Abstract
The production of aromatic compounds by microbial production is a promising and sustainable approach for producing biomolecules for various applications. We describe the metabolic engineering of Corynebacterium glutamicum to increase its production of shikimic acid. Shikimic acid and its precursor-consuming pathways were blocked by the deletion of the shikimate kinase, 3-dehydroshikimate dehydratase, shikimate dehydratase, and dihydroxyacetone phosphate phosphatase genes. Plasmid-based expression of shikimate pathway genes revealed that 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase, encoded by aroG, and DHQ synthase, encoded by aroB, are key enzymes for shikimic acid production in C. glutamicum. We constructed a C. glutamicum strain with aroG, aroB and aroE3 integrated. This strain produced 13.1 g/L of shikimic acid from 50 g/L of glucose, a yield of 0.26 g-shikimic acid/g-glucose, and retained both its phosphotransferase system and its pyruvate kinase activity. We also endowed β-glucosidase secreting ability to this strain. When cellobiose was used as a carbon source, the strain produced shikimic acid at 13.8 g/L with the yield of 0.25 g-shikimic acid/g-glucose (1 g of cellobiose corresponds to 1.1 g of glucose). These results demonstrate the feasibility of producing shikimic acid and its derivatives using an engineered C. glutamicum strain from cellobiose as well as glucose.
Highlights
Shikimic acid is a valuable hydroaromatic compound
We used ATCC13032 MRR as the parent strain for constructing shikimic acid-producing C. glutamicum (Bott et al, 2013)
No shikimic acid was detected in the parental strain, but SA-1 produced 1.3 ± 0.04 g/L of shikimic acid in CGXII medium containing 50 g/L of glucose after 72 h of cultivation (Figure 2A)
Summary
Shikimic acid is a valuable hydroaromatic compound. It is a key metabolic intermediate in the shikimate pathway, a common route for the biosynthesis of a range of aromatic compounds. Inactivation of the phosphotransferase system (PTS) and the redirection of carbon flux from pyruvate to PEP by PEP synthase expression have all been utilized (Chandran et al, 2003) Combining these metabolic approaches, Rodriguez et al (2013) engineered a shikimic acid-producing E. coli strain, which achieved production of 43 g/L of shikimic acid at a yield of 43% (mol/mol) from glucose in a batch fermenter. Kogure et al (2016) achieved a production of 141 g/L of shikimic acid from glucose using the C. glutamicum R strain using growth-arrested cell reactions in fed-batch fermentation These researchers induced the expression of a heterologous gene encoding a feedback-resistant form of DAHP from E. coli (Ger et al, 1994), as well as shikimate pathway-related genes. We constructed a high-producing shikimic acid C. glutamicum strain by rational metabolic engineering approaches without PTS inactivation, pyruvate kinase disruption, or the introduction of feedback-resistant variants of DAHP synthase. The titer of shikimic acid production was 13.8 g/L with the same yield of glucose
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