Formatotrophic Komagataella phaffii expressing recombinant xylanase via metabolic engineering

Formate utilization as a sustainable carbon source for microbial production of high-value chemicals and heterologous proteins presents considerable safety and environmental benefits over conventional feedstocks. As a low-cost, CO₂-derived compound, formate serves as a non-flammable and non-toxic alternative to methanol for induction of recombinant expression in <i>Komagataella phaffii</i>. However, since native <i>K. phaffii</i> only utilizes formate as an energy source rather than a carbon substrate for biomass synthesis, we engineered a synthetic peroxisomal formate assimilation pathway by introducing heterologous acetyl-CoA synthetase (ACS) and acetaldehyde dehydrogenase (ACDH), thereby enabling formatotrophic growth with formate as the sole carbon source. This chassis was further optimized through (i) co-expression of the transcriptional activator Mit1 to enhance the coupling efficiency of the ACS-ACDH module, and (ii) reinforcement of the Xu5P pathway by overexpressing dihydroxyacetone synthase (DAS1) and ribulose-5-phosphate-3-epimerase (RPE) to redirect metabolic flux. The resulting formatotrophic <i>K. phaffi</i> strain achieved a specific growth rate of 0.012 h^-1 in basal salt medium with formate as the sole carbon source, and produced 30.9 U/(mL·OD₆₀₀) of xylanase from <i>Aspergillus niger</i> ATCC 1015 as a model heterologous protein. Furthermore, ¹³C isotopic tracing confirmed the incorporation of formate-derived carbon into central metabolism for the biosynthesis of amino acids, nucleotides, and structural carbohydrates, validating active formate assimilation. This study establishes a microbial platform for formate-based production of heterologous proteins and underscores the potential of metabolic engineering to advance sustainable biomanufacturing from one-carbon feedstocks.