Systematic development of biomass overproducing Scheffersomyces stipitis for high-cell-density fermentations

The development of economically feasible bio-based process requires efficient cell factories capable of producing the desired product at high titer under high-cell-density fermentation. Herein we present a combinatorial approach based on systems metabolic engineering and metabolic evolution for the development of efficient biomass-producing strain. Systems metabolic engineering guided by flux balance analysis (FBA) was first employed to rationally design mutant strains of Scheffersomyces stipitis with high biomass yield. By experimentally implementing these mutations, the biomass yield was improved by 30% in GPD1, 25% in TKL1, 30% in CIT1, and 44% in ZWF1 overexpressed mutants compared to wild-type. These designed mutants were further fine-tuned through metabolic evolution resulting in the maximal biomass yield of 0.49 g-cdw/g-glucose, which matches well with predicted yield phenotype. The constructed mutants are beneficial for biotechnology applications dealing with high cell titer cultivations. This work demonstrates a solid confirmation of systems metabolic engineering in combination with metabolic evolution approach for efficient strain development, which could assist in rapid optimization of cell factory for an economically viable and sustainable bio-based process.