Investigating A Multi-Domain Polyketide Synthase in Amphidinium carterae.

Dinoflagellates are unicellular organisms that are implicated in harmful algal blooms (HABs) caused by potent toxins that are produced through polyketide synthase (PKS) pathways. However, the exact mechanisms of toxin synthesis are unknown due to a lack of genomic segregation of fat, toxins, and other PKS-based pathways. To better understand the underlying mechanisms, the actions and expression of the PKS proteins were investigated using the toxic dinoflagellate Amphidinium carterae as a model. Cerulenin, a known ketosynthase inhibitor, was shown to reduce acetate incorporation into all fat classes with the toxins amphidinol and sulpho-amphidinol. The mass spectrometry analysis of cerulenin-reacted synthetic peptides derived from ketosynthase domains of A. carterae multimodular PKS transcripts demonstrated a strong covalent bond that could be localized using collision-induced dissociation. One multi-modular PKS sequence present in all dinoflagellates surveyed to date was found to lack an AT domain in toxin-producing species, indicating trans-acting domains, and was shown by Western blotting to be post-transcriptionally processed. These results demonstrate how toxin synthesis in dinoflagellates can be differentiated from fat synthesis despite common underlying pathway.

Dynamic cycling of t-SNARE acylation regulates platelet exocytosis.

Platelets regulate vascular integrity by secreting a host of molecules that promote hemostasis and its sequelae. Given the importance of platelet exocytosis, it is critical to understand how it is controlled. The t-SNAREs, SNAP-23 and syntaxin-11, lack classical transmembrane domains (TMDs), yet both are associated with platelet membranes and redistributed into cholesterol-dependent lipid rafts when platelets are activated. Using metabolic labeling and hydroxylamine (HA)/HCl treatment, we showed that both contain thioester-linked acyl groups. Mass spectrometry mapping further showed that syntaxin-11 was modified on cysteine 275, 279, 280, 282, 283, and 285, and SNAP-23 was modified on cysteine 79, 80, 83, 85, and 87. Interestingly, metabolic labeling studies showed incorporation of [3H]palmitate into the t-SNAREs increased although the protein levels were unchanged, suggesting that acylation turns over on the two t-SNAREs in resting platelets. Exogenously added fatty acids did compete with [3H]palmitate for t-SNARE labeling. To determine the effects of acylation, we measured aggregation, ADP/ATP release, as well as P-selectin exposure in platelets treated with the acyltransferase inhibitor cerulenin or the thioesterase inhibitor palmostatin B. We found that cerulenin pretreatment inhibited t-SNARE acylation and platelet function in a dose- and time-dependent manner whereas palmostatin B had no detectable effect. Interestingly, pretreatment with palmostatin B blocked the inhibitory effects of cerulenin, suggesting that maintaining the acylation state is important for platelet function. Thus, our work shows that t-SNARE acylation is actively cycling in platelets and suggests that the enzymes regulating protein acylation could be potential targets to control platelet exocytosis in vivo.

Enterococcus faecalis Responds to Individual Exogenous Fatty Acids Independently of Their Degree of Saturation or Chain Length.

