(R,S)-Tetrahydropapaveroline production by stepwise fermentation using engineered Escherichia coli

Abstract

Tetrahydropapaveroline (THP), a benzylisoquinoline alkaloid (BIA) found in diverse pharmaceutical compounds, is used as a starting material for the production of BIA. THP also has various neurobiological properties but is difficult to synthesize. Therefore, a simple method for THP production is desired. Recent studies have shown that microbes, especially bacteria, can serve as platforms for synthesizing these complex compounds; however, because bacteria lack organelles, the designed synthetic pathway cannot be compartmentalized. Thus, the metabolic flow is frequently inhibited or disrupted by undesirable reactions. Indeed, in the first attempt to synthesize THP using a single strain of engineered Escherichia coli, the yield was quite low (<5 μM), mainly because of the oxidation of THP by tyrosinase, an essential enzyme in our production system. To circumvent these problems, we constructed a stepwise (R,S)-THP production system, in which the dopamine-producing step and the subsequent THP-producing step were separated. The yield of (R,S)-THP reached 1.0 mM (287 mg/L), the highest yielding BIA production method using a microbe reported to date. Furthermore, we demonstrated that (R,S)-THP produced by stepwise fermentation is useful for the production of reticuline, an important BIAs intermediate. Based on these observations, applying the stepwise fermentation method is discussed.

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

“…The whole pathway was divided and transformed into the different strains-dopamine producing strain, THP producing strain, and reticuline producing strain-allowing a three-step fermentation which resulted in S-reticuline titer of 48 mg L −1 (Figure 18c). [284] After the successful production of S-reticuline, additional strains were constructed for the total biosynthesis of opioids. Thus, they have divided the whole pathway into 4 parts, transformed each segment into different strains, and performed a stepwise cultivation, yielding a higher productivity of opiates compared to the previous success in yeast.…”

Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids

“…In another approach, Sato et al focused their efforts on establishing a BIA biosynthetic pathway in E. coli and successfully developed production systems capable of biosynthesizing (R,S)-THP, (R,S)-reticuline, thebaine and hydrocodone ( Figure 16) [70,[81][82][83]. Their strategy relied on supplying the key intermediate reticuline as a racemic mixture using an artificial biosynthetic pathway comprised of tyrosinase, L-3,4-dihydroxyphenylalanine (L-DOPA) decarboxylase and monoamine oxidase (MAO) obtained from various bacterial sources.…”

“…Lastly, addition of P. somniferum T6ODM and P. putida morB genes accomplished the biosynthesis of hydrocodone [83]. This E. coli-based biosynthetic system developed by Sato et al employed a stepwise fermentation technique to avoid tyrosinase from degrading the early intermediate THP [82]. This method also helped minimize product inhibition of pathway enzymes and propagation of undesired byproducts through the biosynthetic steps.…”

“…Initially, an NCS gene was introduced to the system to combine dopamine and 3,4-DHPAA into (S)-THP for subsequent conversion into (S)-reticuline [70,81]. However, NCS was later eliminated to rely on the spontaneous formation of a racemic mixture of THP from dopamine and 3,4-DHPAA via a Pictet-Spengler reaction inside the cells [82]. Then, (R,S)-THP was converted into (R,S)-reticuline by 6OMT, CNMT and 4′OMT [83].…”

“…Figure 16. Heterologous biosynthetic pathway established in E. coli for de novo production of (R,S)-reticuline from simple sugar using a three-step fermentation method [81][82][83]. The three fermentation steps are designated by three different colors: black for the dopamine production step, red for the (R,S)-THP production step and blue for the (R,S)-reticuline production step.…”