| Muconic acid(MA) is a promising C6 block planform chemical due to its extensive industrial applications in the production of adipic acid and other valuable, biodegradable intermediates. MA is heretofore mainly produced from petrochemicals by organic reactions which are not environmentally friendly or renewable. Biological production processes provide a promising alternative for MA production. In this study, two biological approcahs were developed. On one hand, a novel synthetic pathway was designed in E. coli to produce MA from renewable resourses such as glucose. On the other hand, an one-step biocatalysis method was developed for MA production from catechol. The main results were described as follows:(1) Two genes, entX(encoding 2,3-dihydroxybenzoate decarboxylase) and catA(encoding catechol 1,2-dioxygenase) were introduced into pACYCDUET-1 and overexpressed the two genes in E. coli JM109(DE3). As a result, a novel MA biosynthesis approach was developed and the final concentration of MA reached 1.25 mg/L from glucose. After obtaining the MA biosysthetic pathway, we tried some optimization strategies to further improve MA concentration. First, we intensified the enterobactin pathway by overexpressed three related genes, named entC, entB and entA, which resulted in a remarkable improvement to MA concentriation of 102.71 mg/L. To relieving feedback inhibition of aromatic amino acid to shimimate pathway, we overexpressed aroGfbr(which has a D146 N mutation). As a result, the final MA concentration reached 337 mg/L by this strategy along with overexpressing aroL, which encodes shikimate kinase.(2) Relieving metabolic burden to improve MA concentration. Firstly, MA concentriation further improved to 456.77 mg/L by increasing the copy number of ent X and catA in E. coli. Optimization of IPTG induction could relieve growth inhibition and metabolic burden effectively. Finally, the titer of MA reached 605.18 mg/L.(3) MA production in a 3-L fermentor. We attempted to use glycerol instead of glucose as the carbon source for MA biosynthsis in a 3-L fermentor. After 50 h, the final OD660 reached 60.2, and MA concentration reached 2.88 g/L, while 2,3-DHB concentration accumulated to 5.89 g/L and no catechol was detected. We presumed that ampicillinum was degraded in the latter part of fermentation, which caused the plasmid loss. As a result, the recombinant strain no longer had EntX and CatA activities. Then we use a more stable antibiotic, carbenicillin, as a substitute of ampicillinum. The results showed the final MA concentration reached 5.76 g/L with infinitesimal 2,3-DHB and catechol after 85 h fermentation with a final OD660 of 148.(4) One-step biocatalysis of catechol to MA. catA from P. putida KT2440 was chosen as a candidate with a relative higher activity. Then this gene was overexpressed in E. coli BL21(DE3). After optimization, in a 20-mL reaction system, the final MA concetration reachd 50.31 g/L with a yield(mol/mol) of 87.9% when 10 mM catechol was added to the system 30-minutes increments. At pH 2.0 and 5°C, MA readily precipitated from solution and crystals were recovered by vacuum filtration. This method recovered 90% of MA in the purified broth with a high degree of purity(>99%)... |
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