Elsevier

Biotechnology Advances

Volume 36, Issue 4, July–August 2018, Pages 1293-1307
Biotechnology Advances

Research review paper
Toolboxes for cyanobacteria: Recent advances and future direction

https://doi.org/10.1016/j.biotechadv.2018.04.007Get rights and content

Abstract

Photosynthetic cyanobacteria are important primary producers and model organisms for studying photosynthesis and elements cycling on earth. Due to the ability to absorb sunlight and utilize carbon dioxide, cyanobacteria have also been proposed as renewable chassis for carbon-neutral “microbial cell factories”. Recent progresses on cyanobacterial synthetic biology have led to the successful production of more than two dozen of fuels and fine chemicals directly from CO2, demonstrating their potential for scale-up application in the future. However, compared with popular heterotrophic chassis like Escherichia coli and Saccharomyces cerevisiae, where abundant genetic tools are available for manipulations at levels from single gene, pathway to whole genome, limited genetic tools are accessible to cyanobacteria. Consequently, this significant technical hurdle restricts both the basic biological researches and further development and application of these renewable systems. Though still lagging the heterotrophic chassis, the vital roles of genetic tools in tuning of gene expression, carbon flux re-direction as well as genome-wide manipulations have been increasingly recognized in cyanobacteria. In recent years, significant progresses on developing and introducing new and efficient genetic tools have been made for cyanobacteria, including promoters, riboswitches, ribosome binding site engineering, clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease (CRISPR/Cas) systems, small RNA regulatory tools and genome-scale modeling strategies. In this review, we critically summarize recent advances on development and applications as well as technical limitations and future directions of the genetic tools in cyanobacteria. In addition, toolboxes feasible for using in large-scale cultivation are also briefly discussed.

Introduction

Genetic tools are the main undertakers to realize the artificial manipulation using synthetic biology approaches. For decades, genetic tools have been driving the studies related with human, plants and microorganisms, promoting the progresses on medicine, agriculture and microbial engineering (Forsburg, 2001; Liu et al., 2013; Quintana-Murci and Clark, 2013). Among them, a large variety of genetic tools have been developed for both the basic biological researches and strain engineering in bacteria, such as integrative or shuttle vectors, constitutive or inducible promoters, ribosome binding site (RBS) sequences, riboswitches, CRISPR/Cas systems, small RNA (sRNA) regulatory tools and genome-scale modeling strategies etc., which have been applied successfully on studies involving gene transfer, gene expression, gene control and metabolic reconstruction (Farasat et al., 2014; Li et al., 2015; Na et al., 2013; Segall-Shapiro et al., 2018).

Constitutive promoters lead to a continual transcription of their associated genes, while inducible promoters can switch and tune expression of specific genes via controlling the inducers’ concentration. In addition, the RBS related to the recruitment of a ribosome in the initiation of translation is several nucleotides upstream the start codon of an mRNA. It affects the expression level as translational efficiency with different RBS sequences can vary by more than 10000-fold (Farasat et al., 2014). Besides, riboswitches commonly located in the 5’-untranslated region (5’-UTR), transcribes as a portion of the mRNA and have the ability to bind with small molecules called ligands (Breaker, 2011). The conformation of riboswitches would transform in the presence/absence of ligands, mediating a transcription or translation on/off switch or even the self-cleavage of the target mRNA. Moreover, advanced genetic tools developed in recent years like CRISPR/Cas systems and small RNA (sRNA) regulatory tools have aimed at regulating/manipulating multiple genes or pathways simultaneously. Briefly, CRISPR/Cas system is a prokaryotic immune system providing adaptive resistance to the foreign genetic plasmids or phages (Barrangou et al., 2007). Notably, the type-II CRISPR/Cas9 system from Streptococcus pyogenes has been demonstrated suitable for accurate genome editing in both eukaryotic and prokaryotic hosts (Hsu et al., 2014). Traditionally, bacterial sRNAs are a kind of non-coding molecules with a typical length of about 50-300 nt, regulating the target mRNAs via perfect or imperfect base paring (Storz et al., 2011). Based on natural or artificial sRNAs, sRNA regulatory tools have been recently applied in studying gene function, modifying tolerance and production of products among various microbes in recent years (Gaida et al., 2013; Na et al., 2013). Finally, genome-scale network reconstruction based on in silico flux balance analysis (FBA) or 13C flux analysis presents the metabolic capabilities of host cells thus can be used to predict phenotype from genotype (O'Brien et al., 2015), providing insights for rational re-designing of cells. Therefore, the richer the abundance of toolboxes, the better the synthetic biology work could be conducted.

