Production method of recombinant Escherichia coli and high-purity ursodeoxycholic acid

A recombinant Escherichia coli (E. coli), wherein the recombinant E. coli simultaneously expresses 7β-hydroxysteroid dehydrogenase (7β-HSDH) and glucose dehydrogenase (GDH),

• wherein the recombinant E. coli is constructed by using following steps:
• (1) performing a codon optimization on genes of the 7β-HSDH and the GDH, and performing a complete gene synthesis, respectively;
• (2) first connecting the synthesized gene of the 7β-HSDH to a vector pETDuet1, to obtain a recombinant plasmid pETDuet1-7β-HSDH, and then connecting the gene of the GDH to the plasmid pETDuet1-7β-HSDH, to obtain a recombinant plasmid pETDuet1-GDH-7β-HSDH co-expressing the 7β-HSDH and the GDH;
• (3) transferring the pETDuet1-GDH-7β-HSDH into an E. coli BL21(DE3) competent cell, to obtain E. coli BL21(DE3)-pETDuet1-GDH-7β-HSDH carrying the recombinant plasmid;
• and
• (4) culturing the E. coli BL21(DE3)-pETDuet1-GDH-7β-HSDH, to obtain the recombinant E. coli;
• wherein in step (1), after the codon optimization, the 7β-HSDH comprises a sequence of SEQ ID NO:1 and the GDH comprises a sequence of SEQ ID NO:
• 2;
• wherein in step (4), the culture of E. coli comprises the following steps:
• inoculating the E. coli BL21(DE3)-pETDuet1-GDH-7β-HSDH into a lysogeny broth (LB) solid plate culture medium for activation and culture, then picking a single colony from the LB solid plate culture medium, inoculating the single colony into an Erlenmeyer flask with an LB liquid culture medium containing 100 mg/L of ampicillin (Amp+), and performing shaking culture;
• transferring the culture from inoculating the E. coli BL21(DE3)-pETDuet1-GDH-7β-HSDH into a tuberculosis (TB) liquid culture medium containing 100 mg/L of ampicillin (Amp+), performing shaking culture for a period of time, then adding 0.4 mM of isopropyl-β-D-1-thiogalactopyranoside (IPTG), and continuing with the culture;
• and
• after induced expression ends, centrifugally collecting cells, to obtain the recombinant E. coli as engineered bacteria;
• wherein the LB solid plate culture medium comprises the following components:
• 10 g/L of tryptone, 5 g/L of yeast extract, 10 g/L of sodium chloride, and 15.0 g/L of agar;
• the LB liquid culture medium comprises the following components:
• 10 g/L of tryptone, 5 g/L of yeast extract, and 10 g/L of sodium chloride;
• and the TB liquid culture medium comprises the following components:
• 12 g/L of tryptone, 24 g/L of yeast extract, 5 g/L of glycerol, 9.4 g/L of dipotassium phosphate, and 2.2 g/L of monopotassium phosphate;
• and
• wherein the recombinant E. coli is adapted for producing ursodeoxycholic acid (UDCA) using 7-oxo-lithocholic acid with a conversion rate of greater than 99%.