Rational metabolic-flow switching for the production of exogenous secondary metabolites in bamboo suspension cells

Taiji Nomura,Yasuo Kato,Shinjiro Ogita

doi:10.1038/s41598-018-31566-4

Taiji Nomura, Yasuo Kato + Show 1 more

Open Access

https://doi.org/10.1038/s41598-018-31566-4

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Abstract

The synthetic biology-driven production of high-value plant secondary metabolites in microbial hosts has attracted extensive attention despite various challenges, including correct protein expression and limited supplies of starting materials. In contrast, plant cell cultures are rarely used for this purpose owing to their slow proliferation rates and laborious transformation processes. Here, we propose a “rational metabolic-flow switching” strategy to efficiently produce exogenous secondary metabolites using suspension-cultured bamboo (Phyllostachys nigra; Pn) cells as model production hosts. The Pn cells biosynthesise hydroxycinnamic acid amides (HCAAs) of putrescine as major secondary metabolites, which indicates that the phenylpropanoid and polyamine biosynthetic pathways are highly active and that the Pn cells may produce alternative secondary metabolites derived from those pathways. Stable transformants of Pn cells expressing agmatine coumaroyltransferase of barley (Hordeum vulgare) were generated with the expectation of metabolic-flow switching from HCAAs of putrescine to those of agmatine. In the recombinant Pn cells, the levels of HCAAs of putrescine decreased and the HCAAs of agmatine were produced instead. The production titre of the major product, p-coumaroylagmatine, reached approximately 360 mg/L, providing a proof-of-concept for the usefulness of “rational metabolic-flow switching” in synthetic biology using plant cell hosts.

Highlights

Higher plants produce an estimated 200,000 or more secondary metabolites, known as specialised metabolites or natural products[1,2,3]
The lack of intracellular compartments often hampers the efficient expression of eukaryotic enzymes, such as endoplasmic reticulum membrane-bound cytochrome P450s, which are involved in the many aspects of plant secondary metabolite biosynthesis
E. coli does not provide the endogenous precursors required for the biosynthesis of some classes of secondary metabolites, such as those originating from the mevalonate pathway that are needed for terpenoid biosynthesis[19]

Summary

Introduction

Higher plants produce an estimated 200,000 or more secondary metabolites, known as specialised metabolites or natural products[1,2,3]. Eukaryotic S. cerevisiae provides several distinct advantages over E. coli, such as having intracellular compartments, which allows for post-translational modifications, and the functional expression of membrane-bound enzymes, the glycosylation pattern is, in many cases, different from that in plants, which makes it sub-optimal. While the heterologous production of plant secondary metabolites in microbial hosts has been extensively pursued, efforts to use plant suspension cells have not. There are several limitations to using plant cell cultures to produce secondary metabolites in comparison with using microorganisms, including slow proliferation rates and laborious transformation processes, plant cells are essentially the most suitable hosts for the heterologous expression of plant enzymes, and most of the obstacles associated with heterologous expression in microbial hosts can likely be overcome in plant cell cultures. Because the plant cells should be excellent hosts for exogenous gene expression, this concept expands the range of applications of plant cell cultures in high-value metabolite production

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