Systems metabolic engineering of Escherichia coli for production of engineering plastic

Abstract

Systems metabolic engineering has been playing important roles in developing microbial cell factories for the production of various chemicals and materials to achieve sustainable chemical industry. Similarily with other various chemicals, there also has been much interest in microbial production of engineering plastic. In this thesis, E. coli was systems metabolically engineered to produce valuable engineering plastic precursors: 1,3-diaminopropane, ethylene glycol and lactams. In addition, development of E. coli capable of polymerizing various non-proteogenic amino acids has been carried out in order to biosynthesize the engineering plastic itself. To produce 1,3-diaminopropane, the pathway employing Acinetobacter baumannii dat and ddc genes was introduced to E. coli based on genome-scale in silico flux analysis result. Using the rationally engineered E. coli strain, 128 synthetic small RNAs (sRNA) applied in gene knockdown revealed that knocking out pfkA further increases 1,3-diaminopropane production. Next, to produce ethylene glycol in E. coli, the Dahms pathway was introduced to E. coli by expressing xylBCccs genes from Caulobacter crescentus. Various E. coli host strains and aldehyde reductases were screened for efficient ethylene glycol production. The production performance was further improved by fine-tunned knockdown of xylBccs gene expression using sRNA technology. Additionally, new metabolic pathways for production of butyrolactam, valerolactam and caprolactam were designed and constructed. This pathway comprises two steps: activation of ω-amino acids catalyzed by the Clostridium propionicum β-alanine CoA transferase (Act) and following spontaneous cyclization. Using the pathway, three metabolically engineered E. coli strains were developed which allows production of butyrolactam, valerolactam and caprolactam from renewable carbon source. Finally, novel metabolic pathway capable of polymerizing amino acids to produce poly(ester amide) was designed and constructed to directly biosynthesize engineering plastic. This pathway comprises two steps: Activation of amino acids by Act, and polymerization by polyhydroxyalkanoate synthase. Using the platform pathway, E. coli strain capable of polymerizing various amino acids such as 3-aminopropionic acid, 4-aminobutyric acid, 3-aminobutyric acid and 3-aminoisobutyric acid was developed. In conclusion, systems metabolic engineering tools and strategies were applied to construct E. coli strain producing various engineering plastic precursors such 1,3-diaminopropane, butryolactam, valerolactam and caprolactam were developed for the first time using renewable carbon source. In addition, most efficient ethylene glycol producer (higest titer and productivity ever reported) was developed. Finally, we have succeeded in developing an E. coli strain capable of polymerizing amino acids to produce poly(ester amide). It is expected that the systems metabolic engineering strategies employed and E. coli strains developed here can help us move one step closer to the bio-based engineering plastic production.