Carbon source requirements for mating and mating-type switching in the methylotrophic yeasts Ogataea (Hansenula) polymorpha and Komagataella phaffii (Pichia pastoris).

The methylotrophic yeasts Ogataea (Hansenula) polymorpha and Komagataella phaffii (Pichia pastoris) have important industrial applications and are models for several biological processes including peroxisome biology and methanol metabolism. We examined the carbon source requirements for mating-type (MAT) switching and mating in both species. Haploid strains of O. polymorpha and K. phaffii are homothallic, and switch MAT by a flip/flop mechanism in which a chromosomal region containing the MAT genes undergoes an inversion. MAT switching is induced by nitrogen starvation in both species and can be detected 4-6 hr after induction. Both switching and mating require a utilizable carbon source that can be either fermentable or nonfermentable. We further observed that although methanol can be used as a sole carbon source in both species, it does not support the induction of MAT switching or mating. Our results provide insight into the nutritional cues that influence entry into sexual processes in methylotrophic yeasts that undergo flip/flop MAT switching.

Structural and functional analysis of PUR2,5 gene encoding bifunctional enzyme of de novo purine biosynthesis in Ogataea (Hansenula) polymorpha CBS 4732T.

We describe the cloning, sequencing and functional characterization of gene PUR2,5, involved in de novo purine biosynthesis of the yeast Ogataea (Hansenula) polymorpha. This gene (2369 bp) was cloned by genetic complementation of adenine requiring mutation. It encodes a bifunctional enzyme of 789 amino acids (85 kDa) that catalyzes the second and the fifth steps of de novo purine biosynthesis pathway and shows dual enzymatic activity - of glycinamide ribotide synthetase (GARS, EC 6.3.4.13) and of aminoimidazole ribotide synthetase (AIRS, EC 6.3.3.1). Nucleotide sequence analysis revealed the presence of putative regulatory elements located in the adjacent 5' region. Canonical motives that function as binding sites for BAS1 transcription activator were found at positions (-593) and (-389). The putative TAATTA-box was located at (-20) to (-14) and AT-rich heteroduplex was found in the 3'-non-translated region. We compared the amino acid sequence of OpPUR2,5p with those of the corresponding enzymes of other yeast species as well as with distant organisms like bacteria Escherichia coli and human Homo sapiens. A successful disruption of OpPUR2,5 gene was done. It was found that OpPUR2,5::LEU2 replacement affects both mating and sporulation processes. OpPUR2,5 sequence is deposited in the GenBank of NCBI with accession no. JF967633.

Accelerated production of 1,3-propanediol from glycerol by Klebsiella pneumoniae using the method of forced pH fluctuations.

1,3-Propanediol (1,3-PD) is a bivalent alcohol, used in a number of chemical syntheses. It could be produced from glycerol in course of microbial fermentation by Klebsiella pneumoniae along with more than five minor liquid products. With the purpose to enhance 1,3-PD production and to eliminate by-products formation, principally new pH control on the process was applied. The method, named "forced pH fluctuations" was realized by consecutive raisings of pH with definite ΔpH amplitude (ranging from 1.0 to 2.0) at time intervals between 2 and 4 h, during a series of fed batch processes. The fermentation performed by forced pH fluctuations with ΔpH = 1.0, risen at every 3 h was evaluated as the most successful. Increase by 10% of the maximal amount of 1,3-PD (g/l), 22% higher productivity [g/(l h)], and 29% increase in 1,3-PD molar yield were achieved, compared to the referent fed batch (with constant pH = 7.0). In addition, significant decrease in by-products formation was obtained. The most important reduction was observed in the lactic and acetic acids yields, where 50 and 70% decrease were reached. The results suggested the potential of pH to manage the share and quantity of product spectrum in mixed diols-acids fermentations. The application of "forced pH fluctuations method" achieves the desirable increase in 1,3-PD formation and decrease in by-products accumulation at the same time by a comparatively simple approach by adjustment of one bioprocess parameter only.