US-20260125730-A1 - A NEW INDUCER FOR METHANOL-FREE PICHIA PASTORIS EXPRESSION SYSTEM

US20260125730A1US 20260125730 A1US20260125730 A1US 20260125730A1US-20260125730-A1

Abstract

The present invention relates to a method for the expression of genes belonging to different organisms (bacteria, fungi, plants, animals and humans) in P. pastoris cells, comprising the following processing steps; i. Transfer of the gene to be expressed into the plasmid carrying the alcohol oxidase 1 (AOX1) promoter ii. Cloning of the plasmid carrying the gene iii. Transfer of the recombinant plasmid carrying the AOX1 promoter and gene into the expression host iv. Induction of the AOX1 promoter with farnesol and consequent expression of the heterologous gene. The present invention will be used in the field of recombinant protein production.

Inventors

YAGMUR UNVER
Melek ACAR
Seyda YILDIZ
Mesut TASKIN

Assignees

ATATURK UNIVERSITESI REKTORLUGU BILIMSEL ARASTIRMA PROJELERI ( BAP ) KOORDINASYON BIRIMI

Dates

Publication Date: 20260507
Application Date: 20230310
Priority Date: 20220310

Claims (11)

1 . The invention relates with the discovery of a new inducer for the methanol-free Pichia pastoris expression system. The property of the Invention is the use of farnesol in methanol-inducible promoter induction in the P. pastoris expression system for recombinant protein production.
2 . Farnesol, a new inducer for the methanol-free Pichia pastoris expression system according to claim 1 , is used for the induction of the methanol-inducible AOX1 promoter in the P. pastoris expression system for recombinant protein production.
3 . Farnesol, a new inducer for the methanol-free Pichia pastoris expression system according to claim 1 , is used to induce other inducible (AOX2, FLD1 and PEX8) promoters in P. pastoris for recombinant protein production.
4 . Farnesol, a new inducer for the methanol-free Pichia pastoris expression system according to claim 1 , is used to induce promoters such as CUP1, DD12 and MOX for the production of recombinant protein in other yeast species (such as Saccharomyces cerevisiae, Hansenula polymorpha and Schizosaccharomyces pombe ).
5 . Farnesol, a new inducer for the methanol-free Pichia pastoris expression system according to claim 1 , is used as an inducer for recombinant protein production in all bacteria (such as Escherichia coli, Bacillus subtilis and Lactococcus lactis ), molds (such as Aspergillus niger, A. oryzae and Trichoderma reesei ), macrofungi and other organisms (such as algae).
6 . Farnesol, a new inducer for the methanol-free Pichia pastoris expression system according to claim 1 , is used as an Inducer for recombinant protein production in all mammalian (such as Chinese Hamster Ovary; CHO cells) and insect expression systems.
7 . Farnesol precursor molecules such as mevalonic acid, geranyl pyrophosphate (GPP) and farnesyl diphosphate and farnesol conversion products such as farnesol and farnesoic acid according to claim 1 , are also used as inducers for recombinant protein production via yeast, bacteria, molds and algae.
8 . Farnesol derivatives such as farnesyl acetate, α-farnesene, β-farnesene and nerolidol according to claim 1 are also used as inducers for recombinant protein production via yeast, bacteria, molds and algae.
9 . Fungal quorum sensing molecules such as tryptofol, tyrosol, multicholic acid, multicholanic acid and phenylethanol, α-(1,3)-glucan, γ-heptalactone and butyrolactone I according to claim 1 are used as inducers for recombinant protein production via yeast, bacteria, molds and algae.
10 . The use of bacterial quorum sensing molecules (autoinducers) according to claim 1 are used as inducers for recombinant protein production via yeast. bacteria, molds and algae.
11 . Method of using a new inducer for the methanol-free Pichia pastoris expression system, characterized in that, it comprises the following process steps: i. Transfer of the gene to be expressed into the plasmid carrying the alcohol oxidase 1 (AOX1) promoter ii. Cloning of the plasmid carrying the gene iii. Transfer of the recombinant plasmid carrying the AOX1 promoter and gene into the expression host iv. Induction of the AOX1 promoter with farnesol and consequent expression of the heterologous gene.

