Recombinant protein production in Pichia pastoris: from transcriptionally redesigned strains to bioprocess optimization and metabolic modelling.

Pichia pastoris is one of the most widely used host for the production of recombinant proteins. Expression systems that rely mostly on promoters from genes encoding alcohol oxidase 1 or glyceraldehyde-3-phosphate dehydrogenase have been developed together with related bioreactor operation strategies based on carbon sources such as methanol, glycerol, or glucose. Although, these processes are relatively efficient and easy to use, there have been notable improvements over the last twenty years to better control gene expression from these promoters and their engineered variants. Methanol-free and more efficient protein production platforms have been developed by engineering promoters and transcription factors. The production window of P. pastoris has been also extended by using alternative feedstocks including ethanol, lactic acid, mannitol, sorbitol, sucrose, xylose, gluconate, formate or rhamnose. Herein, the specific aspects that are emerging as key parameters for recombinant protein synthesis are discussed. For this purpose, a holistic approach has been considered to scrutinize protein production processes from strain design to bioprocess optimization, particularly focusing on promoter engineering, transcriptional circuitry redesign. This review also considers the optimization of bioprocess based on alternative carbon sources and derived co-feeding strategies. Optimization strategies for recombinant protein synthesis through metabolic modelling are also discussed.

What makes Komagataella phaffii non-conventional?

The important industrial protein production host Komagataella phaffii (syn Pichia pastoris) is classified as a non-conventional yeast. But what exactly makes K. phaffii non-conventional? In this review, we set out to address the main differences to the 'conventional' yeast Saccharomyces cerevisiae, but also pinpoint differences to other non-conventional yeasts used in biotechnology. Apart from its methylotrophic lifestyle, K. phaffii is a Crabtree-negative yeast species. But even within the methylotrophs, K. phaffii possesses distinct regulatory features such as glycerol-repression of the methanol-utilization pathway or the lack of nitrate assimilation. Rewiring of the transcriptional networks regulating carbon (and nitrogen) source utilization clearly contributes to our understanding of genetic events occurring during evolution of yeast species. The mechanisms of mating-type switching and the triggers of morphogenic phenotypes represent further examples for how K. phaffii is distinguished from the model yeast S. cerevisiae. With respect to heterologous protein production, K. phaffii features high secretory capacity but secretes only low amounts of endogenous proteins. Different to S. cerevisiae, the Golgi apparatus of K. phaffii is stacked like in mammals. While it is tempting to speculate that Golgi architecture is correlated to the high secretion levels or the different N-glycan structures observed in K. phaffii, there is recent evidence against this. We conclude that K. phaffii is a yeast with unique features that has a lot of potential to explore both fundamental research questions and industrial applications.

Engineering of alcohol dehydrogenase 2 hybrid-promoter architectures in Pichia pastoris to enhance recombinant protein expression on ethanol.

The aim of this work is to increase recombinant protein expression in Pichia pastoris over the ethanol utilization pathway under novel-engineered promoter variants (NEPVs) of alcohol dehydrogenase 2 promoter (PADH2 ) through the generation of novel regulatory circuits. The NEPVs were designed by engineering of transcription factor binding sites (TFBSs) determined by in silico analyses and manual curation systematically, by (a) single-handedly replacement of specified TFBSs with synthetic motifs for Mxr1, Cat8, and Aca1 binding, and synthetic TATA-box integration; and, (b) nucleosome optimization. PADH2-Cat8-L2 and PADH2-Cat8-L1 designed by the integration of synthetic Cat8 binding sites were superior, and then PADH2-NucOpt . Compared to that with PADH2 at t = 20 hr of the fermentations, PADH2-Cat8-L2 allowed the highest increase in enhanced green fluorescent protein expression as 4.8-fold on ethanol and 3.8-fold on methanol; and, PADH2-NucOpt upregulated the expression 1.5-fold on ethanol and enhanced 3.2-fold on methanol. Using the superior two tools, Cat8-L2 and NucOpt, we designed PADH2-NucOpt-Cat8-L2 . With PADH2-NucOpt-Cat8-L2 , the expression in the fermentation of ethanol was upregulated 3.7-fold that is distinctly higher than that with PADH2-NucOpt but lower than that with PADH2-Cat8-L2 ; while on methanol compared to that with PADH2 , the expression was enhanced 8.8-fold. Extracellular recombinant human serum albumin production was also studied with PADH2-Cat8-L2 and PADH2-NucOpt , and average recombinant human serum albumin yield (YP/X ) on ethanol was 1.13 and 0.38 mg/gWCW, respectively; whereas with PADH2 , YP/X was 0.26 mg/gWCW . We conclude that as upregulation of transcription and enhanced expression correlate with the sequence of synthetic motifs and their location in the hybrid-promoter architectures of NEPVs in coordination with trans-acting factors, which are the design parameters in the engineering of binding sites; the NEPVs generated promising recombinant protein production platforms with a high impact on industrial scale production processes, as well as would open up new avenues for research in P. pastoris.

