Continuous cell recycling in methylotrophic yeast Pichia pastoris to enhance product yields: a case study with Yarrowia lipolytica lipase Lip2

IF 2.5 4区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Shilpa Mohanty, Babbal, Shivani Chauhan, Mohini Talwar, Yogender Pal Khasa
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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 cerevisiae α-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.

Abstract Image

在养甲酵母 Pichia pastoris 中进行连续细胞循环以提高产品产量:利用 Yarrowia lipolytica 脂肪酶 Lip2 进行的案例研究
为了提高具有重要商业价值的脂溶性亚罗酵母脂肪酶 Lip2 的产品滴度,我们建立了一种基于营养甲基酵母 Pichia pastoris 的稳健细胞循环策略。在原核宿主大肠杆菌中表达 Lip2 蛋白会产生包涵体,而利用 SUMO 融合标签并不能提高其溶解度。因此,在甲醇诱导的 AOX1 启动子下,Lip2 通过酿酒酵母的 α 交配信号序列在 P. pastoris 中进行细胞外定向表达。在优化的条件下,对高产 Pichia 克隆进行了摇瓶表达研究,结果显示,在 OD600 为 10 和 20 的诱导条件下,糖基化 Lip2 的产量分别为 438.83 和 420.09 mg/L。高 Lip2 生产率的进一步目标是使用细胞保留技术,即细胞生物量循环利用,以获得更高的产品浓度和更好的产品质量。每 72 小时进行一次生物质循环,可使 Lip2 浓度提高 3.8 倍,产品累积体积浓度为 1,794 mg/L。高特定产品产率(YP/X)在 37.45-47.00 mg/g 干细胞重量(DCW)的范围内也保持到了五个保留周期。在 2.5 升发酵罐中以高细胞密度培养重组 P. pastoris,可获得 5.25 克/升 Lip2 酶,最大特定产率为 51.97 毫克/克 DCW。
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来源期刊
Biotechnology and Bioprocess Engineering
Biotechnology and Bioprocess Engineering 工程技术-生物工程与应用微生物
CiteScore
5.00
自引率
12.50%
发文量
79
审稿时长
3 months
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.
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