Adaptive laboratory evolution and reverse engineering enhances autotrophic growth in Pichia pastoris

Synthetic biology offers several routes for CO2 conversion into biomass or bio-chemicals, helping to avoid unsustainable use of organic feedstocks, which negatively contribute to climate change. The use of well-known industrial organisms, such as the methylotrophic yeast Pichia pastoris (Komagataella phaffii), for the establishment of novel C1-based bioproduction platforms could wean biotechnology from feedstocks with alternative use in food production. Recently, the central carbon metabolism of P. pastoris was re-wired following a rational engineering approach, allowing the resulting strains to grow autotrophically with a mu(max) of 0.008 h(-1), which was further improved to 0.018 h(-1) by adaptive laboratory evolution. Using reverse genetic engineering of single-nucleotide (SNPs) polymorphisms occurring in the genes encoding for phosphoribulokinase and nicotinic acid mononucleotide adenylyltransferase after evolution, we verified their influence on the improved autotrophic phenotypes. The reverse engineered SNPs lead to lower enzyme activities in putative branching point reactions and in reactions involved in energy balancing. Beyond this, we show how further evolution facilitates peroxisomal import and increases growth under autotrophic conditions. The engineered P. pastoris strains are a basis for the development of a platform technology, which uses CO2 for production of value-added products, such as cellular biomass, technical enzymes and chemicals and which further avoids consumption of organic feedstocks with alternative use in food production. Further, the identification and verification of three pivotal steps may facilitate the integration of heterologous CBB cycles or similar pathways into heterotrophic organisms.

Automated summary

Synthetic biology offers multiple pathways for converting CO2 into biomass or bio-chemicals, reducing the unsustainable use of organic feedstocks that contribute to climate change. The use of industrial organisms like Pichia pastoris allows for the development of novel bioproduction platforms with reduced reliance on alternative food production resources. By rewiring the central carbon metabolism and utilizing reverse genetic engineering, it is possible to enhance the autotrophic growth of microbes and explore potential applications for producing value-added products using CO2.