Engineering transport of C1 substrates over yeast membranes

Background.

Engineering transport across biological membranes can direct reaction balances and enhance microbial efficiency. Despite its immense potential, transport engineering is hindered by challenges in protein purification and limited understanding of membrane transporters. While it was previously considered that most compounds passively diffuse through membranes, it is now recognized that specific transporters are required for even the smallest polar molecules (Ishibashi et al., 2011).
C1 feedstocks like methane, methanol, formate, CO₂, and CO offer advantages over traditional carbon sources such as glucose (Cotton et al, 2020). The methylotrophic yeast Komagataella phaffii possesses one of the most efficient methanol catabolism pathways. However, the mechanism by which methanol enters the cell to meet the demand for rapid consumption remains unclear and cannot be attributed solely to passive diffusion.

Aims and methods.

Recent work by Gassler et al. (2020) demonstrated the successful conversion of K. phaffii into an autotrophic yeast, capable of using CO2 as its sole carbon source and methanol as an energy source. While compartmentalization of the CBB cycle in K. phaffii peroxisomes improved CO₂ assimilation efficiency, no additional engineering of CO2 transport or concentration was integrated into the strain yet.
Engineered C1 utilizing K. phaffii strains offer opportunities to establish a circular bioeconomy. Despite significant efforts, current strains do not grow optimally at ambient CO₂ concentrations, and methanol uptake remains poorly characterized. The CiTrY project seeks to address these limitations by focusing on an often-overlooked bottleneck in microbial cell factory research: transport across biological membranes.
The work is based on an analysis of transcriptome and proteome data to identify putative transporters in K. phaffii, as well as a selection of transporter candidate from other microorganisms. Strains will be engineered by CRISPR/Cas9-based gene deletion or insertion and tested in bioreactors for specific uptake and consumption rates, involving ¹³C tracer studies to follow the metabolic fate of methanol and CO₂.

(1) Gassler, T., Sauer, M., Gasser, B., Egermeier, M., Troyer, C., Causon, T., Hann, S., Mattanovich, D., & Steiger, M. G. (2020). The industrial yeast Pichia pastoris is converted from a heterotroph into an autotroph capable of growth on CO2. Nature biotechnology, 38(2), 210–216. https://doi.org/10.1038/s41587-019-0363-0
(2) Ishibashi, K., Kondo, S., Hara, S., & Morishita, Y. (2011). The evolutionary aspects of aquaporin family. American journal of physiology. Regulatory, integrative and comparative physiology, 300(3), R566–R576. https://doi.org/10.1152/ajpregu.90464.2008
(3) Cotton, C. A., Claassens, N. J., Benito-Vaquerizo, S., & Bar-Even, A. (2020). Renewable methanol and formate as microbial feedstocks. Current opinion in biotechnology, 62, 168–180. https://doi.org/10.1016/j.copbio.2019.10.002