Orthologous promoters from related methylotrophic yeasts surpass expression of endogenous promoters of Pichia pastoris.

Methylotrophic yeasts such as Komagataella phaffii (syn. Pichia pastoris, Pp), Hansenula polymorpha (Hp), Candida boidinii (Cb) and Pichia methanolica (Pm) are widely used protein production platforms. Typically, strong, tightly regulated promoters of genes coding for their methanol utilization (MUT) pathways are used to drive heterologous gene expression. Despite highly similar open reading frames in the MUT pathways of the four yeasts, the regulation of the respective promoters varies strongly between species. While most endogenous Pp MUT promoters remain tightly repressed after depletion of a repressing carbon, Hp, Cb and Pm MUT promoters are derepressed to up to 70% of methanol induced levels, enabling methanol free production processes in their respective host background. Here, we have tested a series of orthologous promoters from Hp, Cb and Pm in Pp. Unexpectedly, when induced with methanol, the promoter of the HpMOX gene reached very similar expression levels as the strong methanol, inducible, and most frequently used promoter of the Pp alcohol oxidase 1 gene (PPpAOX1). The HpFMD promoter even surpassed PPpAOX1 up to three-fold, when induced with methanol, and reached under methanol-free/derepressed conditions similar expression as the methanol induced PPpAOX1. These results demonstrate that orthologous promoters from related yeast species can give access to otherwise unobtainable regulatory profiles and may even considerably surpass endogenous promoters in P. pastoris.

Publisher Correction: Engineered bidirectional promoters enable rapid multi-gene co-expression optimization.

The original version of this Article was updated after publication to add the ORCID ID of the author Thomas Vogl, which was inadvertently omitted, and to include a corrected version of the 'Description of Additional Supplementary Files' which originally lacked legends for each file.

Engineered bidirectional promoters enable rapid multi-gene co-expression optimization.

Numerous synthetic biology endeavors require well-tuned co-expression of functional components for success. Classically, monodirectional promoters (MDPs) have been used for such applications, but MDPs are limited in terms of multi-gene co-expression capabilities. Consequently, there is a pressing need for new tools with improved flexibility in terms of genetic circuit design, metabolic pathway assembly, and optimization. Here, motivated by nature's use of bidirectional promoters (BDPs) as a solution for efficient gene co-expression, we generate a library of 168 synthetic BDPs in the yeast Komagataella phaffii (syn. Pichia pastoris), leveraging naturally occurring BDPs as a parts repository. This library of synthetic BDPs allows for rapid screening of diverse expression profiles and ratios to optimize gene co-expression, including for metabolic pathways (taxadiene, ß-carotene). The modular design strategies applied for creating the BDP library could be relevant in other eukaryotic hosts, enabling a myriad of metabolic engineering and synthetic biology applications.

Methanol independent induction in Pichia pastoris by simple derepressed overexpression of single transcription factors.

Carbon source regulated promoters are well-studied standard tools for controlling gene expression. Acquiring control over the natural regulation of promoters is important for metabolic engineering and synthetic biology applications. In the commonly used protein production host Komagataella phaffii (Pichia pastoris), methanol-inducible promoters are used because of their tight regulation and exceptional strength. Yet, induction with toxic and flammable methanol can be a considerable safety risk and cannot be applied in many existing fermentation plants. Here we studied new regulatory circuits based on the most frequently used alcohol oxidase 1 promoter (PAOX1 ), which is tightly repressed in presence of repressing carbon sources and strongly induced by methanol. We compared different overexpression strategies for putative carbon source dependent regulators identified by a homology search in related yeasts and previously published literature in order to convert existing methanol dependent expression strains into methanol free systems. While constitutive overexpression showed only marginal or detrimental effects, derepressed expression (activated when the repressing carbon source is depleted) showed that three transcription factors (TFs) are single handedly suitable to strongly activate PAOX1 in P. pastoris without relying on any specifically engineered host strains. Transcriptome analyses demonstrated that Mxr1, Mit1, and Prm1 regulate partly overlapping and unique sets of genes. Derepressed overexpression of a single TF was sufficient to retrofit existing PAOX1 based expression strains into glucose/glycerol regulated, methanol-free systems. Given the wide applicability of carbon source regulated promoters, the simplicity and low cost of controlling carbon source feed rates in large scale bioreactors, similar approaches as in P. pastoris may also be useful in other organisms.