Description

FIELD OF THE INVENTION The present invention is intended to be used in the field of recombinant protein production wherein the expression of different genes of various microorganisms, plants, animals and humans is performed and thus the production of a large number of different recombinant proteins is carried out. STATE OF THE ART Lately, inducible expression systems have attracted a lot of attention by researchers and many recombinant proteins can be produced with the help of these systems. In this sense, Pichia pastoris (Komagataella phatfii), a methylotrophic yeast (which can use methanol as a carbon and energy source), is one of the most important expression systems used for both intracellular and extracellular recombinant protein production. More than 5000 different proteins have been produced via the P. pastoris expression system in the last 30 years until today, The promoter (PAOX1) of the alcohol oxidase 1 (AOX1) gene, which encodes the alcohol oxidase (AOX) enzyme, is mostly used in the production of high levels of recombinant proteins with this popular expression system. PAOX1 is strongly and tightly controlled by means of the methanol induction. Expression of human, plant, animal and microorganism genes is accomplished in P. pastoris by inducing this promoter with methanol. However, it is known that the use of methanol as an inducer has several disadvantages, In particular for the large-scale fermentation industry: 1) It is flammable and toxic.2) A high oxygen supply is required for its catabolism, and thus this will result in increased production costs due to significant heat release.3) It is not preferred for producing pharmaceutical or edible products.4) Its cost will increase in the event of an oil crisis and5) The by-product of methanol metabolism, hydrogen peroxide (H2O2), causes oxidative stress, thus resulting in proteolytic degradation of recombinant proteins. A methanol-free expression system is required so as to eliminate all these problems. The following patents are encountered which are for obtaining the methanol-free expression system. EP2873734 B1—Method of Eliminating Dependence of Methanol Induced Promoter on Single Methanol Carbon Source EP2106447 B1, U.S. Pat. No. 8,236,528B2—Method for Methanol Independent Induction from Methanol Inducible Promoters in Pichia Although both patents revealed a methanol-free process, they do not contain any description for the use of farnesol. Furthermore, ‘SNP (sodium nitropurisside)’ was used as an inducer of the AOX promoter for methanol-free P. pastoris expression, and a high level of recombinant protein expression was observed with promoter induction in the patent application titled ‘A method for recombinant protein production’, published on 2022 Jul. 14 (Pub. No.: US 2022/0220491 A1). ‘Farnesol’ is proposed as an inducer of AOX promoter for methanol-free expression of P. pastoris within the scope of this invention. DEFINITION OF THE INVENTION Said invention eliminates the disadvantages described in the state of the art and fulfils the needs. Said invention is intended to be used in the field of recombinant protein production wherein the expression of different genes of various microorganisms, plants, animals and humans is performed and thus the production of a large number of different recombinant proteins is carried out. Today, the P. pastoris yeast expression system is used in the production of more than 5000 recombinant proteins, more than 70 of which are therapeutic proteins, available on the market. Although different promoters have been used for protein expression with this system, higher protein production is achieved with alcohol oxidase 1 (AOX1) promoter. The AOX1 promoter (PAOX1) is a methanol-inducible promoter. That is, for the expression of a gene inserted downstream of this promoter, and hence the production of the protein of interest, certain amounts (0.5-4% v/v) and regular intervals (every 12 or 24 hours throughout the cultivation) of methanol must be added to the media of yeast cells. However, the use of methanol as an inducer has numerous disadvantages, as noted above, in particular for the large-scale fermentation industry. The use of methanol was completely eliminated and the protein production was carried out under PAOX1 control in recombinant P. pastoris cells with the help of the present invention. That is, a methanol-free expression method was designed for P. pastoris cells. Thus, we propose to eliminate all the disadvantages of methanol and use a less costly inducing agent instead of methanol. The present invention is to eliminate the disadvantage of using methanol as an inducing agent that allows high amount of protein expression in P. pastoris cells, thus solving the problems it causes in particular in the large-scale fermentation industry. A methanol-free expression system is proposed in this invention so as to solve the problems caused by said disadvantages of using methanol. Therefore, farnesol has been used i