity chemical markets. Phloroglucinol in vivo biocatalysis, from D-glucose, had been shown previously by heterologous expression of phloroglucinol synthase (Type III polyketide synthase, phlD) from Pseudomonas fluorescens Pf-5 in Escherichia coli. Microbial synthesis of phloroglucinol in a controlled fermentation vessel was increased to concentrations approaching 5 g/L at 6% yield (mol phloroglucinol/mol D-glucose) in fed-batch mode with a 1 L working volume scale. Phloroglucinol toxicity is contributive to limited production, leading to an engineered continuous-extractive, two stage microbial synthesis that afforded concentrations up to 38 g/L at 12% yield (mol phloroglucinol/mol D-glucose). Besides continuous extraction to alleviate toxicity issues, transcriptome analysis was another avenue of research. Upregulated genes by transcriptome analysis, along side probable E. coli export machinery, were researched and manipulated in hopes of generating a phloroglucinol tolerant host, to no avail. Genetic advancement of the Pseudomonas fluorescens Pf-5 phlD gene through codon optimization increased the crude lysate specific activity, from malonyl CoA to phloroglucinol, two-fold to 0.045 mumol phloroglucinol/min/mg protein. Downstream purification of biocatalytically produced, phloroglucinol had recovery rates of 91% from production to bottled chemical, that was independently certified to be 99.6% pure.