Increasing efficiency of CO2 assimilation in synthetic autotrophic Pichia pastoris

Background.

Recently a synthetic autotrophic Pichia pastoris strain was developed at acib and BOKU (Gassler et al. 2019). While CO2 assimilation of this strain is highly efficient in terms of mass and energy balance, the rates are limited by the specific activity of the orthologous Calvin-Benson-Bassham cycle setup. Potential bottlenecks are chaperones and cofactors of RuBisCO, and the peroxisomal energy and electron transport.

Aims and methods.

In this respect we plan to evaluate and engineer the role of native cofactors of Thiobacillus denitrificans type II RuBisCO (such as chaperones GroEL/GroES and potential cofactors CbbQ/CbbO), and to overexpress the native yeast peroxisomal ATP transporter gene, as well as components of the malate/oxaloacetate shuttle to exchange electrons from cytosolic to peroxisomal NADH, such as malate dehydrogenase (Visser et al. 2007). As growth of the yeast strain depends fully on the intact CBB cycle and its rate, this offers a unique platform to engineer the optimum recombinant setup of RuBisCO and its cofactors, including evolutionary engineering. This will be addressed also by creating libraries of RuBisCO mutants as well as expression libraries combining variants of expression strength of the CBB genes. By utilizing the growth coupling of the efficiency of CO2 assimilation we can directly select for the best variants, as well as support cell engineering by adaptive laboratory evolution. Selected variants will be characterized by sequencing the respective gene variants or the genomes of mutants, respectively.