Production of beneficial lignans in heterologous host plants

COPYRIGHT © 2022 Koyama, Murata, Horikawa and Satake. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. TYPE Opinion PUBLISHED 11 October 2022 DOI 10.3389/fpls.2022.1026664

Therefore, new plant sources are required to address these issues. The novelty of this article opinion is to highlight the prospects for the development of heterologous production of beneficial lignans such as (+)-sesamin and (-)-podophyllotoxinrelated lignans, as transgenic cells and plants are excellent hosts for heterologous production of other beneficial chemicals (Lu et al., 2016;Arya et al., 2020;Wu et al., 2021). This is underpinned by the identification of the lignan-biosynthetic enzymes, and generation of transgenic plants expressing the lignan-metabolic enzyme genes as heterologous hosts.

The heterologous hosts
Heterologous production of (+)-sesamin and related lignans in Forsythia plants The knowledge of lignan biosynthetic enzymes, from different plants, is necessary for the selection of hosts for heterologous lignan production. The biosynthetic pathways for the sesamin-related lignans have a common beginning in the conversion of phenylalanine to coniferyl alcohol (CA), followed by the dimerization of CA, by dirigent proteins (DIR), to form the lignan precursor (+)-pinoresinol ( Figure 1A) (Davin et al., 1997;Davin and Lewis, 2000;Umezawa, 2003b).
An illustration of the heterologous production of lignans in Forsythia plants: the generation of the transgenic Forsythia plants expressing sesame CYP81Q1 gene results in the production of (+)-sesamin and its intermediate (+)-piperitol ( Figure 1B) (Koyama et al., 2022). These transgenic Forsythia plants produced (+)-sesamin and (+)-piperitol sustainably, even after repeated vegetative propagation via explants (Koyama et al., 2022). Therefore, the transgenic Forsythia plants serve as prototype heterologous host plants, with the potential for mass propagation as alternative sources of (+)-sesamin and (+)-piperitol.
The lignan precursor (+)-pinoresinol is biosynthesized via CA, and CA is produced from phenylalanine (CA pathway, Figure 1D), so to increase the supply of CA in the leaves of N. benthamiana transient co-expression of CA pathway enzymes was carried out (Schultz et al., 2019). Co-expression of 16 genes of the CA pathway, along with the (-)-podophyllotoxinbiosynthetic enzymes, greatly increased the content of (-)-deoxypodophyllotoxin in the leaves of N. benthamiana (Schultz et al., 2019). This co-expression system did not require exogenous application of (+)-pinoresinol, and thus, enabled de novo production of (-)-podophyllotoxin-related lignans in N. benthamiana leaves (Schultz et al., 2019). This study demonstrates the applicability of engineering to supply endogenous precursors to increase the yield of (-)-podophyllotoxin-related lignans in the leaves of N. benthamiana.

Challenges for the heterologous production of beneficial lignans in microbes
Microbes are also important hosts for the heterologous production of plant specialized metabolites (Sato and Kumagai, 2013;Kotopka et al., 2018;Pyne et al., 2019;Birchfield and McIntosh, 2020). Previous studies have suggested the potential capability of bacteria to produce lignans. Two sequential Escherichia coli lines, harboring individual metabolic modules of (-)-matairesinol to (-)-5′desmetyl-yatein, and (−)-5′desmetyl-yatein to (−)-deoxypodophyllotoxin, reportedly metabolized exogenously applied (-)-matairesinol to (-)-deoxypodophyllotoxin (Decembrino et al., 2021). The human gut microbiome also metabolizes exogenously applied (+)-pinoresinol to various lignans (Xie et al., 2003;Espıń et al., 2017;Bess et al., 2020). However, these bacteria require the exogenous application of lignan precursors to produce the beneficial lignans. The lignan precursor (+)-pinoresinol is, apparently, not produced by many bacteria. Therefore, it would be impossible to produce beneficial lignans without exogenous application of precursor lignans. Therein, de novo production of lignans would require synthetic biotechnology approaches that include tremendous bioinformatics and large-scale screening processes (Choi et al., 2019;Birchfield and McIntosh, 2020). In addition, ethical issues may arise in using the gut bacterium for the scalable production of lignans (Choi et al., 2019). Thus, the microbial production of beneficial lignans is more challenging than the plantbased production process.

Discussion
The novelty of this article is to underscore the importance of lignan production in heterologous host plants. Transgenic expression of the lignan-biosynthetic enzyme genes in heterologous hosts, Forsythia and tobacco, is particularly promising for the production of (+)-sesamin and (-)-podophyllotoxin-related lignans, respectively. These heterologous lignan production strategies may circumvent the need for sesame and Podophyllum plants as natural sources of (+)-sesamin and (-)-podophyllotoxin-related lignans in future.
To develop more efficient transgenic plant-based lignan production systems, we emphasize the importance of increasing the lignan content in future studies. Introducing additional genes to activate the enzymes or stimulate the accumulation of the precursor (+)-pinoresinol in transgenic plants will pave the way for increasing the content of beneficial lignans. To activate cytochrome P450 enzymes that function in complex with native reductases (Murata et al., 2017), co-expression of cytochrome P450 enzymes with their native reductases may lead to an increase in the content of (+)-sesamin and (-)-podophyllotoxin-related lignans in transgenic Forsythia and tobacco plants, respectively. Introducing RNAi into the lignan-metabolic pathway stimulates the accumulation of (+)-pinoresinol in transgenic Forsythia cell lines ( Figure 1B) (Kim et al., 2009b;Murata et al., 2015), therefore, co-expression of (-)-podophyllotoxin-metabolic enzymes with an RNAi construct to stimulate the accumulation of (+)-pinoresinol may increase the content of (-)-podophyllotoxin-related lignans in N. benthamiana. Similarly, activation of the CA pathway stimulates the accumulation of (+)-pinoresinol in N. benthamiana ( Figure 1D) (Schultz et al., 2019); thus, coexpression of CYP81Q1 with the CA pathway enzymes may increase the content of (+)-sesamin in transgenic Forsythia plants. Furthermore, since the elicitor treatments and light changes induce the production of beneficial lignans in cultured cells and hairy root lines Changxing et al., 2020;Markulin et al., 2021;Mikac et al., 2021), it is important to optimize the growth conditions also of the heterologous host plants in future studies. Finally, other lignan-rich plants, such as flax species (Markulin et al., 2021), are expected to be useful heterologous hosts for the transgenic expression of lignan biosynthetic enzyme genes.

Author contributions
TK, JM, and HS wrote the manuscript, and TK and MH prepared the figure. All the authors approved the submitted version.

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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