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Title: Complete enzyme set for chlorophyll biosynthesis in Escherichia coli

Abstract

Chlorophylls are essential cofactors for photosynthesis, which sustains global food chains and oxygen production. Billions of tons of chlorophylls are synthesized annually, yet full understanding of chlorophyll biosynthesis has been hindered by the lack of characterization of the Mg–protoporphyrin IX monomethyl ester oxidative cyclase step, which confers the distinctive green color of these pigments. We demonstrate cyclase activity using heterologously expressed enzyme. Next, we assemble a genetic module that encodes the complete chlorophyll biosynthetic pathway and show that it functions in Escherichia coli. Expression of 12 genes converts endogenous protoporphyrin IX into chlorophyll a, turning E. coli cells green. Our results delineate a minimum set of enzymes required to make chlorophyll and establish a platform for engineering photosynthesis in a heterotrophic model organism.

Authors:
ORCiD logo [1];  [1]; ORCiD logo [1];  [1]; ORCiD logo [1]; ORCiD logo [1]; ORCiD logo [2]; ORCiD logo [1]
  1. Univ. of Sheffield (United Kingdom)
  2. Pennsylvania State Univ., University Park, PA (United States)
Publication Date:
Research Org.:
Pennsylvania State Univ., University Park, PA (United States); Washington Univ., St. Louis, MO (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22). Chemical Sciences, Geosciences & Biosciences Division
OSTI Identifier:
1499942
Grant/Contract Number:  
FG02-94ER20137; SC0001035
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Science Advances
Additional Journal Information:
Journal Volume: 4; Journal Issue: 1; Journal ID: ISSN 2375-2548
Publisher:
AAAS
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Chen, Guangyu E., Canniffe, Daniel P., Barnett, Samuel F. H., Hollingshead, Sarah, Brindley, Amanda A., Vasilev, Cvetelin, Bryant, Donald A., and Hunter, C. Neil. Complete enzyme set for chlorophyll biosynthesis in Escherichia coli. United States: N. p., 2018. Web. doi:10.1126/sciadv.aaq1407.
Chen, Guangyu E., Canniffe, Daniel P., Barnett, Samuel F. H., Hollingshead, Sarah, Brindley, Amanda A., Vasilev, Cvetelin, Bryant, Donald A., & Hunter, C. Neil. Complete enzyme set for chlorophyll biosynthesis in Escherichia coli. United States. doi:10.1126/sciadv.aaq1407.
Chen, Guangyu E., Canniffe, Daniel P., Barnett, Samuel F. H., Hollingshead, Sarah, Brindley, Amanda A., Vasilev, Cvetelin, Bryant, Donald A., and Hunter, C. Neil. Fri . "Complete enzyme set for chlorophyll biosynthesis in Escherichia coli". United States. doi:10.1126/sciadv.aaq1407. https://www.osti.gov/servlets/purl/1499942.
@article{osti_1499942,
title = {Complete enzyme set for chlorophyll biosynthesis in Escherichia coli},
author = {Chen, Guangyu E. and Canniffe, Daniel P. and Barnett, Samuel F. H. and Hollingshead, Sarah and Brindley, Amanda A. and Vasilev, Cvetelin and Bryant, Donald A. and Hunter, C. Neil},
abstractNote = {Chlorophylls are essential cofactors for photosynthesis, which sustains global food chains and oxygen production. Billions of tons of chlorophylls are synthesized annually, yet full understanding of chlorophyll biosynthesis has been hindered by the lack of characterization of the Mg–protoporphyrin IX monomethyl ester oxidative cyclase step, which confers the distinctive green color of these pigments. We demonstrate cyclase activity using heterologously expressed enzyme. Next, we assemble a genetic module that encodes the complete chlorophyll biosynthetic pathway and show that it functions in Escherichia coli. Expression of 12 genes converts endogenous protoporphyrin IX into chlorophyll a, turning E. coli cells green. Our results delineate a minimum set of enzymes required to make chlorophyll and establish a platform for engineering photosynthesis in a heterotrophic model organism.},
doi = {10.1126/sciadv.aaq1407},
journal = {Science Advances},
issn = {2375-2548},
number = 1,
volume = 4,
place = {United States},
year = {2018},
month = {1}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Figures / Tables:

Fig. 1. Fig. 1.: Assembly of the Chl biosynthesis pathway in Escherichia coli. (A) Overall reactions from PPIX to Chl a catalyzed by the enzymes introduced to E. coli. The insertion of Fe2+ into PPIX (not shown) creates a biosynthetic branch point (not shown) that yields heme. Colored shading denotes the chemicalmore » change(s) at each step. ATP, adenosine triphosphate; ADP, adenosine diphosphate; SAM, S-adenosine-L-methionine; SAH, S-adenosyl-L-homocysteine; NADP+, nicotinamide adenine dinucleotide phosphate; NADPH, reduced form of NADP+; Pi, inorganic phosphate; PPi, inorganic pyrophosphate. (B) Arrangement and relative size of each gene in the constructed plasmids. The chlI, chlD, chlH, gun4, chlM, acsF, dvr (bciB), and chlG genes were consecutively cloned into a modified pET3a vector with a single T7 promoter upstream of chlI and a ribosome-binding site upstream of each gene using the link-and-lock method (see fig. S2 for details) (40). Colors for genes correspond to those used in (A). Plasmid constructs and gene contents are IM (chlI–chlM), IA (chlI–acsF), ID (chlI–dvr), IG (chlI–chlG), BoP (BoWSCP and chlP), and DE (dxs and crtE). BoP is a pACYCDuet1-based plasmid containing a sequence encoding the BoWSCP protein with a C-terminal His10tag (10) and the Synechocystis chlP gene. DE is a pCOLADuet1-based plasmid containing the E. coli dxs gene and the Rvi. gelatinosus crtE gene.« less

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Works referenced in this record:

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