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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:
; ; ; ; ; ORCiD logo
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:
1619324
Alternate Identifier(s):
OSTI ID: 1505780
Grant/Contract Number:  
FC02-07ER64494; SC0018409
Resource Type:
Journal Article: Published Article
Journal Name:
Planta
Additional Journal Information:
Journal Name: Planta Journal Volume: 249 Journal Issue: 1; Journal ID: ISSN 0032-0935
Publisher:
Springer Science + Business Media
Country of Publication:
Germany
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. Germany: 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. Germany. 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". Germany. doi:10.1007/s00425-018-3053-0.
@article{osti_1619324,
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 = {Germany},
year = {2018},
month = {11}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1007/s00425-018-3053-0

Citation Metrics:
Cited by: 2 works
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Works referenced in this record:

Processing in Arabidopsis thaliana of a heterologous polyprotein resulting in differential targeting of the individual plant defensins
journal, January 2004


δ-Integration of endo/exo-glucanase and β-glucosidase genes into the yeast chromosomes for direct conversion of cellulose to ethanol
journal, July 1999


Physcomitrella patens, a versatile synthetic biology chassis
journal, May 2018


Transposable element origins of epigenetic gene regulation
journal, April 2011


Diterpene resin acids in conifers
journal, November 2006


Engineering Escherichia coli for the synthesis of taxadiene, a key intermediate in the biosynthesis of taxol
journal, September 2001


A vector set for systematic metabolic engineering in Saccharomyces cerevisiae
journal, October 2010

  • Fang, Fang; Salmon, Kirsty; Shen, Michael W. Y.
  • Yeast, Vol. 28, Issue 2
  • DOI: 10.1002/yea.1824

Stable Production of the Antimalarial Drug Artemisinin in the Moss Physcomitrella patens
journal, August 2017

  • Khairul Ikram, Nur Kusaira Binti; Beyraghdar Kashkooli, Arman; Peramuna, Anantha Vithakshana
  • Frontiers in Bioengineering and Biotechnology, Vol. 5
  • DOI: 10.3389/fbioe.2017.00047

Production of taxa-4(5),11(12)-diene by transgenic Physcomitrella patens
journal, February 2009

  • Anterola, Aldwin; Shanle, Erin; Perroud, Pierre-François
  • Transgenic Research, Vol. 18, Issue 4
  • DOI: 10.1007/s11248-009-9252-5

The bryophyte Physcomitrella patens replicates extrachromosomal transgenic elements: RESEARCH Extrachromosomal transgenic elements in a bryophyte
journal, June 2000


Heterologous expression of the isopimaric acid pathway in Nicotiana benthamiana and the effect of N-terminal modifications of the involved cytochrome P450 enzyme
journal, December 2015

  • Gnanasekaran, Thiyagarajan; Vavitsas, Konstantinos; Andersen-Ranberg, Johan
  • Journal of Biological Engineering, Vol. 9, Issue 1
  • DOI: 10.1186/s13036-015-0022-z

Metabolic engineering of isoprenoid biosynthesis in Arabidopsis for the production of taxadiene, the first committed precursor of Taxol : Taxadiene Production in Arabidopsis
journal, September 2004

  • Besumbes, Óscar; Sauret-Güeto, Susanna; Phillips, Michael A.
  • Biotechnology and Bioengineering, Vol. 88, Issue 2
  • DOI: 10.1002/bit.20237

Metabolic Engineering of Synechocystis sp. PCC 6803 for Production of the Plant Diterpenoid Manoyl Oxide
journal, July 2015


Plant diterpene synthases: exploring modularity and metabolic diversity for bioengineering
journal, July 2015


Vectors for gene expression and amplification in the yeastYarrowia lipolytica
journal, January 2001


Sclareol production in the moss Physcomitrella patens and observations on growth and terpenoid biosynthesis
journal, May 2015


Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously
journal, January 2004


The Physcomitrella patens chromosome-scale assembly reveals moss genome structure and evolution
journal, January 2018

