A bacterial platform for fermentative production of plant alkaloids

Abstract

The secondary metabolites of higher plants include diverse chemicals, such as alkaloids, isoprenoids and phenolic compounds (phenylpropanoids and flavonoids). Although these compounds are widely used in human health and nutrition, at present they are mainly obtained by extraction from plants and extraction yields are low because most of these metabolites accumulate at low levels in plant cells. Recent advances in synthetic biology and metabolic engineering have enabled tailored production of plant secondary metabolites in microorganisms, but these methods often require the addition of expensive substrates. Here we develop an Escherichia coli fermentation system that yields plant alkaloids from simple carbon sources, using selected enzymes to construct a tailor-made biosynthetic pathway. In this system, engineered cells cultured in growth medium without additional substrates produce the plant benzylisoquinoline alkaloid, (S)-reticuline (yield, 46.0 mg l−1 culture medium). The fermentation platform described here offers opportunities for low-cost production of many diverse alkaloids.

Benzylisoquinoline alkaloid biosynthesis in opium poppy

“…PR10-type NCS from C. japonica catalyzed the condensation of dopamine and 3,4-DHPAA to (S)-norlaudanosoline, which underwent successive methylations via 6OMT, CNMT and 4′OMT to yield (S)-reticuline. Recent modifications of this platform include the de novo synthesis of dopamine by the expression of two additional enzymes, tyrosinase and DOPA decarboxylase (Nakagawa et al 2011). The use of bacterial enzymes facilitated the linking of BIA metabolism to the primary metabolism of E. coli, enabling a fermentation platform that creates plant products from simple carbon sources.…”

Synthetic RNA devices to expedite the evolution of metabolite-producing microbes

“…M icrobial engineering for the production of chemicals relies on the rational design of metabolic pathways so as to redirect the metabolic flux towards a particular metabolite [1][2][3][4] . Improving product yields or pathway efficiencies, however, requires optimization of various metabolic pathways in the cellular metabolism-a difficult task using rational approaches 5 .…”

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

“…The first production of S-reticuline from simple carbon sources was later achieved in the E. coli system led by the same research group. [263] First, overproduction of tyrosine was sought, which was followed by the conversion of tyrosine to L-DOPA by means of tyrosinase (TYR) isolated from Streptomyces castaneoglobisporus. Whereas TYR only requires copper as its cofactor along with coexpression of an adaptor protein (ORF378), tyrosine hydroxylase which is an alternative to TYR requires a complex cofactor BH 4 .…”

“…Additional expression of further downstream enzymes has enabled the biosynthesis of S-reticuline with the titer of 2.00 mg L −1 from glucose and 5.92 mg L −1 from glycerol. [263] S-reticuline production in the E. coli platform has its advantage over the S. cerevisiae platform in that E. coli has much higher capability of producing the precursor, tyrosine. [264] Production of S-reticuline in S. cerevisiae was also reported by several groups, albeit at a much lower titer than in E. coli.…”