Franz S. Hartner

Regulation of methanol utilisation pathway genes in yeasts

Methylotrophic yeasts such as Candida boidinii, Hansenula polymorpha, Pichia methanolica and Pichia pastoris are an emerging group of eukaryotic hosts for recombinant protein production with an ever increasing number of applications during the last 30 years. Their applications are linked to the use of strong methanol-inducible promoters derived from genes of the methanol utilisation pathway. These promoters are tightly regulated, highly repressed in presence of non-limiting concentrations of glucose in the medium and strongly induced if methanol is used as carbon source. Several factors involved in this tight control and their regulatory effects have been described so far. This review summarises available data about the regulation of promoters from methanol utilisation pathway genes. Furthermore, the role of cis and trans acting factors (e.g. transcription factors, glucose processing enzymes) in the expression of methanol utilisation pathway genes is reviewed both in the context of the native cell environment as well as in heterologous hosts.

Deletion of the Pichia pastoris KU70 Homologue Facilitates Platform Strain Generation for Gene Expression and Synthetic Biology

Targeted gene replacement to generate knock-outs and knock-ins is a commonly used method to study the function of unknown genes. In the methylotrophic yeast Pichia pastoris, the importance of specific gene targeting has increased since the genome sequencing projects of the most commonly used strains have been accomplished, but rapid progress in the field has been impeded by inefficient mechanisms for accurate integration. To improve gene targeting efficiency in P. pastoris, we identified and deleted the P. pastoris KU70 homologue. We observed a substantial increase in the targeting efficiency using the two commonly known and used integration loci HIS4 and ADE1, reaching over 90% targeting efficiencies with only 250-bp flanking homologous DNA. Although the ku70 deletion strain was noted to be more sensitive to UV rays than the corresponding wild-type strain, no lethality, severe growth retardation or loss of gene copy numbers could be detected during repetitive rounds of cultivation and induction of heterologous protein production. Furthermore, we demonstrated the use of the ku70 deletion strain for fast and simple screening of genes in the search of new auxotrophic markers by targeting dihydroxyacetone synthase and glycerol kinase genes. Precise knock-out strains for the well-known P. pastoris AOX1, ARG4 and HIS4 genes and a whole series of expression vectors were generated based on the wild-type platform strain, providing a broad spectrum of precise tools for both intracellular and secreted production of heterologous proteins utilizing various selection markers and integration strategies for targeted or random integration of single and multiple genes. The simplicity of targeted integration in the ku70 deletion strain will further support protein production strain generation and synthetic biology using P. pastoris strains as platform hosts.

New opportunities by synthetic biology for biopharmaceutical production in Pichia pastoris

Graphical abstract Highlights ► The application of Pichia pastoris for biopharmaceutical production is described. ► Synthetic biology approaches and perspectives to improve production are reviewed. ► Glycoengineering efforts to produce humanized, uniform glycoproteins are covered. ► The design and application of synthetic promoter variants are highlighted. ► The molecular toolbox available for synthetic biology in P. pastoris is discussed.

Biochemical Evidence That Berberine Bridge Enzyme Belongs to a Novel Family of Flavoproteins Containing a Bi-covalently Attached FAD Cofactor

Berberine bridge enzyme (BBE) is involved in the transformation of (S)-reticuline to (S)-scoulerine in benzophenanthridine alkaloid biosynthesis of plants. In this report, we describe the high level expression of BBE encoded by the gene from Eschscholzia californica (California poppy) in the methylotrophic yeast Pichia pastoris employing the secretory pathway of the host organism. Using a two-step chromatographic purification protocol, 120 mg of BBE could be obtained from 1 liter of fermentation culture. The purified protein exhibits a turnover number for substrate conversion of 8.2 s-1. The recombinant enzyme is glycosylated and carries a covalently attached FAD cofactor. In addition to the previously known covalent attachment of the 8α-position of the flavin ring system to a histidine (His-104), we could also demonstrate that a covalent linkage between the 6-position and a thiol group of a cysteine residue (Cys-166) is present in BBE. The major evidence for the occurrence of a bi-covalently attached FAD cofactor is provided by N-terminal amino acid sequencing and mass spectrometric analysis of the isolated flavin-containing peptide. Furthermore, it could be shown that anaerobic photoirradiation leads to cleavage of the linkage between the 6-cysteinyl group yielding 6-mercaptoflavin and a peptide with the cysteine residue replaced by alanine due to breakage of the C-S bond. Overall, BBE is shown to exhibit typical flavoprotein oxidase properties as exemplified by the occurrence of an anionic flavin semiquinone species and formation of a flavin N(5)-sulfite adduct.