Enterococcus faecalis is a commensal of the human gastrointestinal tract that can persist in the external environment and is a leading cause of hospital-acquired infections. Given its diverse habitats, the organism has developed numerous strategies to survive a multitude of environmental conditions. Previous studies have demonstrated that E. faecalis will incorporate fatty acids from bile and serum into its membrane, resulting in an induced tolerance to membrane-damaging agents. To discern whether all fatty acids induce membrane stress protection, we examined how E. faecalis responded to individually supplied fatty acids. E. faecalis readily incorporated fatty acids 14 to 18 carbons in length into its membrane but poorly incorporated fatty acids shorter or longer than this length. Supplementation with saturated fatty acids tended to increase generation time and lead to altered cellular morphology in most cases. Further, exogenously supplied saturated fatty acids did not induce tolerance to the membrane-damaging antibiotic daptomycin. Supplementation with unsaturated fatty acids produced variable growth effects, with some impacting generation time and morphology. Exogenously supplied unsaturated fatty acids that are normally produced by E. faecalis and those that are found in bile or serum could restore growth in the presence of a fatty acid biosynthetic inhibitor. However, only the eukaryote-derived fatty acids oleic acid and linoleic acid provided protection from daptomycin. Thus, exogenous fatty acids do not lead to a common physiological effect on E. faecalis The organism responds uniquely to each, and only host-derived fatty acids induce membrane protection.IMPORTANCEEnterococcus faecalis is a commonly acquired hospital infectious agent with resistance to many antibiotics, including those that target its cellular membrane. We previously demonstrated that E. faecalis will incorporate fatty acids found in human fluids, like serum, into its cellular membrane, thereby altering its membrane composition. In turn, the organism is better able to survive membrane-damaging agents, including the antibiotic daptomycin. We examined fatty acids commonly found in serum and those normally produced by E. faecalis to determine which fatty acids can induce protection from membrane damage. Supplementation with individual fatty acids produced a myriad of different effects on cellular growth, morphology, and stress response. However, only host-derived unsaturated fatty acids provided stress protection. Future studies are aimed at understanding how these specific fatty acids induce protection from membrane damage.

fabH deletion increases DHA production in Escherichia coli expressing Pfa genes.

BACKGROUND: Some marine bacteria, such as Moritella marina, produce the nutraceutical docosahexaenoic acid (DHA) thanks to a specific enzymatic complex called Pfa synthase. Escherichia coli heterologously expressing the pfa gene cluster from M. marina also produces DHA. The aim of this study was to find genetic or metabolic conditions to increase DHA production in E. coli. RESULTS: First, we analysed the effect of the antibiotic cerulenin, showing that DHA production increased twofold. Then, we tested a series of single gene knockout mutations affecting fatty acid biosynthesis, in order to optimize the synthesis of DHA. The most effective mutant, fabH, showed a threefold increase compared to wild type strain. The combination of cerulenin inhibition and fabH deletion rendered a 6.5-fold improvement compared to control strain. Both strategies seem to have the same mechanism of action, in which fatty acid synthesis via the canonical pathway (fab pathway) is affected in its first catalytic step, which allows the substrates to be used by the heterologous pathway to synthesize DHA. CONCLUSIONS: DHA-producing E. coli strain that carries a fabH gene deletion boosts DHA production by tuning down the competing canonical biosynthesis pathway. Our approach can be used for optimization of DHA production in different organisms.

OleA Glu117 is key to condensation of two fatty-acyl coenzyme A substrates in long-chain olefin biosynthesis.

In the interest of decreasing dependence on fossil fuels, microbial hydrocarbon biosynthesis pathways are being studied for renewable, tailored production of specialty chemicals and biofuels. One candidate is long-chain olefin biosynthesis, a widespread bacterial pathway that produces waxy hydrocarbons. Found in three- and four-gene clusters, oleABCD encodes the enzymes necessary to produce cis-olefins that differ by alkyl chain length, degree of unsaturation, and alkyl chain branching. The first enzyme in the pathway, OleA, catalyzes the Claisen condensation of two fatty acyl-coenzyme A (CoA) molecules to form a ß-keto acid. In this report, the mechanistic role of Xanthomonas campestris OleA Glu117 is investigated through mutant enzymes. Crystal structures were determined for each mutant as well as their complex with the inhibitor cerulenin. Complemented by substrate modeling, these structures suggest that Glu117 aids in substrate positioning for productive carbon-carbon bond formation. Analysis of acyl-CoA substrate hydrolysis shows diminished activity in all mutants. When the active site lacks an acidic residue in the 117 position, OleA cannot form condensed product, demonstrating that Glu117 has a critical role upstream of the essential condensation reaction. Profiling of pH dependence shows that the apparent pKa for Glu117 is affected by mutagenesis. Taken together, we propose that Glu117 is the general base needed to prime condensation via deprotonation of the second, non-covalently bound substrate during turnover. This is the first example of a member of the thiolase superfamily of condensing enzymes to contain an active site base originating from the second monomer of the dimer.

Mutants of Yarrowia lipolytica NCIM 3589 grown on waste cooking oil as a biofactory for biodiesel production.

