Towards understanding the mechanism of n-hexane tolerance in Synechocystis sp. PCC 6803

doi:10.1016/j.cjche.2022.11.015

中国化学工程学报 ›› 2023, Vol. 59 ›› Issue (7): 128-134.DOI: 10.1016/j.cjche.2022.11.015

• Full Length Article • 上一篇下一篇

Towards understanding the mechanism of n-hexane tolerance in Synechocystis sp. PCC 6803

Tao Sun^1,2,3, Shubin Li^1,2, Guangsheng Pei^1,2, Lei Chen^1,2, Weiwen Zhang^1,2,3

1. Laboratory of Synthetic Microbiology, School of Chemical Engineering & Technology, Tianjin University, Tianjin 300072, China;
2. Frontier Science Center for Synthetic Biology & Key Laboratory of Systems Bioengineering, Ministry of Education of China, Tianjin 300072, China;
3. Center for Biosafety Research and Strategy, Tianjin University, Tianjin 300072, China

收稿日期:2022-10-22 修回日期:2022-11-18 出版日期:2023-07-28 发布日期:2023-10-14
通讯作者: Lei Chen,E-mail:lchen@tju.edu.cn;Weiwen Zhang,E-mail:wwzhang8@tju.edu.cn
基金资助:
This research was supported by grants from the National Key Research and Development Program of China (2020YFA0906800, 2021YFA0909700, 2018YFA0903600 and 2019YFA0904600).

Towards understanding the mechanism of n-hexane tolerance in Synechocystis sp. PCC 6803

Tao Sun^1,2,3, Shubin Li^1,2, Guangsheng Pei^1,2, Lei Chen^1,2, Weiwen Zhang^1,2,3

1. Laboratory of Synthetic Microbiology, School of Chemical Engineering & Technology, Tianjin University, Tianjin 300072, China;
2. Frontier Science Center for Synthetic Biology & Key Laboratory of Systems Bioengineering, Ministry of Education of China, Tianjin 300072, China;
3. Center for Biosafety Research and Strategy, Tianjin University, Tianjin 300072, China

Received:2022-10-22 Revised:2022-11-18 Online:2023-07-28 Published:2023-10-14
Contact: Lei Chen,E-mail:lchen@tju.edu.cn;Weiwen Zhang,E-mail:wwzhang8@tju.edu.cn
Supported by:
This research was supported by grants from the National Key Research and Development Program of China (2020YFA0906800, 2021YFA0909700, 2018YFA0903600 and 2019YFA0904600).

摘要/Abstract

摘要： Synthetic biology efforts have also led to the development of photosynthetic cyanobacteria as “autotrophic cell factories” for biosynthesis of various biofuels directly from CO₂. However, the low tolerance to toxicity of biofuels has restricted the economic application of cyanobacterial hosts. In this study, RNA-seq transcriptomics was employed to reveal stress responses to exogenous n-hexane in Synechocystis sp. PCC 6803. Functional enrichment analysis of the transcriptomic data showed that signal transduction systems were induced significantly. To further identify regulatory genes related to n-hexane tolerance, a library of transcriptional regulators (TRs) deletion mutants was then screened for their roles in n-hexane tolerance. The results showed that a knockout mutant of slr0724 that encodes an HtaR suppressor protein was more tolerant to n-hexane than the wild type, indicating the involvement of slr0724 in n-hexane tolerance. This study provides the foundation for better understanding the cellular responses to n-hexane in Synechocystis sp. PCC 6803, which could contribute to the further engineering of n-hexane tolerance in cyanobacteria.

关键词: Transcriptome, n-Hexane tolerance, Metabolomics, Synechocystis

Abstract: Synthetic biology efforts have also led to the development of photosynthetic cyanobacteria as “autotrophic cell factories” for biosynthesis of various biofuels directly from CO₂. However, the low tolerance to toxicity of biofuels has restricted the economic application of cyanobacterial hosts. In this study, RNA-seq transcriptomics was employed to reveal stress responses to exogenous n-hexane in Synechocystis sp. PCC 6803. Functional enrichment analysis of the transcriptomic data showed that signal transduction systems were induced significantly. To further identify regulatory genes related to n-hexane tolerance, a library of transcriptional regulators (TRs) deletion mutants was then screened for their roles in n-hexane tolerance. The results showed that a knockout mutant of slr0724 that encodes an HtaR suppressor protein was more tolerant to n-hexane than the wild type, indicating the involvement of slr0724 in n-hexane tolerance. This study provides the foundation for better understanding the cellular responses to n-hexane in Synechocystis sp. PCC 6803, which could contribute to the further engineering of n-hexane tolerance in cyanobacteria.