Refined Pichia pastoris reference genome sequence.

Strains of the species Komagataella phaffii are the most frequently used "Pichia pastoris" strains employed for recombinant protein production as well as studies on peroxisome biogenesis, autophagy and secretory pathway analyses. Genome sequencing of several different P. pastoris strains has provided the foundation for understanding these cellular functions in recent genomics, transcriptomics and proteomics experiments. This experimentation has identified mistakes, gaps and incorrectly annotated open reading frames in the previously published draft genome sequences. Here, a refined reference genome is presented, generated with genome and transcriptome sequencing data from multiple P. pastoris strains. Twelve major sequence gaps from 20 to 6000 base pairs were closed and 5111 out of 5256 putative open reading frames were manually curated and confirmed by RNA-seq and published LC-MS/MS data, including the addition of new open reading frames (ORFs) and a reduction in the number of spliced genes from 797 to 571. One chromosomal fragment of 76kbp between two previous gaps on chromosome 1 and another 134kbp fragment at the end of chromosome 4, as well as several shorter fragments needed re-orientation. In total more than 500 positions in the genome have been corrected. This reference genome is presented with new chromosomal numbering, positioning ribosomal repeats at the distal ends of the four chromosomes, and includes predicted chromosomal centromeres as well as the sequence of two linear cytoplasmic plasmids of 13.1 and 9.5kbp found in some strains of P. pastoris.

A Toolbox of Diverse Promoters Related to Methanol Utilization: Functionally Verified Parts for Heterologous Pathway Expression in Pichia pastoris.

The heterologous expression of biosynthetic pathways for pharmaceutical or fine chemical production requires suitable expression hosts and vectors. In eukaryotes, the pathway flux is typically balanced by stoichiometric fine-tuning of reaction steps by varying the transcript levels of the genes involved. Regulated (inducible) promoters are desirable to allow a separation of pathway expression from cell growth. Ideally, the promoter sequences used should not be identical to avoid loss by recombination. The methylotrophic yeast Pichia pastoris is a commonly used protein production host, and single genes have been expressed at high levels using the methanol-inducible, strong, and tightly regulated promoter of the alcohol oxidase 1 gene (PAOX1). Here, we have studied the regulation of the P. pastoris methanol utilization (MUT) pathway to identify a useful set of promoters that (i) allow high coexpression and (ii) differ in DNA sequence to increase genetic stability. We noticed a pronounced involvement of the pentose phosphate pathway (PPP) and genes involved in the defense of reactive oxygen species (ROS), providing strong promoters that, in part, even outperform PAOX1 and offer novel regulatory profiles. We have applied these tightly regulated promoters together with novel terminators as useful tools for the expression of a heterologous biosynthetic pathway. With the synthetic biology toolbox presented here, P. pastoris is now equipped with one of the largest sets of strong and co-regulated promoters of any microbe, moving it from a protein production host to a general industrial biotechnology host.

Synergism of proteomics and mRNA sequencing for enzyme discovery.

Enzyme catalyzed processes are increasingly complementing chemical manufacturing as new enzymes are being discovered. Although, many industrially applied biocatalysts have been identified by functional screenings technological advances in the omics fields have created a different path to access novelty. Here we describe how omics technologies, especially proteomics and transcriptomics, can complement each other in the aim of finding new enzymatic functions. Special emphasis is laid on how mRNA sequencing Zcan improve proteomic experiments by allowing the generation of high quality protein sequence databases, which subsequently facilitates protein identification.