Shikimic acid biosynthesis in microorganisms: Current status and future direction

The biological manufacturing of hydroaromatic compounds has recently attracted extensive attention (Kogure and Inui, 2018). This vital class of chemicals has a huge market and broad industrial prospects as platform chemicals in diverse fields such as polymer materials, pharmaceuticals, and food industry (Averesch and Kromer, 2018). Shikimic acid (SA) is an essential intermediate in the anabolic pathway for hydroaromatic compounds. Owing to its significant pharmaceutical function in the chemical synthesis of the anti-influenza drug oseltamivir phosphate (OSP, an efficient antiviral inhibitor for the treatment of seasonal influenza virus types A and B, human influenza virus H1N1, and avian influenza virus H5N1), pharmaceutical enterprises represented by Roche have escalated their demand for SA (Adelfo Escalante et al., 2014; Martinez et al., 2015). In addition, SA can be assembled into different bioactive compounds, including zeylenone (an antitumor compound) (Zhang et al., 2006), valiolamine (an α-glycosidase inhibitor) (Quan et al., 2013), 3,4-oxo-isopropylidene SA (ulcerative colitis treatment) (Xing et al., 2012), triacetyl SA (an antiplatelet and antithrombotic compound) (Huang et al., 2001), Pt(dach)(SA)2 (an antitumor compound) (Farrell et al., 1991), and 1α, dihydroxy-19-Nor previtamin D3 (for the treatment of osteoporosis and with possible use in the treatment of malignancies) (Candeias et al., 2018; Dı'az et al., 2000) (Fig. 1). Given its significant application potential, it is necessary to develop effective and environmentally friendly approaches for the large-scale production of SA.

Chemical synthesis of SA based on the Diels-Alder reaction has been successfully performed (Ghosh et al., 2012). Nonetheless, chemical methods lack commercial viability because of low yield, production of waste containing environmental pollutants, and required intensive capital (Rawat et al., 2013b). Hence, the commercial production of SA is primarily dependent on extraction from Illicium verum Hook. f., which is still an expensive and cumbersome manufacturing process with the disadvantages of environmental pollution and low yield (Bochkov et al., 2012; Rawat et al., 2013b). With an increase in research on the shikimate pathway in plants and microorganisms, microbial fermentation production of SA is now considered a better alternative to chemical synthesis or plant extraction methods (Kramer et al., 2003). To date, metabolically engineered or mutated Corynebacterium glutamicum (Kogure et al., 2016), Escherichia coli (Chandran et al., 2003), Citrobacter freundii (Rawat et al., 2013c), Bacillus megaterium (Ghosh et al., 2016), Bacillus subtilis (Licona-Cassani et al., 2014), and Saccharomyces cerevisiae (Guo et al., 2018) have been employed for the production of SA. The construction of these engineered strains considerably promoted research related to SA (Table 1, Table 2).

E. coli, owing to the advantages of its unambiguous genetic background and rapid cell growth, is generally employed as a cell factory for natural product biosynthesis (Yang et al., 2020). To date, it is feasible to engineer E. coli for the biosynthesis of SA via the inactivation of aroK and aroL (encoding shikimate kinase) as well as shiA (encoding SA transporter); amplified expression of aroFfbr (encoding feedback-resistance 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase, also known as 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase), tktA (encoding transketolase), ppsA (encoding phosphoenolpyruvate synthase), and aroE (encoding shikimate dehydrogenase); heterologous expression of glf (encoding glucose facilitator) from Zymomonas mobilis and glk (encoding glucose kinase) based on the deletion of PTS (phosphoenolpyruvate: carbohydrate phosphotransferase system); and inserting aroB (encoding 3-dehydroquinate synthase) into the genomic serA (encoding phosphoglycerate dehydrogenase) locus (Chandran et al., 2003; Ding et al., 2021; Draths et al., 1999; Knop et al., 2001; Komera et al., 2022). The SA production titer is highly correlated with the comprehensiveness of these genetic modification strategies and the richness of the culture medium in E. coli. As compared with gene manipulation, the application of nutrient-rich medium had a preferably promoting effect on SA production. For instance, manipulating only the targets of aroK, aroL, pts, tktA, aroG, aroB, and aroE resulted in strain DS7, which produced SA at a titer of 12.63 g/L in an inorganic salt medium (Gao et al., 2019). By contrast, comprehensive modulation (deletion) of aroK, aroL, tyrR, ptsG, pykA, and shiA and chromosome-based overexpression of aroB, aroD, aroG, aroF, ppsA, aroE, galP, and tktA resulted in E. coli Inha 224, which produced 101 g/L of SA during fed-batch cultivation in a nutrient-rich medium (Lee et al., 2021) (Table 1). Additionally, an asymmetry distribution-based synthetic consortium strategy was introduced into E. coli that increased shikimate titer to 30.1 g/L in an inorganic salt medium, while the titers was increased to 82.5 g/L by using nutrient-rich medium (Ding et al., 2022). Furthermore, model ec_iML1515 guided metabolic engineering of strain E. coli, via deletion of aroK aroL ptsH, ptsI, pykF, dhal, and ydiB, overexpression of glyA, talB, aroG, aroD, aroE, and tktA, as well as mutation of aroEL241I/T61W, improved shikimate titer to 126.4 g/L in a nutrient-rich medium (Li et al., 2022). Notably, C. glutamicum is another promising chassis microbe that can produce various chemicals from multiple substrates (Becker and Wittmann, 2019). Recent progress in metabolic engineering of C. glutamicum to produce SA has led to ground-breaking accomplishments. Systems metabolic engineering methods, including disruption of aroK, qsuB (encoding 3-dehydroshikimate dehydratase, also known as DHS dehydratase), qsuD (encoding shikimate dehydrogenase), hdpA (encoding HAD superfamily phosphatase), and iolR (encoding a GntR-type regulator responsible for regulating myo-inositol utilization genes); overexpression of aroB, aroD (encoding 3-dehydroquinate dehydratase, also known as DHQ dehydratase), aroE, tkt, tal (encoding transaldolase), and iolT (encoding a myo-inositol transporter capable of mediating glucose uptake); and introduction of mutated aroGfbr from E. coli have been applied to the shikimate pathway, pentose phosphate pathway (PPP), glycolytic pathway (EMP), and PTS in the wild-type strain. Furthermore, a novel growth-arrested cell cultivation has been applied to SA production, enabling a 141.2 g/L SA production titer in a 1-L fermenter, which is the highest yield reported (Kogure et al., 2016)