BACKGROUND: Oleaginous yeasts are fast emerging as a possible feedstock for biodiesel production. Yarrowia lipolytica, a model oleaginous yeast is known to utilize a variety of hydrophobic substrates for lipid accumulation including waste cooking oil (WCO). Approaches to increase lipid content in this yeast include metabolic engineering which requires manipulation of multiple genes in the lipid biosynthesis pathway. A classical and cost-effective approach, namely, random chemical mutagenesis on the yeast can lead to increased production of biodiesel as is explored here. RESULTS: In this study, chemical mutagenesis using the alkylating agent, N- methyl-N'-nitro-N-nitrosoguanidine (MNNG) as well as an additional treatment with cerulenin, a fatty acid synthase inhibitor generated 800 mutants of Y. lipolytica NCIM 3589 (761 MNNG treated and 39 MNNG + cerulenin treated). A three-stage screening using Sudan Black B plate technique, Nile red fluorimetry and total lipid extraction using solvent was performed, which enabled selection of ten high lipid yielding mutants. Time course studies of all the ten mutants were further undertaken in terms of biomass, lipid yield and lipid content to select three stable mutants (YlB6, YlC7 and YlE1) capable of growing and accumulating lipid on WCO, with lipid contents of 55, 60 and 67% as compared to 45% for the wild type. The mutants demonstrated increased volumetric lipid productivities (0.062, 0.044 and 0.041 g L-1 h-1) as compared to the wild type (0.033 g L-1 h-1). The fatty acid profile of the three mutants consisted of a high content of C16 and C18 saturated and monounsaturated fatty acids and was found to be suitable for biodiesel production. The fuel properties, namely, density, kinematic viscosity, total acid number, iodine value of the three mutants were evaluated and found to lie within the limits specified by internationally accepted standards. Additionally, it was noted that the mutants demonstrated better cetane numbers and higher heating values than the wild type strain. CONCLUSION: The chemical mutagenesis strategy adopted in this study resulted in the successful isolation of three stable high SCO yielding mutants. The mutants, namely, YlB6, YlC7 and YlE1 exhibited a 1.22, 1.33 and 1.49-fold increase in lipid contents when grown on 100 g L-1 waste cooking oil than the parental yeast strain. The fatty acid methyl ester (FAME) profiles of all the three mutants was determined to be suitable for biodiesel suggesting their potential applicability while simultaneously addressing the management of waste cooking oil.

Chemical Genetics in Dissecting Membrane Glycerolipid Functions.

Chemical genetics has emerged as a powerful approach to dissect biological processes, based on the utilization of small molecules disturbing the function of specific target proteins. By analogy with classical genetics, 'reverse chemical genetics' refers to the utilization of drugs acting on a known target, enabling its functional characterization at the levels of the cells, tissues and organisms. Likewise, 'direct chemical genetics' refers to the utilization of a drug of unknown mode of action, but triggering a phenotype of interest. In that case, one has to identify the target(s) possibly blocked (or possibly activated) by the small molecule. This chapter illustrates both approaches, like the analysis of the elongation of fatty acids, the biosynthesis of galactoglycerolipids or the catabolism of phosphoglycerolipids by reverse chemical genetics or the study of the membrane glycerolipid remodeling triggered upon phosphate starvation, by direct chemical genetics.

De novo genome assembly and annotation of rice sheath rot fungus Sarocladium oryzae reveals genes involved in Helvolic acid and Cerulenin biosynthesis pathways.

