Synergistic modular promoter and gene optimization to push cellulase secretion by Pichia pastoris beyond existing benchmarks.

Although successfully used for heterologous gene expression for more than twenty years, general knowledge about all factors influencing protein expression by Pichia pastoris is still lacking. For high titers of protein clones are optimized individually for each target protein. Optimization efforts in this study were focused on the DNA level, evaluating a set of 48 different individual synthetic genes (TrCBH2) coding for the same protein sequence of a Trichoderma reesei cellulase in combination with three different promoter sequences: PGAP (constitutive) and the synthetic AOX1 promoter variants PDeS (derepressed) and PEn (enhanced, inducible). Expression of active secreted enzyme varied from undetectable to ∼300% of the best known gene, as determined by secreted enzyme activity analyses of supernatants from 96 well plate and bioreactor cultivations. Finally, the best optimized gene and new promoters were combined to engineer highly productive P. pastoris CBH2 expression strains. Although no methanol was used for induction a final titer of more than 18g/l of secreted protein was produced under controlled conditions in small scale bioreactor cultivations after 60-70h of growth limiting glycerol feed. This is the highest concentration of secreted enzyme in P. pastoris published so far and single parts of the expression cassette could be independently optimized showing additive effects for improvements in protein production by P. pastoris.

Pichia pastoris Aft1--a novel transcription factor, enhancing recombinant protein secretion.

BACKGROUND: The methylotrophic yeast Pichia pastoris is frequently used for the production of recombinant proteins. However, expression levels can vary depending on the target protein. Allowing for simultaneous regulation of many genes, which may elicit a desired phenotype like increased protein production, overexpression of transcription factors can be used to overcome expression bottlenecks. Here, we present a novel P. pastoris transcription factor currently annotated as Aft1, activator of ferrous transport. RESULTS: The promoter regions of key secretory P. pastoris genes were screened for fungal transcription factor binding sites, revealing Aft1 as an interesting candidate for improving secretion. Genome wide analysis of transcription factor binding sites suggested Aft1 to be involved in the regulation of many secretory genes, but also indicated possible novel functions in carbohydrate metabolism. No Aft binding sites were found in promoters of characteristic iron homeostasis genes in P. pastoris. Microarrays were used to study the Aft1 regulon in detail, confirming Aft1 involvement in the regulation of carbon-responsive genes, and showing that iron regulation is dependent on FEP1, but not AFT1 expression levels. The positive effect of AFT1 overexpression on recombinant protein secretion was demonstrated for a carboxylesterase from Sphingopyxis sp. MTA144, for which secretion was improved 2.5-fold in fed batch bioreactor cultivations. CONCLUSION: This study demonstrates that the transcription factor Aft1 can be used to improve recombinant protein secretion in P. pastoris. Furthermore, we discovered possible novel functions of Aft1 in carbohydrate metabolism and provide evidence arguing against a direct role of Aft1 in P. pastoris iron regulation.

Strains and molecular tools for recombinant protein production in Pichia pastoris.

Within the last two decades, the methylotrophic yeast Pichia pastoris has become an important alternative to E. coli or mammalian cell lines for the production of recombinant proteins. Easy handling, strong promoters, and high cell density cultivations as well as the capability of posttranslational modifications are some of the major benefits of this yeast. The high secretion capacity and low level of endogenously secreted proteins further promoted the rapid development of a versatile Pichia pastoris toolbox. This chapter reviews common and new "Pichia tools" and their specific features. Special focus is given to expression strains, such as different methanol utilization, protease-deficient or glycoengineered strains, combined with application highlights. Different promoters and signal sequences are also discussed.

Synthetic core promoters for Pichia pastoris.

Synthetic promoters are commonly used tools for circuit design or high level protein production. Promoter engineering efforts in yeasts, such as Saccharomyces cerevisiae and Pichia pastoris have mostly been focused on altering upstream regulatory sequences such as transcription factor binding sites. In higher eukaryotes synthetic core promoters, directly needed for transcription initiation by RNA Polymerase II, have been successfully designed. Here we report the first synthetic yeast core promoter for P. pastoris, based on natural yeast core promoters. Furthermore we used this synthetic core promoter sequence to engineer the core promoter of the natural AOX1 promoter, thereby creating a set of core promoters providing a range of different expression levels. As opposed to engineering strategies of the significantly longer entire promoter, such short core promoters can directly be added on a PCR primer facilitating library generation and are sufficient to obtain variable expression yields.

Spore germination of Trichoderma atroviride is inhibited by its LysM protein TAL6.

