Transcriptomic analysis of cyanobacterial alkane overproduction reveals stress-related genes and inhibitors of lipid droplet formation

Abstract

The cyanobacterium Nostoc punctiforme can form lipid droplets (LDs), internal inclusions containing triacylglycerols, carotenoids and alkanes. LDs are enriched for a 17 carbon-long alkane in N. punctiforme , and it has been shown that the overexpression of the aar and ado genes results in increased LD and alkane production. To identify transcriptional adaptations associated with increased alkane production, we performed comparative transcriptomic analysis of an alkane overproduction strain. RNA-seq data identified a large number of highly upregulated genes in the overproduction strain, including genes potentially involved in rRNA processing, mycosporine-glycine production and synthesis of non-ribosomal peptides, including nostopeptolide A. Other genes encoding helical carotenoid proteins, stress-induced proteins and those for microviridin synthesis were also upregulated. Construction of N. punctiforme strains with several upregulated genes or operons on multi-copy plasmids resulted in reduced alkane accumulation, indicating possible negative regulators of alkane production. A strain containing four genes for microviridin biosynthesis completely lost the ability to synthesize LDs. This strain exhibited wild-type growth and lag phase recovery under standard conditions, and slightly faster growth under high light. The transcriptional changes associated with increased alkane production identified in this work will provide the basis for future experiments designed to use cyanobacteria as a production platform for biofuel or high-value hydrophobic products.

Insights into cyanobacterial alkane biosynthesis

“…Genome-scale modelling data suggested that the higher CEF pressurises the cells to use less energy-efficient pathway, which in turn leads to the lower quantum efficiency of photosynthesis and result in slower growth (Berla et al, 2015 ). It was also suggested that alkanes are essential for maintaining proper membrane fluidity and flexibility (Arias et al, 2020 ; Lea-Smith et al, 2016 ). Further, data suggest that alkanes are essential for cyanobacteria to tolerate abiotic stresses including cold temperature (20°C) (Berla et al, 2015 ) and salt (Yamamori et al, 2018 ).…”

“…A comparative transcriptomic analysis was performed recently for the cyanobacterium ( N. punctiforme ) harbouring alkane-overproducing plasmid and control plasmid (Arias et al, 2020 ). The analysis suggested that 177 genes were upregulated and 121 genes were downregulated.…”

“…Intriguingly, it was found that alkane production was reduced significantly. This suggests that the genes involved in cellular stress might alarm the cells towards the overproduced alkane and act as negative feedback regulators of alkane production (Arias et al, 2020 ).…”

“…As fatty aldehydes are toxic to the cells (Gibbons et al, 2018 ), it further leads to decarboxylation or deformylation into alkane and formate with one carbon less using ADO enzyme (Kaiser et al, 2013 ). ADO belongs to a family of ferritin-like non-heme di-iron oxygenase which requires oxygen and four electrons for its reducing activity (Arias et al, 2020 ). One oxygen atom and aldehyde hydrogen are retained by the co-product formate (Jia et al, 2015 ).…”

“…Biofuel production by the recycling of released carbon dioxide by cyanobacteria is a novel approach that allows liberty from fossil fuel dependency (Arias et al, 2020 ). In some of the cyanobacteria, alkane biosynthesis is essential for normal physiological growth (Lea-Smith et al, 2016 ), however, precise mechanism is not well understood (Coates et al, 2014 ).…”