HetN and patS Mutations Enhance Accumulation of Fatty Alcohols in the hglT Mutants of Anabaena sp. PCC 7120

Heli Siti Halimatul Munawaroh,Egi Tritya Apdila,Koichiro Awai

doi:10.3389/fpls.2020.00804

Heli Siti Halimatul Munawaroh, Egi Tritya Apdila + Show 1 more

Open Access

https://doi.org/10.3389/fpls.2020.00804

Copy DOI

Abstract

The heterocysts present in filamentous cyanobacteria such as Anabaena sp. PCC 7120 are known to be regulated by HetN and PatS, the repressors of heterocyst differentiation; therefore, the inactivation of these proteins will result in the formation of multiple heterocysts. To enhance the accumulation of fatty alcohols synthesized in the heterocyst, we introduced mutations of these repressors to increase heterocyst frequency. First, we isolated double mutants of hetN and patS and confirmed that the null mutation of these genes promoted higher frequencies of heterocyst formation and higher accumulation of heterocyst-specific glycolipids (Hgls) compared with its wild type. Next, we combined hetN and patS mutations with an hglT (encoding glycosyltransferase, an enzyme involved in Hgl synthesis) mutation to increase the accumulation of fatty alcohols since knockout mutation of hglT results in accumulation of very long chain fatty alcohol, the precursor of Hgl. We also observed retarded growth, lower chlorophyll content and up to a five-fold decrease in photosynthetic activity of the hetN/patS/hglT triple mutants. In contrast, the triple mutants showed three times higher heterocyst formation frequencies than the hglT single mutant and wild type. The production rate of fatty alcohol in the triple mutants attained a value 1.41 nmol/mL OD730, whereas accumulation of Hgls in the wild type was 0.90 nmol/mL OD730. Aeration of culture improved the accumulation of fatty alcohols in hetN/patS/hglT mutant cells up to 2.97 nmol/mL OD730 compared with cells cultured by rotation. Our study outlines an alternative strategy for fatty alcohol production supported by photosynthesis and nitrogen fixation.

Highlights

We demonstrated that increased ratio of heterocyst per filament in the hglT mutants of Anabaena enhances the accumulation of fatty alcohols
The mutants cultured with continuous aeration accumulated fatty alcohol with a maximum production rate, over twice that obtained by rotary shaking (Figure 5B)
Another possibility is that aeration led to oxygen saturation in the culture, which led the thickening of the heterocyst envelope, especially the glycolipid layer

Summary

INTRODUCTION

PatS diffuses laterally to inhibit the differentiation of neighboring cells by hampering the DNA binding activity of HetR (Golden and Yoon, 2003; Zhang et al, 2006) Another gene, hetN, is required for the maintenance of the heterocyst pattern (Callahan and Buikema, 2001). Unlike patS mutants, the deletion of hetN in Anabaena shows no alteration in the heterocyst formation pattern and the vegetative cell interval This null mutant forms multiple heterocysts after the normal pattern of heterocyst formation (Fan et al, 2005), indicating that HetN is not required for the formation of the initial heterocyst pattern in response to nitrogen-starved conditions. We demonstrated that increased ratio of heterocyst per filament in the hglT mutants of Anabaena enhances the accumulation of fatty alcohols

MATERIALS AND METHODS

RESULTS

DISCUSSION

Findings

DATA AVAILABILITY STATEMENT