# A Synthetic Approach for Biosynthesis of Miquelianin and Scutellarin A in _Escherichia coli_
## 2 Materials and Methods
### Cloning and Expression of Recombinant Proteins  
Restriction enzymes, _LaTaq_ polymerase, and T4 DNA ligase were purchased from Takara Bio Inc. (Shiga, Japan). UDP-glycosyltransferases, _VvGT5_ (grapevine; _Vitis vinifera_ UDP-glucuron acid: flavonol 3-O-glucurononsyltransferase) and _UGT8D8_ (the blue flowers of _Veronica persica_ poiret; _Lamiales, Scrophulariaceae_ structurally flavonoid 7-O-specific glucuronosyltransferases (F7 GAT)) were codon optimized, synthesized, cloned and expressed in restriction site dependent multi-mono-cistronic vector, piBR181. Oligonucleotide pr primers were synthesized from Genotech (South Korea). All PCR size and primers used in this study are described in Table 2. To increase the pool of UDP-glucuron acid, its pathway specific genes were individually cloned and assembled together in the piBR181 vector. The UDP-glucuron acid sugar pathway genes glucokinase (_gkl_: from _Zymomonas mobilisi_, GenBank ID: AE008692.2), a vector pVWEx1-_glf-glk_ provided by Dr. [PERSON] (Institut fur Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Julich, Julich, \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Primers** & **Oligonucleotide Sequences (5\({}^{\prime}\)-3\({}^{\prime}\))** & **Total PCR Size** & **Restriction Sites** \\ \hline glk-F & TCTAGAATGGAAATTGTCCGATTGACTCGGT & 984 bp & _Xhal_ \\ \hline glk-R & AAGCTITTATTCAACTTCAAGAAATTTGTGTGTGC & 984 bp & _Hind_III \\ \hline pgm2-F & TCTAGAATGAGACTGGAGAACGACTATGAACC & 1749 bp & _[PERSON]_ \\ \hline pgm2-R & AAGCTTTTCAGAGATTGGAGCTTTTCAAT & 1749 bp & _Hind_III \\ \hline galU-F & TCTAGAGGTCCATTATTACGAATCAAGTCAAA & 908 bp & _[PERSON]_ \\ \hline galU-R & AAGCLITTATTCTTATTATCCATCCCTTCTTCTTCTT & 908 bp & _Hind_III \\ \hline ugd-F & TCTAGAATGAAAATCCCTTCCGGTACTGGCC & 1167 bp & _[PERSON]_ \\ \hline ugd-R & AAGCTTTTACTCCTCCAAAAGAGATCCGGT & 1167 bp & _Hind_III \\ \hline UGT8D8-F & TCTAGAATGGAAGACACAGATTCTGATGC & & _[PERSON]_ \\ \hline UGT8D8-R & AAGCTTTGGAACTTATCCGATTCCACACACGC & & _Hind_III \\ \hline VvGT5-F & TCTAGAATGCACAGACCGCCTCCTCCTCAATGGAT & & _Xhal_ \\ \hline VvGT5-R & AAGCTTCGTTCTCACCGGCAGGTTTTAGAGGTGT & & _Hind_III \\ \hline \hline \end{tabular}
\end{table} 
Table 1: List of primers used in this study. Figure 1: Schematic diagram of the uridine diphosphate (UDP)-glucuron acid biosynthesis pathway and its utilization by UGT8D8 and VvGT5 to transfer glucuronic moiety to 7-OH position of apigenin and 3-OH position of quercetin, respectively. Germany), phosphoglucomutase (_pgm2_; from _Bacillus licheniformis_ DSM 13, GenBank accession No. YP_006712377.1), glucose 1-phosphate uridylyltransferase (_gallU_; from _E. coli_ K-12, GenBank accession No. CP001509.3), and UDP-glucose 6-dehydrogenase (_ugd_; GenBank accession No. YP_490271.1 from _E. coli_ BL21(DE3) were selected for overexpression. All these genes were amplified from the genomic DNA of respective strains using a pair of primer that had _XbaI_/_Hind_III restriction sites, as shown in Table 1. Finally, the sugar cassette was assembled with codon optimized regiospecific glycurunosyltransferase (_UGT88D8_ or _VvGT5_) separately, as the physical scheme explains in Figure 1. These two plasmids were used for enhanced biosynthesis of two molecules. In all cases, construction of each recombinant and assembly of each pathway gene were verified with restriction digestion, PCR, and direct nucleotide sequencing of PCR-amplified products of the respective genes in the recombinant plasmid. Each recombinant plasmid containing UDP-glucuronic acid sugar pathway genes was transformed into _E. coli_ BL21 (DE3) with the heat shock transformation technique. Later, the restriction-digestion-confirmed single transformant was cultured for further studies. Sodium dodecyl sulfate-polyacrylamideamide gel electrophoresis (SDS-PAGE) analysis was performed to check the expression of each gene and was compared with the standard protein marker. To check the expression of each gene, we followed the same methodology as described previously [22] with the same concentration of IPTG.