Dynamic Flux Balance Analysis to Evaluate the Strain Production Performance on Shikimic Acid Production in Escherichia coli

Flux balance analysis (FBA) is used to improve the microbial production of useful compounds. However, a large gap often exists between the FBA solution and the experimental yield, because of growth and byproducts. FBA has been extended to dynamic FBA (dFBA), which is applicable to time-varying processes, such as batch or fed-batch cultures, and has significantly contributed to metabolic and cultural engineering applications. On the other hand, the performance of the experimental strains has not been fully evaluated. In this study, we applied dFBA to the production of shikimic acid from glucose in Escherichia coli, to evaluate the production performance of the strain as a case study. The experimental data of glucose consumption and cell growth were used as FBA constraints. Bi-level FBA optimization with maximized growth and shikimic acid production were the objective functions. Results suggest that the shikimic acid concentration in the high-shikimic-acid-producing strain constructed in the experiment reached up to 84% of the maximum value by simulation. Thus, this method can be used to evaluate the performance of strains and estimate the milestones of strain improvement.

Glyphosate as a tool to produce shikimic acid in plants

Oseltamivir phosphate is a potent viral inhibitor produced from shikimic acid extracted from seeds of Ilicium verum, the most important natural source. With the site of action 5-enolpyruvylshikimate-3-phosphate synthase (EPSP), glyphosate is the only compound capable of inhibiting its activity with the consequent accumulation of shikimic acid in plants. Corn and soybean plants were sprayed with reduced rates of glyphosate (0.0 to 230.4 g a.i. ha¹) and shikimic acid content in the dry mass was determined by HPLC 3, 7 and 10 days after application. Results showed shikimic acid accumulation in dry mass with increases of up to 969% in corn and 33,000% on soybeans, with peak concentrations 3 days after treatment (DAT). Industrial feasibility for shikimic acid production, combined with favorable climatic conditions for growing corn and soybean in virtually all over Brazil, favor the use of reduced rates of glyphosate in shikimic acid biosynthesis, with potential for use as an inducer in exploration of alternative sources for production of oseltamivir phosphate with low environmental impact.

Shikimic Acid Production from Ginkgo biloba via Callus Culture

Shikimic acid is a very important precursor for industrial synthesis of oseltamivir (Tamiflu®) which is used for the antiviral treatment. In this study, callus culture of Ginkgo biloba for shikimic acid production was reported. Callus induced from either leaves or nodal stems of sterilized ginkgo was grown on MS medium supplemented with a combination of plant growth regulators as followed: MS+KD, MS+BD, MS+KN and MS+BN for 90 days. Morphological changes, fresh weight and shikimic acid content of callus in each medium were monitored every 30 days. The result showed that callus cultures from each treatment were morphologically different. It is likely due to explant used for callus induction and type of plant growth regulators added into the medium. Browning effect was noticeably detected from 60 days to 90 days. Moreover, fresh weight and shikimic acid content of callus culture depended on cultivation time, cultivation medium and type of explants used for callus induction. Callus induced from nodal stem grown on MS+BN for 30 days offered the highest fresh weight. For shikimic acid production, the most satisfied quantity of shikimic acid was achieved from callus cultured on MS+KN for 30 days by exploiting nodal stem as explant.