Optimization of dilution rate and mixed carbon feed for continuous production of recombinant plant sucrose:sucrose 1-fructosyltransferase in Komagataella phaffii.

The trisaccharide 1-kestose, a major constituent of commercial fructooligosaccharide (FOS) formulations, shows a superior prebiotic effect compared to higher-chain FOS. The plant sucrose:sucrose 1-fructosyltransferases (1-SST) are extensively used for selective synthesis of lower chain FOS. In this study, enhanced recombinant (r) 1-SST production was achieved in Komagataella phaffii (formerly Pichia pastoris) containing three copies of a codon-optimized Festuca arundinacea 1-SST gene. R1-SST production reached 47 U/mL at the shake-flask level after a 96-h methanol induction phase. A chemostat-based strain characterization methodology was adopted to assess the influence of specific growth rate (µ) on cell-specific r1-SST productivity (Qp) and cell-specific oxygen uptake rate (Qo) under two different feeding strategies across dilution rates from 0.02 to 0.05 h-1. The methanol-sorbitol co-feeding strategy significantly reduced Qo by 46 ± 2.4% compared to methanol-only feeding without compromising r1-SST productivity. Based on the data, a dilution rate of 0.025 h-1 was applied for continuous cultivation of recombinant cells to achieve a sustained r1-SST productivity of 5000 ± 64.4 U/L/h for 15 days.

High level production of stable human serum albumin in Pichia pastoris and characterization of the recombinant product.

Human serum albumin (HSA) is an important therapeutic used in clinical settings for restoration of blood volume and treatment of chemotherapy induced neutropenia. Currently sourced from human serum, it carries the risk of contamination with viruses. The production of stable extracellular recombinant (r)HSA was achieved at nearly 1 g/L at shake-flask level in Pichia pastoris (syn. Komagataella phaffii) containing a three-copy containing HSA expression cassette, prepared in vitro. The HSA specific transcripts were increased by 1.82- to 2.46-fold in the three-copy containing clones indicating increased transcript levels to result in enhanced production of extracellular rHSA. The purified rHSA displayed secondary structure, zeta potential, size distribution and biological efficacy that matched with that of the commercial HSA. Cultivation strategy was developed at bioreactor level for the single HSA expression cassette containing recombinant which led to productivity of 300 mg/L/d of rHSA with minimum proteolytic cleavage.