Promising approaches for simultaneous enhancement of medicinally significant benzylisoquinoline alkaloids in opium poppy

Abstract

Benzylisoquinoline alkaloids (BIAs) produced in opium poppy have been evidenced to heal patients suffering from various diseases. They, therefore, hold an integral position in the herbal drug industry. Despite the adoption of several approaches for the large-scale production of BIAs, opium poppy remains the only platform in this purpose. The only disadvantage associated with producing BIAs in the plant is their small quantity. Thus, recruiting strategies that boost their levels is deemed necessary. All the methods which have been employed so far are just able to enhance a maximum of two BIAs. Thus, if these methods are utilized, a sizable amount of time and budget must be spent on the synthesis of all BIAs. Hence, the exploitation of strategies which increase the content of all BIAs at the same time is more commercially effective and time-saving, avoiding the laborious step of resolving the biosynthetic pathway of each compound. Exposure to biotic and abiotic elicitors, development of a synthetic auto-tetraploid, overexpression of a WRKY transcription factor, formation of an artificial metabolon, and suppression of a gene in the shikimate pathway and miRNA are strategies that turn opium poppy into a versatile bioreactor for the concurrent and massive production of BIAs. The last three strategies have never been applied for BIA biosynthetic pathways.

Keywords: CRISPR/Cas9; PsWRKY; autopolyploidy; elicitor; opium poppy; synthetic metabolon.

Figure 1

Woven events which are triggered by an external elicitor. BIAs, benzylisoquinoline alkaloids; MAPK; mitogen-activated protein kinase; MeJA, methyl jasmonate; PsLOX, lipoxygenase; PsPLA₂, phospholipases A₂; ROS, reactive oxygen species; SA, salicylic acid.

Figure 2

Synthetic autotetraploid developed from a diploid cell through disruption in the mitosis cycle mediated by colchicine, enhancing the biomass and secondary metabolites contents in the resulting plant. 1, prophase; 2, metaphase; 3, anaphase; 4, telophase; 5, S-phase. C, codeine; M, morphine; N, noscapine; P, papaverine; S, sanguinarine; T, thebaine.

Figure 3

Transcription of the genes whose enzymes are involved in the BIA biosynthetic pathways. (A) Binding of one copy of transcription factor PsWRKY to one W-box (TTGACC/T) in the promoter and subsequently the synthesis of one set of BIAs. (B) Overproduction of BIAs results from the overexpression of PsWRKY and multiple occupancy of W-boxes. BIAs, benzylisoquinoline alkaloids; PsWRKY, Papaver somniferum WRKY transcription factor.

Figure 4

The production of (S)-norcoclaurine allows the biosynthesis of L-tyrosine-derived benzylisoquinoline alkaloids occurring in opium poppy. L-DOPA, 3,4-dihydroxy-L-phenylalanine; 3HOase, L-tyrosine/tyramine 3-hydroxylase; 4-HPAA, 4-hydroxyphenylacetaldehyde; 4-HPP, 4-hydroxyphenylpyruvate; 4HPPDC, 4-hydroxyphenylpuruvate decarboxylase; NCS, norcoclaurine synthase; PPO, polyphenol oxidase; TAT, L-tyrosine aminotransferase; TYDC, L-tyrosine/L-DOPA decarboxylase.

Figure 5

Artificially synthesized metabolon consisting of key enzymes involved in benzylisoquinoline alkaloid biosynthesis. NCS, norcoclaurine synthase; TYDC, _L-tyrosine/_L-DOPA decarboxylase; , _L-DOPA; , dopamine; , 4-hydroxyphenylacetaldehyde; , (S)-norcoclaurine.

Figure 6

Acquiring higher BIA synthesis through directing more carbon from chorismate to _L-tyrosine with the aid of the CRISPR/Cas9-mediated downregulation of ADT. ADH, arogenate dehydrogenase; ADT, arogenate dehydratase; BIAs, benzylisoquinoline alkaloids; CM, chorismate mutase; PAM, protospacer-adjacent motif; PPY-AT, phenylpyruvate aminotransferase.