BACKGROUND: Sheath rot disease caused by Sarocladium oryzae is an emerging threat for rice cultivation at global level. However, limited information with respect to genomic resources and pathogenesis is a major setback to develop disease management strategies. Considering this fact, we sequenced the whole genome of highly virulent Sarocladium oryzae field isolate, Saro-13 with 82x sequence depth. RESULTS: The genome size of S. oryzae was 32.78 Mb with contig N50 18.07 Kb and 10526 protein coding genes. The functional annotation of protein coding genes revealed that S. oryzae genome has evolved with many expanded gene families of major super family, proteinases, zinc finger proteins, sugar transporters, dehydrogenases/reductases, cytochrome P450, WD domain G-beta repeat and FAD-binding proteins. Gene orthology analysis showed that around 79.80 % of S. oryzae genes were orthologous to other Ascomycetes fungi. The polyketide synthase dehydratase, ATP-binding cassette (ABC) transporters, amine oxidases, and aldehyde dehydrogenase family proteins were duplicated in larger proportion specifying the adaptive gene duplications to varying environmental conditions. Thirty-nine secondary metabolite gene clusters encoded for polyketide synthases, nonribosomal peptide synthase, and terpene cyclases. Protein homology based analysis indicated that nine putative candidate genes were found to be involved in helvolic acid biosynthesis pathway. The genes were arranged in cluster and structural organization of gene cluster was similar to helvolic acid biosynthesis cluster in Metarhizium anisophilae. Around 9.37 % of S. oryzae genes were identified as pathogenicity genes, which are experimentally proven in other phytopathogenic fungi and enlisted in pathogen-host interaction database. In addition, we also report 13212 simple sequences repeats (SSRs) which can be deployed in pathogen identification and population dynamic studies in near future. CONCLUSIONS: Large set of pathogenicity determinants and putative genes involved in helvolic acid and cerulenin biosynthesis will have broader implications with respect to Sarocladium disease biology. This is the first genome sequencing report globally and the genomic resources developed from this study will have wider impact worldwide to understand Rice-Sarocladium interaction.

Stalk cell differentiation without polyketides in the cellular slime mold.

Polyketides induce prestalk cell differentiation in Dictyostelium. In the double-knockout mutant of the SteelyA and B polyketide synthases, most of the pstA cells-the major part of the prestalk cells-are lost, and we show by whole mount in situ hybridization that expression of prestalk genes is also reduced. Treatment of the double-knockout mutant with the PKS inhibitor cerulenin gave a further reduction, but some pstA cells still remained in the tip region, suggesting the existence of a polyketide-independent subtype of pstA cells. The double-knockout mutant and cerulenin-treated parental Ax2 cells form fruiting bodies with fragile, single-cell layered stalks after cerulenin treatment. Our results indicate that most pstA cells are induced by polyketides, but the pstA cells at the very tip of the slug are induced in some other way. In addition, a fruiting body with a single-cell layered, vacuolated stalk can form without polyketides.

Identification of a mutation causing a defective spindle assembly checkpoint in high ethyl caproate-producing sake yeast strain K1801.

In high-quality sake brewing, the cerulenin-resistant sake yeast K1801 with high ethyl caproate-producing ability has been used widely; however, K1801 has a defective spindle assembly checkpoint (SAC). To identify the mutation causing this defect, we first searched for sake yeasts with a SAC-defect like K1801 and found that K13 had such a defect. Then, we searched for a common SNP in only K1801 and K13 by examining 15 checkpoint-related genes in 23 sake yeasts, and found 1 mutation, R48P of Cdc55, the PP2A regulatory B subunit that is important for the SAC. Furthermore, we confirmed that the Cdc55-R48P mutation was responsible for the SAC-defect in K1801 by molecular genetic analyses. Morphological analysis indicated that this mutation caused a high cell morphological variation. But this mutation did not affect the excellent brewing properties of K1801. Thus, this mutation is a target for breeding of a new risk-free K1801 with normal checkpoint integrity.

Improved pinocembrin production in Escherichia coli by engineering fatty acid synthesis.

The development of efficient microbial processes for pinocembrin production has attracted considerable attention. However, pinocembrin biosynthetic efficiency is greatly limited by the low availability of the malonyl-CoA cofactor in Escherichia coli. Fatty acid biosynthesis is the only metabolic process in E. coli that consumes malonyl-CoA; therefore, we overexpressed the fatty acid biosynthetic pathway enzymes ß-ketoacyl-ACP synthase III (FabH) and ß-ketoacyl-ACP synthase II (FabF) alone and in combination, and investigated the effect on malonyl-CoA. Interestingly, overexpressing FabH, FabF or both enzymes in E. coli BL21 (DE3) decreased fatty acid synthesis and increased cellular malonyl-CoA levels 1.4-, 1.6-, and 1.2-fold, respectively. Furthermore, pinocembrin production was increased 10.6-, 31.8-, and 5.87-fold in recombinant strains overexpressing FabH, FabF and both enzymes, respectively. Overexpression of FabF, therefore, triggered the highest pinocembrin production and malonyl-CoA levels. The addition of cerulenin further increased pinocembrin production in the FabF-overexpressing strain, from 25.8 to 29.9 mg/L. These results demonstrated that overexpressing fatty acid synthases can increase malonyl-CoA availability and improve pinocembrin production in a recombinant E. coli host. This strategy may hold promise for the production of other important natural products in which cellular malonyl-CoA is rate limiting.

