Fatty Acid Activation in Cyanobacteria Mediated by Acyl-Acyl Carrier Protein Synthetase Enables Fatty Acid Recycling

Kaczmarzyk, Danuta; Fulda, Martin

doi:10.1104/pp.109.148007

Cited by 159 publications

(175 citation statements)

References 39 publications

Supporting

Mentioning

166

Contrasting

Order By: Relevance

“…Intracellular accumulation of free fatty acids (FFAs) is uncommon in cyanobacteria, because they are activated and recycled into membrane lipids in an ATP-dependent reaction catalyzed by acyl-ACP synthetase (AAS) (38). However, in ΔrpaA, we observed a large increase in the abundance of palmitic acid, palmitoleic acid, and myristic acid at the 4-to 6-h time points after dark exposure ( Figs.…”

Section: Darkness Initiates Pigmentation Changes and Rapid Cell Deathmentioning

confidence: 75%

Redox crisis underlies conditional light–dark lethality in cyanobacterial mutants that lack the circadian regulator, RpaA

Diamond

Rubin

Shultzaberger

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Cyanobacteria evolved a robust circadian clock, which has a profound influence on fitness and metabolism under daily lightdark (LD) cycles. In the model cyanobacterium Synechococcus elongatus PCC 7942, a functional clock is not required for diurnal growth, but mutants defective for the response regulator that mediates transcriptional rhythms in the wild-type, regulator of phycobilisome association A (RpaA), cannot be cultured under LD conditions. We found that rpaA-null mutants are inviable after several hours in the dark and compared the metabolomes of wild-type and rpaA-null strains to identify the source of lethality. Here, we show that the wild-type metabolome is very stable throughout the night, and this stability is lost in the absence of RpaA. Additionally, an rpaA mutant accumulates excessive reactive oxygen species (ROS) during the day and is unable to clear it during the night. The rpaA-null metabolome indicates that these cells are reductant-starved in the dark, likely because enzymes of the primary nighttime NADPH-producing pathway are direct targets of RpaA. Because NADPH is required for processes that detoxify ROS, conditional LD lethality likely results from inability of the mutant to activate reductant-requiring pathways that detoxify ROS when photosynthesis is not active. We identified second-site mutations and growth conditions that suppress LD lethality in the mutant background that support these conclusions. These results provide a mechanistic explanation as to why rpaA-null mutants die in the dark, further connect the clock to metabolism under diurnal growth, and indicate that RpaA likely has important unidentified functions during the day.C yanobacteria are both key agents of global carbon and nitrogen cycles and promising platforms for renewable chemicals, fuels, and nutraceuticals (1-3). Understanding the control mechanisms that govern the flow of carbon and nitrogen through these organisms is crucial for predicting their behavior in natural environments as well as for improving engineering strategies. Although the basic pathways for carbon and nitrogen metabolism, and their regulation, are well understood in heterotrophic bacteria, cyanobacteria exhibit important deviations in these core metabolic pathways (4-7). Additionally, metabolic control mechanisms in cyanobacteria evolved to be compatible with photoautotrophic metabolism and the dramatic shifts that are imposed on those pathways by predictable daily light-dark (LD) cycles. Examples include enzymatic activity that responds to light-dependent cellular redox changes (8-11); the preference for NADPH, the reductant produced by the photochemical reactions, over NADH by many biosynthetic enzymes (12, 13); and a circadian clock that drives 24-h transcriptional rhythms in most genes (14-16).A daily LD cycle presents a strong metabolic driver for the photosynthetic cyanobacteria, but a circadian clock also imposes daily cycles in transcription and redox regulatory systems (17, 18). Circadian measurements historically have been performed in...

show abstract

Section: Darkness Initiates Pigmentation Changes and Rapid Cell Deathmentioning

confidence: 75%

Redox crisis underlies conditional light–dark lethality in cyanobacterial mutants that lack the circadian regulator, RpaA

Diamond

Rubin

Shultzaberger

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…PCC 6803 as a model. Previous studies had revealed the presence of only a single protein in Synechocystis homologous to higher plant acyl-activating enzymes (Kaczmarzyk and Fulda, 2010). These studies also established that the Synechocystis acyl-acyl carrier protein synthetase (SynAas) specifically uses ACP as a cosubstrate to recycle free fatty acids occurring naturally in Synechocystis cells (Kaczmarzyk and Fulda, 2010).…”

Section: O]acetate or [mentioning

confidence: 79%

“…PCC 6803. Analogous to Kaczmarzyk and Fulda (2010), we identified Slr1609 as the only homologous protein through the BLASTP function of the CyanoBase Web site (http:// genome.kazusa.or.jp/cyanobase). Slr1609 had been named Aas (SynAas throughout this paper).…”

