RNA-Seq analysis and targeted mutagenesis for improved free fatty acid production in an engineered cyanobacterium

Ruffing, Anne M.

doi:10.1186/1754-6834-6-113

RNA-Seq analysis and targeted mutagenesis for improved free fatty acid production in an engineered cyanobacterium

Journal Article · Tue Jan 01 00:00:00 EST 2013 · Biotechnology for Biofuels

DOI:https://doi.org/10.1186/1754-6834-6-113· OSTI ID:1626656

Ruffing, Anne M. ^[1]

Sandia National Lab. (SNL-NM), Albuquerque, NM (United States). dept. of Bioenergy and Defense Technologies; DOE/OSTI

Background: High-energy-density biofuels are typically derived from the fatty acid pathway, thus establishing free fatty acids (FFAs) as important fuel precursors. FFA production using photosynthetic microorganisms like cyanobacteria allows for direct conversion of carbon dioxide into fuel precursors. Recent studies investigating cyanobacterial FFA production have demonstrated the potential of this process, yet FFA production was also shown to have negative physiological effects on the cyanobacterial host, ultimately limiting high yields of FFAs. Results: Cyanobacterial FFA production was shown to generate reactive oxygen species (ROS) and lead to increased cell membrane permeability. To identify genetic targets that may mitigate these toxic effects, RNA-seq analysis was used to investigate the host response of Synechococcus elongatus PCC 7942. Stress response, nitrogen metabolism, photosynthesis, and protein folding genes were up-regulated during FFA production while genes involved in carbon and hydrogen metabolisms were down-regulated. Select genes were targeted for mutagenesis to confirm their role in mitigating FFA toxicity. Gene knockout of two porins and the overexpression of ROS-degrading proteins and hypothetical proteins reduced the toxic effects of FFA production, allowing for improved growth, physiology, and FFA yields. Comparative transcriptomics, analyzing gene expression changes associated with FFA production and other stress conditions, identified additional key genes involved in cyanobacterial stress response. Conclusions: A total of 15 gene targets were identified to reduce the toxic effects of FFA production. While single-gene targeted mutagenesis led to minor increases in FFA production, the combination of these targeted mutations may yield additional improvement, advancing the development of high-energy-density fuels derived from cyanobacteria.

View Accepted Manuscript (DOE)

Research Organization:: Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences & Biosciences Division

Grant/Contract Number:: AC04-94AL85000

OSTI ID:: 1626656

Journal Information:: Biotechnology for Biofuels, Journal Name: Biotechnology for Biofuels Journal Issue: 1 Vol. 6; ISSN 1754-6834

Publisher:: BioMed CentralCopyright Statement

Country of Publication:: United States

Language:: English

References (43)