Isolation of a spontaneous cerulenin-resistant sake yeast with both high ethyl caproate-producing ability and normal checkpoint integrity.

In the brewing of high-quality sake such as Daiginjo-shu, the cerulenin-resistant sake yeast strains with high producing ability to the flavor component ethyl caproate have been used widely. Genetic stability of sake yeast would be important for the maintenance of both fermentation properties of yeast and quality of sake. In eukaryotes, checkpoint mechanisms ensure genetic stability. However, the integrity of these mechanisms in sake yeast has not been examined yet. Here, we investigated the checkpoint integrity of sake yeasts, and the results suggested that a currently used cerulenin-resistant sake yeast had a defect in spindle assembly checkpoint (SAC). We also isolated a spontaneous cerulenin-resistant sake yeast FAS2-G1250S mutant, G9CR, which showed both high ethyl caproate-producing ability and integrity/intactness of the checkpoint mechanisms. Further, morphological phenotypic robustness analysis by use of CalMorph supported the genetic stability of G9CR. Finally, we confirmed the high quality of sake from G9CR in an industrial sake brewing setting.

Sustainable production of biopesticides for common bean root rot control within the biorefinery approach: a Technology Readiness Level 3 experimental proof of concept.

Biopesticides are recognized as an efficient alternative to synthetic pesticides for pest and disease crop management. However, their commercial production processes use grains, generating large amounts of organic waste, even when agriculture waste or byproducts are the feedstock of choice. Frequently, these organic wastes are rich in nutrients that, after adequate treatment, can be used as nitrogen and carbohydrate sources for secondary metabolite production produced by microorganisms during submerged fermentation. In this sense, this study aimed to prove the concept that biopesticides could be produced under a full biorefinery process, using the entire biomass of an underexplored agroindustrial waste-damaged bean-as the main feedstock. A combination of sequential processes, including solid state fermentation, hydrolysis, and submerged fermentation, were designed for the production of two biopesticides (conventional-fungal conidia and second-generation secondary metabolite-cerulenin) from a high potential biological control agent strain Sarocladium oryzae BRM 59907. The combined processes, using damaged common bean grain as the main feedstock, provided biopesticides and organic fertilizer production that successfully controlled common bean root rot disease. This work proved to be possible the biopesticide production using a full biorefinery concept, inside the same productive chain, contributing to a sustainable environment and economy, together with animal and human health safety.

Cerulenin suppresses ErbB2-overexpressing breast cancer by targeting ErbB2/PKM2 pathway.

Cerulenin is a fungal metabolite and a specific inhibitor of fatty acid synthase (FASN), which has shown a potential anticancer activity. 20-25% of breast cancer patients with ErbB2-overexpressing develop resistance to treatment. Therefore, it is urgent to find an effective new target for the treatment of ErbB2-overexpressing breast cancer. Our previous study found that cerulenin inhibits the glycolysis and migration of SK-BR-3 cells, but the effect of cerulenin on other malignant phenotypes of breast cancer is still unknown. Furthermore, the mechanism by which cerulenin displays its inhibitory effects is not fully understood. In this study, we systematically investigate the inhibitory effects of cerulenin on proliferation, migration, invasion and glycolysis of ErbB2-overexpressing breast cancer cells and its molecular mechanism. We found that cerulenin obviously suppresses the proliferation, migration, invasion as well as glycolysis. Through bioinformatic analyses, we found that PKM2 might be a target of cerulenin. In addition, ErbB2 and its signaling pathway upregulated PKM2 protein levels. Furthermore, we demonstrated that cerulenin downregulated the protein levels of ErbB2, PKM2 and EMT markers (MMP9, MMP2 and Snail2) in a dose- and time-dependent manner. Finally, the inhibitory of cerulenin on colony formation, migration, invasion and glycolysis, as well as protein levels of EMT markers were rescued by replenishing with PKM2. These findings illustrated that cerulenin inhibits proliferation, migration, invasion and glycolysis by targeting ErbB2/PKM2 pathway in ErbB2-overexpressing breast cancer cells.

