Fifty‐four bacterial strains belonging to 37 species were tested for their ability to assimilate short chain and/or medium chain liquid n‐alkanes. A gene probe derived from the alkB gene of Pseudomonas oleovorans ATCC 29347 was utilized in hybridization experiments. Results of Southern hybridization of PCR‐amplificates were compared with those of colony hybridization and dot blot hybridization. Strongest signals were received only from Gram‐negative bacteria growing solely with short n‐alkanes (C10). Hybridization results with soil isolates growing with n‐alkanes of different chain lengths suggested as well that alkB genes seem to be widespread only in solely short‐chain n‐alkane‐degrading pseudomonads. PCR products of Rhodococcus sp., Nocardioides sp., Gordona sp. and Sphingomonas sp. growing additionally or solely with medium‐chain n‐alkane as hexadecane had only few sequence identity with alkB though hybridizing with the gene probe. The derived amino acid sequence of the alkB‐amplificate of Pseudomonas aureofaciens showed high homology (95%) with AlkB from Ps. oleovorans. alkB gene disruptants were not able to grow with decane.
Itaconic acid is a valuable platform compound for the production of bio‐based polymers, chemicals, and fuels. Ustilago maydis is a promising host for the production of itaconic acid from biomass‐derived substrates due to its unicellular growth pattern and its potential to utilize biomass‐derived sugar monomers and polymers. The potential of U. maydis for industrial itaconate production was assessed in pH‐controlled batch fermentations with varying medium compositions. Using 200 g/L glucose and 75 mM ammonium, 44.5 g/L of itaconate was produced at a maximum rate of 0.74 g L−1 h−1. By decreasing the substrate concentrations to 50 g/L glucose and 30 mM ammonium, a yield of 0.34 g/g (47 mol%) could be achieved. Itaconate production from xylose was also feasible. These results indicate that high itaconic acid titers can be achieved with U. maydis. However, further optimization of the biocatalyst itself through metabolic engineering is still needed in order to achieve an economically feasible process, which can be used to advance the development of a bio‐based economy.
We introduced a reporter gene system into Pichia stipitis using the gene for the artificial green fluorescent protein (GFP), variant yEGFP. This system was used to analyse hypoxia-dependent PsADH2 regulation. Reporter gene activity was only found under oxygen limitation on a fermentable carbon source. The promoter was not induced by oxygen limitation in the Crabtree-positive yeast Saccharomyces cerevisiae. Promoter deletions revealed that a region of 15 bp contained the essential site for hypoxic induction. This motif was different from the known hypoxia response elements of S. cerevisiae but showed some similarity to the mammalian HIF-1 binding site. Electrophoretic mobility shift assays demonstrated specific protein binding to this region under oxygen limitation. Similar to the S. cerevisiae heme sensor system, the promoter was induced by Co 2+ . Cyanide was not able to mimic the effect of oxygen limitation. The activation mechanism of PsADH2 also, in this respect, has similarities to the mammalian HIF-1 system, which is inducible by Co 2+ but not by cyanide. Thus, the very first promoter analysis in P. stipitis revealed a hitherto unknown mechanism of oxygen sensing in yeast.
Two Pichia stipitis ADH genes (PsADH1 and PsADH2) were isolated by complementation of a Saccharomyces cerevisiae Adh(-)-mutant. The genes enabled the transformants to grow in the presence of antimycin A on glucose, to use ethanol as sole carbon source and made them sensitive to allylalcohol. The sequences of the genes showed similarities of 70-77% to sequences of ADH genes of Candida albicans, Kluyveromyces lactis, K. marxianus, and S. cerevisiae and about 60% homology to those of Schizosaccharomyces pombe and Aspergillus flavus. Southern hybridization experiments suggested that P. stipitis has only these two ADH genes. Both genes are located on the largest chromosome of P. stipitis. PsADH2 encodes for the ADH activity that is responsible for ethanol formation at oxygen limitation. The gene is regulated at the transcriptional level. Moreover, also in cells grown on ethanol, only PsADH2 transcript was found. PsADH1 transcript was detected under aerobic conditions on fermentable carbon sources.
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