Systematic assessment of Pichia pastoris system for optimized β -galactosidase production

Sun, Haijie; Bankefa, Olufemi Emmanuel; Ijeoma, Ijeoma Onyinyechi; Miao, Liangtian; Zhu, Tong; Li, Yin

doi:10.1016/j.synbio.2017.04.001

Cited by 5 publications

(2 citation statements)

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“…β-galactosidase coding genes were successfully amplified from the four fungal isolates. Their appropriate gene sizes 2,900 and 3,000 bp which correspond to the size of this enzyme [13,22], were successfully mapped with the marker size while the negative control revealed no band size (Figure 1). For efficient metabolic processes, microorganisms must be subjected to optimal conditions [23].…”

Section: Table 2 Morphological Features Of the Fungal Isolatesmentioning

confidence: 99%

Improved β-galactosidase Secretion in Fresh Water Fungi through Cultural Optimization

et al. 2022

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The wide range of applications of β-galactosidase has made it a candidate enzyme of study for improved yield. Most studies focused on expressing this enzyme in expressible vector(s) while sources of this enzyme are rarely investigated. Aquatic habitats harbor arrays of microorganisms that can participate in demineralization, nutrient restoration, circulation and hence confer significant properties on the habitats and significantly influence microbial interactions and metabolism. In view of this, aquatic fungal isolates were qualitatively and quantitatively screened for intracellular and extracellular β-galactosidase production. Four fungal isolates tagged SB01 and SC02 from stream identified as Aspergillus oryzae and Aspergillus niger and SC01 and SA01 from pond identified as A. oryzae and A. niger were studied. β-galactosidase gene was successfully amplified from the four isolates and their enzyme production levels were further studied. All the isolates showed considerably higher extracellular secretion than the intracellular synthesis on glucose-based medium. The production level was probed by xylose supplementation but surprisingly, the intracellular synthesis was higher than the extracellular secretion for all the isolates. In an attempt to boost the enzyme production, temperature was varied and 25 °C showed improved β-galactosidase synthesis and secretion than the 30 °C. This study revealed these filamentous fungi as candidates for β-galactosidase production and broaden the understanding of harnessing microorganisms from an aquatic reservoir for bio-production, and more importantly, revealed the intracellular accumulation of the enzyme as a bottleneck for industrial production. HIGHLIGHTS The wide range of applications of β-galactosidase has made it a candidate enzyme of study for improved yield Most studies focused on expressing this enzyme in expressible vector(s) while sources of this enzyme are rarely investigated Aquatic fungal isolates were qualitatively and quantitatively screened for intracellular and extracellular β-galactosidase production All the isolates showed considerable higher extracellular secretion than the intracellular synthesis on glucose-based medium, and vice-versa on xylose-supplemented medium, with the highest β-galactosidase synthesis and secretion efficiency occurring at 25 °C in both instances This study revealed these filamentous fungi as candidates for β-galactosidase production and broaden the understanding of harnessing microorganisms from an aquatic reservoir for bio-production GRAPHICAL ABSTRACT

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Section: Table 2 Morphological Features Of the Fungal Isolatesmentioning

confidence: 99%

Improved β-galactosidase Secretion in Fresh Water Fungi through Cultural Optimization

et al. 2022

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“…При синтезе гетерологичных белков повышается уровень экспрессии как гена PpKar2, так и гена PpPdI1 [12]. С другой стороны, известно, что сверхэкспрессия этих генов может повышать продукцию гетерологичных белков [17,18], в частности интерферонов [19].…”

Section: методология экологической генетикиunclassified

Cloning of PpKAR2 and PpPDI1 genes promoters from yeast Pichia pastoris, an evaluation of their activity and efficacy for the heterologous genes expression

Tsygankov

Александрович²,

Падкина

et al. 2018

Ecological genetics

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Background. Yeast Pichia pastoris is successfully used in biotechnology, with their help synthesized various compounds. Promoters are a key factor in the productivity of an expression system, since they determine the expression level of a heterologous gene. The aim of our work was to study the promoter regions of the PpKAR2 and PpPDI1 genes and to evaluate their use for effective expression of heterologous genes. Materials and Methods. To evaluate the activity of promoters, we used a reporter system based on the structural gene of acid phosphatase of yeast Saccharomyces cerevisiae – PHO5. To determine the effect of overproduction of native and heterologous protein on the activity of the promoters under study, we used the producer strains of P. pastoris protein disulfide isomerase and maize delta-zein. To evaluate the effectiveness of the use of the promoters under study for the expression of heterologous genes, we have expressed under their control a gene encoding human interferon-alpha16. Results. The promoters of the yeast genes – PpKAR2 and PpPDI1 were cloned. Their activity was compared with the promoter of the PpAOX1 gene in the native strains, as well as in strains with overproduction of native and heterologous proteins. Under the control of these promoters, the gene encoding human interferon-alpha 16 is expressed. Conclusion. The promoters studied were weaker than the promoter of the AOX1 gene, but increase their activity in response to the production of heterologous proteins and can be used to express heterologous genes.

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Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins

Keikha

Rezaee

Farsiani

2020

Journal Cellular Physiology

377

220

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One of the most important branches of genetic engineering is the expression of recombinant proteins using biological expression systems. Nowadays, different expression systems are used for the production of recombinant proteins including bacteria, yeasts, molds, mammals, plants, and insects. Yeast expression systems such as Saccharomyces cerevisiae (S. cerevisiae) and Pichia pastoris (P. pastoris) are more popular. P. pastoris expression system is one of the most popular and standard tools for the production of recombinant protein in molecular biology. Overall, the benefits of protein production by P. pastoris system include appropriate folding (in the endoplasmic reticulum) and secretion (by Kex2 as signal peptidase) of recombinant proteins to the external environment of the cell. Moreover, in the P. pastoris expression system due to its limited production of endogenous secretory proteins, the purification of recombinant protein is easy. It is also considered a unique host for the expression of subunit vaccines which could significantly affect the growing market of medical biotechnology. Although P. pastoris expression systems are impressive and easy to use with well-defined process protocols, some degree of process optimization is required to achieve maximum production of the target proteins. Methanol and sorbitol concentration, Mut forms, temperature and incubation time have to be adjusted to obtain optimal conditions, which might vary among different strains and externally expressed protein. Eventually, optimal conditions for the production of a recombinant protein in P. pastoris expression system differ according to the target protein.

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Systematic assessment of Pichia pastoris system for optimized β -galactosidase production

Cited by 5 publications

References 24 publications

Improved β-galactosidase Secretion in Fresh Water Fungi through Cultural Optimization

Improved β-galactosidase Secretion in Fresh Water Fungi through Cultural Optimization

Cloning of PpKAR2 and PpPDI1 genes promoters from yeast Pichia pastoris, an evaluation of their activity and efficacy for the heterologous genes expression

Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins

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