Benzylisoquinoline Alkaloid Production in Yeast via Norlaudanosoline Improves Titer, Selectivity, and Yield

TL;DR

Scientists improved yeast-based production of benzylisoquinoline alkaloids, achieving higher yields and purity for pharmaceutical compounds like reticuline and sanguinarine.

Contribution

A new pathway using norlaudanosoline improves yeast production of benzylisoquinoline alkaloids with higher titer, selectivity, and yield.

Findings

Yeast produced 4.8 g/L of (S)-reticuline using the norlaudanosoline pathway, a 0.2 g/L increase over prior methods.

Yield improved from 17 to 24 mg/g sucrose in fed-batch fermentations.

Dihydrosanguinarine and sanguinarine were produced at 635 mg/L, a 40-fold increase over prior reports.

Abstract

The benzylisoquinoline alkaloid (BIA) family of tetrahydroisoquinolines
(THIQs) comprises >2,500 members, including the pharmaceuticals
morphine,
codeine, and papaverine, as well as the antibiotics sanguinarine and
chelerythrine. Agricultural cultivation can supply the demand for
the BIAs that accumulate in plants, but broader access to the BIA
family would facilitate additional research and commercialization.
Microbial synthesis presents an attractive option due to cheap feedstock,
genetic tractability, and ease of scale-up. Previously, we reported
titers of the branch-point BIA (S)-reticuline of
4.6 g/L in yeast, which was achieved through leveraging the Ehrlich
pathway 2-oxoacid decarboxylase Aro10 to generate the intermediate
4-hydroxyphenylacetaldehyde (4-HPAA). Here, we establish a superior
route to (S)-reticuline by switching the pathway
intermediate from 4-HPAA to 3,4-dihydroxyphenylacetaldehyde (3,4-dHPAA)
using monoamine oxidase A (MAO). The resulting (S)-norlaudanosoline route to (S)-reticuline synthesis
is more selective, resolving prior issues with off-pathway THIQs synthesized
due to cascading enzyme promiscuity, and more efficient, enabling
titers of 4.8 g/L (S)-reticuline while improving
yields by over 40%, from 17 to 24 mg/g sucrose in fed-batch fermentations.
Finally, we extend de novo (S)-reticuline
synthesis to dihydrosanguinarine, achieving 635 mg/L dihydrosanguinarine
and sanguinarine in fed-batch fermentation, the highest reported titer
of these BIAs by a factor of 40.

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