Manipulating the Expression of Glycogen Phosphorylase in Synechococcus elongatus PCC 7942 to Mobilize Glycogen Storage for Sucrose Synthesis

Abstract

Cyanobacteria are a promising photosynthetic chassis to produce biofuels, biochemicals, and pharmaceuticals at the expense of CO2 and light energy. Glycogen accumulation represents a universal carbon sink mechanism among cyanobacteria, storing excess carbon and energy from photosynthesis and may compete with product synthesis. Therefore, the glycogen synthesis pathway is often targeted to increase cyanobacterial production of desired carbon-based products. However, these manipulations caused severe physiological and metabolic impairments and often failed to optimize the overall performance of photosynthetic production. Here, in this work, we explored to mobilize the glycogen storage by strengthening glycogen degradation activities. In Synechococcus elongatus PCC 7942, we manipulated the abundances of glycogen phosphorylase (GlgP) with a theophylline dose-responsive riboswitch approach, which holds control over the cyanobacterial glycogen degradation process and successfully regulated the glycogen contents in the recombinant strain. Taking sucrose synthesis as a model, we explored the effects of enhanced glycogen degradation on sucrose production and glycogen storage. It is confirmed that under non-hypersaline conditions, the overexpressed glgP facilitated the effective mobilization of glycogen storage and resulted in increased secretory sucrose production. The findings in this work provided fresh insights into the area of cyanobacteria glycogen metabolism engineering and would inspire the development of novel metabolic engineering approaches for efficient photosynthetic biosynthesis.

Cyanobacteria as cell factories for the photosynthetic production of sucrose

“…SPS 7942 is bidomainal and bifunctional (i.e., possessing active GTD and PHD domains), in contrast to SPS 6803 which is also bidomainal, but has a non-functional PHD domain and is regulated distinctly from SPS 7942 ( Curatti et al, 1998 ; Lunn et al, 1999 ; Gibson et al, 2002 ). However, the partial-functionality of SPS 6803 does not mean it is less effective, as heterologous co-overexpression of SPS 6803 and CscB in S. elongatus PCC 7942 increases sucrose production relative to overexpression of the native SPS 7942 ( Abramson et al, 2016 ; Dan et al, 2022 ; Table 1 ). It is curious that SPS 6803 is a more effective enzyme for rerouting carbon flux towards sucrose bioproduction, given that it lacks a functional SPP domain ( S. elongatus PCC 7942 encodes other endogenous SPP proteins in the examples above), so it is possible that this observation is related either to the manner in which salt-ions can regulate the function of some SPS domains ( Liang et al, 2020 ), or to other unknown functions for SPP and/or SPP-like domains other than S6P phosphatase activity (see 2.1.3.…”

“…GlgP is responsible for hydrolyzing glycosidic bonds in glycogen to release glucose-1-phosphate, so it was theorized that increasing GlgP activity would mobilize carbon from the glycogen pool for sucrose biosynthesis. However, when GlgP was overexpressed in sucrose-secreting strains of S. elongatus PCC 7942 with its native SPS, there were no changes in glycogen content and a decrease in sucrose was observed ( Ducat et al, 2012 ; Dan et al, 2022 ), while heterologous expression of both SPS 6803 and GlgP overexpression reduced glycogen content while increasing sucrose secretion by 2.4-fold ( Dan et al, 2022 ). The variability in sucrose production of glycogen-deficient strains might be related to the pleotropic cellular deficiencies of these strains, including reduced growth, reduced O 2 evolution and consumption, abnormal pigmentation, and light sensitivity ( Suzuki et al, 2010 ; Ducat et al, 2012 ; Gründel et al, 2012 ; Xu et al, 2013 ; Qiao et al, 2018 ).…”

Impact of irradiance and inorganic carbon availability on heterologous sucrose production in Synechococcus elongatus PCC 7942

“…We and others have found that expression of sucrose permease ( cscB ), a symporter of sucrose and protons, will lead to the export of cytosolic sucrose to the medium ( Ducat et al., 2012 ; Santos-Merino et al., 2023 ). In S. elongatus PCC 7942 strains with inducible copies of both sps and cscB , sucrose synthesis and export can be achieved in the absence of external osmotic pressure ( Abramson et al., 2016 , 2018 ; Lin et al., 2020 ; Dan et al., 2022 ). We therefore selected this strain to minimize the requirement for addition of salt to the growth medium.…”

Generation and comprehensive analysis of Synechococcus elongatus–Aspergillus nidulans co-culture system for polyketide production

“…5 B). The overexpressed glgP is recently demonstrated to facilitate glycogen degradation and result in increased sucrose production in S. elongatus PCC 7942 [ 32 ]. As a heterotrophic partner, filamentous fungus TWY1.1 seems to be a carbon sink for cyanobacterium FL130, making the phototrophic partner convert more polymer carbohydrates (glycogen) into transportable sugars, such as sucrose.…”