Fig. 1: Strain development strategy for enhanced recombinant secretory protein production by K. phaffii. | Communications Biology

Fig. 1: Strain development strategy for enhanced recombinant secretory protein production by K. phaffii.

From: A streamlined strain engineering workflow with genome-wide screening detects enhanced protein secretion in Komagataella phaffii

Fig. 1

a Outline of our strain development scheme. Genome-wide high-throughput screening (HTS) for gene-disruption-type effective factors, and combining perturbations, can improve secretion of a target protein (e.g. small antibody), and adaptive laboratory evolution (ALE) can be used to improve cell growth and further increase protein production. A random genome-disruption library was constructed using restriction enzyme-mediated integration (REMI) and effective factors that increase the secretion titer of an anti-lysozyme scFv antibody (a model of difficult-to-secrete proteins) were identified. Red cross(es), genes whose disruption has beneficial effects on protein secretion. The factors can be combined to further improve protein secretion. The strains can then be subjected to ALE for further improvement. b Schematic of the high-throughput, genome-wide screening workflow. Linearized plasmids carrying resistance genes were electroporated into wild-type K. phaffii to generate a random genome-disruption library. These insertional genome-disrupted mutants were then arrayed to 96-well format on square agarose plates using an automated colony picker. The 96-well format transformants were then inoculated into glycerol media (BMGY) in 96-deep-well plates for cultivation, followed by inoculation into an induction medium (BMMY). The small antibody titers of individual genome-disruption strains (>19,000 strains) were determined from cleared culture supernatants by ELISA. Genome-disruption sites of positives were identified as follows: genomic DNA from positives was extracted, digested with 8 restriction enzymes, then self-ligated and transformed into E. coli. The sites of insertion were then determined by Sanger sequencing of plasmids extracted from E. coli. Gene-disruption strains independently generated via targeted homologous recombination were then evaluated to confirm correct identification of the gene-disruption-type effective factors.

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