A multistrategy approach for improving the expression of <i>E. coli</i> phytase in <i>Pichia pastoris</i>

Helian, Yuankun; Gai, Yuanming; Fang, Huan; Sun, Yumei; Zhang, Dawei

doi:10.1007/s10295-020-02311-6

Cited by 11 publications

(6 citation statements)

References 56 publications

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“…The present study obtained a final phytase activity of 12.65 MU/L which represented a 36-fold improvement compared to flask fermentations (Navone et al, 2021a). In instrumented fermenters, the reported commercial phytase production comprises levels from 5 MU/L to 30 MU/L (Helian et al, 2020;Luo et al, 2007;Mittal et al, 2013;Yao et al, 1998), suggesting the capability of our P.…”

Section: Ph Profile and Stability Differential Scanning Calorimetry (...supporting

confidence: 47%

“…The present study obtained a final phytase activity of 12.65 MU/L which represented a 36‐fold improvement compared to flask fermentations (Navone et al, 2021a). In instrumented fermenters, the reported commercial phytase production comprises levels from 5 MU/L to 30 MU/L (Helian et al, 2020; Luo et al, 2007; Mittal et al, 2013; Yao et al, 1998), suggesting the capability of our P. pastoris P1 strain to produce commercially relevant levels of phytase and the potential to continue improving phytase production by metabolic engineering optimization such as the improvement of the protein secretion capability of the α‐mating factor secretion signal (Helian et al, 2020). The specific activity of E. coli phytase has been reported to be around 3.6 MU/g (of pure phytase) (Li et al, 2015), which was similar to the specific phytase activity of 4 MU/g obtained in this study after the de‐repression phase (Figure 2 and Table 3).…”

Section: Discussionmentioning

confidence: 99%

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Improving phytase production in Pichia pastoris fermentations through de‐repression and methanol induction optimization

Luna‐Flores,

Weng,

Wang

et al. 2023

Biotech & Bioengineering

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Pichia pastoris (Komagataella phaffii) is a fast‐growing methylotrophic yeast with the ability to assimilate several carbon sources such as methanol, glucose, or glycerol. It has been shown to have outstanding secretion capability with a variety of heterologous proteins. In previous studies, we engineered P. pastoris to co‐express Escherichia coli AppA phytase and the HAC1 transcriptional activator using a bidirectional promoter. Phytase production was characterized in shake flasks and did not reflect industrial conditions. In the present study, phytase expression was explored and optimized using instrumented fermenters in continuous and fed‐batch modes. First, the production of phytase was investigated under glucose de‐repression in continuous culture at three dilution factors, 0.5 d−1, 1 d−1, and 1.5 d−1. The fermenter parameters of these cultures were used to inform a kinetic model in batch and fed‐batch modes for growth and phytase production. The kinetic model developed aided to design the glucose‐feeding profile of a fed‐batch culture. Kinetic model simulations under glucose de‐repression and fed‐batch conditions identified optimal phytase productivity at the specific growth rate of 0.041 h−1. Validation of the model simulation with experimental data confirmed the feasibility of the model to predict phytase production in our newly engineered strain. Methanol was used only to induce the expression of phytase at high cell densities. Our results showed that high phytase production required two stages, the first stage used glucose under de‐repression conditions to generate biomass while expressing phytase, and stage two used methanol to induce phytase expression. The production of phytase was improved 3.5‐fold by methanol induction compared to the expression with glucose alone under de‐repression conditions to a final phytase activity of 12.65 MU/L. This final volumetric phytase production represented an approximate 36‐fold change compared to the flask fermentations. Finally, the phytase protein produced was assayed to confirm its molecular weight, and pH and temperature profiles. This study highlights the importance of optimizing protein production in P. pastoris when using novel promoters and presents a general approach to performing bioprocess optimization in this important production host.