Photosynthetic cyanobacteria are a large group of gram-negative prokaryotes capable of respectively taking solar energy and CO2 as the sole energy and carbon source for growth (R Y Stanier and Bazine, 1977). Besides the traditional roles as primary producers (Giordano et al., 2005), cyanobacteria are considered as model organisms for studying photosynthesis process as well as carbon and nitrogen cycling on earth (Campbell et al., 1998; Herrero et al., 2001; Lea-Smith et al., 2015; Raven and Allen, 2003). More recently, to meet the challenges of increasing energy cost and environmental pollution, cyanobacteria have been recently developed to be photosynthetic “microbial cell factories” to produce renewable fuels and chemicals, as a promising alternative to traditional petroleum-based production (Gao et al., 2016; Oliver and Atsumi, 2014). With the completion of whole-genome sequencing of more than 80 cyanobacterial species since 1996 (http://genome.microbedb.jp/cyanobase), significant progresses have been made on synthetic biology researches of cyanobacteria. For example, Atsumi et al. (2009) directed photosynthetic recycling of CO2 to isobutyraldehyde in Synechococcus elongatus PCC7942 (hereafter Synechococcus 7942), reaching a production of 1.1 g/L in 8 days. In addition, Gao et al. (2012) systematically optimized the ethanol production in Synechocystis sp. PCC6803 (hereafter Synechocystis 6803) and improved the production up to 5.5 g/L in 26 days. These studies clearly demonstrated the feasibility of using cyanobacteria for producing fuels and chemicals. Nevertheless, a high level of gene expression had been challenging for Synechocystis 6803 as only promoters with middle strength like Prbc (Gao et al., 2012), PpsbA2 (Anfelt et al., 2013) and PpetE (Tan et al., 2011) were available previously. Besides, controlling the gene expression was hard due to the lack of efficient inducible systems. In addition, comprehensive metabolic regulation targeting multiple genes or pathways in cyanobacterial cells can’t be carried out, as only a limited number of selection markers are available for most cyanobacterial species. Moreover, elucidation of essential genes or pathways was difficult due to the lethal phenotype after gene deletion with the conventional method. Therefore, the lack of genetic tools has severely restricted the basic research, development, optimization and application of cyanobacterial chassis.

Currently the development and application of genetic tools in cyanobacteria are largely lagging the toolboxes widely available for E. coli, Bacillus subtilis and S. cerevisiae, whose promoters have been recorded as standard biological parts in databases like iGEM (http://parts.igem.org/Promoters/Catalog), RBS calculating tools like “RBS Calculator” have been developed (Espah Borujeni and Salis, 2016; Farasat et al., 2014; Salis et al., 2009), CRISPR/Cas systems and sRNA regulatory tools have been widely used (Dong and Zhang, 2014; Jakočiūnas et al., 2015; Jensen and Keasling, 2015; Jiang et al., 2015; Na et al., 2013). With the booming studies on cyanobacterial synthetic biology in last 5 years, the number of publications in this area has more than doubled from that of 2002 to 2012, as shown by a PubMed keyword search. Meanwhile, significant efforts were also made in extending toolboxes in cyanobacteria, which in return greatly stimulated the studies on cyanobacterial metabolic engineering and basic researches on cyanobacterial physiology and genetics. In this article, we critically review recent advances and applications of genetic tools in cyanobacteria, with a focus mainly on tools newly developed in the past 5 years, such as new constitutive/inducible promoters, wide-range RBS sequences, tightly induced riboswitches, CRISPR/Cas systems and sRNA tools and genome-scale modeling strategies in cyanobacteria. In addition, a comparison between different tools, their current limitations, the directions for future development as well as toolboxes suitable for using in large-scale cultivation are also critically discussed. The review here aims at providing not only the latest progresses but also insightful perspectives for further development of genetic tools in cyanobacteria.