Suppressing fatty acid synthase by type I interferon and chemical inhibitors as a broad spectrum anti-viral strategy against SARS-CoV-2.

SARS-CoV-2 is an emerging viral pathogen and a major global public health challenge since December of 2019, with limited effective treatments throughout the pandemic. As part of the innate immune response to viral infection, type I interferons (IFN-I) trigger a signaling cascade that culminates in the activation of hundreds of genes, known as interferon stimulated genes (ISGs), that collectively foster an antiviral state. We report here the identification of a group of type I interferon suppressed genes, including fatty acid synthase (FASN), which are involved in lipid metabolism. Overexpression of FASN or the addition of its downstream product, palmitate, increased viral infection while knockout or knockdown of FASN reduced infection. More importantly, pharmacological inhibitors of FASN effectively blocked infections with a broad range of viruses, including SARS-CoV-2 and its variants of concern. Thus, our studies not only suggest that downregulation of metabolic genes may present an antiviral strategy by type I interferon, but they also introduce the potential for FASN inhibitors to have a therapeutic application in combating emerging infectious diseases such as COVID-19.

Effects of fatty acid synthase-inhibitors on polyunsaturated fatty acid production in marine diatom Fistulifera solaris JPCC DA0580.

Omega-3 polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA, C20:5ω-3) are essential for human health and fish growth especially in aquaculture sector. However, with the growing of aquaculture, the demand of PUFA supply also has been increasing. Fistulifera solaris, a marine diatom, is known for its ability to accumulate 65% of lipid content per dry cell weight, and can produce the high content of EPA. Thus, this diatom shows a great potential to be a feedstock of omega-3 PUFAs for fish feeds. In this study, in order to further understand and enhance the metabolism of PUFA biosynthesis in the diatom, the impacts of ketoacyl-ACP synthase (KAS) and ketoacyl-CoA synthase (KCS) inhibition on the PUFA production were analyzed by adding the specific inhibitors. KAS and KCS enzymes both play a role in the fatty acid elongation. As a result, the inhibition of KAS showed an increase in EPA content without arresting the cell growth. On the other hand, inhibition of KCS did not show a significant impact on the PUFA content in F. solaris. Our finding suggests that the specific suppression of KAS function can be a promising way to enhance the omega-3 PUFA production in F. solaris.

Comparative Proteomics Reveals Evidence of Enhanced EPA Trafficking in a Mutant Strain of Nannochloropsis oculata.

The marine microalga Nannochloropsis oculata is a bioproducer of eicosapentaenoic acid (EPA), a fatty acid. EPA is incorporated into monogalactosyldiacylglycerol within N. oculata thylakoid membranes, and there is a biotechnological need to remodel EPA synthesis to maximize production and simplify downstream processing. In this study, random mutagenesis and chemical inhibitor-based selection method were devised to increase EPA production and accessibility for improved extraction. Ethyl methanesulfonate was used as the mutagen with selective pressure achieved by using two enzyme inhibitors of lipid metabolism: cerulenin and galvestine-1. Fatty acid methyl ester analysis of a selected fast-growing mutant strain had a higher percentage of EPA (37.5% of total fatty acids) than the wild-type strain (22.2% total fatty acids), with the highest EPA quantity recorded at 68.5 mg/g dry cell weight, while wild-type cells had 48.6 mg/g dry cell weight. Label-free quantitative proteomics for differential protein expression analysis revealed that the wild-type and mutant strains might have alternative channeling pathways for EPA synthesis. The mutant strain showed potentially improved photosynthetic efficiency, thus synthesizing a higher quantity of membrane lipids and EPA. The EPA synthesis pathways could also have deviated in the mutant, where fatty acid desaturase type 2 (13.7-fold upregulated) and lipid droplet surface protein (LDSP) (34.8-fold upregulated) were expressed significantly higher than in the wild-type strain. This study increases the understanding of EPA trafficking in N. oculata, leading to further strategies that can be implemented to enhance EPA synthesis in marine microalgae.

