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Title: A Genetic Toolbox for Modulating the Expression of Heterologous Genes in the Cyanobacterium Synechocystis sp. PCC 6803

Abstract

Cyanobacteria, genetic models for photosynthesis research for decades, have recently become attractive hosts for producing renewable fuels and chemicals, owing to their genetic tractability, relatively fast growth, and their ability to utilize sunlight, fix carbon dioxide, and in some cases, fix nitrogen. Despite significant advances, there is still an urgent demand for synthetic biology tools in order to effectively manipulate genetic circuits in cyanobacteria. In this study, we have compared a total of 17 natural and chimeric promoters, focusing on expression of the ethylene-forming enzyme (EFE) in the cyanobacterium Synechocystis sp. PCC 6803. We report the finding that the E. coli σ 70 promoter Ptrc is superior compared to the previously reported strong promoters, such as PcpcB and PpsbA, for the expression of EFE. In addition, we found that the EFE expression level was very sensitive to the 5'-untranslated region upstream of the open reading frame. A library of ribosome binding sites (RBSs) was rationally designed and was built and systematically characterized. We demonstrate a strategy complementary to the RBS prediction software to facilitate the rational design of an RBS library to optimize the gene expression in cyanobacteria. Our results show that the EFE expression level is dramatically enhanced throughmore » these synthetic biology tools and is no longer the rate-limiting step for cyanobacterial ethylene production. Furthermore, these systematically characterized promoters and the RBS design strategy can serve as useful tools to tune gene expression levels and to identify and mitigate metabolic bottlenecks in cyanobacteria.« less

Authors:
ORCiD logo [1]; ORCiD logo [2];  [1];  [1]
  1. National Renewable Energy Lab. (NREL), Golden, CO (United States)
  2. National Renewable Energy Lab. (NREL), Golden, CO (United States); Univ. of Colorado, Boulder, CO (United States)
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (EE-3B); USDOE Office of Energy Efficiency and Renewable Energy (EERE), NREL Laboratory Directed Research and Development (LDRD); USDOE Office of Energy Efficiency and Renewable Energy (EERE), Hydrogen and Fuel Cell Technologies Program (EE-3F)
OSTI Identifier:
1416722
Report Number(s):
NREL/JA-2700-70604
Journal ID: ISSN 2161-5063
Grant/Contract Number:
AC36-08GO28308
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
ACS Synthetic Biology
Additional Journal Information:
Journal Volume: 7; Journal Issue: 1; Journal ID: ISSN 2161-5063
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; 59 BASIC BIOLOGICAL SCIENCES; cyanobacteria; ethylene-forming enzyme; promoter; protein expression; RBS; synthetic biology

Citation Formats

Wang, Bo, Eckert, Carrie, Maness, Pin -Ching, and Yu, Jianping. A Genetic Toolbox for Modulating the Expression of Heterologous Genes in the Cyanobacterium Synechocystis sp. PCC 6803. United States: N. p., 2017. Web. doi:10.1021/acssynbio.7b00297.
Wang, Bo, Eckert, Carrie, Maness, Pin -Ching, & Yu, Jianping. A Genetic Toolbox for Modulating the Expression of Heterologous Genes in the Cyanobacterium Synechocystis sp. PCC 6803. United States. doi:10.1021/acssynbio.7b00297.
Wang, Bo, Eckert, Carrie, Maness, Pin -Ching, and Yu, Jianping. 2017. "A Genetic Toolbox for Modulating the Expression of Heterologous Genes in the Cyanobacterium Synechocystis sp. PCC 6803". United States. doi:10.1021/acssynbio.7b00297.
@article{osti_1416722,
title = {A Genetic Toolbox for Modulating the Expression of Heterologous Genes in the Cyanobacterium Synechocystis sp. PCC 6803},
author = {Wang, Bo and Eckert, Carrie and Maness, Pin -Ching and Yu, Jianping},
abstractNote = {Cyanobacteria, genetic models for photosynthesis research for decades, have recently become attractive hosts for producing renewable fuels and chemicals, owing to their genetic tractability, relatively fast growth, and their ability to utilize sunlight, fix carbon dioxide, and in some cases, fix nitrogen. Despite significant advances, there is still an urgent demand for synthetic biology tools in order to effectively manipulate genetic circuits in cyanobacteria. In this study, we have compared a total of 17 natural and chimeric promoters, focusing on expression of the ethylene-forming enzyme (EFE) in the cyanobacterium Synechocystis sp. PCC 6803. We report the finding that the E. coli σ70 promoter Ptrc is superior compared to the previously reported strong promoters, such as PcpcB and PpsbA, for the expression of EFE. In addition, we found that the EFE expression level was very sensitive to the 5'-untranslated region upstream of the open reading frame. A library of ribosome binding sites (RBSs) was rationally designed and was built and systematically characterized. We demonstrate a strategy complementary to the RBS prediction software to facilitate the rational design of an RBS library to optimize the gene expression in cyanobacteria. Our results show that the EFE expression level is dramatically enhanced through these synthetic biology tools and is no longer the rate-limiting step for cyanobacterial ethylene production. Furthermore, these systematically characterized promoters and the RBS design strategy can serve as useful tools to tune gene expression levels and to identify and mitigate metabolic bottlenecks in cyanobacteria.},
doi = {10.1021/acssynbio.7b00297},
journal = {ACS Synthetic Biology},
number = 1,
volume = 7,
place = {United States},
year = 2017,
month =
}

