Phthalates are esters of phthalic acid that give flexibility to polyvinyl chloride. Diverse studies have reported that these compounds might be carcinogenic, mutagenic and/or teratogenic. Radial growth rate, biomass, hyphal thickness of Neurospora sitophyla, Trichoderma harzianum and Aspergillus niger, grown in two different concentrations of dibutyl phthalate (DBP) (500 and 1,000 mg/l) in agar and in submerged fermentation were studied. The inhibitory concentration (IC50) and the constant of biodegradation of dibutyl phthalate in Escherichia coli cultures were used to evaluate toxicity. The radial growth rate and thickness of the hypha were positively correlated with the concentration of phthalate. The pH of the cultures decreased as the fermentation proceeded. It is shown that these fungi are able to degrade DBP to non-toxic compounds and that these can be used as sole carbon and energy sources by this bacterium. It is demonstrated that the biodegradation of the DBP is directly correlated with the IC50. This is the first study that reports a method to determine the biodegradation of DBP on the basis of the IC50 and fungal growth, and the effect of this phthalate on the growth and thickness of hyphae of filamentous fungi in agar and in submerged fermentation.

Thiolases catalyze the condensation of acyl-CoA thioesters through the Claisen condensation reaction. The best described enzymes usually yield linear condensation products. Using a combined computational/experimental approach, and guided by structural information, we have studied the potential of thiolases to synthesize branched compounds. We have identified a bulky residue located at the active site that blocks proper accommodation of substrates longer than acetyl-CoA. Amino acid replacements at such a position exert effects on the activity and product selectivity of the enzymes that are highly dependent on a protein scaffold. Among the set of five thiolases studied, Erg10 thiolase from Saccharomyces cerevisiae showed no acetyl-CoA/butyryl-CoA branched condensation activity, but variants at position F293 resulted the most active and selective biocatalysts for this reaction. This is the first time that a thiolase has been engineered to synthesize branched compounds. These novel enzymes enrich the toolbox of combinatorial (bio)chemistry, paving the way for manufacturing a variety of α-substituted synthons. As a proof of concept, we have engineered Clostridium's 1-butanol pathway to obtain 2-ethyl-1-butanol, an alcohol that is interesting as a branched model compound.

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The photosynthetic protist Euglena gracilis contains chloroplasts surrounded by three membranes which arise from secondary endosymbiosis. The genes petA and petD, encoding cytochrome f and subunit IV of the cytochrome bf complex, normally present in chloroplast genomes, are lacking from the chloroplast DNA (cpDNA) of E. gracilis. The bf complex of E. gracilis was isolated, and the identities of cytochrome f and subunit IV were established immunochemically, by heme-specific staining, and by Edman degradation. Based on N-terminal and conserved internal protein sequences, primers were designed and used for PCR gene amplification and cDNA sequencing. The complete sequence of the petA cDNA and the partial sequence of the petD cDNA from E. gracilis are described. Evidence is provided that in this protist, the petA and petD genes have migrated from the chloroplast to the nucleus. Both genes exhibit a typical nuclear codon usage, clearly distinct from the usage of chloroplast genes. The petA gene encodes an atypical cytochrome f, with a unique insertion of 62 residues not present in other f-type cytochromes. The petA gene also acquired a region that encodes a large tripartite chloroplast transit peptide (CTP), which is thought to allow the import of apocytochrome f through the three-membrane envelope of E. gracilis chloroplasts. This is the first description of petA and petD genes that are nucleus-localized.

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