A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

Murphy, Katherine M.; Chung, Siwon; Fogla, Shruti; Minsky, Hana B.; Zhu, Karen Yong; Zerbe, Philipp

doi:10.3791/59992

Title: A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

Abstract

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be usedmore »« less

Authors:

Murphy, Katherine M. ^[1]; Chung, Siwon ^[1]; Fogla, Shruti ^[1]; Minsky, Hana B. ^[1]; Zhu, Karen Yong ^[1]; Zerbe, Philipp ^[1]

Univ. of California, Davis, CA (United States)

Publication Date:: Fri Oct 04 00:00:00 EDT 2019

Research Org.:: Univ. of California, Davis, CA (United States)

Sponsoring Org.:: USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division; National Science Foundation (NSF)

OSTI Identifier:: 1633650

Grant/Contract Number:: SC0019178

Resource Type:: Accepted Manuscript

Journal Name:: Journal of Visualized Experiments

Additional Journal Information:: Journal Issue: 152; Journal ID: ISSN 1940-087X

Publisher:: MyJoVE Corp.

Country of Publication:: United States

Language:: English

Subject:: 59 BASIC BIOLOGICAL SCIENCES; 09 BIOMASS FUELS

Citation Formats


                    Murphy, Katherine M., Chung, Siwon, Fogla, Shruti, Minsky, Hana B., Zhu, Karen Yong, and Zerbe, Philipp. A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products.  United States: N. p., 2019. 
Web.  doi:10.3791/59992.

Copy to clipboard


                    Murphy, Katherine M., Chung, Siwon, Fogla, Shruti, Minsky, Hana B., Zhu, Karen Yong, & Zerbe, Philipp. A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products.  United States.  https://doi.org/10.3791/59992

Copy to clipboard


                    Murphy, Katherine M., Chung, Siwon, Fogla, Shruti, Minsky, Hana B., Zhu, Karen Yong, and Zerbe, Philipp. Fri .  
"A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products".  United States.  https://doi.org/10.3791/59992.  https://www.osti.gov/servlets/purl/1633650.

Copy to clipboard


                    
@article{osti_1633650,

  title        = {A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products},

  author       = {Murphy, Katherine M. and Chung, Siwon and Fogla, Shruti and Minsky, Hana B. and Zhu, Karen Yong and Zerbe, Philipp},

  abstractNote = {Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.},

  doi          = {10.3791/59992},

  journal      = {Journal of Visualized Experiments},

  number       = 152,

  volume       = ,

  place        = {United States},

  year         = {Fri Oct 04 00:00:00 EDT 2019},

  month        = {Fri Oct 04 00:00:00 EDT 2019}

}

Copy to clipboard

Journal Article:

Free Publicly Available Full Text

Accepted Manuscript (DOE)

Publisher's Version of Record

https://doi.org/10.3791/59992

Other availability

Search WorldCat to find libraries that may hold this journal

Citation Metrics:

Cited by: 3 works

Citation information provided by
Web of Science

Figures / Tables:

Table 1: Antibiotic concentrations for plasmid co-expression in E. coli or N. benthamiana.

All figures and tables (5 total)

Save / Share:

Export Metadata

Save to My Library

Figures / Tables found in this record:

Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.

Similar Records in DOE PAGES and OSTI.GOV collections:

Bioactive diterpenoids impact the composition of the root-associated microbiome in maize (Zea mays)

Journal Article Murphy, Katherine M. ; Edwards, Joseph ; Louie, Katherine B. ; ... - Scientific Reports

Abstract Plants deploy both primary and species-specific, specialized metabolites to communicate with other organisms and adapt to environmental challenges, including interactions with soil-dwelling microbial communities. However, the role of specialized metabolites in modulating plant-microbiome interactions often remains elusive. In this study, we report that maize ( Zea mays ) diterpenoid metabolites with known antifungal bioactivities also influence rhizosphere bacterial communities. Metabolite profiling showed that dolabralexins, antibiotic diterpenoids that are highly abundant in roots of some maize varieties, can be exuded from the roots. Comparative 16S rRNA gene sequencing determined the bacterial community composition of the maize mutant Zman2 ( anthermore »« less
https://doi.org/10.1038/s41598-020-79320-z
Production of Putative Diterpene Carboxylic Acid Intermediates of Triptolide in Yeast

