A Biological OR(XNOR) Logic Gate Couples Carbon Source and Transgene Expression Switching in a Komagataella phaffii (Pichia pastoris) Strain Co-producing Process-Enhancing Lipase and a Virus- like Particle (VLP) Vaccine

We constructed a three-input biological logic gate: S OR (G XNOR M), where S is sorbitol, G is glycerol, and M is methanol, to optimize co-expression of two transgenes in Komagataella phaffii using batch-mode carbon source switching (CSS). K. phaffii was engineered to harbor transgenes encoding a Candida rugosa triacylglycerol lipase, which can enhance downstream processing by removing host cell lipids from homogenates, and the hepatitis B virus surface antigen (HBsAg), a protein that self assembles into a virus-like particle (VLP) vaccine. Using the native alcohol oxidase 1 (PAOX1) and enolase 1 (PENO1) promoters to direct VLP vaccine and lipase expression, respectively, successfully provided an OR(XNOR) gate function with double-repression as the output. This logic gate functionality enabled use of CSS to ensure that approximately 80% of total VLP yield was accumulated before cells were burdened with lipase expression in 250 mL DasGip bioreactor cultivation.

Automated summary

We constructed a three-input biological logic gate, S OR (G XNOR M), to optimize co-expression of two transgenes in Komagataella phaffii using batch-mode carbon source switching. This logic gate functionality enabled use of CSS to accumulate approximately 80% of total virus-like particle (VLP) yield before cells were burdened with lipase expression in 250 mL DasGip bioreactor cultivation.