Double promoter expression systems for recombinant protein production by industrial microorganisms.

Using double promoter expression systems is a promising approach to increase heterologous protein production. In this review, current double promoter expression systems for the production of recombinant proteins (r-proteins) by industrially important bacteria, Bacillus subtilis and Escherichia coli; and yeasts, Saccharomyces cerevisiae and Pichia pastoris, are discussed by assessing their potentials and drawbacks. Double promoter expression systems need to be designed to maintain a higher specific product formation rate within the production domain. While bacterial double promoter systems have been constructed as chimeric tandem promoters, yeast dual promoter systems have been developed as separate expression cassettes. To increase production and productivity, the optimal transcriptional activity should be justified either by simultaneously satisfying the requirements of both promoters, or by consecutively stimulating the changeover from one to another in a biphasic process or via successive-iterations. Thus, considering the dynamics of a fermentation process, double promoters can be classified according to their operational mechanisms, as: i) consecutively operating double promoter systems, and ii) simultaneously operating double promoter systems. Among these metabolic design strategies, extending the expression period with two promoters activated under different conditions, or enhancing the transcriptional activity with two promoters activated under similar conditions within the production domain, can be applied independently from the host. Novel studies with new insights, which aim a rational systematic design and construction of dual promoter expression vectors with tailored transcriptional activity, will empower r-protein production with enhanced production and productivity. Finally, the current state-of-the-art review emphasizes the advantages of double promoter systems along with the necessity for discovering new promoters for the development of more effective and adaptive processes to meet the increasing demand of r-protein industry.

Lignocellulose degrading extremozymes produced by Pichia pastoris: current status and future prospects.

In this review article, extremophilic lignocellulosic enzymes with special interest on xylanases, ß-mannanases, laccases and finally cellulases, namely, endoglucanases, exoglucanases and ß-glucosidases produced by Pichia pastoris are reviewed for the first time. Recombinant lignocellulosic extremozymes are discussed from the perspectives of their potential application areas; characteristics of recombinant and native enzymes; the effects of P. pastoris expression system on recombinant extremozymes; and their expression levels and applied strategies to increase the enzyme expression yield. Further, effects of enzyme domains on activity and stability, protein engineering via molecular dynamics simulation and computational prediction, and site-directed mutagenesis and amino acid modifications done are also focused. Superior enzyme characteristics and improved stability due to the proper post-translational modifications and better protein folding performed by P. pastoris make this host favourable for extremozyme production. Especially, glycosylation contributes to the structure, function and stability of enzymes, as generally glycosylated enzymes produced by P. pastoris exhibit better thermostability than non-glycosylated enzymes. However, there has been limited study on enzyme engineering to improve catalytic efficiency and stability of lignocellulosic enzymes. Thus, in the future, studies should focus on protein engineering to improve stability and catalytic efficiency via computational modelling, mutations, domain replacements and fusion enzyme technology. Also metagenomic data need to be used more extensively to produce novel enzymes with extreme characteristics and stability.

Second generation Pichia pastoris strain and bioprocess designs.

Yeast was the first microorganism used by mankind for biotransformation processes that laid the foundations of industrial biotechnology. In the last decade, Pichia pastoris has become the leading eukaryotic host organism for bioproduct generation. Most of the P. pastoris bioprocess operations has been relying on toxic methanol and glucose feed. In the actual bioeconomy era, for sustainable value-added bioproduct generation, non-conventional yeast P. pastoris bioprocess operations should be extended to low-cost and renewable substrates for large volume bio-based commodity productions. In this review, we evaluated the potential of P. pastoris for the establishment of circular bioeconomy due to its potential to generate industrially relevant bioproducts from renewable sources and waste streams in a cost-effective and environmentally friendly manner. Furthermore, we discussed challenges with the second generation P. pastoris platforms and propose novel insights for future perspectives. In this regard, potential of low cost substrate candidates, i.e., lignocellulosic biomass components, cereal by-products, sugar industry by-products molasses and sugarcane bagasse, high fructose syrup by-products, biodiesel industry by-product crude glycerol, kitchen waste and other agri-food industry by products were evaluated for P. pastoris cell growth promoting effects and recombinant protein production. Further metabolic pathway engineering of P. pastoris to construct renewable and low cost substrate utilization pathways was discussed. Although, second generation P. pastoris bioprocess operations for valorisation of wastes and by-products still in its infancy, rapidly emerging synthetic biology tools and metabolic engineering of P. pastoris will pave the way for more sustainable environment and bioeconomy. From environmental point of view, second generation bioprocess development is also important for waste recycling otherwise disposal of carbon-rich effluents creates environmental concerns. P. pastoris high tolerance to toxic contaminants found in lignocellulosic biomass hydrolysate and industrial waste effluent crude glycerol provides the yeast with advantages to extend its applications toward second generation P. pastoris strain design and bioprocess engineering, in the years to come.

