掲載種別：研究論文（学術雑誌）   出版者・発行元：Japanese Society for Plant Cell and Molecular Biology  

DOI： 10.5511/plantbiotechnology.24.0312b

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Advancements in synthetic biology have facilitated the microbial production of valuable plant metabolites. However, constructing complete biosynthetic pathways within a single host organism remains challenging. To solve this problem, modular co-culture systems involving host organisms with partial pathways have been developed. We focused on Escherichia coli, a general host for metabolite production, and Pichia pastoris (Komagataella phaffii), a novel synthetic biology host due to its high expression of biosynthetic enzymes. Previously, we reported the co-culture of E. coli cells, which produce reticuline (an important intermediate for various alkaloids) from glycerol, with P. pastoris cells, which produce the valuable alkaloid stylopine from reticuline. However, Pichia cells inhibited E. coli growth and reticuline production. Therefore, we aimed to improve this co-culture system. We investigated the pre-culture time before co-culture to enhance E. coli growth and reticuline production. Additionally, we examined the optimal concentration of Pichia cells inoculated for co-culture and methanol addition during co-culture for the continuous expression of biosynthetic enzymes in Pichia cells. We successfully established an improved co-culture system that exhibited an 80-fold increase in productivity compared to previous methods. This enhanced system holds great potential for the rapid and large-scale production of various valuable plant metabolites.

DOI： 10.1248/bpb.b23-00473

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Microorganisms can be metabolically engineered to produce specialized plant metabolites. However, these methods are limited by low productivity and intracellular accumulation of metabolites. We sought to use transport engineering for producing reticuline, an important intermediate in the alkaloid biosynthetic pathway. In this study, we established a reticuline-producing Escherichia coli strain into which the multidrug and toxic compound extrusion transporter Arabidopsis AtDTX1 was introduced. AtDTX1 was selected due to its suitable expression in E. coli and its reticuline-transport activity. Expression of AtDTX1 enhanced reticuline production by 11-fold, and the produced reticuline was secreted into the medium. AtDTX1 expression also conferred high plasmid stability and resulted in upregulation or downregulation of several genes associated with biological processes, including metabolic pathways for reticuline biosynthesis, leading to the production and secretion of high levels of reticuline. The successful employment of a transporter for alkaloid production suggests that the proposed transport engineering approach may improve the biosynthesis of specialized metabolites via metabolic engineering.

DOI： 10.1016/j.mec.2021.e00184

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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.

DOI： 10.1186/s12934-021-01687-z

PubMed

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担当区分：筆頭著者   記述言語：日本語   掲載種別：記事・総説・解説・論説等（学術雑誌）   出版者・発行元：公益社団法人 日本薬学会  

微生物は，安価な材料から高付加価値な化合物を生産する工場として活用されている．従来型酵母と呼ばれるSaccharomyces cerevisiaeはそのゲノム情報が完全に解読され，また遺伝子編集ツールも豊富に揃えられていることから，真核モデル微生物として広く利用されている．一方，高温や低pH環境に強く，多様な基質を利用でき，優れたタンパク質発現能力などを持つ非従来型酵母が近年注目されている．例えばYarrowia lipolyticaは，細胞内に多くの脂肪滴を蓄積し疎水性環境を自然に形成するため，テルペノイド，フラボノイド，油脂などの生産に適している．しかし，相同組換え効率の低さと遺伝子編集ツールの不足が課題である．本稿では，Cas9の改良や遺伝子導入の影響が小さい場所(中立部位)の網羅的探索により，高効率かつ高発現する遺伝子挿入部位を予測し，Y. lipolyticaでの植物由来アルカロイドとフラボノイドの合成により，本プラットフォームの実用性を証明したZhangらの研究を紹介する．

なお，本稿は下記の文献に基づいて，その研究成果を紹介するものである.

1) Zhang C. et al., ACS Synth. Biol., 14, 585-597(2025).

2) Pyne M. E. et al., Nat. Commun., 11, 3337(2020).

3) Qin Z. et al., J. Agric. Food Chem., 72, 5348-5357(2024).

DOI： 10.14894/faruawpsj.61.12_1110

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担当区分：筆頭著者   記述言語：日本語   掲載種別：研究発表ペーパー・要旨（全国大会，その他学術会議）  

J-GLOBAL

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担当区分：筆頭著者   記述言語：日本語   掲載種別：研究発表ペーパー・要旨（全国大会，その他学術会議）  

J-GLOBAL

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記述言語：日本語   掲載種別：研究発表ペーパー・要旨（全国大会，その他学術会議）  

J-GLOBAL

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担当区分：筆頭著者   記述言語：日本語   掲載種別：研究発表ペーパー・要旨（全国大会，その他学術会議）  

J-GLOBAL

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開催年月日： 2025年5月31日 - 2025年6月1日

会議種別：ポスター発表  

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開催年月日： 2024年11月28日 - 2024年11月29日

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開催年月日： 2024年7月6日 - 2024年7月7日

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会議種別：口頭発表（一般）  

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会議種別：口頭発表（一般）  

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