Biology subjects

Ata, O.

Publications and source records attributed to Ata, O..

4 recordsLinked to original sources

bioRxiv2025

Synthetic autotrophic yeast enables high itaconic acid production from CO2 via integrated pathway and process design

Single carbon (C1) substrates are gaining importance as future feedstocks for the production of bio-based chemicals. Carbon dioxide, a major greenhouse gas, offers a promising alternative to the traditional feedstocks to shift towards C1-based, sustainable processes. Here, we present a synthetic autotrophic Komagataella phaffii (Pichia pastoris) that is able to produce itaconic acid by the direct conversion of CO2, achieving final titers of approximately 12 g L-1 in bioreactor cultivations. We show that a combined approach that integrates balancing the flux between the Calvin-Benson-Bassham (CBB) cycle and itaconic acid metabolism with process design was essential to enhance the production. Our study demonstrates the potential of K. phaffii as a microbial platform using CO2 as the direct carbon source, aligning with the future goals of establishing sustainable bioprocesses.

synthetic biology↗

bioRxiv2025

Integration of strain and process optimization to increase autotrophic growth of engineered Komagataella phaffii

Synthetic autotrophs are a promising platform for sustainable bioproduction using CO2 as substrate. The methylotrophic yeast Komagataella phaffii has been engineered to use CO2 as the sole carbon source by integration of the Calvin-Benson-Bassham (CBB) cycle, based on its native methanol assimilating xylulose monophosphate pathway (XuMP) cycle. Initial growth rates were low, but could be doubled by adaptive laboratory evolution (ALE). Beneficial mutations led to a decrease of CBB cycle reactions, indicating further limitations. During this study, temperature was identified as one of the key process parameters to improve autotrophic growth. For this reason, a new round of adaptive laboratory evolution was performed at the identified optimal cultivation temperature of 25{degrees}C, resulting in isolates growing up to 50 % faster compared to the control strain. Whole genome resequencing followed by reverse engineering helped to identify first key mutations of the evolved strains. In addition, targeted engineering was performed by increasing the copy number of the key gene of the CBB cycle RuBisCO, which is the bottleneck of carbon fixation. Combining this with the optimal cultivation temperature boosted maximum specific growth rates of the autotrophic K. phaffii strain. In comparison to ALE, the targeted engineering still is lagging behind a bit. Starting from the initial condition, growth was boosted more than 2.5-fold in this study to a maximum of 0.025 h-1.

synthetic biology↗

bioRxiv2024

Efficient production of itaconic acid from the single carbon substrate methanol with engineered Komagataella phaffii

BackgroundAmidst the escalating carbon dioxide levels resulting from fossil fuel consumption, there is a pressing need for sustainable, bio-based alternatives to underpin future global economies. Single carbon feedstocks, derived from CO2, represent promising substrates for biotechnological applications. Especially methanol is gaining prominence for bio-production of commodity chemicals. ResultsIn this study, we show the potential of Komagataella phaffii as a production platform for itaconic acid using methanol as the carbon source. Successful integration of heterologous genes from Aspergillus terreus (cadA, mttA and mfsA) alongside fine-tuning of the mfsA gene expression, led to promising initial itaconic acid titers of 28 g{middle dot}L-1 after five days of fed-batch cultivation. Through the combined efforts of process optimization and strain engineering strategies we further boosted the itaconic acid production reaching titers of 55 g{middle dot}L-1 after less than five days of methanol feed, whilst increasing the product yield on methanol from 0.06 g{middle dot}g- 1 to 0.24 g{middle dot}g-1. ConclusionOur results highlight the potential of K. phaffii as a methanol-based platform organism for sustainable biochemical production.

microbiology↗

bioRxiv2023

A native phosphoglycolate salvage pathway of the synthetic autotrophic yeast Komagataella phaffii

Synthetic autotrophs can serve as chassis strains for bioproduction from CO2 as a feedstock to take measures against the climate crisis. Integration of the Calvin-Benson-Bassham (CBB) cycle into the methylotrophic yeast Komagataella phaffii (Pichia pastoris) enabled it to use CO2 as the sole carbon source. The key enzyme in this cycle is ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzing the carboxylation step. However, this enzyme is error prone to perform an oxygenation reaction leading to the production of toxic 2-phosphoglycolate. Native autotrophs have evolved different recycling pathways for 2-phosphoglycolate. However, for synthetic autotrophs, no information is available for the existence of such pathways. Deletion of CYB2 in the autotrophic K. phaffii strain led to the accumulation of glycolate, an intermediate in phosphoglycolate salvage pathways, suggesting that such a pathway is enabled by native K. phaffii enzymes. 13C tracer analysis with labeled glycolate indicated that the yeast pathway recycling phosphoglycolate is similar to the plant salvage pathway. This orthogonal yeast pathway may serve as a sensor for RuBisCO oxygenation, and as an engineering target to boost autotrophic growth rates in K. phaffii.

microbiology↗