Section snippets

Promoters

A list of promoters characterized in recent years for cyanobacteria was summarized in Table 1. Meanwhile, readers may also refer to the promoters summed up in several excellent reviews published early (Berla et al., 2013; Heidorn et al., 2011; Wang et al., 2012a).

Future directions of cyanobacterial toolboxes

Even with the great progresses made in recent years, the standard libraries of bio-bricks are not available for cyanobacteria due to the non-established disciplines for most of the genetic tools. In addition, the difficulties for biologists in performing FBA analysis limited the application of the genome-scale modeling analysis. To address the issues, the following aspects might deserve more attention in the future:

Applicable toolboxes for large-scale cultivation of cyanobacteria in the future

Compared to heterotrophic microorganisms like E. coli, metabolism of cyanobacteria has unique characteristics. As phototrophic organisms, most cyanobacteria rely on indigenous compounds like glycogen (the most prevalent), cyanophycin and poly-beta-hydroxybutyrate as storage to maintain cellular function during the night and darkness (Beck et al., 2012). In addition, a relatively powerful sugar phosphate pathway but weak tricarboxylic acid cycle (TCA) were demonstrated via 13C flux analysis in

Conclusions

In this review, we critically summarized the recent advances on genetic tools including promoters, riboswitches, RBS, CRISPR/Cas, sRNA tools as well as the genome modeling strategies in cyanobacteria. Though exciting progresses have been made, more work still need to be carried out in the future. Our review here provided not only the latest progresses but also useful insights on further development and application of the genetic tools in cyanobacteria.

Acknowledgements

We sincerely thank Profs. Pia Lindberg and Elias Englund of Uppsala University for providing the original experimental data for YFP and mTagBFP using different RBS sequences. The work was supported by the National Science Foundation of China (NSFC) [No. 21621004, 31770100, 31170043, 31270086, and 31370115], and the National Science Foundation of Tianjin, China [No. 13JCQNJC09900].

References (193)

  • P.D. Hsu et al.

    Development and applications of CRISPR-Cas9 for genome engineering

    Cell

    (2014)
  • T. Jakočiūnas et al.

    Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae

    Metab. Eng.

    (2015)
  • L.J. Jazmin et al.

    Isotopically nonstationary (13)C flux analysis of cyanobacterial isobutyraldehyde production

    Metab. Eng.

    (2017)
  • D. Kaczmarzyk et al.

    Diversion of the long-chain acyl-ACP pool in Synechocystis to fatty alcohols through CRISPRi repression of the essential phosphate acyltransferase PlsX

    Metab. Eng.

    (2018)
  • E.I. Lan et al.

    Metabolic engineering of cyanobacteria for 1-butanol production from carbon dioxide

    Metab. Eng.

    (2011)
  • T.C. Lee et al.

    Engineered xylose utilization enhances bio-products productivity in the cyanobacterium Synechocystis sp. PCC 6803

    Metab. Eng.

    (2015)
  • Y. Li et al.

    Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing

    Metab. Eng.

    (2015)
  • H. Li et al.

    CRISPR-Cas9 for the genome engineering of cyanobacteria and succinate production

    Metab. Eng.

    (2016)
  • P. Lindberg et al.

    Engineering a platform for photosynthetic isoprene production in cyanobacteria, using Synechocystis as the model organism

    Metab. Eng.

    (2010)
  • D. Liu et al.

    The nitrogen-regulated response regulator NrrA controls cyanophycin synthesis and glycogen catabolism in the cyanobacterium Synechocystis sp. PCC 6803

    J. Biol. Chem.

    (2014)
  • S. Abalde-Cela et al.

    High-throughput detection of ethanol-producing cyanobacteria in a microdroplet platform

    J. R. Soc. Interface

    (2015)
  • K. Abe et al.

    Design of riboregulators for control of cyanobacterial (Synechocystis) protein expression

    Biotechnol. Lett.

    (2014)
  • M.H. Abernathy et al.

    Deciphering cyanobacterial phenotypes for fast photoautotrophic growth via isotopically nonstationary metabolic flux analysis

    Biotechnol. Biofuels

    (2017)
  • O.O. Abudayyeh et al.

    C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector

    Science

    (2016)
  • A.O. Adebiyi et al.

    13C flux analysis of cyanobacterial metabolism

    Photosynth. Res.

    (2015)
  • J. Anfelt et al.