Physiological effects of free fatty acid production in genetically engineered Synechococcus elongatus PCC 7942 Ruffing, Anne M.; Jones, Howland D. T. Biotechnology and Bioengineering, Vol. 109, Issue 9 https://doi.org/10.1002/bit.24509	journal	April 2012
Diatom/Copepod Interactions in Plankton: The Indirect Chemical Defense of Unicellular Algae Pohnert, Georg ChemBioChem, Vol. 6, Issue 6 https://doi.org/10.1002/cbic.200400348	journal	May 2005
Salt Stress and Hyperosmotic Stress Regulate the Expression of Different Sets of Genes in Synechocystis sp. PCC 6803 Kanesaki, Yu; Suzuki, Iwane; Allakhverdiev, Suleyman I. Biochemical and Biophysical Research Communications, Vol. 290, Issue 1 https://doi.org/10.1006/bbrc.2001.6201	journal	January 2002
Cyanobacterial Phycobilisomes MacColl, Robert Journal of Structural Biology, Vol. 124, Issue 2-3 https://doi.org/10.1006/jsbi.1998.4062	journal	December 1998
Borrowing genes from Chlamydomonas reinhardtii for free fatty acid production in engineered cyanobacteria Ruffing, Anne M. Journal of Applied Phycology, Vol. 25, Issue 5 https://doi.org/10.1007/s10811-013-9993-7	journal	January 2013
Cooperation of group 2 σ factors, SigD and SigE for light-induced transcription in the cyanobacterium Synechocystis sp. PCC 6803 Yoshimura, Tsutomu; Imamura, Sousuke; Tanaka, Kan FEBS Letters, Vol. 581, Issue 7 https://doi.org/10.1016/j.febslet.2007.03.010	journal	March 2007
Overproduction of free fatty acids in E. coli: Implications for biodiesel production Lu, Xuefeng; Vora, Harmit; Khosla, Chaitan Metabolic Engineering, Vol. 10, Issue 6, p. 333-339 https://doi.org/10.1016/j.ymben.2008.08.006	journal	November 2008
Targeting redox metabolism: the perfect storm induced by acrylamide poisoning in the brain Raldúa, Demetrio; Casado, Marta; Prats, Eva Scientific Reports, Vol. 10, Issue 1 https://doi.org/10.1038/s41598-019-57142-y	journal	January 2020
Quantitative analysis of expression of two circadian clock-controlled gene clusters coding for the bidirectional hydrogenase in the cyanobacterium Synechococcus sp. PCC7942 Schmitz, Oliver; Boison, Gudrun; Bothe, Hermann Molecular Microbiology, Vol. 41, Issue 6 https://doi.org/10.1046/j.1365-2958.2001.02612.x	journal	September 2001
The histidine kinase Hik33 perceives osmotic stress and cold stress in Synechocystis sp. PCC 6803: Osmostress and cold sensor Hik33 in Synechocystis Mikami, Koji; Kanesaki, Yu; Suzuki, Iwane Molecular Microbiology, Vol. 46, Issue 4 https://doi.org/10.1046/j.1365-2958.2002.03202.x	journal	November 2002
Fatty acid production in genetically modified cyanobacteria Liu, X.; Sheng, J.; Curtiss III, R. Proceedings of the National Academy of Sciences, Vol. 108, Issue 17, p. 6899-6904 https://doi.org/10.1073/pnas.1103014108	journal	April 2011
The High Light-inducible Polypeptides in Synechocystis PCC6803 : EXPRESSION AND FUNCTION IN HIGH LIGHT He, Qingfang; Dolganov, Nadia; Björkman, Olle Journal of Biological Chemistry, Vol. 276, Issue 1 https://doi.org/10.1074/jbc.m008686200	journal	October 2000
RNA Polyadenylation and Degradation in Cyanobacteria Are Similar to the Chloroplast but Different from Escherichia coli Rott, Ruth; Zipor, Gadi; Portnoy, Victoria Journal of Biological Chemistry, Vol. 278, Issue 18 https://doi.org/10.1074/jbc.m211571200	journal	February 2003
Quality control and preprocessing of metagenomic datasets Schmieder, R.; Edwards, R. Bioinformatics, Vol. 27, Issue 6 https://doi.org/10.1093/bioinformatics/btr026	journal	January 2011
Response to Oxidative Stress Involves a Novel Peroxiredoxin Gene in the Unicellular Cyanobacterium Synechocystis sp. PCC 6803 Kobayashi, Mari; Ishizuka, Tomokazu; Katayama, Mitsunori Plant and Cell Physiology, Vol. 45, Issue 3 https://doi.org/10.1093/pcp/pch034	journal	March 2004
Effects of high light on transcripts of stress-associated genes for the cyanobacteria Synechocystis sp. PCC 6803 and Prochlorococcus MED4 and MIT9313 Mary, Isabelle; Tu, Chao-Jung; Grossman, Arthur Microbiology, Vol. 150, Issue 5 https://doi.org/10.1099/mic.0.27014-0	journal	May 2004
Osmotic stress in Synechocystis sp. PCC 6803: low tolerance towards nonionic osmotic stress results from lacking activation of glucosylglycerol accumulation Marin, K. Microbiology, Vol. 152, Issue 7 https://doi.org/10.1099/mic.0.28771-0	journal	July 2006
The Histidine Kinase Hik34 Is Involved in Thermotolerance by Regulating the Expression of Heat Shock Genes in Synechocystis Suzuki, Iwane; Kanesaki, Yu; Hayashi, Hidenori Plant Physiology, Vol. 138, Issue 3 https://doi.org/10.1104/pp.104.059097	journal	June 2005
Fatty Acid Activation in Cyanobacteria Mediated by Acyl-Acyl Carrier Protein Synthetase Enables Fatty Acid Recycling Kaczmarzyk, Danuta; Fulda, Martin Plant Physiology, Vol. 152, Issue 3 https://doi.org/10.1104/pp.109.148007	journal	January 2010
C-di-GMP: the dawning of a novel bacterial signalling system: C-di-GMP signalling in bacteria Römling, Ute; Gomelsky, Mark; Galperin, Michael Y. Molecular Microbiology, Vol. 57, Issue 3 https://doi.org/10.1111/j.1365-2958.2005.04697.x	journal	June 2005
Identification of Transport Proteins Involved in Free Fatty Acid Efflux in Escherichia coli Lennen, R. M.; Politz, M. G.; Kruziki, M. A. Journal of Bacteriology, Vol. 195, Issue 1 https://doi.org/10.1128/jb.01477-12	journal	October 2012
Regulation and sequence of the Synechococcus sp. strain PCC 7942 groESL operon, encoding a cyanobacterial chaperonin Webb, R.; Reddy, K. J.; Sherman, L. A. Journal of Bacteriology, Vol. 172, Issue 9 https://doi.org/10.1128/jb.172.9.5079-5088.1990	journal	September 1990