  • Lang, Daniel; Ullrich, Kristian K.; Murat, Florent
  • The Plant Journal, Vol. 93, Issue 3
  • DOI: 10.1111/tpj.13801

Metabolic engineering of the moss Physcomitrella patens to produce the sesquiterpenoids patchoulol and α/β-santalene
journal, November 2014


Redirection of carotenoid metabolism for the efficient production of taxadiene [taxa-4(5),11(12)-diene] in transgenic tomato fruit
journal, November 2006


Novel clades of chromodomain-containing Gypsy LTR retrotransposons from mosses (Bryophyta)
journal, November 2008


Efficient gene targeting in the moss Physcomitrella patens
journal, June 1997


Additional diterpenes from Physcomitrella patens synthesized by copalyl diphosphate/kaurene synthase ( Pp CPS/KS)
journal, November 2015


Phytozome: a comparative platform for green plant genomics
journal, November 2011

  • Goodstein, David M.; Shu, Shengqiang; Howson, Russell
  • Nucleic Acids Research, Vol. 40, Issue D1
  • DOI: 10.1093/nar/gkr944

Rescue and characterization of episomally replicating DNA from the moss Physcomitrella
journal, November 2009

  • Muren, E.; Nilsson, A.; Ulfstedt, M.
  • Proceedings of the National Academy of Sciences, Vol. 106, Issue 46
  • DOI: 10.1073/pnas.0908037106

Expanding the Landscape of Diterpene Structural Diversity through Stereochemically Controlled Combinatorial Biosynthesis
journal, January 2016

  • Andersen-Ranberg, Johan; Kongstad, Kenneth Thermann; Nielsen, Morten Thrane
  • Angewandte Chemie International Edition, Vol. 55, Issue 6
  • DOI: 10.1002/anie.201510650

Exploring plant biodiversity: the Physcomitrella genome and beyond
journal, October 2008


In vivo assembly of DNA-fragments in the moss, Physcomitrella patens
journal, April 2016

  • King, Brian Christopher; Vavitsas, Konstantinos; Ikram, Nur Kusaira Binti Khairul
  • Scientific Reports, Vol. 6, Issue 1
  • DOI: 10.1038/srep25030

High-level diterpene production by transient expression in Nicotiana benthamiana
journal, January 2013


Evolution of Diterpene Metabolism: Sitka Spruce CYP720B4 Catalyzes Multiple Oxidations in Resin Acid Biosynthesis of Conifer Defense against Insects
journal, October 2011

  • Hamberger, Björn; Ohnishi, Toshiyuki; Hamberger, Britta
  • Plant Physiology, Vol. 157, Issue 4
  • DOI: 10.1104/pp.111.185843

Opportunities for yeast metabolic engineering: Lessons from synthetic biology
journal, February 2011

  • Krivoruchko, Anastasia; Siewers, Verena; Nielsen, Jens
  • Biotechnology Journal, Vol. 6, Issue 3
  • DOI: 10.1002/biot.201000308

Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss <em>Physcomitrella patens</em>
journal, January 2011

  • Liu, Yen-Chun; Vidali, Luis
  • Journal of Visualized Experiments, Issue 50
  • DOI: 10.3791/2560

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


Engineering plant metabolism into microbes: from systems biology to synthetic biology
journal, April 2013


Moss-made pharmaceuticals: from bench to bedside
journal, May 2015

  • Reski, Ralf; Parsons, Juliana; Decker, Eva L.
  • Plant Biotechnology Journal, Vol. 13, Issue 8
  • DOI: 10.1111/pbi.12401

Arabidopsis ent -Kaurene Oxidase Catalyzes Three Steps of Gibberellin Biosynthesis
journal, February 1999

  • Helliwell, Chris A.; Poole, Andrew; James Peacock, W.
  • Plant Physiology, Vol. 119, Issue 2
  • DOI: 10.1104/pp.119.2.507

    Works referencing / citing this record:

    High-titer production of 13R-manoyl oxide in metabolically engineered Saccharomyces cerevisiae
    journal, April 2019