Key words: Transcriptome, n-Hexane tolerance, Metabolomics, Synechocystis

Tao Sun, Shubin Li, Guangsheng Pei, Lei Chen, Weiwen Zhang. Towards understanding the mechanism of n-hexane tolerance in Synechocystis sp. PCC 6803[J]. 中国化学工程学报, 2023, 59(7): 128-134.

Tao Sun, Shubin Li, Guangsheng Pei, Lei Chen, Weiwen Zhang. Towards understanding the mechanism of n-hexane tolerance in Synechocystis sp. PCC 6803[J]. Chinese Journal of Chemical Engineering, 2023, 59(7): 128-134.

参考文献

[1] S. Gudmundsson, J. Nogales, Cyanobacteria as photosynthetic biocatalysts: A systems biology perspective, Mol. BioSyst. 11 (1) (2015) 60-70.
[2] A. Röttig, L. Wenning, D. Bröker, A. Steinbüchel, Fatty acid alkyl esters: Perspectives for production of alternative biofuels, Appl Microbiol Biotechnol 85 (6) (2010) 1713-1733.
[3] Z. Fatma, Model-assisted metabolic engineering of Escherichia coli for long chain alkane and alcohol production, Metab. Eng. 46 (2018) 1-12.
[4] J.B. Li, Y.S. Ma, N. Liu, B.E. Eser, Z. Guo, P.R. Jensen, G. Stephanopoulos, Synthesis of high-titer alka(e)nes in Yarrowia lipolytica is enabled by a discovered mechanism, Nat. Commun. 11 (2020) 6198.
[5] X.Y. Gao, T. Sun, G.S. Pei, L. Chen, W.W. Zhang, Cyanobacterial chassis engineering for enhancing production of biofuels and chemicals, Appl Microbiol Biotechnol 100 (8) (2016) 3401-3413.
[6] D. Noreña-Caro, Cyanobacteria as photoautotrophic biofactories of high-value chemicals, J. CO₂ Util. 28 (2018) 335-366.
[7] B.K. Kaiser, M. Carleton, J.W. Hickman, C. Miller, D. Lawson, M. Budde, P. Warrener, A. Paredes, S. Mullapudi, P. Navarro, F. Cross, J.M. Roberts, Fatty aldehydes in cyanobacteria are a metabolically flexible precursor for a diversity of biofuel products, PLoS One 8 (3) (2013) e58307.
[8] I.S. Yunus, Synthetic metabolic pathways for photobiological conversion of CO₂ into hydrocarbon fuel, Metab. Eng. 49 (2018) 201-211.
[9] M.J. Dunlop, Engineering microbes for tolerance to next-generation biofuels, Biotechnol. Biofuels 4 (2011) 32.
[10] J. Kämäräinen, Physiological tolerance and stoichiometric potential of cyanobacteria for hydrocarbon fuel production, J. Biotechnol. 162 (1) (2012) 67-74.
[11] J. Liu, L. Chen, J.X. Wang, J.J. Qiao, W.W. Zhang, Proteomic analysis reveals resistance mechanism against biofuel hexane in Synechocystis sp. PCC 6803, Biotechnol. Biofuels 5 (1) (2012) 68.
[12] J.L. Ramos, E. Duque, M.T. Gallegos, P. Godoy, M.I. Ramos-Gonzalez, A. Rojas, W. Teran, A. Segura, Mechanisms of solvent tolerance in gram-negative bacteria, Annu. Rev. Microbiol. 56 (2002) 743-768.
[13] X.X. Tian,,, Quantitative proteomics reveals dynamic responses of Synechocystis sp. PCC 6803 to next-generation biofuel butanol, J. Proteom. 78 (2013) 326-345.
[14] J.J. Qiao, J.X. Wang, L. Chen, X.X. Tian, S.Q. Huang, X.Y. Ren, W.W. Zhang, Quantitative iTRAQ LC-MS/MS proteomics reveals metabolic responses to biofuel ethanol in cyanobacterial Synechocystis sp. PCC 6803, J. Proteome Res. 11 (11) (2012) 5286-5300.
[15] T. Matsusako, Y. Toya, K. Yoshikawa, H. Shimizu, Identification of alcohol stress tolerance genes of Synechocystis sp. PCC 6803 using adaptive laboratory evolution, Biotechnol. Biofuels 10 (2017) 307.