Section: Resultsmentioning

confidence: 99%

“…Previous studies had revealed the presence of only a single protein in Synechocystis homologous to higher plant acyl-activating enzymes (Kaczmarzyk and Fulda, 2010). These studies also established that the Synechocystis acyl-acyl carrier protein synthetase (SynAas) specifically uses ACP as a cosubstrate to recycle free fatty acids occurring naturally in Synechocystis cells (Kaczmarzyk and Fulda, 2010). Here, we provide evidence that SynAas is also involved in the transfer of free fatty acids across membranes by vectorial acylation and discuss an analogous mechanism for fatty acid transfer across the chloroplast envelope.…”

Section: O]acetate or [mentioning

confidence: 99%

See 1 more Smart Citation

The Acyl-Acyl Carrier Protein Synthetase from Synechocystis sp. PCC 6803 Mediates Fatty Acid Import

et al. 2012

View full text Add to dashboard Cite

The transfer of fatty acids across biological membranes is a largely uncharacterized process, although it is essential at membranes of several higher plant organelles like chloroplasts, peroxisomes, or the endoplasmic reticulum. Here, we analyzed loss-of-function mutants of the unicellular cyanobacterium Synechocystis sp. PCC 6803 as a model system to circumvent redundancy problems encountered in eukaryotic organisms. Cells deficient in the only cytoplasmic Synechocystis acyl-acyl carrier protein synthetase (SynAas) were highly resistant to externally provided α-linolenic acid, whereas wild-type cells bleached upon this treatment. Bleaching of wild-type cells was accompanied by a continuous increase of α-linolenic acid in total lipids, whereas no such accumulation could be observed in SynAas-deficient cells (Ɗsynaas). When SynAas was disrupted in the tocopherol-deficient, α-linolenic acid-hypersensitive Synechocystis mutant Ɗslr1736, double mutant cells displayed the same resistance phenotype as Ɗsynaas. Moreover, heterologous expression of SynAas in yeast (Saccharomyces cerevisiae) mutants lacking the major yeast fatty acid import protein Fat1p (Ɗfat1) led to the restoration of wild-type sensitivity against exogenous α-linolenic acid of the otherwise resistant Ɗfat1 mutant, indicating that SynAas is functionally equivalent to Fat1p. In addition, liposome assays provided direct evidence for the ability of purified SynAas protein to mediate α-[14C]linolenic acid retrieval from preloaded liposome membranes via the synthesis of [14C]linolenoyl-acyl carrier protein. Taken together, our data show that an acyl-activating enzyme like SynAas is necessary and sufficient to mediate the transfer of fatty acids across a biological membrane.

show abstract

“…PCC 6803 was also expressed in the cells. ORF slr1609 codes for an acyl-ACP synthetase whose proposed role is the reactivation of FAs to fatty acyl-ACP (23). When ORF slr1609 and the N. punctiforme AR/AD pathway were coexpressed, cells produced branched pentadecane from exogenous branched FA.…”

Section: Resultsmentioning

confidence: 99%

Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli

Howard

Middelhaufe

Moore

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

163

142

View full text Add to dashboard Cite

Biofuels are the most immediate, practical solution for mitigating dependence on fossil hydrocarbons, but current biofuels (alcohols and biodiesels) require significant downstream processing and are not fully compatible with modern, mass-market internal combustion engines. Rather, the ideal biofuels are structurally and chemically identical to the fossil fuels they seek to replace (i.e., aliphatic n-and iso-alkanes and -alkenes of various chain lengths). Here we report on production of such petroleum-replica hydrocarbons in Escherichia coli. The activity of the fatty acid (FA) reductase complex from Photorhabdus luminescens was coupled with aldehyde decarbonylase from Nostoc punctiforme to use free FAs as substrates for alkane biosynthesis. This combination of genes enabled rational alterations to hydrocarbon chain length (C n ) and the production of branched alkanes through upstream genetic and exogenous manipulations of the FA pool. Genetic components for targeted manipulation of the FA pool included expression of a thioesterase from Cinnamomum camphora (camphor) to alter alkane C n and expression of the branched-chain α-keto acid dehydrogenase complex and β-keto acyl-acyl carrier protein synthase III from Bacillus subtilis to synthesize branched (iso-) alkanes. Rather than simply reconstituting existing metabolic routes to alkane production found in nature, these results demonstrate the ability to design and implement artificial molecular pathways for the production of renewable, industrially relevant fuel molecules.branched fatty acid biosynthesis | lux genes | metabolic engineering | synthetic biology

show abstract

Fatty Acid Activation in Cyanobacteria Mediated by Acyl-Acyl Carrier Protein Synthetase Enables Fatty Acid Recycling

Cited by 159 publications

References 39 publications

Redox crisis underlies conditional light–dark lethality in cyanobacterial mutants that lack the circadian regulator, RpaA

Redox crisis underlies conditional light–dark lethality in cyanobacterial mutants that lack the circadian regulator, RpaA

The Acyl-Acyl Carrier Protein Synthetase from Synechocystis sp. PCC 6803 Mediates Fatty Acid Import

Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli

Contact Info

Product

Resources

About