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 Ca...

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Bibliographic Details
Published in:ACS synthetic biology 2023-03, Vol.12 (3), p.657-663
Main Authors: Bandyopadhyay, Sushobhan, Pavlika, Vasos, Bracewell, Daniel G., Nesbeth, Darren N.
Format: Article
Language:English
Subjects:
Carbon - metabolism
Letter
Lipase - genetics
Lipase - metabolism
Methanol - metabolism
Pichia - metabolism
Vaccines, Virus-Like Particle - metabolism
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Summary: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.
ISSN:2161-5063
2161-5063
DOI:10.1021/acssynbio.2c00342