BACKGROUND:The yeast Pichia pastoris is a popular host organism for production of a range of biological products, several of which are intracellular. The disruption of yeast cells by homogenization also releases large quantities of lipids, which can foul the downstream membranes and chromatography matrices used for purification. This work examines lipid removal from yeast cells following homogenization by enzymatic degradation and its impact on the performance of the subsequent centrifugation and filtration.RESULTS: Lipase treatment of cell homogenate at 37°C for 2 h, followed by clarification using a scaled-down mimic of disc stack centrifugation, resulted in a 6.5-fold improvement in solids removal when compared to untreated feed material. The lipasetreated and untreated materials that had undergone initial centrifugation were then tested for filtration performance by passing the material through a 0.45 ∼m polyethylene sulfone membrane under constant flux. A 50% increase in throughput was observed in comparison to the untreated material.CONCLUSION: These proof-of-concept data suggest enzymatic digestion of lipids, analogous to the widely performed DNA reduction using nucleases, could be a valuable process improvement strategy.
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.
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