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Title: Engineering modular diterpene biosynthetic pathways in Physcomitrella patens

Abstract

Modular assembly and heterologous expression in the moss Physcomitrella patens of pairs of diterpene synthases results in accumulation of modern land plant diterpenoids. Physcomitrella patens is a representative of the ancient bryophyte plant lineage with a genome size of 511 Mb, dominant haploid life cycle and limited chemical and metabolic complexity. For these plants, exceptional capacity for genome editing through homologous recombination is met with recently demonstrated in vivo assembly of multiple heterologous DNA fragments. These traits earlier made P. patens an attractive choice as a biotechnological chassis for photosynthesis-driven production of recombinant peptides. The lack of diterpene gibberellic acid phytohormones in P. patens combined with the recent targeted disruption of the single bifunctional diterpene synthase yielded lines devoid of endogenous diterpenoid metabolites and well-suited for engineering of terpenoid production. Here, we mimicked the modular nature of diterpene biosynthetic pathways found in modern land plants by developing a flexible pipeline to install three combinations of class II and class I diterpene synthases in P. patens to access industrially relevant diterpene biomaterials. In addition to a well-established neutral locus for targeted integration, we also explored loci created by a class of Long Terminal Repeat Retrotransposon present at moderate number in themore » genome of P. patens. Assembly of the pathways and production of the enzymes from the neutral locus led to accumulation of diterpenes matching the reported activities in the angiosperm sources. In contrast, insights gained with the retrotransposon loci indicate their suitability for targeting, but reveal potentially inherent complications which may require adaptation of the experimental design.« less

Authors:
 [1];  [2];  [3];  [2];  [2]; ORCiD logo [1]
  1. Michigan State Univ., East Lansing, MI (United States); Wisconsin Energy Inst., Madison, WI (United States)
  2. Michigan State Univ., East Lansing, MI (United States)
  3. Michigan State Univ., East Lansing, MI (United States); Heinrich-Heine-Univ. Düsseldorf (Germany)
Publication Date:
Research Org.:
Great Lakes Bioenergy Research Center, Madison, WI (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1505780
Grant/Contract Number:  
SC0018409
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Planta
Additional Journal Information:
Journal Volume: 249; Journal Issue: 1; Journal ID: ISSN 0032-0935
Publisher:
Springer
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Diterpenoids, Synthetic biology, Homologous recombination, Photosynthetic production, Neutral locus, Retrotransposon

Citation Formats

Banerjee, Aparajita, Arnesen, Jonathan A., Moser, Daniel, Motsa, Balindile B., Johnson, Sean R., and Hamberger, Bjoern. Engineering modular diterpene biosynthetic pathways in Physcomitrella patens. United States: N. p., 2018. Web. doi:10.1007/s00425-018-3053-0.
Banerjee, Aparajita, Arnesen, Jonathan A., Moser, Daniel, Motsa, Balindile B., Johnson, Sean R., & Hamberger, Bjoern. Engineering modular diterpene biosynthetic pathways in Physcomitrella patens. United States. doi:10.1007/s00425-018-3053-0.
Banerjee, Aparajita, Arnesen, Jonathan A., Moser, Daniel, Motsa, Balindile B., Johnson, Sean R., and Hamberger, Bjoern. Fri . "Engineering modular diterpene biosynthetic pathways in Physcomitrella patens". United States. doi:10.1007/s00425-018-3053-0.
@article{osti_1505780,
title = {Engineering modular diterpene biosynthetic pathways in Physcomitrella patens},
author = {Banerjee, Aparajita and Arnesen, Jonathan A. and Moser, Daniel and Motsa, Balindile B. and Johnson, Sean R. and Hamberger, Bjoern},
abstractNote = {Modular assembly and heterologous expression in the moss Physcomitrella patens of pairs of diterpene synthases results in accumulation of modern land plant diterpenoids. Physcomitrella patens is a representative of the ancient bryophyte plant lineage with a genome size of 511 Mb, dominant haploid life cycle and limited chemical and metabolic complexity. For these plants, exceptional capacity for genome editing through homologous recombination is met with recently demonstrated in vivo assembly of multiple heterologous DNA fragments. These traits earlier made P. patens an attractive choice as a biotechnological chassis for photosynthesis-driven production of recombinant peptides. The lack of diterpene gibberellic acid phytohormones in P. patens combined with the recent targeted disruption of the single bifunctional diterpene synthase yielded lines devoid of endogenous diterpenoid metabolites and well-suited for engineering of terpenoid production. Here, we mimicked the modular nature of diterpene biosynthetic pathways found in modern land plants by developing a flexible pipeline to install three combinations of class II and class I diterpene synthases in P. patens to access industrially relevant diterpene biomaterials. In addition to a well-established neutral locus for targeted integration, we also explored loci created by a class of Long Terminal Repeat Retrotransposon present at moderate number in the genome of P. patens. Assembly of the pathways and production of the enzymes from the neutral locus led to accumulation of diterpenes matching the reported activities in the angiosperm sources. In contrast, insights gained with the retrotransposon loci indicate their suitability for targeting, but reveal potentially inherent complications which may require adaptation of the experimental design.},
doi = {10.1007/s00425-018-3053-0},
journal = {Planta},
issn = {0032-0935},
number = 1,
volume = 249,
place = {United States},
year = {2018},
month = {11}
}

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Works referenced in this record:

Isoprenoid Pathway Optimization for Taxol Precursor Overproduction in Escherichia coli
journal, September 2010