Journal Article:
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  • The existence of active transport systems (permeases) operating on amino acids in the photoautotrophic cyanobacterium Synechocystis sp. strain 6803 was demonstrated by following the initial rates of uptake with /sup 14/C-labeled amino acids, measuring the intracellular pools of amino acids, and isolating mutants resistant to toxic amino acids. One class of mutants (Pfa1) corresponds to a regulatory defect in the biosynthesis of the aromatic amino acids, but two other classes (Can1 and Aza1) are defective in amino acid transport. The Can1 mutants are defective in the active transport of three basic amino acids (arginine, histidine, and lysine) and in onemore » of two transport systems operating on glutamine. The Aza1 mutants are not affected in the transport of the basic amino acids but have lost the capacity to transport all other amino acids except glutamate. The latter amino acid is probably transported by a third permease which could be identical to the Can1-independent transport operating on glutamine. Thus, genetic evidence suggests that strain 6803 has only a small number of amino acid transport systems with fairly broad specificity and that, with the exception of glutamine, each amino acid is accumulated by only one major transport system. Compared with heterotrophic bacteria such as Escherichia coli, these permeases are rather inefficient in terms of affinity (apparent K/sub m/ ranging from 6 to 60 ..mu..M) and of V/sub max/.« less
  • The authors have utilized the unicellular cyanobacterium Synechocystis sp. PCC 6803 to incorporate site-directed amino acid substitutions into the photosystem I (PSI) reaction-center protein PsaB. A cysteine residue (position 565 of PSaB) proposed to serve as a ligand to the [4Fe-4S] center F[sub x] was changed to serine, histidine, and aspartate. These three mutants - C565S, C565H, and C565D - all exhibited greatly reduced accumulation of PSI reaction-center proteins and failed to grow autotrophically, indicating that this cysteine most likely does coordinate F[sub x], which is crucial for PSI biogenesis. Interestingly, the strain C565S accumulated significantly more PSI than themore » other two cysteine mutants and displayed photoreduction of the [4Fe-4S] terminal electron acceptors F[sub A] and F[sub B]. Mutations were also introduced into a leucine zipper motif of PsaB, proposed to participate in reaction-center dimerization. The mutants L522V, L536M, and L522V/L536M all exhibited wild-type characteristics and grew autotrophically, whereas the L522P mutation prevented PSI accumulation. These data do not provide support for a major structural role of the leucine zipper in reaction-center dimerization or in assembly of F[sub x]. However, the amino acid substitutions incorporated were conservative and might not have perturbed the leucine zipper. 31 refs., 4 figs., 1 tab.« less
  • A new open reading frame ORF242, coding for a 26.47-kDa polypeptide, was found in a DNA fragment of the cyanobacterium Synechocystis 6803, transforming a photosynthetic mutant to photoautotrophy and having homology with plant chloroplast DNA. In the 5{prime} flanking region of ORF242, consensus sequences characteristic of a functioning gene were found. One copy of ORF242 is present in the Synechocystis 6803 genome. Insertion inactivation of ORF242 does not lead to a decrease in photosynthetic activity in cells of cyanobacteria but may influence the ratio between active complexes of photosystems I and II. 22 refs., 6 figs., 2 tabs.
  • Oscillation patterns of the oxygen yield per flash induced by a train of single-turnover flashes were measured as a function of dark incubation and different pre-illumination conditions in several autotrophic mutant strains of Synechocystis sp. PCC 6803 carrying short deletions within the large, lumen-exposed hydrophilic region (loop E) of the chlorophyll a-binding photosystem II protein CP47. A physiological and biochemical characterization of these mutant strains has been presented previously, and some functional properties were described recently. The present study shows that in several mutants the water-oxidizing complex (WOC) became inactivated during prolonged dark incubation, whereas the WOC of the wild-typemore » strain remained active. The rate and extent of the inactivation in the mutants depend on the domain of loop E, where 3-8 amino acid residues were deleted. The most pronounced effects are observed in mutants {Delta}(A373-D380) and {Delta}(R384-V392). A competent WOC can be restored from the fully inactivated state by illumination with short saturating flashes. The number of flashes required for this process strongly depends on the site at which a deletion has been introduced into loop E. Again, the most prominent effects were found in mutants {Delta}(A373-D380) and {Delta}(R384-V392). Interestingly, the number of flashes required for activation was reduced by more than an order of magnitude in both mutants by the addition of 10 mM CaCl{sub 2} to the cell suspension. On the basis of a model for photoactivation proposed by Tamura and Cheniae (1987), a scheme is presented for the process of dark inactivation and photoactivation in these mutants. The results presented here corroborate an important role if the large hydrophilic domain (loop E) of CP47 in a functional and stable WOC. 64 refs., 9 figs.« less