Research review paperToolboxes for cyanobacteria: Recent advances and future direction
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].
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