Hybrid-architectured promoter design to deregulate expression in yeast.

Hybrid-architectured promoter design to deregulate expression in yeast under modulating power of carbon sources involves replacing native cis-acting DNA sequence(s) with de novo synthetic tools in coordination with master regulator transcription factor (TF) to alter crosstalk between signaling pathways, and consequently, transcriptionally rewire the expression. Hybrid-promoter architectures can be designed to mimic native promoter architectures in yeast's preferred carbon source utilization pathway. The method aims to generate engineered promoter variants (EPVs) that combine the advantages of being an exceptionally stronger EPV(s) than the naturally occurring promoters and permit "green-and-clean" production on a non-toxic carbon source. To implement the method, a predetermined essential part of the general transcription machinery is targeted. This targeting involves cis-acting DNA sequences to be replaced with synthetic cis-acting DNA sites in coordination with the targeted TF that must bind for transcription machinery activation. The method needs genomic and functional information that can lead to the discovery of the master TF(s) and synthetic cis-acting DNA elements, which enable the engineering of binding of master regulator TF(s). By introducing our recent work on the engineering of Pichia pastoris (syn. Komagataella phaffii) alcohol oxidase 1 (AOX1) hybrid-promoter architectures, we provide the method and protocol for the hybrid-architectured EPV design to deregulate expression in yeast. The method can be adapted to other promoters in different substrate utilization pathways in P. pastoris, as well as in other yeasts.

Hybrid-architectured promoter design to engineer expression in yeast.

Engineered promoters are key components that allow engineered expression of genes in the cell-factory design. Promoters having exceptional strength are attractive candidates for designing metabolic engineering strategies for tailoring de novo production strategies that require directed evolution methods by engineering with de novo synthetic biology tools. Engineered promoter variants (EPVs) of naturally occurring promoters (NOPs) can be designed using metabolic engineering strategies and synthetic biology tools if the genes encoding the activating transcription factors (TFs) exist in the genome and are expressed and synthesized at non-limiting concentrations within the cell. The hybrid-architectured EPV design method targets an essential and predetermined part of the general transcription machinery. That is cis-acting DNA site(s) in coordination with the trans-acting factor(s) that must bind for the regulated transcription machinery activation. The method needs genomic and functional information that can lead to the discovery of the master TF(s) and synthetic cis-acting DNA elements, enabling the engineering of binding of master regulator TF(s). The method aims to generate EPVs that combine the advantages of being an exceptional stronger EPV(s) than the NOPs and permit "green-and-clean production" on a non-toxic carbon source, such as ethanol or glucose. By introducing our recent work on the engineering of ADH2 hybrid-promoter architectures to enhance recombinant protein expression on ethanol, we provide the method and protocol for the design of ADH2 hybrid-promoter architectures that can be adapted to other promoters in different substrate utilization pathways in Pichia pastoris (syn. Komagataella phaffii), as well as in other yeasts.

Engineered Deregulation of Expression in Yeast with Designed Hybrid-Promoter Architectures in Coordination with Discovered Master Regulator Transcription Factor.

Engineered promoters are key components in the cell-factory design, allowing precise and enhanced expression of genes. Promoters having exceptional strength are attractive candidates for designing metabolic engineering strategies for tailoring de novo production strategies that require directed evolution methods by engineering with de novo synthetic biology tools. Here, the custom-designed AOX1 hybrid-promoter architectures in coordination with targeted transcription factors are shown, transcriptionally rewired the expression over methanol-free substrate-utilization pathway(s) and converted methanol-dependent Pichia pastoris alcohol oxidase 1(AOX1) promoter (PAOX1 ) expression into a non-toxic carbon-source-regulated system. AOX1 promoter variants are engineered by replacing specified cis-regulatory DNA elements with synthetic Adr1, Cat8, and Aca2 cis-acting DNA elements for Mxr1, Cat8, and Aca1 binding, respectively. Applications of the engineered-promoters are validated for eGFP expression and extracellular human serum albumin production. The hybrid-promoter architecture designed with single Cat8 cis-acting DNA element deregulates the expression on ethanol. Compared with PAOX1 on methanol, the expression on ethanol is increased with i) PAOX1/Cat8-L3 (designed with single Cat8 cis-acting element) to 74%, ii) PAOX1/Adr1-L3/Cat8-L3 (designed with single- Cat8 and Adr1 cis-acting elements) to 85%, and for further consolidation of deregulated expression iii) PeAOX1 (designed with triplet- Cat8 and Adr1 cis-acting elements) 1.30-fold, at t = 20 h of batch cultivations.