Cargando…

Establishment of a co-culture system using Escherichia coli and Pichia pastoris (Komagataella phaffii) for valuable alkaloid production

BACKGROUND: Plants produce a variety of specialized metabolites, many of which are used in pharmaceutical industries as raw materials. However, certain metabolites may be produced at markedly low concentrations in plants. This problem has been overcome through metabolic engineering in recent years,...

Descripción completa

Detalles Bibliográficos
Autores principales: Urui, Miya, Yamada, Yasuyuki, Ikeda, Yoshito, Nakagawa, Akira, Sato, Fumihiko, Minami, Hiromichi, Shitan, Nobukazu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8522034/
https://www.ncbi.nlm.nih.gov/pubmed/34663314
http://dx.doi.org/10.1186/s12934-021-01687-z
_version_ 1784585013485895680
author Urui, Miya
Yamada, Yasuyuki
Ikeda, Yoshito
Nakagawa, Akira
Sato, Fumihiko
Minami, Hiromichi
Shitan, Nobukazu
author_facet Urui, Miya
Yamada, Yasuyuki
Ikeda, Yoshito
Nakagawa, Akira
Sato, Fumihiko
Minami, Hiromichi
Shitan, Nobukazu
author_sort Urui, Miya
collection PubMed
description BACKGROUND: Plants produce a variety of specialized metabolites, many of which are used in pharmaceutical industries as raw materials. However, certain metabolites may be produced at markedly low concentrations in plants. This problem has been overcome through metabolic engineering in recent years, and the production of valuable plant compounds using microorganisms such as Escherichia coli or yeast cells has been realized. However, the development of complicated pathways in a single cell remains challenging. Additionally, microbial cells may experience toxicity from the bioactive compounds produced or negative feedback effects exerted on their biosynthetic enzymes. Thus, co-culture systems, such as those of E. coli–E. coli and E. coli-Saccharomyces cerevisiae, have been developed, and increased production of certain compounds has been achieved. Recently, a co-culture system of Pichia pastoris (Komagataella phaffii) has gained considerable attention due to its potential utility in increased production of valuable compounds. However, its co-culture with other organisms such as E. coli, which produce important intermediates at high concentrations, has not been reported. RESULTS: Here, we present a novel co-culture platform for E. coli and P. pastoris. Upstream E. coli cells produced reticuline from a simple carbon source, and the downstream P. pastoris cells produced stylopine from reticuline. We investigated the effect of four media commonly used for growth and production of P. pastoris, and found that buffered methanol-complex medium (BMMY) was suitable for P. pastoris cells. Reticuline-producing E. coli cells also showed better growth and reticuline production in BMMY medium than that in LB medium. De novo production of the final product, stylopine from a simple carbon source, glycerol, was successful upon co-culture of both strains in BMMY medium. Further analysis of the initial inoculation ratio showed that a higher ratio of E. coli cells compared to P. pastoris cells led to higher production of stylopine. CONCLUSIONS: This is the first report of co-culture system established with engineered E. coli and P. pastoris for the de novo production of valuable compounds. The co-culture system established herein would be useful for increased production of heterologous biosynthesis of complex specialized plant metabolites. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-021-01687-z.
format Online
Article
Text
id pubmed-8522034
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-85220342021-10-21 Establishment of a co-culture system using Escherichia coli and Pichia pastoris (Komagataella phaffii) for valuable alkaloid production Urui, Miya Yamada, Yasuyuki Ikeda, Yoshito Nakagawa, Akira Sato, Fumihiko Minami, Hiromichi Shitan, Nobukazu Microb Cell Fact Research BACKGROUND: Plants produce a variety of specialized metabolites, many of which are used in pharmaceutical industries as raw materials. However, certain metabolites may be produced at markedly low concentrations in plants. This problem has been overcome through metabolic engineering in recent years, and the production of valuable plant compounds using microorganisms such as Escherichia coli or yeast cells has been realized. However, the development of