Diversion of the long-chain acyl-ACP pool in Synechocystis to fatty alcohols through CRISPRi repression of the essential phosphate acyltransferase PlsX.

Fatty alcohol production in Synechocystis sp. PCC 6803 was achieved through heterologous expression of the fatty acyl-CoA/ACP reductase Maqu2220 from the bacteria Marinobacter aquaeolei VT8 and the fatty acyl-ACP reductase DPW from the rice Oryza sativa. These platform strains became models for testing multiplex CRISPR-interference (CRISPRi) metabolic engineering strategies to both improve fatty alcohol production and to study membrane homeostasis. CRISPRi allowed partial repression of up to six genes simultaneously, each encoding enzymes of acyl-ACP-consuming pathways. We identified the essential phosphate acyltransferase enzyme PlsX (slr1510) as a key node in C18 fatty acyl-ACP consumption, repression of slr1510 increased octadecanol productivity threefold over the base strain and gave the highest specific titers reported for this host, 10.3mgg-1 DCW. PlsX catalyzes the first committed step of phosphatidic acid synthesis, and has not been characterized in Synechocystis previously. We found that accumulation of fatty alcohols impaired growth, altered the membrane composition, and caused a build-up of reactive oxygen species.

Toll-like receptors in maraena whitefish: Evolutionary relationship among salmonid fishes and patterns of response to Aeromonas salmonicida.

Toll-like receptors (TLRs) interact directly with particular pathogenic structures and are thus highly important to innate immunity. The present manuscript characterises a suite of 14 TLRs in maraena whitefish (Coregonus maraena), a salmonid species with increasing importance for aquaculture. Whitefish TLRs were structurally and evolutionary analysed. The results revealed a close relationship with TLRs from salmonid fish species rainbow trout and Atlantic salmon. Profiling the baseline expression of TLR genes in whitefish indicated that mainly members of the TLR11 family were highly expressed across all investigated tissues. A stimulation model with inactivated Aeromonas salmonicida was used to induce inflammation in the peritoneal cavity of whitefish. This bacterial challenge induced the expression of pro-inflammatory cytokine genes and evoked a strong influx of granulated cells of myeloid origin into the peritoneal cavity. As a likely consequence, the abundance of TLR-encoding transcripts increased moderately in peritoneal cells, with the highest levels of transcripts encoding non-mammalian TLR22a and a soluble TLR5 variant. In the course of inflammation, the proportion of granulated cells increased in peripheral blood accompanied by elevated TLR copy numbers in spleen and simultaneously reduced TLR copy numbers in head kidney at day 3 post-stimulation. Altogether, the present study provides in-vivo evidence for relatively modest TLR response patterns, but marked trafficking of myeloid cells as an immunophysiological consequence of A. salmonicida inflammation in whitefish. The present results contribute to improved understanding of the host-pathogen interaction in salmonid fish.

Arabidopsis acyl-acyl carrier protein synthetase AAE15 with medium chain fatty acid specificity is functional in cyanobacteria.

Cyanobacteria are potential hosts for the biosynthesis of oleochemical compounds. The metabolic precursors for such compounds are fatty acids and their derivatives, which require chemical activation to become substrates in further conversion steps. We characterized the acyl activating enzyme AAE15 of Arabidopsis encoded by At4g14070, which is a homologue of a cyanobacterial acyl-ACP synthetase (AAS). We expressed AAE15 in insect cells and demonstrated its AAS activity with medium chain fatty acid (C10-C14) substrates in vitro. Furthermore, we used AAE15 to complement a Synechocystis aas deletion mutant and showed that the new strain preferentially incorporates supplied medium chain fatty acids into internal lipid molecules. Based on this data we propose that AAE15 can be utilized in metabolic engineering strategies for cyanobacteria that aim to produce compounds based on medium chain fatty acids.

Genetic and nutrient modulation of acetyl-CoA levels in Synechocystis for n-butanol production.

BACKGROUND: There is a strong interest in using photosynthetic cyanobacteria as production hosts for biofuels and chemicals. Recent work has shown the benefit of pathway engineering, enzyme tolerance, and co-factor usage for improving yields of fermentation products. RESULTS: An n-butanol pathway was inserted into a Synechocystis mutant deficient in polyhydroxybutyrate synthesis. We found that nitrogen starvation increased specific butanol productivity up to threefold, but cessation of cell growth limited total n-butanol titers. Metabolite profiling showed that acetyl-CoA increased twofold during nitrogen starvation. Introduction of a phosphoketolase increased acetyl-CoA levels sixfold at nitrogen replete conditions and increased butanol titers from 22 to 37 mg/L at day 8. Flux balance analysis of photoautotrophic metabolism showed that a Calvin-Benson-Bassham-Phosphoketolase pathway had higher theoretical butanol productivity than CBB-Embden-Meyerhof-Parnas and a reduced butanol ATP demand. CONCLUSION: These results demonstrate that phosphoketolase overexpression and modulation of nitrogen levels are two attractive routes toward increased production of acetyl-CoA derived products in cyanobacteria and could be implemented with complementary metabolic engineering strategies.

Overexpression of sigma factor SigB improves temperature and butanol tolerance of Synechocystis sp. PCC6803.

Among phenotypes of interest for an industrial cyanobacteria host are improved tolerance to temperature, salt, and solvent stress. Cellular responses to many stresses are controlled by the network of sensory receptors and downstream regulatory proteins. We applied transcription factor engineering to Synechocystis and tested mutant strains for tolerance to temperature and the biofuel 1-butanol. Histidine kinases (Hik), response regulators (Rre), and an RNA polymerase sigma factor (SigB) were overexpressed or deleted. Overexpression of SigB increased both temperature and butanol tolerance and lowered the intracellular concentration of reactive oxygen species. This report demonstrates that alteration of regulatory proteins in a cyanobacterium can be a useful tool to improve stress tolerance.

Fatty acid activation in cyanobacteria mediated by acyl-acyl carrier protein synthetase enables fatty acid recycling.

In cyanobacteria fatty acids destined for lipid synthesis can be synthesized de novo, but also exogenous free fatty acids from the culture medium can be directly incorporated into lipids. Activation of exogenous fatty acids is likely required prior to their utilization. To identify the enzymatic activity responsible for activation we cloned candidate genes from Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 and identified the encoded proteins as acyl-acyl carrier protein synthetases (Aas). The enzymes catalyze the ATP-dependent esterification of fatty acids to the thiol of acyl carrier protein. The two protein sequences are only distantly related to known prokaryotic Aas proteins but they display strong similarity to sequences that can be found in almost all organisms that perform oxygenic photosynthesis. To investigate the biological role of Aas activity in cyanobacteria, aas knockout mutants were generated in the background of Synechocystis sp. PCC 6803 and S. elongatus PCC 7942. The mutant strains showed two phenotypes characterized by the inability to utilize exogenous fatty acids and by the secretion of endogenous fatty acids into the culture medium. The analyses of extracellular and intracellular fatty acid profiles of aas mutant strains as well as labeling experiments indicated that the detected free fatty acids are released from membrane lipids. The data suggest a considerable turnover of lipid molecules and a role for Aas activity in recycling the released fatty acids. In this model, lipid degradation represents a third supply of fatty acids for lipid synthesis in cyanobacteria.