Journal Article Forman, Victor ; Callari, Roberta ; Folly, Christophe ; ... - Molecules

The development of medical applications exploiting the broad bioactivities of the diterpene therapeutic triptolide from Tripterygium wilfordii is limited by low extraction yields from the native plant. Furthermore, the extraordinarily high structural complexity prevents an economically attractive enantioselective total synthesis. An alternative production route of triptolide through engineered Saccharomyces cerevisiae (yeast) could provide a sustainable source of triptolide. A potential intermediate in the unknown biosynthetic route to triptolide is the diterpene dehydroabietic acid. Here, we report a biosynthetic route to dehydroabietic acid by transient expression of enzymes from T. wilfordii and Sitka spruce (Picea sitchensis) in Nicotiana benthamiana. The combinationmore »« less
Cited by 14
https://doi.org/10.3390/molecules22060981

Full Text Available
Cytochrome P450–catalyzed biosynthesis of furanoditerpenoids in the bioenergy crop switchgrass (Panicum virgatum L.)

Journal Article Muchlinski, Andrew ; Jia, Meirong ; Tiedge, Kira ; ... - The Plant Journal

Specialized diterpenoid metabolites are important mediators of plant-environment interactions in monocot crops. To understand metabolite functions in plant environmental adaptation that ultimately can enable crop improvement strategies, a deeper knowledge of the underlying species-specific biosynthetic pathways is required. Furthermore, we report the genomics-enabled discovery of five cytochrome P450 monooxygenases (CYP71Z25-CYP71Z29) that form previously unknown furanoditerpenoids in the monocot bioenergy crop Panicum virgatum (switchgrass). Combinatorial pathway reconstruction showed that CYP71Z25-CYP71Z29 catalyze furan ring addition directly to primary diterpene alcohol intermediates derived from distinct class II diterpene synthase products. Transcriptional co-expression patterns and the presence of select diterpenoids in switchgrass roots supportmore »« less
https://doi.org/10.1111/tpj.15492

Full Text Available
The foxtail millet (Setaria italica) terpene synthase gene family

Journal Article Karunanithi, Prema S. ; Berrios, David I. ; Wang, Sadira ; ... - The Plant Journal

Terpenoid metabolism plays vital roles in stress defense and the environmental adaptation of monocot crops. Here, we describe the identification of the terpene synthase (TPS) gene family of the panicoid food and bioenergy model crop foxtail millet (Setaria italica). The diploid S. italica genome contains 32 TPS genes, 17 of which were biochemically characterized in this study. Unlike other thus far investigated grasses, S. italica contains TPSs producing all three ent-, (+)- and syn-copalyl pyrophosphate stereoisomers that naturally occur as central building blocks in the biosynthesis of distinct monocot diterpenoids. Conversion of these intermediates by the promiscuous TPS SiTPS8 yieldedmore »« less
https://doi.org/10.1111/tpj.14771

Full Text Available
Engineering Nannochloropsis oceanica for the production of diterpenoid compounds

Journal Article Du, Zhi‐Yan ; Bhat, Wajid W. ; Poliner, Eric ; ... - mLife

Abstract Photosynthetic microalgae like Nannochloropsis hold enormous potential as sustainable, light‐driven biofactories for the production of high‐value natural products such as terpenoids. Nannochloropsis oceanica is distinguished as a particularly robust host with extensive genomic and transgenic resources available. Its capacity to grow in wastewater, brackish, and sea waters, coupled with advances in microalgal metabolic engineering, genome editing, and synthetic biology, provides an excellent opportunity. In the present work, we demonstrate how N. oceanica can be engineered to produce the diterpene casbene—an important intermediate in the biosynthesis of pharmacologically relevant macrocyclic diterpenoids. Casbene accumulated after stably expressing and targeting the casbenemore »« less
https://doi.org/10.1002/mlf2.12097

Similar Records