Development of a New High-Cell Density Fermentation Strategy for Enhanced Production of a Fungus β-Glucosidase in Pichia pastoris

Liu, Wan-Cang; Xiang, Haibo; Zhang, Tao; Pang, Xu; Su, Jing; Liu, Hongyu; B, Ma; Yu, Liyan

doi:10.3389/fmicb.2020.01988

Cited by 23 publications

(13 citation statements)

References 72 publications

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“…obtained a specific activity of about 9 U mg –1 (pNPG) by recombinant expression of β-glucosidase in K. phaffii . Compared to the recent reports, the K.…”

Section: Resultsmentioning

confidence: 83%

Combinatorial Enzymatic Catalysis for Bioproduction of Ginsenoside Compound K

Yang

Zhou

et al. 2023

J. Agric. Food Chem.

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Ginsenoside compound K (CK) is an emerging functional food or pharmaceutical product. To date, there are still challenges to exploring effective catalytic enzymes for enzyme-catalyzed manufacturing processes and establishing enzyme-catalyzed processes. Herein, we identified three ginsenoside hydrolases BG07 (glucoamylase), BG19 (β-glucosidase), and BG23 (β-glucosidase) from Aspergillus tubingensis JE0609 by transcriptome analysis and peptide mass fingerprinting. Among them, BG23 was expressed in Komagataella phaffii with a high volumetric activity of 235.73 U mL–1 (pNPG). Enzymatic property studies have shown that BG23 is an acidic (pH adaptation range of 4.5–7.0) and mesophilic (thermostable < 50 °C) enzyme. Moreover, a one-pot combinatorial enzyme-catalyzed strategy based on BG23 and BGA35 (β-galactosidase from Aspergillus oryzae) was established, with a high CK yield of 396.7 mg L–1 h–1. This study explored the ginsenoside hydrolases derived from A. tubingensis at the molecular level and provided a reference for the efficient production of CK.

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“…obtained a specific activity of about 9 U mg –1 (pNPG) by recombinant expression of β-glucosidase in K. phaffii . Compared to the recent reports, the K.…”

Section: Resultsmentioning

confidence: 83%

Combinatorial Enzymatic Catalysis for Bioproduction of Ginsenoside Compound K

Yang

Zhou

et al. 2023

J. Agric. Food Chem.

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“…Agitation and aeration may often cause excessive foam formation and thus influence cell growth and biotransformation efficiency. In our previous study, a certain quantity of antifoam reagent was added into the fermentation medium and reaction broth for the SmF of yeast and recombinant enzyme catalysis, respectively. − The antifoam reagent has been used commonly in SmF, and the mechanisms of action were also summarized, such as bridging-stretching, spreading fluid entrainment, bridging-dewetting, etc . However, there is very little knowledge on the effect of antifoam reagent addition on the growth of Fusarium strains.…”

Section: Resultsmentioning

confidence: 99%

Development of a New Bioprocess for Clean Diosgenin Production through Submerged Fermentation of an Endophytic Fungus

et al. 2021

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Diosgenin is used widely to synthesize steroidal hormone drugs in the pharmaceutical industry. The conventional diosgenin production process, direct acid hydrolysis of the root of Dioscorea zingiberensis C. H. Wright (DZW), causes large amounts of wastewater and severe environmental pollution. To develop a clean and effective method, the endophytic fungus Fusarium sp. CPCC 400226 was screened for the first time for the microbial biotransformation of DZW in submerged fermentation (SmF). Statistical design and response surface methodology (RSM) were implemented to develop the diosgenin production process using the Fusarium strains. The environmental variables that significantly affected diosgenin yield were determined by the two-level Plackett–Burman design (PBD) with nine factors. PBD indicates that the fermentation period, culture temperature, and antifoam reagent addition are the most influential variables. These three variables were further optimized using the response surface design (RSD). A quadratic model was then built by the central composite design (CCD) to study the impact of interaction and quadratic effect on diosgenin yield. The values of the coefficient of determination for the PBD and CCD models were all over 0.95. P -values for both models were 0.0024 and <0.001, with F -values of ∼414 and ∼2215, respectively. The predicted results showed that a maximum diosgenin yield of 2.22% could be obtained with a fermentation period of 11.89 days, a culture temperature of 30.17 °C, and an antifoam reagent addition of 0.20%. The experimental value was 2.24%, which was in great agreement with predicted value. As a result, over 80% of the steroidal saponins in DZW were converted into diosgenin, presenting a ∼3-fold increase in diosgenin yield. For the first time, we report the SmF of a Fusarium strain used to produce diosgenin through the microbial biotransformation of DZW. A practical diosgenin production process was established for the first time for Fusarium strains. This bioprocess is acid-free and wastewater-free, providing a promising environmentally friendly alternative to diosgenin production in industrial applications. The information provided in the current study may be applicable to produce diosgenin in SmF by other endophytic fungi and lays a solid foundation for endophytic fungi to produce natural products.

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“…Other P AOX1 -regulated P. pastoris systems for secretory recombinant protein production normally claim a protein yield ranging from milligrams to grams per liter of culture with methanol induction. [29,30] For example, Gurramkonda et al [31] reported a yield of 3.1 g/L insulin precursor in the broth of a 15 L bioreactor through fedbatch fermentation; Werten et al [32] presented a study on improved secretion of recombinant gelatins through fedbatch fermentation in bioreactors ranging from 1-140 L, and the 15-copy transformant was able to produce an unprecedented high yield of 14.8 g gelatin per liter of clarified broth. Therefore, P. pastoris is a very robust expression system for high-yield recombinant protein production.…”

Section: Statements and Declarationsmentioning

confidence: 99%

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2022

BPJ

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Nowadays, therapeutic monoclonal antibodies (mAbs) are predominantly produced with mammalian cell culture systems such as those using Chinese hamster ovary (CHO) cells. Efforts are underway to reduce the costs of this process to meet the increasing global demand in biopharmaceuticals; meanwhile, cheaper and faster expression systems are being investigated as alternatives. The yeast, Pichia pastoris, has become a substantial workhorse for recombinant protein production. However, the N-linked glycosylation in P. pastoris, namely high mannose glycosylation, is significantly different from that in CHO or other mammalian cells, including human cells. In this study, a SuperMan5 strain of P. pastoris was constructed using Pichia GlycoSwitch® technology to successfully produce a more mammalian-like immunoglobulin G (IgG) fragment crystallizable (Fc), which showcases the potential of P. pastoris as a next-generation mAb production platform. Importantly, in this study, a strong methanol-independent promoter, P UPP , was applied, which only requires glycerol feeding for protein production. Most P. pastoris promoters used for protein expression are derived from genes in the methanol metabolism pathway, creating safety concerns due to the flammable nature of methanol, especially at large scale. Here, a fed-batch SuperMan5 P. pastoris fermentation was carried out in which methanol induction, as well as its affiliated safety risks, were eliminated. Overall, this study provides insights into the development of safe and cost-effective industrial mAb production approaches independent of mammalian cell culture.

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Development of a New High-Cell Density Fermentation Strategy for Enhanced Production of a Fungus β-Glucosidase in Pichia pastoris

Cited by 23 publications

References 72 publications

Combinatorial Enzymatic Catalysis for Bioproduction of Ginsenoside Compound K

Combinatorial Enzymatic Catalysis for Bioproduction of Ginsenoside Compound K

Development of a New Bioprocess for Clean Diosgenin Production through Submerged Fermentation of an Endophytic Fungus

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