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Title: Zn 2+ -Inducible Expression Platform for Synechococcus sp. Strain PCC 7002 Based on the smtA Promoter/Operator and smtB Repressor

Abstract

ABSTRACT Synechococcussp. strain PCC 7002 has been gaining significance as both a model system for photosynthesis research and for industrial applications. Until recently, the genetic toolbox for this model cyanobacterium was rather limited and relied primarily on tools that only allowed constitutive gene expression. This work describes a two-plasmid, Zn 2+-inducible expression platform that is coupled with azurAmutation, providing enhanced Zn 2+uptake. The control elements are based on the metal homeostasis system of a class II metallothionein gene (smtA 7942) and its cognate SmtB 7942repressor fromSynechococcus elongatusstrain PCC 7942. Under optimal induction conditions, yellow fluorescent protein (YFP) levels were about half of those obtained with the strong, constitutive phycocyanin (cpcBA 6803) promoter ofSynechocystissp. strain PCC 6803. This metal-inducible expression system inSynechococcussp. strain PCC 7002 allowed the titratable gene expression of YFP that was up to 19-fold greater than the background level. This system was utilized successfully to control the expression of theDrosophila melanogasterβ-carotene 15,15′-dioxygenase, NinaB, which is toxic when constitutively expressed from a strong promoter inSynechococcussp. strain PCC 7002. Together, these properties establish this metal-inducible system as an additional useful tool that is capable of controlling gene expression for applications ranging from basic research to synthetic biology inSynechococcussp. strain PCCmore » 7002. IMPORTANCEThis is the first metal-responsive expression system in cyanobacteria, to our knowledge, that does not exhibit low sensitivity for induction, which is one of the major hurdles for utilizing this class of genetic tools. In addition, high levels of expression can be generated that approximate those of established constitutive systems, with the added advantage of titratable control. Together, these properties establish this Zn 2+-inducible system, which is based on thesmtA 7942operator/promoter andsmtB 7942repressor, as a versatile gene expression platform that expands the genetic toolbox ofSynechococcussp. strain PCC 7002.« less

Authors:
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Publication Date:
Research Org.:
Pennsylvania State Univ., University Park, PA (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1536858
DOE Contract Number:  
FG02-05ER46222
Resource Type:
Journal Article
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 83; Journal Issue: 3; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
Biotechnology & Applied Microbiology; Microbiology

Citation Formats

Pérez, Adam A., Gajewski, John P., Ferlez, Bryan H., Ludwig, Marcus, Baker, Carol S., Golbeck, John H., Bryant, Donald A., and Löffler, Frank E. Zn 2+ -Inducible Expression Platform for Synechococcus sp. Strain PCC 7002 Based on the smtA Promoter/Operator and smtB Repressor. United States: N. p., 2016. Web. doi:10.1128/aem.02491-16.
Pérez, Adam A., Gajewski, John P., Ferlez, Bryan H., Ludwig, Marcus, Baker, Carol S., Golbeck, John H., Bryant, Donald A., & Löffler, Frank E. Zn 2+ -Inducible Expression Platform for Synechococcus sp. Strain PCC 7002 Based on the smtA Promoter/Operator and smtB Repressor. United States. doi:10.1128/aem.02491-16.
Pérez, Adam A., Gajewski, John P., Ferlez, Bryan H., Ludwig, Marcus, Baker, Carol S., Golbeck, John H., Bryant, Donald A., and Löffler, Frank E. Fri . "Zn 2+ -Inducible Expression Platform for Synechococcus sp. Strain PCC 7002 Based on the smtA Promoter/Operator and smtB Repressor". United States. doi:10.1128/aem.02491-16.
@article{osti_1536858,
title = {Zn 2+ -Inducible Expression Platform for Synechococcus sp. Strain PCC 7002 Based on the smtA Promoter/Operator and smtB Repressor},
author = {Pérez, Adam A. and Gajewski, John P. and Ferlez, Bryan H. and Ludwig, Marcus and Baker, Carol S. and Golbeck, John H. and Bryant, Donald A. and Löffler, Frank E.},
abstractNote = {ABSTRACT Synechococcussp. strain PCC 7002 has been gaining significance as both a model system for photosynthesis research and for industrial applications. Until recently, the genetic toolbox for this model cyanobacterium was rather limited and relied primarily on tools that only allowed constitutive gene expression. This work describes a two-plasmid, Zn2+-inducible expression platform that is coupled with azurAmutation, providing enhanced Zn2+uptake. The control elements are based on the metal homeostasis system of a class II metallothionein gene (smtA7942) and its cognate SmtB7942repressor fromSynechococcus elongatusstrain PCC 7942. Under optimal induction conditions, yellow fluorescent protein (YFP) levels were about half of those obtained with the strong, constitutive phycocyanin (cpcBA6803) promoter ofSynechocystissp. strain PCC 6803. This metal-inducible expression system inSynechococcussp. strain PCC 7002 allowed the titratable gene expression of YFP that was up to 19-fold greater than the background level. This system was utilized successfully to control the expression of theDrosophila melanogasterβ-carotene 15,15′-dioxygenase, NinaB, which is toxic when constitutively expressed from a strong promoter inSynechococcussp. strain PCC 7002. Together, these properties establish this metal-inducible system as an additional useful tool that is capable of controlling gene expression for applications ranging from basic research to synthetic biology inSynechococcussp. strain PCC 7002. IMPORTANCEThis is the first metal-responsive expression system in cyanobacteria, to our knowledge, that does not exhibit low sensitivity for induction, which is one of the major hurdles for utilizing this class of genetic tools. In addition, high levels of expression can be generated that approximate those of established constitutive systems, with the added advantage of titratable control. Together, these properties establish this Zn2+-inducible system, which is based on thesmtA7942operator/promoter andsmtB7942repressor, as a versatile gene expression platform that expands the genetic toolbox ofSynechococcussp. strain PCC 7002.},
doi = {10.1128/aem.02491-16},
journal = {Applied and Environmental Microbiology},
issn = {0099-2240},
number = 3,
volume = 83,
place = {United States},
year = {2016},
month = {11}
}