A promoter library consisting of several AOX1
promoter variants fused to GFP displayed a broad range of activities (from ~6% to >160%) with small increments between the individual promoter variants. Although we started with one of the most powerful promoters described to date, the PAOX1
), we obtained some variants which were even stronger. Several important cis
-acting elements responsible for high PAOX1
activity were identified and characterized. Using our approach starting with computational TFBS prediction, we: (i) achieved a high success rate in changing the promoter strength by introducing mutations within the TFBSs and (ii) localized the cis
-acting elements and their individual effects precisely by the first deletion series.
We identified seven regions within the AOX1
promoter where a deletion of either small core sequences of the putative TFBS or the deletion of a larger stretch covering the whole putative TFBS resulted in a decrease in promoter activity of >50%. Additionally, the prediction of putative TFBSs suggests possible target TFs, where a search for homologues in the P. pastoris
genome might facilitate future analyses about PAOX1
regulation (and regulation of other genes of the methanol utilization pathway). While we expected to strongly downregulate the promoter activity upon deletion of the putative TATA box, we did not expect such a large number of regions where a mutation can decrease promoter activity by >50%, some of the effects being even more dramatic than in the case of the TATA box deletion. For instance, when we deleted (or mutated) nucleotides surrounding position −209 to −210, the promoter activity dropped to ~6–30% of the wild-type activity, depending on the mutation (B). In addition, these results are consistent with previous studies where promoter activity dropped with stepwise truncations (20
) and which effects observed in the case of larger deletions of the whole AOX1
promoter sequence (56
Our approach of predicting putative TFBSs followed by deletion or duplication of the respective region within the promoter sequence was highly successful in generating a library spanning a broad range of activities. This not only delivered mutants with strongly altered activity but also suggested which TFs might be involved in the regulation of the AOX1 promoter. Our results also demonstrated the complex architecture of the AOX1 promoter with several activator/repressor sites, which might explain its strong and tight regulation.
To identify TFs involved in methanol-sensitive gene regulation, past studies have focused on the behaviour of these types of promoters in heterologous hosts that are better characterized than most methylotrophic fungi. Pichia pastoris AOX1
and H. polymorpha MOX
promoter regions were shown to promote expression of the reporter enzyme β-galactosidase (lacZ
of E. coli
) in S. cerevisiae
). The regulation patterns of these genes in S. cerevisiae
are similar, though not the same, as in their natural hosts: glucose represses gene expression, under carbon starvation conditions expression is slightly derepressed, and glycerol as carbon source induces expression [however, the latter is contradictory to what happens in P. pastoris
promoter driven expression was induced by ethanol, methanol and oleic acid in S. cerevisiae
). Furthermore, Adr1p, a positive effector of ADH2
(alcohol dehydrogenase 2) and some peroxisomal proteins in S. cerevisiae
), was shown to be an activator of the MOX
promoter in S. cerevisiae
when glucose is lacking in the medium (25
). Interestingly, the only identified TF involved in AOX1
regulation, Mxr1p, shows similarity at the structural level to the S. cerevisiae
). However, Mxr1p was identified to bind to a region between −170 and −411, but the putative Adr1p-binding site we found with MatInspector is located at −576 (5′ end of the binding site). Thus, the exact localization of the Mxr1p-binding site in the AOX1
promoter remains to be elucidated.
We introduced deletions into the promoter sequence, which possibly modified positioning of trans-acting factors that work in a concerted fashion to achieve full induction (e.g. facilitating the binding of partner factors or bending of DNA). Thus, the observed effects are not necessarily caused by the deletion of a cis-acting element, but may result simply from changes in spacing between authentic elements. Whether this characteristic PAOX1 regulation pattern (glucose repression, derepression in absence of carbon source and methanol induction) is caused by the presence of one or several repressor proteins, by inaccessibility of the promoter packed in nucleosomes or by the absence of activator proteins, or a combination of those possibilities remains to be elucidated in further studies.
However, a rough model can be presented based on our preliminary results in addition to other previous studies. Cells lacking MXR1 function cannot induce the AOX1 promoter at all. Mxr1p has many similarities to S. cerevisiae Adr1p and can be considered a homologue. Having several domains, Adr1p not only binds to and activates target promoters but also modifies the activity or expression level of other TFs which modulate the same target promoters. Adr1p can be considered both a global and local regulator of transcription. In parallel fashion, we hypothesize that Mxr1p not only binds the AOX1 promoter but also can regulate at least one additional P. pastoris TF protein additionally involved in regulation of the AOX1 promoter. Such a model helps to explain the presence of so many different binding sites in the same promoter.
Furthermore, we demonstrated the transferability of promoter strength of the new promoter variants to the expression of industrial enzymes that are produced by P. pastoris in a wide variety of concentrations either intracellularly (HbHNL) or extracellularly (PaHNL5α, HRP).
Since P. pastoris
is one of the major protein production host, a detailed understanding of the regulation of its most commonly used promoter at a molecular level will have a dramatic impact on the optimization of protein production processes in the future. This should be supported further by the availability of the P. pastoris
genome sequence, which could enable a straight-forward isolation and study of trans
-acting factors involved in regulation of PAOX1
-driven gene expression. There is no general optimal promoter: the regulatory properties of the promoter variant, the target protein and the production process have to be seen as a whole. The new promoter variants are especially useful for tailoring a production strategy that is specific to the needs of the individual protein. For example, stronger variants would be useful for the expression of non-toxic proteins, as in the case of reporters where fewer copies of the expression cassette would be sufficient to obtain a maximum of expression. Weaker variants could be favourable for the expression of proteins, which cause deleterious effects to the host cell. Within this group, we include proteins which are toxic to the cell per se due to their biological activity (e.g. proteases, toxins) or which induce an unfolded protein response (UPR) or ER-associated degradation (ERAD) due to incomplete folding in the ER during secretion (58–61
) or which face other bottlenecks where an excess of transcript would ultimately end up in less active protein. Another group of ‘difficult’ proteins that needs optimization of expression are integral membrane proteins, which are intrinsically hard to produce (62
For the first time, we succeeded in the generation of promoter mutants which displayed increased promoter activity without the need for any methanol for strong expression while, in contrast to constitutive promoters like PGAP, still are strongly repressed in presence of glucose. Such an expression profile was also found for short synthetic promoter variants, which were constructed using PAOX1-derived cis-acting elements. Such promoters can be used to express proteins in carbon starvation phases like the stationary phase in batch cultures, under carbon-limited conditions like fedbatch or by continuous cultivation below the maximum specific growth rate, as demonstrated by HRP production employing a glucose-limited fedbatch process.
Briefly, we demonstrated that specific mutations introduced at putative TFBSs can be employed to generate a promoter library with a broad range of activities and thereby, identified regulatory regions that provide a basis for designing short, synthetic promoter variants for metabolic engineering and synthetic biology. Further work is needed to understand the biological role of the different cis-acting elements so that a complete characterization of the molecular basis of PAOX1 regulation can be achieved. Nevertheless, considering the results obtained during this study and the advantages of P. pastoris in recombinant protein production processes, especially proteins needed in the pharmaceutical, chemical and biofuel industry, we are convinced that our library will be broadly applicable to optimize production yields.