Abstract
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 μmax 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.
Highlights
From the seven currently known pathways for carbon dioxide (CO2) fixation in nature, the Calvin-Benson-Bassham (CBB) cycle can be seen as the main driver of primary production on Earth
The best performing isolates from our earlier study grew significantly better under autotrophic conditions, compared to the parental strain (0.008 h− 1) which had not gone through adaptive laboratory evolution (Gassler et al, 2020)
The presence of SNPs in the coding sequence (CDS) of the PRK gene after adaptive laboratory evolu tion (ALE) prompted us to analyze the specific activity of phosphor ibulokinase (Prk) of the parental and evolved strains
Summary
From the seven currently known pathways for carbon dioxide (CO2) fixation in nature, the Calvin-Benson-Bassham (CBB) cycle can be seen as the main driver of primary production on Earth. A lower activity upon introduction of the NMA1 1076 C > G mutation restored the improved growth phenotype of the evolved strain (Fig. 7) As seen in this experiment, and routinely in other cultivations, the reverse engineered NMA1 strain grew 1.4 times faster in terms of maximum obtainable growth rate compared to the parent (μmax = 0.011 ± 0.001 h− 1), which matches with evolved NMA1 strains. With the reverse engineering approach it is clearly shown that integration of the 1076 C > G mutation leading to the amino acid change Thr358Ile restores the improved autotrophic phenotype of the evolved strain by decreasing the specific Nma activity
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
