Add time:07/13/2019         Source:sciencedirect.com

Dicarboxylic acids, such as the phthalic acids and their derivatives, are monomeric components in several important polyesters and polyamides. In most cases, these compounds are derived from fossil fuels and are not easily biodegradable. Dipicolinic acid (DPA) is a biologically derived aromatic di-acid that has a similar structure to isophthalic acid. Furthermore, DPA has been shown to give rise to polyesters, is readily biodegradable, and is non-toxic. DPA is naturally produced by Bacillus and Clostridium species during sporulation and can comprise up to 15% of the dry weight of bacterial spores. In this paper we demonstrate the first heterologous production of DPA and identify the genes appropriate for gram-scale production in the industrial workhorse organism, E. coli. Initially, several combinations of genes from the lysine pathway, including lysC, asd, dapA, and dapB, were overexpressed to determine which genes are necessary for recombinant production in E. coli. The in vitro activity of dipicolinate synthase was then compared between Bacillus subtilis and Clostridium perfringens. Next, in order to improve DPA production from glucose, an optimized strain was created that lacked several genes (lysA, tdh, and metA), resulting in 5.21 g/L DPA when 5 g/L of aspartate was supplied. Then, several aspartate kinases and dipicolinate synthases were screened for optimal activity in E. coli. The optimal genes were combined with the overexpression of phosphoenolpyruvate carboxylase to develop a full biosynthetic pathway capable of producing a titer of 4.7 g/L DPA directly from glucose. In summary, we have performed a detailed biochemical study of the key pathway enzyme dipicolinate synthase and achieved scalable heterogeneous production of DPA in the workhorse organism E. coli.

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