Abstract
Muconic acid (MA) is a valuable compound for adipic acid production, which is a precursor for the synthesis of various polymers such as plastics, coatings, and nylons. Although MA biosynthesis has been previously reported in several bacteria, the engineered strains were not satisfactory owing to low MA titers. Here, we generated an engineered Corynebacterium cell factory to produce a high titer of MA through 3-dehydroshikimate (DHS) conversion to MA, with heterologous expression of foreign protocatechuate (PCA) decarboxylase genes. To accumulate key intermediates in the MA biosynthetic pathway, aroE (shikimate dehydrogenase gene), pcaG/H (PCA dioxygenase alpha/beta subunit genes) and catB (chloromuconate cycloisomerase gene) were disrupted. To accomplish the conversion of PCA to catechol (CA), a step that is absent in Corynebacterium, a codon-optimized heterologous PCA decarboxylase gene was expressed as a single operon under the strong promoter in a aroE-pcaG/H-catB triple knock-out Corynebacterium strain. This redesigned Corynebacterium, grown in an optimized medium, produced about 38 g/L MA and 54 g/L MA in 7-L and 50-L fed-batch fermentations, respectively. These results show highest levels of MA production demonstrated in Corynebacterium, suggesting that the rational cell factory design of MA biosynthesis could be an alternative way to complement petrochemical-based chemical processes.
Highlights
The aroE-encoding shikimate dehydrogenase was deleted from the chromosome to block the conversion of DHS, causing DHS to accumulate in the cells
The cg1835 gene showed 98% identity with aroE, which is known as the main shikimate dehydrogenase in the C. glutamicum R strain[31]
It was previously reported that the introduction of three heterologous biosynthetic genes such as aroZ, aroY, and catA into the aroE-deleted E. coli, led to the accumulation of Muconic acid (MA) as a final product, through the DHS-PCA-CA-MA route
Summary
Han-Na Lee[1,3], Woo-Shik Shin[2], Seung-Yeul Seo[3,4], Si-Sun Choi[1], Ji-soo Song[1], Ji-yeon Kim[1], Ji-Hoon Park[1], Dohoon Lee 2,5, SangYong Kim[2,5], Sang Joung Lee[3], Gie-Taek Chun4 & Eung-Soo Kim[1]. To accomplish the conversion of PCA to catechol (CA), a step that is absent in Corynebacterium, a codon-optimized heterologous PCA decarboxylase gene was expressed as a single operon under the strong promoter in a aroEpcaG/H-catB triple knock-out Corynebacterium strain This redesigned Corynebacterium, grown in an optimized medium, produced about 38 g/L MA and 54 g/L MA in 7-L and 50-L fed-batch fermentations, respectively. Frost and Draths successfully biosynthesized MA from glucose for the first time using E. coli[14] They introduced three foreign genes (aroZ, aroY, and catA) into the aromatic amino acid biosynthetic pathway, to produce MA via stepwise. We engineered a C. glutamicum strain to produce MA from glucose through redesign of the aromatic amino acid biosynthetic pathway. Optimization of culture media and processes using the MA-producing C. glutamicum strain were performed to achieve a significantly increased titer of MA, suggesting that the rational cell factory design in Corynebacterium could be an efficient method for MA production
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