Incorporation of Exogenous Fatty Acids Enhances the Salt Tolerance of Food Yeast Zygosaccharomyces rouxii.

Fatty acids have great effects on the maintenance of the cell membrane structure, cell viability, and cell metabolisms. In this study, we sought to elucidate the effects of exogenous fatty acids on the salt tolerance of food yeast Zygosaccharomyces rouxii. Results showed that Z. rouxii can grow by using exogenous fatty acids (C12:0, C14:0, C16:0, C16:1, C18:0, C18:1, and C18:2) as the sole carbon source. Four fatty acids (C12:0, C16:0, C16:1, and C18:1) can improve the salt tolerance of cells, enhance the formation of the cell biofilm, regulate the chemical compositions, restore growth in the presence of cerulenin, regulate the contents of membrane fatty acids, and control the expression of key genes in the fatty acid metabolism. Our results reveal that Z. rouxii can synthesize membrane fatty acids from exogenous fatty acids and the supplementation of these fatty acids can override the need for de novo fatty acid biosynthesis.

Survival of the fittest: how the rice microbial community forces Sarocladium oryzae into pathogenicity.

The fungus Sarocladium oryzae (Sawada) causes rice sheath rot and produces the phytotoxins cerulenin and helvolic acid. Both toxins show antimicrobial activity but only helvolic acid production in the rice sheath correlates with virulence. Sarocladium oryzae isolates that differ in their toxin production were used to study their interaction with the rice culturable bacterial endophyte community. The diversity and community structure was defined in the edge of sheath rot lesions, followed by a null model-based co-occurrence analysis to discover pairwise interactions. Non-random pairs were co-cultured to study the nature of the interactions and the role of the toxins herein. Compared to healthy sheaths, endophyte diversity strongly increased when infected with the least virulent S. oryzae isolates producing low amounts of toxins. Virulent S. oryzae isolates did not affect diversity but caused strong shifts in species composition. The endophyte community of healthy rice plants was dominated by B. cereus. This bacterium was enriched in lesions produced by low-virulent S. oryzae isolates and caused hyphal lysis. Contrarily, helvolic acid producers eliminated this bacterium from the sheath endosphere. We conclude that S. oryzae needs to produce antibiotics to defend itself against antagonistic rice endophytes to successfully colonize and infect the rice sheath.

Morphological, Pathogenic and Toxigenic Variability in the Rice Sheath Rot Pathogen Sarocladium Oryzae.

Sheath rot is an emerging rice disease that leads to considerable yield losses. The main causal agent is the fungus Sarocladium oryzae. This pathogen is known to produce the toxins cerulenin and helvolic acid, but their role in pathogenicity has not been clearly established. S. oryzea isolates from different rice-producing regions can be grouped into three phylogenetic lineages. When grown in vitro, isolates from these lineages differed in growth rate, colour and in the ability to form sectors. A diverse selection of isolates from Rwanda and Nigeria, representing these lineages, were used to further study their pathogenicity and toxin production. Liquid chromatography high-resolution mass spectrometry analysis was used to measure cerulenin and helvolic acid production in vitro and in planta. The three lineages clearly differed in pathogenicity on the japonica cultivar Kitaake. Isolates from the least pathogenic lineage produced the highest levels of cerulenin in vitro. Helvolic acid production was not correlated with the lineage. Sectorisation was observed in isolates from the two least pathogenic lineages and resulted in a loss of helvolic acid production. In planta, only the production of helvolic acid, but not of cerulenin, correlated strongly with disease severity. The most pathogenic isolates all belonged to one lineage. They were phenotypically stable, shown by the lack of sectorisation, and therefore maintained high helvolic acid production in planta.