complicated pathways in a single cell remains challenging. Additionally, microbial cells may experience toxicity from the bioactive compounds produced or negative feedback effects exerted on their biosynthetic enzymes. Thus, co-culture systems, such as those of E. coli–E. coli and E. coli-Saccharomyces cerevisiae, have been developed, and increased production of certain compounds has been achieved. Recently, a co-culture system of Pichia pastoris (Komagataella phaffii) has gained considerable attention due to its potential utility in increased production of valuable compounds. However, its co-culture with other organisms such as E. coli, which produce important intermediates at high concentrations, has not been reported. RESULTS: Here, we present a novel co-culture platform for E. coli and P. pastoris. Upstream E. coli cells produced reticuline from a simple carbon source, and the downstream P. pastoris cells produced stylopine from reticuline. We investigated the effect of four media commonly used for growth and production of P. pastoris, and found that buffered methanol-complex medium (BMMY) was suitable for P. pastoris cells. Reticuline-producing E. coli cells also showed better growth and reticuline production in BMMY medium than that in LB medium. De novo production of the final product, stylopine from a simple carbon source, glycerol, was successful upon co-culture of both strains in BMMY medium. Further analysis of the initial inoculation ratio showed that a higher ratio of E. coli cells compared to P. pastoris cells led to higher production of stylopine. CONCLUSIONS: This is the first report of co-culture system established with engineered E. coli and P. pastoris for the de novo production of valuable compounds. The co-culture system established herein would be useful for increased production of heterologous biosynthesis of complex specialized plant metabolites. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-021-01687-z. BioMed Central 2021-10-18 /pmc/articles/PMC8522034/ /pubmed/34663314 http://dx.doi.org/10.1186/s12934-021-01687-z Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Urui, Miya
Yamada, Yasuyuki
Ikeda, Yoshito
Nakagawa, Akira
Sato, Fumihiko
Minami, Hiromichi
Shitan, Nobukazu
Establishment of a co-culture system using Escherichia coli and Pichia pastoris (Komagataella phaffii) for valuable alkaloid production
title Establishment of a co-culture system using Escherichia coli and Pichia pastoris (Komagataella phaffii) for valuable alkaloid production
title_full Establishment of a co-culture system using Escherichia coli and Pichia pastoris (Komagataella phaffii) for valuable alkaloid production
title_fullStr Establishment of a co-culture system using Escherichia coli and Pichia pastoris (Komagataella phaffii) for valuable alkaloid production
title_full_unstemmed Establishment of a co-culture system using Escherichia coli and Pichia pastoris (Komagataella phaffii) for valuable alkaloid production
title_short Establishment of a co-culture system using Escherichia coli and Pichia pastoris (Komagataella phaffii) for valuable alkaloid production
title_sort establishment of a co-culture system using escherichia coli and pichia pastoris (komagataella phaffii) for valuable alkaloid production
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8522034/
https://www.ncbi.nlm.nih.gov/pubmed/34663314
http://dx.doi.org/10.1186/s12934-021-01687-z
work_keys_str_mv AT uruimiya establishmentofacoculturesystemusingescherichiacoliandpichiapastoriskomagataellaphaffiiforvaluablealkaloidproduction
AT yamadayasuyuki establishmentofacoculturesystemusingescherichiacoliandpichiapastoriskomagataellaphaffiiforvaluablealkaloidproduction
AT ikedayoshito establishmentofacoculturesystemusingescherichiacoliandpichiapastoriskomagataellaphaffiiforvaluablealkaloidproduction
AT nakagawaakira establishmentofacoculturesystemusingescherichiacoliandpichiapastoriskomagataellaphaffiiforvaluablealkaloidproduction
AT satofumihiko establishmentofacoculturesystemusingescherichiacoliandpichiapastoriskomagataellaphaffiiforvaluablealkaloidproduction
AT minamihiromichi establishmentofacoculturesystemusingescherichiacoliandpichiapastoriskomagataellaphaffiiforvaluablealkaloidproduction
AT shitannobukazu establishmentofacoculturesystemusingescherichiacoliandpichiapastoriskomagataellaphaffiiforvaluablealkaloidproduction