LysM motifs are carbohydrate-binding modules found in prokaryotes and eukaryotes. They have general N-acetylglucosamine binding properties and therefore bind to chitin and related carbohydrates. In plants, plasma-membrane-bound proteins containing LysM motifs are involved in plant defence responses, but also in symbiotic interactions between plants and microorganisms. Filamentous fungi secrete LysM proteins that contain several LysM motifs but no enzymatic modules. In plant pathogenic fungi, for LysM proteins roles in dampening of plant defence responses and protection from plant chitinases were shown. In this study, the carbohydrate-binding specificities and biological function of the LysM protein TAL6 from the plant-beneficial fungus Trichoderma atroviride were investigated. TAL6 contains seven LysM motifs and the sequences of its LysM motifs are very different from other fungal LysM proteins investigated so far. The results showed that TAL6 bound to some forms of polymeric chitin, but not to chito-oligosaccharides. Further, no binding to fungal cell wall preparations was detected. Despite these rather weak carbohydrate-binding properties, a strong inhibitory effect of TAL6 on spore germination was found. TAL6 was shown to specifically inhibit germination of Trichoderma spp., but interestingly not of other fungi. Thus, this protein is involved in self-signalling processes during fungal growth rather than fungal-plant interactions. These data expand the functional repertoire of fungal LysM proteins beyond effectors in plant defence responses and show that fungal LysM proteins are also involved in the self-regulation of fungal growth and development.

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.

Combined use of fluorescent dyes and flow cytometry to quantify the physiological state of Pichia pastoris during the production of heterologous proteins in high-cell-density fed-batch cultures.

Matching both the construction of a recombinant strain and the process design with the characteristics of the target protein has the potential to significantly enhance bioprocess performance, robustness, and reproducibility. The factors affecting the physiological state of recombinant Pichia pastoris Mut(+) (methanol utilization-positive) strains and their cell membranes were quantified at the individual cell level using a combination of staining with fluorescent dyes and flow cytometric enumeration. Cell vitalities were found to range from 5 to 95% under various process conditions in high-cell-density fed-batch cultures, with strains producing either porcine trypsinogen or horseradish peroxidase extracellularly. Impaired cell vitality was observed to be the combined effect of production of recombinant protein, low pH, and high cell density. Vitality improved when any one of these stress factors was excluded. At a pH value of 4, which is commonly applied to counter proteolysis, recombinant strains exhibited severe physiological stress, whereas strains without heterologous genes were not affected. Physiologically compromised cells were also found to be increasingly sensitive to methanol when it accumulated in the culture broth. The magnitude of the response varied when different reporters were combined with either the native AOX1 promoter or its d6* variant, which differ in both strength and regulation. Finally, the quantitative assessment of the physiology of individual cells enables the implementation of innovative concepts in bioprocess development. Such concepts are in contrast to the frequently used paradigm, which always assumes a uniform cell population, because differentiation between the individual cells is not possible with methods commonly used.

Perspectives on synthetic promoters for biocatalysis and biotransformation.

Acting on the transcriptional level, synthetic promoters have been useful tools for controlling gene expression and have applications in many fields. Here, we discuss synthetic promoters and libraries in regard to current and future applications in the field of biocatalysis or biotransformation. We also focus on synthetic promoter design principles and distinguish between prokaryotic and eukaryotic destinations. The natural toolboxes available for tuneable gene expression and the regulation of enzyme function are limited and primarily host specific. Synthetic biology offers generally applicable concepts and quick implementation. Smart alternatives to transcriptional regulation enrich the engineer's tool box for optimizing industrial enzyme production and host-cell physiology for whole-cell processes. Industrially applicable, tuneable enzyme cascades and artificial circuits for iterative up- and down-regulation will soon be achieved.

Promoter library designed for fine-tuned gene expression in Pichia pastoris.

Although frequently used as protein production host, there is only a limited set of promoters available to drive the expression of recombinant proteins in Pichia pastoris. Fine-tuning of gene expression is often needed to maximize product yield and quality. However, for efficient knowledge-based engineering, a better understanding of promoter function is indispensable. Consequently, we created a promoter library by deletion and duplication of putative transcription factor-binding sites within the AOX1 promoter (P(AOX1)) sequence. This first library initially spanned an activity range between approximately 6% and >160% of the wild-type promoter activity. After characterization of the promoter library employing a green fluorescent protein (GFP) variant, the new regulatory toolbox was successfully utilized in a 'real case', i.e. the expression of industrial enzymes. Characterization of the library under repressing, derepressing and inducing conditions displayed at least 12 cis-acting elements involved in P(AOX1)-driven high-level expression. Based on this deletion analysis, novel short artificial promoter variants were constructed by combining cis-acting elements with basal promoter. In addition to improving yields and quality of heterologous protein production, the new P(AOX1) synthetic promoter library constitutes a basic toolbox to fine-tune gene expression in metabolic engineering and sequential induction of protein expression in synthetic biology.