Real‐time PCR‐based determination of gene copy numbers in Pichia pastoris

Pichia pastoris is a preferred host for heterologous protein production. Expression cassettes are usually integrated into the genome of this methylotrophic yeast. This manuscript describes a method for fast and reliable gene copy number determinations for P. pastoris expression strains. We believe that gene copy number determinations are important for all researchers working with P. pastoris and also many other research groups using similar gene integration techniques for the transformation of other yeasts. The described method uses real‐time PCR to quantify the integrated expression cassettes. Similar methods were employed previously for other host systems such as animal and plant cells but no such method comparing different detection methods and describing details for yeast analysis by quantitative PCR is known to us, especially for methylotrophic yeasts such as P. pastoris. Neglecting gene copy numbers can easily lead to false interpretations of experimental results from codon optimization or promoter studies and co‐expression of helper proteins as demonstrated in an application example, which is also described here.

Laboratory evolved biocatalysts for stereoselective syntheses of substituted benzaldehyde cyanohydrins.

Hydroxynitrile lyases (HNLs) are biocatalysts employed industrially for the asymmetric addition of HCN to aldehydes or ketones. The resulting optically active cyanohydrins are important building blocks for pharmaceuticals and agrochemicals. Several genes of Rand S-selective HNLs have been cloned and expressed in suitable host systems, and can thus be ACHTUNGTRENNUNGengineered to improve their catalytic properties. The almond (Prunus amygdalus) (R)-HNL isoenzyme 5 (PaHNL5) is particularly suited as a starting point for engineering approaches. PaHNL5 can be heterologously expressed in Pichia pastoris and is highly stable even at pH values lower than 3.0. It is efficiently secreted into the culture supernatant, which can be directly employed for biocatalysis in aqueous or biphasic systems. Reduction of steric hindrance by structure-guided design has resulted in the simple and quick generation of new enzyme variants with significantly improved catalytic rates and enhanced stereoselectivity in cyanohydrin syntheses with employment of non-natural substrates. We wanted to explore whether the so far best, highly active, and stereoselective enzyme variant for the production of (R)-2-chloromandelonitrile (2a, Scheme 1), PaHNL5/L1Q/A111G, already industrially used, can reach an activity similar to that of the WT enzyme with its preferred substrate benzaldehyde. Compound 2a is a key intermediate for the production of a widely administered anticoagulant that reduces the risk of cardiovascular events in patients with acute coronary syndromes. As computational methods did not provide any indications for further improvement, we addressed the challenge of applying directed evolution. Bacteria, especially E. coli, are widely used for laboratory evolution, because of their simple genetic molecular manipulability, high transformation efficiency, and rapid growth rates. However, for eukaryotic proteins their use is often limited because of misfolding and the lack of typical eukaryotic posttranslational modifications. P. pastoris, an efficient system for heterologous protein production, can compensate for such disadvantages. Drawbacks, however, are a high input of linearized plasmid DNA and the concomitant relatively low integration rate into the genome. In addition, we have observed varying copy numbers or incomplete cassette integration, which seemed to impede the utilization of P. pastoris for highthroughput expression, screening, and laboratory evolution of proteins. Although expression of PaHNL in E. coli has been described, large amounts of highly active PaHNL5 could only be obtained with P. pastoris. Consequently, it was necessary to devise a new method for the efficient construction of reliable expression libraries in P. pastoris and the uniform expression of thousands of individual transformants, while circumventing time-consuming ligation and cloning steps in E. coli, which lead to a loss of diversity and library efficiency. Furthermore, there are no stable plasmids for Pichia transformation available. A concise procedure to generate PaHNL5/L1Q/A111G libraries by integration of linear expression cassettes produced by an overlap extension PCR (OE-PCR) strategy was thus designed, and so the randomly mutated Pahnl5 gene, a fragment consisting of a partial GAP (glyceraldehyde 3-phosphate dehydrogenase) promoter, a 5’ secretion signal sequence for the a-mating factor from bakers’ yeast and a 3’ zeocin resistance cassette were linked together (Figure 1). Both flanking arms were generated by proof-reading polymerases. To improve the efficiency of the OE-PCR, the overlapping region between the mutated gene and the selection marker (overlap 2) was redesigned. Then, without any prior ligation step, P. pastoris X33 was directly transformed with the linear PCR-based integration cassettes. In order to test this new strategy, the Pahnl5/L1Q gene was used to reassemble a linear integration cassette by overlap extension PCR. Since an L1Q mutation seemed to enhance exScheme 1. Synthesis of halogen-substituted (R)-mandelonitriles (TBME: tertbutyl methyl ether).

