Shilpa Mohanty, Babbal, Shivani Chauhan, Mohini Talwar, Yogender Pal Khasa
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引用次数: 0
Abstract
A robust cell recycling strategy based on the methylotrophic yeast, Pichia pastoris, was established to enhance product titers of the commercially important Yarrowia lipolytica lipase Lip2. The expression of Lip2 protein in the prokaryotic host Escherichia coli resulted in inclusion bodies, whereas utilization of SUMO fusion tag could not improve its solubility. Therefore, Lip2 extracellular expression was targeted via the Saccharomyces cerevisiaeα-mating signal sequence in P. pastoris under methanol-inducible AOX1 promoter. Shake flask expression studies of hyper-producer Pichia clone under optimized conditions resulted in 438.83 and 420.09 mg/L of glycosylated Lip2 production after induction at an OD600 of 10 and 20, respectively. A high Lip2 productivity was further targeted using a cell retention technique where the cell biomass was recycled to obtain higher product concentration with improved product quality. The biomass recycling at every 72 h followed a 3.8-fold enhanced Lip2 concentration with a cumulative volumetric product concentration of 1,794 mg/L. A high specific product yield (YP/X) in the range of 37.45–47.00 mg/g dry cell weight (DCW) was also maintained up to five retention cycles. Furthermore, higher cumulative protein yields were obtained from the 5-time recycled cells compared to five individual batch runs at shake flask up to 72 h. High cell density cultivation of recombinant P. pastoris in a 2.5-L fermenter yielded 5.25 g/L of Lip2 enzyme with a maximum specific yield of 51.97 mg/g DCW.
期刊介绍:
Biotechnology and Bioprocess Engineering is an international bimonthly journal published by the Korean Society for Biotechnology and Bioengineering. BBE is devoted to the advancement in science and technology in the wide area of biotechnology, bioengineering, and (bio)medical engineering. This includes but is not limited to applied molecular and cell biology, engineered biocatalysis and biotransformation, metabolic engineering and systems biology, bioseparation and bioprocess engineering, cell culture technology, environmental and food biotechnology, pharmaceutics and biopharmaceutics, biomaterials engineering, nanobiotechnology, and biosensor and bioelectronics.