Total biosynthesis of opiates by stepwise fermentation using engineered Escherichia coli

Abstract

Opiates such as morphine and codeine are mainly obtained by extraction from opium poppies. Fermentative opiate production in microbes has also been investigated, and complete biosynthesis of opiates from a simple carbon source has recently been accomplished in yeast. Here we demonstrate that Escherichia coli serves as an efficient, robust and flexible platform for total opiate synthesis. Thebaine, the most important raw material in opioid preparations, is produced by stepwise culture of four engineered strains at yields of 2.1 mg l−1 from glycerol, corresponding to a 300-fold increase from recently developed yeast systems. This improvement is presumably due to strong activity of enzymes related to thebaine synthesis from (R)-reticuline in E. coli. Furthermore, by adding two genes to the thebaine production system, we demonstrate the biosynthesis of hydrocodone, a clinically important opioid. Improvements in opiate production in this E. coli system represent a major step towards the development of alternative opiate production systems.

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

“…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. [279] This stepwise multistrain cultivation resulted in 2.1 mg L −1 of thebaine and 0.36 mg L −1 of hydrocodone production. Although the multistep cultivation successfully improved target compounds production, fundamental constraints still remain.…”

“…SalSyn was also unable to be functionally expressed in E. coli. [279] For functional expression of P450s in E. coli, N-terminal deletion was attempted for this enzyme, with the resultant enzyme denoted as SalSNcut. It turned out that the SalSNcut was successfully expressed in a functional form in the cell (Figure 18b).…”

“…It turned out that the SalSNcut was successfully expressed in a functional form in the cell (Figure 18b). [279] Not only for the N-terminal deletion, but also N-terminal tagging can be useful. For the production of noscapine, N-terminus of MSH was fused with CYP82Y1, thus significantly enhancing its activity.…”

“…In the E. coli system, in contrast with the previous reports, Nakagawa et al were able to produce both S and R forms of reticuline by the incorporation of only two methyltransferases-CNMT and 4′OMT-without expressing 6OMT. [279] Expression of the recently discovered REP was rather ineffective in the E. coli platform. Heterologous natural product biosynthesis sometimes even elucidates the exact functions or cooperative behaviors of enzymes.…”

“…Recently, de novo production of thebaine and hydrocodone in E. coli was also reported, at much higher titers of 2.1 and 0.36 mg L −1 . [279] Higher titer was possible since E. coli has a higher capability for the production of intermediates including tyrosine, L-DOPA, norlaudanosoline, and S-reticuline. Although the alkaloid titers were still relatively low, it has been considered as a huge triumph since it has opened up a new era of producing complex alkaloids from simple carbon sources.…”