methanol-independent-expression-pichia-pastoris-employing-de

Methanol Independent Expression by <em>Pichia Pastoris</em> Employing De-repression Technologies

Methanol is a well-established carbon source and inducer for efficient protein production employing Pichia pastoris (P. pastoris) as a host on micro-, lab ...

bisy e.U.

Technical University of Graz

Cellulose is a highly available and renewable carbon source in nature. However, it cannot be directly metabolized by most microbes including Komagataella phaffii (formerly Pichia pastoris), which is a frequently employed host for heterologous protein expression and production of high-value compounds. A K. phaffii strain was engineered that constitutively co-expresses an endoglucanase and a β-glucosidase both from Aspergillus niger and an exoglucanase from Trichoderma reesei under the control of bidirectional promoters. This engineered strain was able to grow on cellobiose and carboxymethyl cellulose (CMC) but not on Avicel. However, the detected release of cellobiose from Avicel by using the produced mixture of endoglucanase and exoglucanase as well as the released glucose from Avicel by using the produced mixture of all three cellulases at 50 °C indicated the production of exoglucanase under the liquid culture conditions. The successful expression of three cellulases in K. phaffii demonstrated the feasibility to enable K. phaffii to directly use cellulose as a carbon source for producing recombinant proteins or other high-value compounds.

Construction of a cellulose-metabolizing Komagataella phaffii (Pichia pastoris) by co-expressing glucanases and β-glucosidase.

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, β-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.

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.

Anton Glieder

bisy e.U.

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies