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TMT-labeled differential proteomics study of the glycosyltransferase slr1064 mutant in cyanobacteria

ABSTRACT: Glycosyltransferases (GTs) are enzymes that play important roles in various biological processes, such as photosynthesis, cell motility, exopolysaccharides, and lipid metabolism, by transferring sugars to different targets. However, the regulation of carbon metabolism by GTs in Synechocystis has not been reported yet. To identify novel GTs proteins involved in carbon metabolism, we performed a large-scale screening of GT gene-deletion mutants in Synechocystis. Our results revealed that the glycosyltransferase Slr1064, which is mainly distributed in the membrane compartment, is essential for the growth of Synechocystis under heterotrophic and mixotrophic conditions (HT and MT), but not under autotrophic conditions (AT). Integrated proteomic and metabolic analyses showed that Slr1064 regulates the Calvin cycle by modulating the levels of UDP-GlcNAc, which have been reported to mediate O-GlcNAc modification of Gap2. Further analyses demonstrated that the Δslr1064 mutant has a lower turnover rate of Gap2 under MT condition and shows dissociation of the PRK/GAPDH/CP12 complex under dark culture conditions. These findings provide new insights into the role of GTs in carbon metabolism in Synechocystis and expand our understanding of the regulatory mechanism of carbon metabolism in this important model organism.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Synechocystis Sp. Pcc 6803

TISSUE(S): Photosynthetic Cell, Whole Body

SUBMITTER: Longfa Fang

LAB HEAD: Longfa Fang

PROVIDER: PXD047088 | Pride | 2024-05-28

REPOSITORIES: pride

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			Action	DRS
	Synechocystis_Cyanobase10262011.fasta	Fasta
	checksum.txt	Txt
	flf_tmt_23_F10_desalt_20150713.raw	Raw
	flf_tmt_23_F11_desalt_20150713.raw	Raw
	flf_tmt_23_F12_desalt_20150713.raw	Raw

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Project description:Cyanobacteria are phototrophic prokaryotes that can convert inorganic carbon as CO2 into organic carbon compounds at the expense of light energy. In addition, they need only a few inorganic nutrients and can be cultivated in high densities using non-arable land and seawater. This features qualified cyanobacteria as attractive organisms for the production of third generation biofuels as part of the development of future CO2-neutral energy production. Synechocystis sp. PCC 6803 represents one of the most widely used cyanobacterial model strains. On the basis of its available genome sequence and genetic tools, many strains of Synechocystis have been generated that produce different biotechnological products. Efficient isoprene production is an attractive goal, since this compound represents not only an energy-rich biofuel but is also used as chemical feedstock. Here, we report on our attempts to generate isoprene-producing strains of Synechocystis. The cDNA of a codon-optimized plant isoprene synthase (IspS) was cloned under the control of different Synechocystis promoters, which ensure strong constitutive or light-regulated ispS expression. The expression of the ispS gene was quantified by qPCR, whereas the amount of isoprene was quantified using GC-MS. Incubation of our strains at different salt conditions had marked impact on the isoprene production rates. Under low salt conditions, a good correlation was found between ispS expression and isoprene production rate. However, the cultivation of isoprene production strains under salt-supplemented conditions decreased isoprene production despite the fact that ispS expression was salt-stimulated. The characterization of the metabolome of isoprene producing strains indicated that isoprene production might be limited by insufficient precursor levels. Our isoprene production rates under low salt conditions were 2 - 6.5times higher compared to the previous report of Lindberg et al. (2010). These results can be used to guide future attempts establishing the isoprene production with cyanobacterial host systems.

Project description:The trophic condition related proteomes of an oxygenic photosynthetic cyanobacterium Synechocystis sp. PCC 6803 (glucose tolerant strain) were analyzed to get further insights into the metabolic traits governing the early exponential growth under photoautotrophic, mixotrophic and heterotrophic conditions. The cells were grown under constant light autotrophically (AT) in low (air-level) CO2 (ATLC) and in high CO2 (ATHC), mixotrophically (MT) in low (air-level) CO2 in the presence of glucose (MTLC) and heterotrophically in low (air-level) CO2 in the presence of glucose and only 10 min exposure to light every 24 hours (Light-Activated Heterotrophic Growth; LAHGLC). The proteomes of ATHC, MTLC and LAHGLC differed substantially from that of ATLC, particularly by downregulation of the inducible carbon concentrating mechanisms (CCM) and photorespiration related enzymes. On the contrary, under carbon-rich conditions (ATHC, MTLC, LAHGLC) the cells accumulated respiratory NAD(P)H dehydrogenase I complexes in the thylakoid membrane. In glucose supplemented cultures (MTLC, LAHGLC) a distinct NDH-2 protein, NdbA, additionally accumulated in the thylakoid membrane whilst the plasma membrane localized NdbC and ARTO decreased in abundance. The light-harvesting proteins together with the photosystem I and II subunits were uniquely depleted under LAHGLC condition, accumulated under ATHC and in MTLC remained at the level of the reference ATLC condition. The accumulation of P-transporters was unique, revealing only minor differences between ATLC and ATHC but strong up-regulation in MTLC and down-regulation in LAHGLC. This likely implies that MTLC stores energy surplus in the highly energetic bonds of polyphosphate polymer that can be used under unfavorable growth conditions. It is concluded that the rigor of cell growth in MTLC condition results to a great extent from high intracellular inorganic carbon conditions created upon glucose breakdown and release of CO2, beside the direct utilization of glucose-derived carbon skeletons for growth. This combination provides the MTLC cultures with excellent conditions for growth that often exceeds that of the mere photoautotrophic growth at high CO2.

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TMT-labeled differential proteomics study of the glycosyltransferase slr1064 mutant in cyanobacteria

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