Knock-out of the major regulator Flo8 in Komagataella phaffii results in unique host strain performance for methanol-free recombinant protein production.

Flo8 is a main transcriptional regulator of flocculation and pseudohyphal growth in yeast. Disruption of FLO8 in the popular recombinant protein production host Komagataella phaffii (Pichia pastoris) prevents pseudohyphal growth and reduces cell-to-surface adherence, making the mutant an interesting platform for research and industry. However, knowledge of the physiological impact of the mutation remained scarce.

Comparative Analysis of the Conversion of Mandelonitrile and 2-Phenylpropionitrile by a Large Set of Variants Generated from a Nitrilase Originating from EBC191.

The arylacetonitrilase from the bacterium EBC191 has been intensively studied as a model to understand the molecular basis for the substrate-, reaction-, and enantioselectivity of nitrilases. The nitrilase converts various aromatic and aliphatic nitriles to the corresponding acids and varying amounts of the corresponding amides. The enzyme has been analysed by site-specific mutagenesis and more than 50 different variants have been generated and analysed for the conversion of (,)-mandelonitrile and (,)-2-phenylpropionitrile.

Superior protein titers in half the fermentation time: Promoter and process engineering for the glucose-regulated GTH1 promoter of Pichia pastoris.

Protein production in Pichia pastoris is often based on the methanol-inducible P promoter which drives the expression of the target gene. The use of methanol has major drawbacks, so there is a demand for alternative promoters with good induction properties such as the glucose-regulated P promoter which we reported recently. To further increase its potential, we investigated its regulation in more details by the screening of promoter variants harboring deletions and mutations.

Induction without methanol: novel regulated promoters enable high-level expression in Pichia pastoris.

Background: Inducible high-level expression is favoured for recombinant protein production in Pichia pastoris. Therefore, novel regulated promoters are desired, ideally repressing heterologous gene expression during initial growth and enabling it in the production phase. In a typical large scale fed-batch culture repression is desired during the batch phase where cells grow on a surplus of e.

Construction and application of variants of the Pseudomonas fluorescens EBC191 arylacetonitrilase for increased production of acids or amides.

The arylacetonitrilase from Pseudomonas fluorescens EBC191 differs from previously studied arylacetonitrilases by its low enantiospecificity during the turnover of mandelonitrile and by the large amounts of amides that are formed in the course of this reaction. In the sequence of the nitrilase from P. fluorescens, a cysteine residue (Cys163) is present in direct neighborhood (toward the amino terminus) to the catalytic active cysteine residue, which is rather unique among bacterial nitrilases.

Identification of amino acid residues responsible for the enantioselectivity and amide formation capacity of the Arylacetonitrilase from Pseudomonas fluorescens EBC191.

The nitrilase from Pseudomonas fluorescens EBC191 converted (R,S)-mandelonitrile with a low enantioselectivity to (R)-mandelic acid and (S)-mandeloamide in a ratio of about 4:1. In contrast, the same substrate was hydrolyzed by the homologous nitrilase from Alcaligenes faecalis ATCC 8750 almost exclusively to (R)-mandelic acid. A chimeric enzyme between both nitrilases was constructed, which represented in total 16 amino acid exchanges in the central part of the nitrilase from P.

Influence of different carboxy-terminal mutations on the substrate-, reaction- and enantiospecificity of the arylacetonitrilase from Pseudomonas fluorescens EBC191.

Different members of the nitrilase superfamily (D-carbamoylases, Nit-Fhit proteins, amidases, cyanide dihydratases and nitrilases) were compared by multiple sequence alignments and a long carboxy-terminal extension (about 50 amino acids) identified in all nitrilases and cyanide dihydratases which was not present in other members of the nitrilase superfamily. The function of this C-terminal part was experimentally analysed in the arylacetonitrilase of Pseudomonas fluorescens EBC191 by the construction of various deletion mutants, chimeric enzymes with other bacterial nitrilases and site-specific mutagenesis. The enzyme variants were tested with the substrates 2-phenylpropionitrile and mandelonitrile and compared regarding specific activities, degree of amide formation and enantioselectivity.

Nitrilase from Pseudomonas fluorescens EBC191: cloning and heterologous expression of the gene and biochemical characterization of the recombinant enzyme.

The gene encoding an enantioselective arylacetonitrilase was identified on a 3.8 kb DNA fragment from the genomic DNA of Pseudomonas fluorescens EBC191. The gene was isolated, sequenced and cloned into the L-rhamnose-inducible expression vector pJOE2775.

Cloning of a nitrilase gene from the cyanobacterium Synechocystis sp. strain PCC6803 and heterologous expression and characterization of the encoded protein.

The gene encoding a putative nitrilase was identified in the genome sequence of the photosynthetic cyanobacterium Synechocystis sp. strain PCC6803. The gene was amplified by PCR and cloned into an expression vector.