Developing benzylisoquinoline alkaloid-enriched opium poppy via CRISPR-directed genome editing: A review

Abstract

Among plant-derived secondary metabolites are benzylisoquinoline alkaloids (BIAs) that play a vital role in medicine. The most conspicuous BIAs frequently found in opium poppy are morphine, codeine, thebaine, papaverine, sanguinarine, and noscapine. BIAs have provided abundant clinically useful drugs used in the treatment of various diseases and ailments With an increasing demand for these herbal remedies, genetic improvement of poppy plants appears to be essential to live up to the expectations of the pharmaceutical industry. With the advent of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated9 (Cas9), the field of metabolic engineering has undergone a paradigm shift in its approach due to its appealing attributes, such as the transgene-free editing capability, precision, selectivity, robustness, and versatility. The potentiality of the CRISPR system for manipulating metabolic pathways in opium poppy was demonstrated, but further investigations regarding the use of CRISPR in BIA pathway engineering should be undertaken to develop opium poppy into a bioreactor synthesizing BIAs at the industrial-scale levels. In this regard, the recruitment of RNA-guided genome editing for knocking out miRNAs, flower responsible genes, genes involved in competitive pathways, and base editing are described. The approaches presented here have never been suggested or applied in opium poppy so far.

Keywords: Benzylisoquinoline alkaloids; CRISPR/Cas9; Genome editing; Opium poppy; SNPs; miRNA.

Fig. 1

The biosynthetic pathways of dominant BIAs in opium poppy, leading to the formation of papaverine, noscapine, sanguinarine, thebaine, codeine, and morphine. All enzymes have been purified from opium poppy and functionally characterized. AT1, 1,13-dihydroxy-N-methylcanadine 13-O-acetyltransferase; BBE, berberine bridge enzyme; CAS, canadine synthase; CEX1, 3-O-acetylpapaveroxine carboxylesterase; CFS, cheilanthifoline synthase; CNMT, coclaurine N-methyltransferase; CODM, codeine O-demethylase; COR, codeinone reductase; CYP82X1, 1-hydroxy-13-O-acetyl-N-methylcanadine 8-hydroxylase; CYP82X2, 1-hydroxy-N-methylcanadine 13-O-hydroxylase; CYP82Y1, N-methylcanadine 1-hydroxylase; DBOX, dihydrobenzophenanthridine oxidase; 3HOase, _L-tyrosine/tyramine 3-hydroxylase; 4-HPAA, 4-hydroxyphenylacetaldehyde; 4-HPP, 4-hydroxyphenylpyruvate; 4HPPDC, 4-hydroxyphenylpuruvate decarboxylase; MSH, N-methylstylopine 14-hydroxylase; NCS, norcoclaurine synthase; NISO, neopinone isomerase; NMCH, N-methylcoclaurine 3′-hydroxylase; NOS, noscapine synthase; OMT2/3, 4′-O-desmethyl-3-O-acetylpapaveroxine 4′-O-methyltransferase; 4′OMT, 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase; 6OMT, norcoclaurine 6-O-methyltransferase; 7OMT, norreticuline 7-O-methyltransferase; PAAS, phenylacetaldehyde synthase; P6H, protopine 6-hydroxylase; PPO, polyphenol oxidase; REPI, reticuline epimerase; RNMT, reticuline N-methyltransferase; SalAT, salutaridinol 7-O-acetyltransferase; SalR, salutaridine reductase; SalSyn, salutaridine synthase; SanR, sanguinarine reductase; SOMT, scoulerine 9-O-methyltransferase; SPS, stylopine synthase; STOX, tetrahydroprotoberberine oxidase; T6ODM, thebaine 6-O-demethylase; THS, thebaine synthase; TNMT, tetrahydroprotoberberine N-methyltransferase; TYDC, _L-tyrosine/DOPA decarboxylase; TAT, _L-tyrosine aminotransferase

Fig. 2

The shikimate pathway supports the synthesis of numerous primary metabolites. The final product of this pathway, chorismate, is channeled into the pathways generating K and B9 vitamins (Vits), _L-tyrosine (Tyr), phenylalanine (Phe), salicylic acid (SA), and tryptophan (Trp). _L-tyrosine is further converted to benzylisoquinoline alkaloids (BIAs), cyanogenic glycosides (CNgles), and tocopherol. The cleavage of CYP79A1 involved in the biosynthesis pathway of CNgles by the means of the molecular scissor, Cas9, results in InDels in the given gene, generating loss-of-function allele. As a consequence, more _L-tyrosine is directed toward BIA pathway

Fig. 3

a genetic information archived in DNA is transferred to mRNA transcript and to protein that finally gives instruction to secondary metabolism responsible for BIA biosynthesis. b mature mRNA processed by 5′ capping and 3′ polyadenylation is directed to the RNA-silencing pathway mediated by miRNAs. The miRNA complementary to mRNA inhibits its translation via cleavage the transcript. c CRISPR-based miRNA editing causes the modifications in the mRNA sequence. The resulting miRNA is incapable to bind with the target mRNA, culminating in the continuous protein synthesis and an improvement in the BIA accumulation. Adenine;  Uracil;  Cytosine;  Guanine

Fig. 4

The schematic overview of base editing. a cytosine base editor (CBE) enables the conversion of C to uracil (U) directed by cytidine deaminase (1). The resulting unmatched U*G is transformed to U*A pair after DNA repair on the non-edited strand (2). Ultimately, T*A pair is resulted from DNA replication (3). b adenine base editor (ABE) makes use of adenosine deaminase instead. The enzyme converts A to inosine (I) (1). The I*T base mismatch is corrected to I*C pair by repair mechanism on the non-edited strand (2). The final G*C pair is produced during DNA replication (3)