    Using transcriptomics to improve butanol tolerance of Synechocystis sp strain PCC 6803

    Appl. Environ. Microbiol.

    (2013)
  • P. Armshaw et al.

    Utilising the native plasmid, pCA2.4, from the cyanobacterium Synechocystis sp. strain PCC6803 as a cloning site for enhanced product production

    Biotechnol. Biofuels

    (2015)
  • S. Atsumi et al.

    Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde

    Nat. Biotechnol.

    (2009)
  • R. Barrangou et al.

    CRISPR provides acquired resistance against viruses in prokaryotes

    Science

    (2007)
  • C. Beck et al.

    The diversity of cyanobacterial metabolism: genome analysis of multiple phototrophic microorganisms

    BMC Genomics

    (2012)
  • C.L. Beisel et al.

    Design principles for riboswitch function

    PLoS Comput. Biol.

    (2009)
  • B.M. Berla et al.

    Synthetic biology of cyanobacteria: unique challenges and opportunities

    Front. Microbiol.

    (2013)
  • O. Borirak et al.

    Quantitative proteomics analysis of an ethanol- and a lactate-producing mutant strain of Synechocystis sp. PCC6803

    Biotechnol. Biofuels

    (2015)
  • J.T. Broddrick et al.

    Unique attributes of cyanobacterial metabolism revealed by improved genome-scale metabolic modeling and essential gene analysis

    Proc. Natl. Acad. Sci. U. S. A.

    (2016)
  • F. Cai et al.

    Evidence for the widespread distribution of CRISPR-Cas system in the Phylum Cyanobacteria

    RNA Biol.

    (2013)
  • D. Campbell et al.

    Chlorophyll Fluorescence Analysis of Cyanobacterial Photosynthesis and Acclimation

    Microbiol. Mol. Biol. Rev.

    (1998)
  • D. Camsund et al.

    Design and analysis of LacI-repressed promoters and DNA-looping in a cyanobacterium

    J. Biol. Eng.

    (2014)
  • Y.Q. Cao et al.

    AraBAD Based Toolkit for Gene Expression and Metabolic Robustness Improvement in Synechococcus elongatus

    Sci. Rep.

    (2017)
  • C. Cassier-Chauvat et al.

    Comparative Genomics of DNA Recombination and Repair in Cyanobacteria: Biotechnological Implications

    Front. Microbiol.

    (2016)
  • P. Ceres et al.

    Engineering modular 'ON' RNA switches using biological components

    Nucleic Acids Res.

    (2013)
  • L. Chen et al.

    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.

    (2014)
  • Y. Chen et al.

    Self-replicating shuttle vectors based on pANS, a small endogenous plasmid of the unicellular cyanobacterium Synechococcus elongatus PCC 7942

    Microbiology

    (2016)
  • D. Dienst et al.

    The cyanobacterial homologue of the RNA chaperone Hfq is essential for motility of Synechocystis sp. PCC 6803

    Microbiology

    (2008)
  • D. Dienst et al.

    Transcriptomic response to prolonged ethanol production in the cyanobacterium Synechocystis sp. PCC6803

    Biotechnol. Biofuels

    (2014)
  • G. Domin et al.

    Applicability of a computational design approach for synthetic riboswitches

    Nucleic Acids Res.

    (2017)
  • H. Dong et al.

    Current development in genetic engineering strategies of Bacillus species

    Microb. Cell Factories

    (2014)
  • U. Duhring et al.

    An internal antisense RNA regulates expression of the photosynthesis gene isiA

    Proc. Natl. Acad. Sci. U. S. A.

    (2006)
  • E. Englund et al.

    Evaluation of promoters and ribosome binding sites for biotechnological applications in the unicellular cyanobacterium Synechocystis sp. PCC 6803

    Sci. Rep.

    (2016)
  • A. Espah Borujeni et al.

    Translation Initiation is Controlled by RNA Folding Kinetics via a Ribosome Drafting Mechanism

    J. Am. Chem. Soc.

    (2016)
  • J. Espinosa et al.

    PipX, the coactivator of NtcA, is a global regulator in cyanobacteria

    Proc. Natl. Acad. Sci. U. S. A.

    (2014)
  • Cited by (89)

    View all citing articles on Scopus
    View full text