Biology subjects

Fickers, P.

Publications and source records attributed to Fickers, P..

4 recordsLinked to original sources

bioRxiv2026

Engineering reduced nicotinamide cofactor metabolism for enhanced cell growth and succinic acid production in a succinate dehydrogenase deficient Yarrowia lipolytica strain

BackgroundSuccinic acid (SA) is a four-carbon dicarboxylic acid of considerable industrial relevance, with applications spanning the food, chemical, and pharmaceutical sectors. The remarkable acid tolerance of the yeast Yarrowia lipolytica makes it a promising microbial cell factory for SA production. Numerous metabolic engineering strategies have focused on disrupting genes encoding the succinate dehydrogenase (SDH) complex to enhance SA accumulation. However, such a modification is associated with impaired growth and the accumulation of by-products, notably acetic acid (AA). ResultsTo improve growth capacity, SA productivity, and reduce AA formation in Y. lipolytica SDH5-deficient strains (Sdh5{Delta}), carbon flux from glycolysis was partially redirected toward the pentose phosphate pathway by overexpression of the native genes encoding glucose-6-phosphate dehydrogenase (ZWF1) and 6-phosphogluconate dehydrogenase (GND1), thereby enhancing NADPH generation. The resulting strain was further engineered to increase NADH availability for the mitochondrial electron transport chain by overexpressing genes encoding either a mutated NADPH-dependent malate dehydrogenase (TfMdh) from Thermus flavus or the soluble transhydrogenase (EcSthA) from Escherichia coli, enabling indirect conversion of NADPH to NADH. This strategy resulted in 2-fold and 2.2-fold increase in SA productivity and titre, respectively, compared to the Sdh5{Delta}-ALE strain during bioreactor cultivation on glucose-based media. Moreover, AA accumulation was reduced 1.2-fold, while growth rates were significantly improved. ConclusionsThe proposed engineering strategies, especially heterologous expression of EcSthA, partly alleviated energy limitations in Y. lipolytica Sdh5{Delta} strain, resulting in improved SA productivity and growth performance.

molecular biology↗

bioRxiv2025

Engineering serine metabolism to enhance AOX1 promoter self-induction in a formate dehydrogenase-deficient Komagataella phaffii

The methylotrophic yeast Komagataella phaffii is a premier host for recombinant protein (rProt) production, traditionally relying on methanol induction of the alcohol oxidase 1 promoter (pAOX1). However, methanol flammability and associated industrial limitations have motivated the search for methanol-free induction systems. We recently demonstrated that disruption of formate dehydrogenase (FDH) in K. phaffii allows endogenous formate, derived from tetrahydrofolate (THF)-mediated C1 metabolism, to induce pAOX1 without addition of external inducers. Building on this, we hypothesized that increasing intracellular formate production by enhancing serine biosynthesis could further improve promoter induction and rProt productivity. Overexpression of SER3, encoding 3-phosphoglycerate dehydrogenase, the rate-limiting enzyme in serine synthesis, significantly increased pAOX1-driven expression of an intracellular reporter protein (eGFP) and secreted glucose oxidase (Gox) from Aspergillus niger, without compromising cell fitness. Enhanced formate accumulation and stronger pAOX1 induction were observed in both micro- and bioreactor cultivations using sorbitol or glycerol-sorbitol mixtures. In bioprocess conditions, SER3 overexpression led to a 30% increase in specific Gox activity compared to the parental FdhKO strain. This study provides a cost-effective metabolic engineering strategy for methanol-free, self-inducible expression systems in K. phaffii based on pAOX1, enabling safer and more sustainable industrial rProt production.

bioengineering↗

bioRxiv2025

Sorbitol uptake and oxygen transfer shape AOX1 promoter induction under methanol-free conditions in Komagataella phaffii lacking formate dehydrogenase

For decades, expression systems based on the methanol-regulated AOX1 promoter (pAOX1) from the alcohol oxidase 1 gene have served as a benchmark for recombinant protein (rProt) production in Komagataella phaffii. However, methanol-free processes are increasingly being developed to overcome the drawbacks of methanol utilization, particularly its toxicity and flammability. The use of formate as a pAOX1 inducer in combination with sorbitol, a non-repressive carbon source, has emerged as a promising alternative to methanol-based expression systems. Recently, we demonstrated that formate derived from the tetrahydrofolate-mediated one-carbon (THF-C1) metabolism accumulates in K. phaffii cells deficient in formate dehydrogenase (FdhKO) when grown in sorbitol-based methanol-free medium. Using the lipase CalB from Candida antarctica as a model protein, we observed that rProt productivity in an FdhKO strain grown on sorbitol was comparable to that of an Fdh-proficient strain grown on methanol. However, sorbitol is inefficiently metabolized in K. phaffii, leading to a low growth rate and potentially limiting rProt productivity due to insufficient energy and carbon supply. Here, we increased sorbitol uptake rate, and thus improved sorbitol metabolism, by overexpressing the gene encoding sorbitol dehydrogenase (SOR1) in an FdhKO strain. Our results demonstrate that while increased sorbitol metabolism promotes biomass formation, it reduces pAOX1 induction, as evidenced by lower formate accumulation and decreased rProt productivity, both for intracellular eGFP and secreted proteins namely CalB lipase and glucose oxidase GOx from Aspergillus niger in SOR1-overexpressing strains. Additionally, oxygen availability for cells influences these dynamics, with lower oxygen transfer favoring higher pAOX1 induction due to increased formate accumulation in an FdhKO strain. Our data also suggests that at low oxygen transfer and low sorbitol uptake rate, the proportion of cells in an induced state increased significantly. This work provides valuable insights into the interplay between sorbitol metabolism and oxygen transfer conditions, contributing to the development of improved recombinant protein production strategies in K. phaffii.

bioengineering↗

bioRxiv2024

Formate from THF-C1 metabolism induces the AOX1 promoter in formate dehydrogenase-deficient Pichia pastoris

In Pichia pastoris (Komagataella phaffii), formate is a recognized alternative inducer to methanol for expression systems based on the AOX1 promoter (pAOX1). By disrupting the formate dehydrogenase encoding FDH1 gene, we converted such a system into a self-induced one, as adding any inducer in the culture medium is no longer requested for pAOX1 induction. In cells, formate is generated from serine through the THF-C1 metabolism, and it cannot be converted into carbon dioxide in an fdh1{Delta} strain. Under non-repressive culture conditions, such as on sorbitol, the intracellular formate generated from the THF-C1 metabolism is sufficient to induce pAOX1 and initiate protein synthesis. This was evidenced for two model proteins, namely intracellular eGFP and secreted CalB lipase from C. antarctica. Similar protein productivities were obtained for an fdh1{Delta} strain on sorbitol and a non-disrupted strain on sorbitol-methanol. Considering a P. pastoris fdh1{Delta} strain as a workhorse for recombinant protein synthesis paves the way for the further development of methanol-free processes in P. pastoris.

microbiology↗