Engineering the Pichia pastoris methanol oxidation pathway for improved NADH regeneration during whole-cell biotransformation.

Industrial biocatalytic reduction processes require the efficient regeneration of reduced cofactors for the asymmetric reduction of prochiral compounds to chiral intermediates which are needed for the production of fine chemicals and drugs. Here, we present a new engineering strategy for improved NADH regeneration based on the Pichia pastoris methanol oxidation pathway. Studying the kinetic properties of alcohol oxidase (AOX), formaldehyde dehydrogenase (FLD) and formate dehydrogenase (FDH) and using the derived kinetic data for subsequent kinetic simulations of NADH formation rates led to the identification of FLD activity to constitute the main bottleneck for efficient NADH recycling via the methanol dissimilation pathway. The simulation results were confirmed constructing a recombinant P. pastoris strain overexpressing P. pastoris FLD and the highly active NADH-dependent butanediol dehydrogenase from S. cerevisiae. Employing the engineered strain, significantly improved butanediol production rates were achieved in whole-cell biotransformations.

Enhancing stress resistance and production phenotypes through transcriptome engineering.

As Saccharomyces cerevisiae is engineered further as a microbial factory for industrially relevant but potentially cytotoxic molecules such as ethanol, issues of cell viability arise that threaten to place a biological limit on output capacity and/or the use of less refined production conditions. Evidence suggests that one naturally evolved mode of survival in deleterious environments involves the complex, multigenic interplay between disparate stress response and homeostasis mechanisms. Rational engineering of such resistance would require a systems-level understanding of cellular behavior that is, in general, not yet available. To circumvent this limitation, we have developed a phenotype discovery approach termed global transcription machinery engineering (gTME) that allows for the generation and selection of nonphysiological traits. We alter gene expression on a genome-wide scale by selecting for dominant mutations in a randomly mutagenized general transcription factor. The gene encoding the mutated transcription factor resides on a plasmid in a strain carrying the unaltered chromosomal allele. Thus, although the dominant mutations may destroy the essential function of the plasmid-borne variant, alteration of the transcriptome with minimal perturbation to normal cellular processes is possible via the presence of the native genomic allele. Achieving a phenotype of interest involves the construction and diversity evaluation of yeast libraries harboring random sequence variants of a chosen transcription factor and the subsequent selection and validation of mutant strains. We describe the rationale and procedures associated with each step in the context of generating strains possessing enhanced ethanol tolerance.

Engineering Pichia pastoris for improved NADH regeneration: A novel chassis strain for whole-cell catalysis

Summary Many synthetically useful reactions are catalyzed by cofactor-dependent enzymes. As cofactors represent a major cost factor, methods for efficient cofactor regeneration are required especially for large-scale synthetic applications. In order to generate a novel and efficient host chassis for bioreductions, we engineered the methanol utilization pathway of Pichia pastoris for improved NADH regeneration. By deleting the genes coding for dihydroxyacetone synthase isoform 1 and 2 (DAS1 and DAS2), NADH regeneration via methanol oxidation (dissimilation) was increased significantly. The resulting Δdas1 Δdas2 strain performed better in butanediol dehydrogenase (BDH1) based whole-cell conversions. While the BDH1 catalyzed acetoin reduction stopped after 2 h reaching ~50% substrate conversion when performed in the wild type strain, full conversion after 6 h was obtained by employing the knock-out strain. These results suggest that the P. pastoris Δdas1 Δdas2 strain is capable of supplying the actual biocatalyst with the cofactor over a longer reaction period without the over-expression of an additional cofactor regeneration system. Thus, focusing the intrinsic carbon flux of this methylotrophic yeast on methanol oxidation to CO2 represents an efficient and easy-to-use strategy for NADH-dependent whole-cell conversions. At the same time methanol serves as co-solvent, inductor for catalyst and cofactor regeneration pathway expression and source of energy.