# A Synthetic Approach for Biosynthesis of Miquelianin and Scutellarin A in _Escherichia coli_
## 3 Results and Discussion
### Enhanced Synthesis of Miquelianin and Scutellarin A  
After a successful confirmation of target products through spectroscopic analyses, we used other recombinants generated for this study. Two single recombinant vectors generated after assembly of UDP-glucuron acid biosynthesis pathway genes and regiospecific UGTs were compared for increased production of respective products. Based on previous experiences, we hypothesized that the production of glycosides should increase upon overexpression of UDP-glucuron acid pathway specific genes, along with _UGT88D8_ and _VvGT5_. As in previous experiments, 50 mL cultures were grown and allowed the functional expression of protein for 20 h at 15 \({}^{\circ}\)C. For biotransformation reaction, the same 200 \(\upmu\)mol/L concentration of substrates apigenin and quercetin were supplemented in respective recombinant strains. The cultures were harvested in 48 h incubation and analysed by HPLC-PDA. The production of each glycoside was increased approximately twice. The conversion of apigenin to scutellarin A was \(\sim\)14% (28 \(\upmu\)mol/L; 12 mg/L) and that of quercetin to miquelianin was \(\sim\)31% (62 \(\upmu\)mol/L; 30 mg/L). However, the increment was not satisfactory. We optimized the experiments with the addition of glucose as a carbon source. The production did not change significantly, although the cell growth was reasonably high (Figure 4).  
Figure 3: HPLC-PDA analysis of bio-catalysis reaction mixtures showing the generation of apigenin 7-\(O\)-glucuronoid (scutellarin A) and quercetin 3-\(O\)-glucuronoid (miquelianin) peak(s) by a circle in each chromatogram compared with their standard reference peak(s). Additionally, the mass spectrum of each reaction mixtures confirmed the target products.  
Several plant metabolites, including flavonoids and their glycoside derivatives, have been produced from engineered _E. coli_ cells as a microbial cell factory. Such microbial production has attracted extensive interest in the development of a novel system for the modification and efficient production of highly useful value-added compounds [14]. To produce glycosylated flavonoids from engineered _E. coli_, the pool of activated NDP-sugars, the donor substrates of UGTs and limiting factor in the cell cytosol is enhanced by introducing heterologous genes encoding the target sugar pathway genes, or by blocking the indigenous competing genes [14]. Using this approach, several flavonoid glycosides are produced from _E. coli_. For example, 3-O-rhamosyl quercetin, 3-O-rhamosyl kaempfer, and 3-O-allosyl quercetin [27], astragalin [28], quercetin-3-O-4-deoxy-4-formamido-l-arabinose [29], quercetin 3-O-N-acetylglucosamine [30], quercetin-3-O xyloside [31] and quercetin 3-O-arabinoside [32], flavonoid 3-O-rhamosides [33; 34], flavone 6-C-glucosidase [35] are some representative flavonoid glycosides produced from _E. coli_. Using a similar approach, several polyketide glycosides have been produced from engineered microbial hosts [36]. Most of these systems used multiple vectors for overexpression of genes, which required the addition of multiple antibiotics to the culture broth for selection pressure. Thus, to reduce the antibiotic stress on the growing cell, we used a single vector system in which multiple genes were assembled for biosynthesis of two bioactive molecules; muquelianin and scutellarin A. Previously, muquelianin was produced using an approach similar to the approach that produced engineered _E. coli_ cell [37]. However, this is the first report to produce scutellarin A from _E. coli_ system. The production of muquelianin and scutellarin A from the systems developed in this study is relatively low compared to previous studies that reported biosynthesis of flavonoid glycosides [14]. However, this study opens possible paths for further harnessing of systems and synthetic biology tools to engineer central carbon metabolic pathways, to dam up the flow of UDP-glucuron acid, and to develop an efficient system to produce glucuronic acid conjugated flavonoids.