[16] T. Lopes da Silva,,, Evaluation of the ethanol tolerance for wild and mutant Synechocystis strains by flow cytometry, Biotechnol. Rep. 17 (2018) 137-147.
[17] Z.L. Lin,,, Engineering of transcriptional regulators enhances microbial stress tolerance, Biotechnol. Adv. 31 (6) (2013) 986-991.
[18] D. Kaczmarzyk, Overexpression of sigma factor SigB improves temperature and butanol tolerance of Synechocystis sp. PCC6803, J. Biotechnol. 182-183 (2014) 54-60.
[19] L. Chen, L.N. Wu, J.X. Wang, W.W. Zhang, Butanol tolerance regulated by a two-component response regulator Slr1037 in photosynthetic Synechocystis sp. PCC 6803, Biotechnol. Biofuels 7 (2014) 89.
[20] X.F. Niu, Y. Zhu, G.S. Pei, L.N. Wu, L. Chen, W.W. Zhang, Elucidating butanol tolerance mediated by a response regulator Sll0039 in Synechocystis sp, Appl Microbiol Biotechnol 99 (4) (2015) 1845-1857.
[21] Z.D. Song,,, A transcriptional regulator Sll0794 regulates tolerance to biofuel ethanol in photosynthetic Synechocystis sp. PCC 6803*, Mol. Cell. Proteom. 13 (12) (2014) 3519-3532.
[22] Y. Zhu, G.S. Pei, X.F. Niu, M.L. Shi, M.Y. Zhang, L. Chen, W.W. Zhang, Metabolomic analysis reveals functional overlapping of three signal transduction proteins in regulating ethanol tolerance in cyanobacterium Synechocystis sp. PCC 6803, Mol. BioSyst. 11 (3) (2015) 770-782.
[23] Y.Q. Bi, G.S. Pei, T. Sun, Z.X. Chen, L. Chen, W.W. Zhang, Regulation mechanism mediated by Trans-encoded sRNA Nc117 in short chain alcohols tolerance in Synechocystis sp. PCC 6803, Front. Microbiol. 9 (2018) 863.
[24] H.J. Zhu, X.Y. Ren, J.X. Wang, Z.D. Song, M.L. Shi, J.J. Qiao, X.X. Tian, J. Liu, L. Chen, W.W. Zhang, Integrated OMICS guided engineering of biofuel butanol-tolerance in photosynthetic Synechocystis sp. PCC 6803, Biotechnol. Biofuels 6 (1) (2013) 106.
[25] R.K. Patel, M. Jain, NGS QC Toolkit: A toolkit for quality control of next generation sequencing data, PLoS One 7 (2) (2012) e30619.
[26] B. Langmead, C. Trapnell, M. Pop, S.L. Salzberg, Ultrafast and memory-efficient alignment of short DNA sequences to the human genome, Genome Biol. 10 (3) (2009) R25.
[27] S. Anders, P.T. Pyl, W. Huber, HTSeq—a Python framework to work with high-throughput sequencing data, Bioinformatics 31 (2) (2014) 166-169.
[28] M.I. Love, W. Huber, S. Anders, Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2, Genome Biol. 15 (12) (2014) 550.
[29] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method, Methods 25 (4) (2001) 402-408.
[30] H.L. Wang, B.L. Postier, R.L. Burnap, Optimization of fusion PCR for in vitro construction of gene knockout fragments, BioTechniques 33 (1) (2002) 26, 28, 30passim.
[31] J. Anfelt, B. Hallström, J. Nielsen, M. Uhlén, E.P. Hudson, Using transcriptomics to improve butanol tolerance of Synechocystis sp. strain PCC 6803, Appl. Environ. Microbiol. 79 (23) (2013) 7419-7427.
[32] S. Powell, K. Forslund, D. Szklarczyk, K. Trachana, A. Roth, J. Huerta-Cepas, T. Gabaldón, T. Rattei, C. Creevey, M. Kuhn, L.J. Jensen, C. von Mering, P. Bork, eggNOG v4.0: Nested orthology inference across 3686 organisms, Nucleic Acids Res 42 (D1) (2013) D231-D239.
[33] M.D. Young, M.J. Wakefield, G.K. Smyth, A. Oshlack, Gene ontology analysis for RNA-seq: Accounting for selection bias, Genome Biol. 11 (2) (2010) R14.
