Re-direction of carbon flux to key precursor malonyl-CoA via artificial small RNAs in photosynthetic Synechocystis sp. PCC 6803

Abstract

BackgroundPhotosynthetic cyanobacteria have attracted a significant attention as promising chassis to produce renewable fuels and chemicals due to their capability to utilizing solar energy and CO2. Notably, the enhancing supply of key precursors like malonyl-CoA would benefit the production of many bio-compounds. Nevertheless, the lacking of genetic tools in cyanobacteria, especially the knockdown strategies for essential pathways, has seriously restricted the attempts to re-direct carbon flux from the central carbohydrate metabolism to the synthesis of bioproducts.ResultsAiming at developing new genetic tools, two small RNA regulatory tools are reported for the model cyanobacterium Synechocystis sp. PCC6803, based on paired termini RNAs as well as the exogenous Hfq chaperone and MicC scaffold (Hfq-MicC) previously developed in Escherichia coli. Both regulatory tools functioned well in regulating exogenous reporter gene lacZ and endogenous glgC gene in Synechocystis sp. PCC6803, achieving a downregulation of gene expression up to 90% compared with wildtype. In addition, the Hfq-MicC tool was developed to simultaneously regulate multiple genes related to essential fatty acids biosynthesis, which led to decreased fatty acids content by 11%. Furthermore, aiming to re-direct the carbon flux, the Hfq-MicC tool was utilized to interfere the competing pathway of malonyl-CoA, achieving an increased intracellular malonyl-CoA abundance up to 41% (~ 698.3 pg/mL/OD730 nm) compared to the wildtype. Finally, the Hfq-MicC system was further modified into an inducible system based on the theophylline-inducible riboswitch.ConclusionsIn this study, two small RNA regulatory tools for manipulating essential metabolic pathways and re-directing carbon flux are reported for Synechocystis sp. PCC6803. The work introduces efficient and valuable metabolic regulatory strategies for photosynthetic cyanobacteria.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1032-0) contains supplementary material, which is available to authorized users.

Emerging Technologies in Algal Biotechnology: Toward the Establishment of a Sustainable, Algae-Based Bioeconomy

“…Multiple molecular techniques are available to modify native gene expression or target specific areas of the genome in microalgae. Gene knockdown by introduction of antisense, artificial small RNAs, and CRISPRi has been implemented in multiple systems (De Riso et al, 2009;Zhao et al, 2009;Yao et al, 2016;Wei et al, 2017;Sun et al, 2018). Site-specific genetic manipulation by homologous recombination (HR) is routine in cyanobacteria (Zang et al, 2007), the chloroplast genome of C. reinhardtii (Esland et al, 2018), and the nuclear genome of Nannochloropsis (Kilian et al, 2011).…”

Regulatory systems for gene expression control in cyanobacteria

“…In the absence of the ligand, the riboswitch forms an anti-terminator hairpin, consequently allowing full-length transcription of the target gene. Upon binding of the ligand and conformational changes of the riboswitch, a terminator hairpin is formed and the RNAP is dissociated, thus resulting in an offset of transcription an inducing system (Sun et al 2018b). As another example, Riboswitch F was applied as an additional control element for dCas9 expression in order to improve the dynamic range of glnA repression in a CRISPRi study (Higo et al 2018a).…”

“…Two further sRNA regulatory tools have recently been established in Sy_6803; both were originally developed in E. coli (Sun et al 2018b). One tool is based on paired termini trans-sRNAs (PTRNAs) (Nakashima et al 2006), which works similar to the IS10 system.…”

“…The other tool, namely Hfq/MicC, makes use of the scaffold RNA MicC, which is fused to a target-specific antisense sequence and recognised by the Hfq chaperon (Na et al 2013). Via target-specific binding of MicC, Hfq is directed to the final location and causes mRNA degradation by the recruitment of the major endoribonuclease RNase E. Both tools allowed an approximately 90% repression of target gene expression (Sun et al 2018b). In both cases however, induction control was leaky.…”

Modifying the Cyanobacterial Metabolism as a Key to Efficient Biopolymer Production in Photosynthetic Microorganisms

“…It caused mutant S. sp. PCC 6803 decrease in expression of that gene by 75% [ 47 ]. Theophylline-responsive riboswitch has also been used, this resulted in a 90% decrease in glycogen accumulation in S. sp.…”