Differential Gene Expression in Response to Hydrogen Peroxide and the Putative PerR Regulon of Synechocystis sp. Strain PCC 6803 Li, H.; Singh, A. K.; McIntyre, L. M. Journal of Bacteriology, Vol. 186, Issue 11 https://doi.org/10.1128/jb.186.11.3331-3345.2004	journal	May 2004
Regulation of Fatty Acid Synthesis Ohlrogge, John B.; Jaworski, Jan G. Annual Review of Plant Physiology and Plant Molecular Biology, Vol. 48, Issue 1, p. 109-136 https://doi.org/10.1146/annurev.arplant.48.1.109	journal	June 1997
REACTIVE OXYGEN SPECIES: Metabolism, Oxidative Stress, and Signal Transduction Apel, Klaus; Hirt, Heribert Annual Review of Plant Biology, Vol. 55, Issue 1 https://doi.org/10.1146/annurev.arplant.55.031903.141701	journal	June 2004
Hydrogen production by Cyanobacteria Dutta, Debajyoti; De, Debojyoti; Chaudhuri, Surabhi Microbial Cell Factories, Vol. 4, Issue 1 https://doi.org/10.1186/1475-2859-4-36	journal	December 2005
Integrative analysis of large scale expression profiles reveals core transcriptional response and coordination between multiple cellular processes in a cyanobacterium Singh, Abhay K.; Elvitigala, Thanura; Cameron, Jeffrey C. BMC Systems Biology, Vol. 4, Issue 1 https://doi.org/10.1186/1752-0509-4-105	journal	August 2010
Extracellular Secretion of Free Fatty Acids by Disruption of a Fatty Acyl-CoA Synthetase Gene in Saccharomyces cerevisiae Michinaka, Yasunari; Shimauchi, Toshitsugu; Aki, Tsunehiro Journal of Bioscience and Bioengineering, Vol. 95, Issue 5 https://doi.org/10.1263/jbb.95.435	journal	January 2003
Chloroplast-Encoded chlB Is Required for Light-Independent Protochlorophyllide Reductase Activity in Chlamydomonas reinhardtii Li, Jianming; Goldschmidt-Clermont, Michel; Timko, Michael P. The Plant Cell, Vol. 5, Issue 12 https://doi.org/10.2307/3869697	journal	December 1993
DNA Microarray Analysis of Cyanobacterial Gene Expression during Acclimation to High Light Hihara, Yukako; Kamei, Ayako; Kanehisa, Minoru The Plant Cell, Vol. 13, Issue 4 https://doi.org/10.2307/3871341	journal	April 2001
Natural Antibiotic Resistance and Contamination by Antibiotic Resistance Determinants: The Two Ages in the Evolution of Resistance to Antimicrobials Martínez, José L. Frontiers in Microbiology, Vol. 3 https://doi.org/10.3389/fmicb.2012.00001	journal	January 2012
Antibacterial free fatty acids: activities, mechanisms of action and biotechnological potential Desbois, Andrew P.; Smith, Valerie J. Applied Microbiology and Biotechnology, Vol. 85, Issue 6 https://doi.org/10.1007/s00253-009-2355-3	journal	December 2009
Cytotoxic effects of free fatty acids on phytoplankton algae and cyanobacteria Wu, Jiunn-Tzong; Chiang, Yin-Ru; Huang, Wen-Ya Aquatic Toxicology, Vol. 80, Issue 4 https://doi.org/10.1016/j.aquatox.2006.09.011	journal	December 2006
Homogeneous, heterogeneous and enzymatic catalysis for transesterification of high free fatty acid oil (waste cooking oil) to biodiesel: A review Lam, Man Kee; Lee, Keat Teong; Mohamed, Abdul Rahman Biotechnology Advances, Vol. 28, Issue 4 https://doi.org/10.1016/j.biotechadv.2010.03.002	journal	July 2010
SPOCD1 is an essential executor of piRNA-directed de novo DNA methylation Zoch, Ansgar; Auchynnikava, Tania; Berrens, Rebecca V. Nature, Vol. 584, Issue 7822 https://doi.org/10.1038/s41586-020-2557-5	journal	July 2020
Cold-regulated genes under control of the cold sensor Hik33 in Synechocystis: Gene expression at low temperature in Synechocystis Suzuki, Iwane; Kanesaki, Yu; Mikami, Koji Molecular Microbiology, Vol. 40, Issue 1 https://doi.org/10.1046/j.1365-2958.2001.02379.x	journal	April 2001
RNA Polyadenylation and Degradation in Cyanobacteria Are Similar to the Chloroplast but Different from Escherichia coli Rott, Ruth; Zipor, Gadi; Portnoy, Victoria Journal of Biological Chemistry, Vol. 278, Issue 18 https://doi.org/10.1074/jbc.M211571200	journal	February 2003
Regulation of Fatty Acid Synthesis Zakim, David; Herman, Robert H. The American Journal of Clinical Nutrition, Vol. 22, Issue 2 https://doi.org/10.1093/ajcn/22.2.200	journal	February 1969
edgeR: a Bioconductor package for differential expression analysis of digital gene expression data Robinson, M. D.; McCarthy, D. J.; Smyth, G. K. Bioinformatics, Vol. 26, Issue 1 https://doi.org/10.1093/bioinformatics/btp616	journal	November 2009
Identification of Transport Proteins Involved in Free Fatty Acid Efflux in Escherichia coli Lennen, R. M.; Politz, M. G.; Kruziki, M. A. Journal of Bacteriology, Vol. 195, Issue 1 https://doi.org/10.1128/JB.01477-12	journal	October 2012
Differential Gene Expression in Response to Hydrogen Peroxide and the Putative PerR Regulon of Synechocystis sp. Strain PCC 6803 Li, H.; Singh, A. K.; McIntyre, L. M. Journal of Bacteriology, Vol. 186, Issue 11 https://doi.org/10.1128/JB.186.11.3331-3345.2004	journal	May 2004
RNA-seq based identification and mutant validation of gene targets related to ethanol resistance in cyanobacterial Synechocystis sp. PCC 6803 Wang, Jiangxin; Chen, Lei; Huang, Siqiang Biotechnology for Biofuels, Vol. 5, Issue 1 https://doi.org/10.1186/1754-6834-5-89	journal	January 2012
Ultrafast and memory-efficient alignment of short DNA sequences to the human genome Langmead, Ben; Trapnell, Cole; Pop, Mihai Genome Biology, Vol. 10, Issue 3 https://doi.org/10.1186/gb-2009-10-3-r25	journal	January 2009

