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The methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii) is a successful host widely used in recombinant protein production. The widespread use of a methanol-regulated alcohol oxidase 1 (AOX1) promoter for recombinant protein production has directed studies particularly about methanol metabolism in this yeast. Although there is comprehensive knowledge about methanol metabolism, there are other mechanisms in P. pastoris that have not been investigated yet, such as ethanol metabolism. The gene responsible for the consumption of ethanol ADH2 (XM_002491337, known as ADH3) was identified and characterized in our previous study. In this study, the ADH genes (XM_002489969, XM_002491163, XM_002493969) in P. pastoris genome were investigated to determine their roles in ethanol production by gene disruption analysis. We report that the ADH900 (XM_002491163) is the main gene responsible for ethanol production in P. pastoris. The ADH2 gene, previously identified as the only gene responsible for ethanol consumption, also plays a minor role in ethanol production in the absence of the ADH900 gene. The investigation of the carbon source regulation mechanism has also revealed that the ADH2 gene exhibit similar expression behaviours with ADH900 on glucose, glycerol, and methanol, however, it is strongly induced by ethanol.

Background The methylotrophic, Crabtree-negative yeast Pichia pastoris is widely used as a heterologous protein production host. Strong inducible promoters derived from methanol utilization genes or constitutive glycolytic promoters are typically used to drive gene expression. Notably, genes involved in methanol utilization are not only repressed by the presence of glucose, but also by glycerol. This unusual regulatory behavior prompted us to study the regulation of carbon substrate utilization in different bioprocess conditions on a genome wide scale. Results We performed microarray analysis on the total mRNA population as well as mRNA that had been fractionated according to ribosome occupancy. Translationally quiescent mRNAs were defined as being associated with single ribosomes (monosomes) and highly-translated mRNAs with multiple ribosomes (polysomes). We found that despite their lower growth rates, global translation was most active in methanol-grown P. pastoris cells, followed by excess glycerol- or glucose-grown cells. Transcript-specific translational responses were found to be minimal, while extensive transcriptional regulation was observed for cells grown on different carbon sources. Due to their respiratory metabolism, cells grown in excess glucose or glycerol had very similar expression profiles. Genes subject to glucose repression were mainly involved in the metabolism of alternative carbon sources including the control of glycerol uptake and metabolism. Peroxisomal and methanol utilization genes were confirmed to be subject to carbon substrate repression in excess glucose or glycerol, but were found to be strongly de-repressed in limiting glucose-conditions (as are often applied in fed batch cultivations) in addition to induction by methanol. Conclusions P. pastoris cells grown in excess glycerol or glucose have similar transcript profiles in contrast to S. cerevisiae cells, in which the transcriptional response to these carbon sources is very different. The main response to different growth conditions in P. pastoris is transcriptional; translational regulation was not transcript-specific. The high proportion of mRNAs associated with polysomes in methanol-grown cells is a major finding of this study; it reveals that high productivity during methanol induction is directly linked to the growth condition and not only to promoter strength. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1393-8) contains supplementary material, which is available to authorized users.