Production of 10-methyl branched fatty acids in yeast

Background: Despite the environmental value of biobased lubricants, they account for less than 2% of global lubricant use due to poor thermo-oxidative stability arising from the presence of unsaturated double bonds. Methyl branched fatty acids (BFAs), particularly those with branching near the acyl-chain mid-point, are a high-performance alternative to existing vegetable oils because of their low melting temperature and full saturation. Results: We cloned and characterized two pathways to produce 10-methyl BFAs isolated from actinomycetes and γ-proteobacteria. In the two-step bfa pathway of actinomycetes, BfaB methylates Δ9 unsaturated fatty acids to form 10-methylene BFAs, and subsequently, BfaA reduces the double bond to produce a fully saturated 10-methyl branched fatty acid. A BfaA-B fusion enzyme increased the conversion efficiency of 10-methyl BFAs. The ten-methyl palmitate production (tmp) pathway of γ-proteobacteria produces a 10-methylene intermediate, but the TmpA putative reductase was not active in E. coli or yeast. Comparison of BfaB and TmpB activities revealed a range of substrate specificities from C14-C20 fatty acids unsaturated at the Δ9, Δ10 or Δ11 position. We demonstrated efficient production of 10-methylene and 10-methyl BFAs in S. cerevisiae by secretion of free fatty acids and in Y. lipolytica as triacylglycerides, which accumulated to levels more than 35% of total cellular fatty acids. Conclusions: We report here the characterization of a set of enzymes that can produce position-specific methylene and methyl branched fatty acids. Yeast expression of bfa enzymes can provide a platform for the large-scale production of branched fatty acids suitable for industrial and consumer applications

The CDK-Activating Kinase (CAK) Csk1 Is Required for Normal Levels of Homologous Recombination and Resistance to DNA Damage in Fission Yeast

BACKGROUND: Cyclin-dependent kinases (CDKs) perform essential roles in cell division and gene expression in all eukaryotes. The requirement for an upstream CDK-activating kinase (CAK) is also universally conserved, but the fission yeast Schizosaccharomyces pombe appears to be unique in having two CAKs with both overlapping and specialized functions that can be dissected genetically. The Mcs6 complex--orthologous to metazoan Cdk7/cyclin H/Mat1--activates the cell-cycle CDK, Cdk1, but its non-redundant essential function appears to be in regulation of gene expression, as part of transcription factor TFIIH. The other CAK is Csk1, an ortholog of budding yeast Cak1, which activates all three essential CDKs in S. pombe--Cdk1, Mcs6 and Cdk9, the catalytic subunit of positive transcription elongation factor b (P-TEFb)--but is not itself essential. METHODOLOGY/PRINCIPAL FINDINGS: Cells lacking csk1(+) are viable but hypersensitive to agents that damage DNA or block replication. Csk1 is required for normal levels of homologous recombination (HR), and interacts genetically with components of the HR pathway. Tests of damage sensitivity in csk1, mcs6 and cdk9 mutants indicate that Csk1 acts pleiotropically, through Cdk9 and at least one other target (but not through Mcs6) to preserve genomic integrity. CONCLUSIONS/SIGNIFICANCE: The two CAKs in fission yeast, which differ with respect to their substrate range and preferences for monomeric CDKs versus CDK/cyclin complexes as substrates, also support different functions of the CDK network in vivo. Csk1 plays a non-redundant role in safeguarding genomic integrity. We propose that specialized activation pathways dependent on different CAKs might insulate CDK functions important in DNA damage responses from those capable of triggering mitosis

Nuclear localised more sulphur accumulation1 epigenetically regulates sulphur homeostasis in Arabidopsis thaliana

Sulphur (S) is an essential element for all living organisms. The uptake, assimilation and metabolism of S in plants are well studied. However, the regulation of S homeostasis remains largely unknown. Here, we report on the identification and characterisation of the more sulphur accumulation1 (msa1-1) mutant. The MSA1 protein is localized to the nucleus and is required for both S adenosylmethionine (SAM) production and DNA methylation. Loss of function of the nuclear localised MSA1 leads to a reduction in SAM in roots and a strong S-deficiency response even at ample S supply, causing an over- accumulation of sulphate, sulphite, cysteine and glutathione. Supplementation with SAM suppresses this high S phenotype. Furthermore, mutation of MSA1 affects genome-wide DNA methylation, including the methylation of S-deficiency responsive genes. Elevated S accumulation in msa1-1 requires the increased expression of the sulphate transporter genes SULTR1;1 and SULTR1;2 which are also differentially methylated in msa1-1. Our results suggest a novel function for MSA1 in the nucleus in regulating SAM biosynthesis and maintaining S homeostasis epigenetically via DNA methylation

Identification of ORC1/CDC6-Interacting Factors in Trypanosoma brucei Reveals Critical Features of Origin Recognition Complex Architecture

DNA Replication initiates by formation of a pre-replication complex on sequences termed origins. In eukaryotes, the pre-replication complex is composed of the Origin Recognition Complex (ORC), Cdc6 and the MCM replicative helicase in conjunction with Cdt1. Eukaryotic ORC is considered to be composed of six subunits, named Orc1–6, and monomeric Cdc6 is closely related in sequence to Orc1. However, ORC has been little explored in protists, and only a single ORC protein, related to both Orc1 and Cdc6, has been shown to act in DNA replication in Trypanosoma brucei. Here we identify three highly diverged putative T. brucei ORC components that interact with ORC1/CDC6 and contribute to cell division. Two of these factors are so diverged that we cannot determine if they are eukaryotic ORC subunit orthologues, or are parasite-specific replication factors. The other we show to be a highly diverged Orc4 orthologue, demonstrating that this is one of the most widely conserved ORC subunits in protists and revealing it to be a key element of eukaryotic ORC architecture. Additionally, we have examined interactions amongst the T. brucei MCM subunits and show that this has the conventional eukaryotic heterohexameric structure, suggesting that divergence in the T. brucei replication machinery is limited to the earliest steps in origin licensing