[34] S. Anders, W. Huber, Differential expression analysis for sequence count data, Nat. Preced. (2010) 1.
[35] D.A. Los, A. Zorina, M. Sinetova, S. Kryazhov, K. Mironov, V.V. Zinchenko, Stress sensors and signal transducers in cyanobacteria, Sensors (Basel) 10 (3) (2010) 2386-2415.
[36] L. Tripathi, Y. Zhang, Z.L. Lin, Bacterial sigma factors as targets for engineered or synthetic transcriptional control, Front. Bioeng. Biotechnol. 2 (2014) 33.
[37] L. Chen, L.N. Wu, Y. Zhu, Z.D. Song, J.X. Wang, W.W. Zhang, An orphan two-component response regulator Slr1588 involves salt tolerance by directly regulating synthesis of compatible solutes in photosynthetic Synechocystis sp. PCC 6803, Mol. BioSyst. 10 (7) (2014) 1765-1774.
[38] D. Bhaya, D. Vaulot, P. Amin, A.W. Takahashi, A.R. Grossman, Isolation of regulated genes of the cyanobacterium Synechocystis sp. strain PCC 6803 by differential display, J. Bacteriol. 182 (20) (2000) 5692-5699.
[39] A. Ishii, Y. Hihara, An AbrB-like transcriptional regulator, Sll0822, is essential for the activation of nitrogen-regulated genes in synechocystis sp. PCC 6803, Plant Physiol 148 (1) (2008) 660-670.
[40] A. Marchler-Bauer, M.K. Derbyshire, N.R. Gonzales, S.N. Lu, F. Chitsaz, L.Y. Geer, R.C. Geer, J. He, M. Gwadz, D.I. Hurwitz, C.J. Lanczycki, F. Lu, G.H. Marchler, J.S. Song, N. Thanki, Z.X. Wang, R.A. Yamashita, D.C. Zhang, C.J. Zheng, S.H. Bryant, CDD: NCBI's conserved domain database, Nucleic Acids Res 43 (D1) (2014) D222-D226.
[41] O. Schmidt, prlF and yhaV encode a new toxin-antitoxin system in Escherichia coli, J. Mol. Biol. 372 (4) (2007) 894-905.
[42] P. Oliveira, P. Lindblad, An AbrB-Like protein regulates the expression of the bidirectional hydrogenase in Synechocystis sp. strain PCC 6803, J. Bacteriol. 190 (3) (2008) 1011-1019.
[43] Y. Kaniya, A. Kizawa, A. Miyagi, M. Kawai-Yamada, H. Uchimiya, Y. Kaneko, Y. Nishiyama, Y. Hihara, Deletion of the transcriptional regulator cyAbrB2 deregulates primary carbon metabolism in synechocystis sp. PCC 6803, Plant Physiol 162 (2) (2013) 1153-1163.
[44] A. Agervald, X.H. Zhang, K. Stensjö, E. Devine, P. Lindblad, CalA, a cyanobacterial AbrB protein, interacts with the upstream region of hypC and acts as a repressor of its transcription in the cyanobacterium Nostoc sp. strain PCC 7120, Appl. Environ. Microbiol. 76 (3) (2010) 880-890.
[45] A. Higo, E. Nishiyama, K. Nakamura, Y. Hihara, S. Ehira, cyAbrB transcriptional regulators as safety devices to inhibit heterocyst differentiation in Anabaena sp. strain PCC 7120, J. Bacteriol. 201 (17) (2019) e00244-e00219.
[46] H. Ling, B. Chen, A. Kang, J.M. Lee, M.W. Chang, Transcriptome response to alkane biofuels in Saccharomyces cerevisiae: Identification of efflux pumps involved in alkane tolerance, Biotechnol. Biofuels 6 (1) (2013) 95.
[47] H. Ling, B. Chen, A. Kang, J.M. Lee, M.W. Chang, Transcriptome response to alkane biofuels in Saccharomyces cerevisiae: Identification of efflux pumps involved in alkane tolerance, Biotechnol. Biofuels 6(1) (2013) 1-10.

Towards understanding the mechanism of n-hexane tolerance in Synechocystis sp. PCC 6803

Towards understanding the mechanism of n-hexane tolerance in Synechocystis sp. PCC 6803

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 1

编辑推荐

Metrics

本文评价