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Additional file 3 of Cross-feeding between cyanobacterium Synechococcus and Escherichia coli in an artificial autotrophic–heterotrophic coculture system revealed by integrated omics analysis Ma, Jiajia; Guo, Taohong; Ren, Meijin figshare https://doi.org/10.6084/m9.figshare.20131177.v1	image	January 2022
Additional file 4 of Cross-feeding between cyanobacterium Synechococcus and Escherichia coli in an artificial autotrophic–heterotrophic coculture system revealed by integrated omics analysis Ma, Jiajia; Guo, Taohong; Ren, Meijin figshare https://doi.org/10.6084/m9.figshare.20131180	image	January 2022
Additional file 5 of Cross-feeding between cyanobacterium Synechococcus and Escherichia coli in an artificial autotrophic–heterotrophic coculture system revealed by integrated omics analysis Ma, Jiajia; Guo, Taohong; Ren, Meijin figshare https://doi.org/10.6084/m9.figshare.20131183	image	January 2022
Additional file 6 of Cross-feeding between cyanobacterium Synechococcus and Escherichia coli in an artificial autotrophic–heterotrophic coculture system revealed by integrated omics analysis Ma, Jiajia; Guo, Taohong; Ren, Meijin figshare https://doi.org/10.6084/m9.figshare.20131186.v1	image	January 2022
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