Inactivation of Pyruvate Kinase or the Phosphoenolpyruvate: Sugar Phosphotransferase System Increases Shikimic and Dehydroshikimic Acid Yields from Glucose in <b><i>Bacillus subtilis</i></b>

Abstract

The glycolytic intermediate phosphoenolpyruvate (PEP) is a precursor of several cellular components, including various aromatic compounds. Modifications to the PEP node such as PEP:sugar phosphotransferase system (PTS) or pyruvate kinase inactivation have been shown to have a positive effect on aromatics production capacity in Escherichia coli and Bacillus subtilis. In this study, pyruvate kinase and PTS-deficient B. subtilis strains were employed for the construction of derivatives lacking shikimate kinase activity that accumulate two industrially valuable chemicals, the intermediates of the common aromatic pathway, shikimic and dehydroshikimic acids. The pyruvate kinase-deficient strain (CLC6-PYKA) showed the best production parameters under resting-cell conditions. Compared to the PTS-deficient strain, the shikimic and dehydroshikimic acids specific production rates for CLC6-PYKA were 1.8- and 1.7-fold higher, respectively. A batch fermentor culture using complex media supplemented with 83 g/l of glucose was developed with strain CLC6-PYKA, where final titers of 4.67 g/l (shikimic acid) and 6.2 g/l (dehydroshikimic acid) were produced after 42 h.

Studies on the production of shikimic acid using the aroK knockout strain of Bacillus megaterium

“…It shows the importance of blocking the pathway downstream of shikimic acid by knocking out the aroK gene. The aroK knock out strain showed less formation of side products as compared to the similar knock out strains reported in other microorganisms (Chen et al 2012;Liu et al 2014;Licona-Cassani et al 2014). It suggests that by generating aroK knock out mutant in B. megaterium, one can have better metabolic control than other microbial platforms used for shikimic acid production.…”

“…This shows enhancement in shikimic acid yield over the previously reported aroK knockout strain of Bacillus sp. (Licona-Cassani et al 2014) and makes it a better process in terms of substrate utilization. The cellmass concentration started increasing from the very beginning and a maximum of 4.03 g/L cellmass was obtained at 40 h. DO concentration showed the downward trend initially along the course of growth and reached almost zero at the end of fermentation.…”

“…Simultaneous overexpression of genes for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (aroA) and SA dehydrogenase (aroD) in Bacillus subtilis was mentioned in literature which resulted in SA titer of 3.2 g/L . A modified B. subtilis strain with inactivation of pyruvate kinase and shikimate kinase was also reported to produce 4.67 g/L titer of shikimate (Licona-Cassani et al 2014).…”

Transcription Factor Engineering for High-Throughput Strain Evolution and Organic Acid Bioproduction: A Review

“…Shikimic acid is an important metabolic intermediate of the shikimate pathway. Various microorganisms have been engineered to produce SA (Licona-Cassani et al, 2014;Martínez et al, 2015;Kogure et al, 2016). developed an SA biosensor constructed from a LysR-type transcriptional regulator ShiR to monitor the SA production of different Corynebacterium glutamicum strains (Schulte et al, 2017).…”

Current perspectives on applications of shikimic and aminoshikimic acids in pharmaceutical chemistry

“…76 A B. subtilis strain carrying a defective shikimate kinase enzyme, deletion of the aroE gene encoding for EPSP synthase with enhanced shikimate dehydrogenase, and DAHPS activities successfully accumulated SA, while a Pyk − , PTS − , and shikimate kinase-deficient B. subtilis strain successfully accumulated DHS and SA. 77,78 In addition to microbial fermentative processes, SA has been produced from glucose in a two-step procedure involving the cell-dried or membrane fractions from Gluconobacter oxydans with the initial conversion of QA → DHQ → DHS catalyzed by quinate dehydrogenase and DHQ dehydratase enzymes. This reaction is then coupled to a second step catalyzed by cytoplasmic enzymes: SA dehydrogenase (NADPH [dihydronicotinamide-adenine dinucleotide phosphate] + DHS → SA + NADP [nicotinamide adenine dinucleotide phosphate]) and the NADPH regenerating glutamate dehydrogenase (glucose + NADP → NADPH + glucono-δ-lactone), with a conversion efficiency of 57%-77% from QA.…”