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Section: Ph Profile and Stability Differential Scanning Calorimetry (...supporting

confidence: 47%

Section: Discussionmentioning

confidence: 99%

Improving phytase production in Pichia pastoris fermentations through de‐repression and methanol induction optimization

Luna‐Flores,

Weng,

Wang

et al. 2023

Biotech & Bioengineering

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“…After that, the ER-associated degradation pathway can be activated and then reduce the production of heterologous proteins. Based on the above rationales, to prevent the degradation of overexpressed heterologous protein by the proteasome, the disruption of Pep4 protease increases phytase secretion in P. pastoris [ 12 ]. Lipase is widely used in the production of biodiesels [ 13 ], detergents [ 14 ], chiral compounds [ 15 ], etc.…”

Section: Introductionmentioning

confidence: 99%

Oxidative stress tolerance contributes to heterologous protein production in Pichia pastoris

Lin

et al. 2021

Biotechnol Biofuels

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Background Pichia pastoris (syn. Komagataella phaffii) is an important yeast system for heterologous protein expression. A robust P. pastoris mutant with oxidative and thermal stress cross-tolerance was acquired in our previous study. The robust mutant can express a 2.5-fold higher level of lipase than its wild type (WT) under methanol induction conditions. Results In this study, we found that the robust mutant not only can express a high level of lipase, but also can express a high level of other heterogeneous proteins (e.g., green fluorescence protein) under methanol induction conditions. Additionally, the intracellular reactive oxygen species (ROS) levels in the robust mutant were lower than that in the WT under methanol induction conditions. To figure out the difference of cellular response to methanol between the WT and the robust mutant, RNA-seq was detected and compared. The results of RNA-seq showed that the expression levels of genes related to antioxidant, MAPK pathway, ergosterol synthesis pathway, transcription factors, and the peroxisome pathway were upregulated in the robust mutant compared to the WT. The upregulation of these key pathways can improve the oxidative stress tolerance of strains and efficiently eliminate cellular ROS. Hence, we inferred that the high heterologous protein expression efficiency in the robust mutant may be due to its enhanced oxidative stress tolerance. Promisingly, we have indeed increased the expression level of lipase up to 1.6-fold by overexpressing antioxidant genes in P. pastoris. Conclusions This study demonstrated the impact of methanol on the expression levels of genes in P. pastoris and emphasized the contribution of oxidative stress tolerance on heterologous protein expression in P. pastoris. Our results shed light on the understanding of protein expression mechanism in P. pastoris and provided an idea for the rational construction of robust yeast with high expression ability.

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“…One unit of pectinase activity is the amount of enzyme that releases 1 μmol of d ‐(+)‐galacturonic acid per min in the buffer (pH 8.0) at 40 °C. The activities of tannase and phytase were determined by Ma’s (Ma et al ., 2015) and Helian’s (Helian et al ., 2020) methods.…”

Section: Methodsmentioning

confidence: 99%

Metaproteomic analysis of enzymatic composition in Baobaoqu fermentation starter for Wuliangye baijiu

Fan

Zhao

et al. 2021

Int J of Food Sci Tech

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Wuliangye is one of the most famous brands of baijiu in China. The typical flavours of Wuliangye baijiu are mainly formed by the multifarious enzymes in fermentation starter (Baobaoqu, BBQ). As the enzymes remained poorly understood, the detailed enzymatic composition of BBQ was analysed by metaproteomics for the first time. It is worth noting that the enzymes from plant, animals and micro-organisms were detected together and oxidoreductases were the dominant enzymes. Next, the analysis of function showed that the enzymes were mainly involved in carbon metabolism and the biosynthesis of amino acids. Additionally, the relationship between differential abundant enzymes and formation of typical flavour substances in premium and normal BBQ was established. Furthermore, the activities of classic enzymes in BBQ were examined to verify the reliability of metaproteomic results. This study will provide clues to further improve the manufacturing technology for Wuliangye baijiu.

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A multistrategy approach for improving the expression of E. coli phytase in Pichia pastoris

Cited by 11 publications

References 56 publications

Improving phytase production in Pichia pastoris fermentations through de‐repression and methanol induction optimization

Improving phytase production in Pichia pastoris fermentations through de‐repression and methanol induction optimization

Oxidative stress tolerance contributes to heterologous protein production in Pichia pastoris

Metaproteomic analysis of enzymatic composition in Baobaoqu fermentation starter for Wuliangye baijiu

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