Increasing the Expression of FAD-dependent Glucose Dehydrogenase by Recombinant Pichia pastoris Using a Combined Strategy

doi:10.13560/j.cnki.biotech.bull.1985.2022-1255

Abstract

Abstract:

In order to increase the expression of FAD-dependent glucose dehydrogenase（FAD-GDH）in Pichia pastoris, the recombinant strains of co-expressing different copy numbers of molecular chaperone genes were constructed to achieve high-efficiency expression through the G418 concentration gradient screening, and the shake flask fermentation conditions were optimized. One high-yielding recombinant strain was obtained by G418 concentration gradient screening. On this basis, the recombinant strains co-expressing 1-4 copies of three molecular chaperones, HAC1, Ero1 and PDI, were constructed respectively, and the results of RT-qPCR were in line with expectations. The enzyme activity at the test tube level showed that 3 copies of PDI, 3 copies of HAC1 and 2 copies of Ero1 increased by 83%, 77% and 51%, respectively, compared with 1 copy. The recombinant strain with 3 copies of PDI had the highest enzyme activity, which was 198% higher than the control. The optimal conditions of the recombinant strain were as follows: initial pH 7.0, liquid loading volume 50 mL, the methanol addition amount 1%, and induction temperature 28℃. The efficient expression of FAD-GDH in P. pastoris is achieved by screening of antibiotic concentration and increasing the gene copy number of molecular chaperones.

Key words: FAD-dependent glucose dehydrogenase, Pichia pastoris, antibiotic gradient screening, multiple copies of molecular chaperones

DONG Cong, GAO Qing-hua, WANG Yue, LUO Tong-yang, WANG Qing-qing. Increasing the Expression of FAD-dependent Glucose Dehydrogenase by Recombinant Pichia pastoris Using a Combined Strategy[J]. Biotechnology Bulletin, 2023, 39(6): 316-324.

Figures/Tables 10

References 29

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引物名称 Primer name	基因 Gene	引物序列 Primer sequence（5'-3'）
H- F	HAC1	CTCGCAGATCCTTGACTGAGGATCTGGACGAAG
H- R	HAC1	CCTCAGTCAAGGATCTGCGAGTGGATGTAGATG
E- F	Ero1	TGGAGAAACAAATCTTTTACCGATTGGTTTCTG
E- R	Ero1	GGTAAAAGATTTGTTTCTCCAAACAGAGGTCTTC
P1-F	PDI	CTGCTGCCGAAATCTTAAAGGACAATGAGCAGG
P1-R		CCTTTAAGATTTCGGCAGCAGAAACAAGTTCAG
P2-F		TTGACGGATCTACCTTCAAATCATATGCTGAAG
P2-F		ATTTGAAGGTAGATCCGTCAATGTCTCCAAATAG

引物名称 Primer name	基因 Gene	引物序列 Primer sequence（5'-3'）
H- F	HAC1	CTCGCAGATCCTTGACTGAGGATCTGGACGAAG
H- R	HAC1	CCTCAGTCAAGGATCTGCGAGTGGATGTAGATG
E- F	Ero1	TGGAGAAACAAATCTTTTACCGATTGGTTTCTG
E- R	Ero1	GGTAAAAGATTTGTTTCTCCAAACAGAGGTCTTC
P1-F	PDI	CTGCTGCCGAAATCTTAAAGGACAATGAGCAGG
P1-R		CCTTTAAGATTTCGGCAGCAGAAACAAGTTCAG
P2-F		TTGACGGATCTACCTTCAAATCATATGCTGAAG
P2-F		ATTTGAAGGTAGATCCGTCAATGTCTCCAAATAG

引物名称Primer name	引物序列Primer sequence（5'-3'）
RT GAP up	CCAGCGGCAACAAGATCAAC
RT GAP down	CTCCTCGTTGACACCGACAA
RT GDH up	GGCAATACCAATCTGTCAACCAACCTTA
RT GDH down	AACCAGTGTTACCGATAGTTTCCCAAGC
RT HAC1 up	CTGAATATGACGACGAAGAA
RT HAC1 down	CTCCTGCTTGATAGATGTG
RT PDI up	ACCACATTTTACGGAGTTGCCGGT
RT PDI down	CCTCGCCAGGTCTGACAAGCA
RT Ero1 up	ATGAGGATAGTAAGGAGCGTAGC
RT Ero1 down	GTTGACCAGTTCCACCAGTTTG

引物名称Primer name	引物序列Primer sequence（5'-3'）
RT GAP up	CCAGCGGCAACAAGATCAAC
RT GAP down	CTCCTCGTTGACACCGACAA
RT GDH up	GGCAATACCAATCTGTCAACCAACCTTA
RT GDH down	AACCAGTGTTACCGATAGTTTCCCAAGC
RT HAC1 up	CTGAATATGACGACGAAGAA
RT HAC1 down	CTCCTGCTTGATAGATGTG
RT PDI up	ACCACATTTTACGGAGTTGCCGGT
RT PDI down	CCTCGCCAGGTCTGACAAGCA
RT Ero1 up	ATGAGGATAGTAAGGAGCGTAGC
RT Ero1 down	GTTGACCAGTTCCACCAGTTTG

菌株 Strain	GDH基因拷贝数 Copy number of GDH	HAC1基因拷贝数 Copy number of HAC1	PDI基因拷贝数 Copy number of PDI	Ero1基因拷贝数 Copy number of Ero1
CK（X33/GDH）	3.20±0.20	Endogenous 1	Endogenous 1	Endogenous 1
CK-H	3.20±0.20	1.10±0.09	Endogenous 1	Endogenous 1
CK-2H	3.20±0.20	2.08±0.26	Endogenous 1	Endogenous 1
CK-3H	3.20±0.20	2.95±0.11	Endogenous 1	Endogenous 1
CK-4H	3.20±0.20	4.35±0.21	Endogenous 1	Endogenous 1
CK-P	3.20±0.20	Endogenous 1	1.02±0.11	Endogenous 1
CK-2P	3.20±0.20	Endogenous 1	2.32±0.19	Endogenous 1
CK-3P（X33/GDH-3PDI）	3.20±0.20	Endogenous 1	3.006±0.20	Endogenous 1
CK-4P	3.20±0.20	Endogenous 1	4.42±0.29	Endogenous 1
CK-E	3.20±0.20	Endogenous 1	Endogenous 1	1.21±0.13
CK-2E	3.20±0.20	Endogenous 1	Endogenous 1	1.98±0.34
CK-3E	3.20±0.20	Endogenous 1	Endogenous 1	3.22±0.17
CK-4E	3.20±0.20	Endogenous 1	Endogenous 1	4.19±0.20