Effect of Co-expression of HAC1 and Molecular Chaperone Genes on the Expression of Mannanase in Pichia pastoris

doi:10.13560/j.cnki.biotech.bull.1985.2019-0718

Abstract

Abstract: For the higher activity of expressed mannanase ManA in Pichia pastoris,the unfolded protein response（UPR）activation factor HAC1 and 5 reported endoplasmic reticulum protein folding related chaperones ERO1,PDI,PDI1,CPR5,and BiP in P. pastoris were constructed to have 6 intracellular-expressing recombinant plasmids,then each was electro-transformed into recombinant strain P. pastoris secreting ManA for intracellular co-expression respectively,and the influence of the recombinant strain on the expression of ManA during the shake-flask fermentation were analyzed. The results showed that the enzyme activity of ManA in the supernatant of the recombinant strain with co-expressing HAC1,ERO1 and PDI increased by 26%,15% and 20%,and the enzyme activity reached 1 014 U/mL,925 U/mL and 965 U/mL at the shake-flask fermentation. By analyzing the data of enzyme activity in supernatant,intracellular-retention enzyme activity,and protein concentration in supernatant as well as further selecting HAC1,ERO1,PDI to have 2 genes or 3 genes combinations,which then co-expressed in the recombinant strains secreting and expressing ManA,while the enzyme activity in the fermentation supernatants of each co-expressed recombinant strain was not further increased. Co-expression of HAC1 or molecular chaperone ERO1,and PDI separately enhanced the correct folding of ManA and thus increased the expression of the protein.

Key words: mannanase, Pichia pastoris, gene HAC1, molecular chaperones, protein expression

MIN Qi, GAO Zi-han, YAO Yin, ZHANG Hua-shan, XIONG Hai-rong, ZHANG Li. Effect of Co-expression of HAC1 and Molecular Chaperone Genes on the Expression of Mannanase in Pichia pastoris[J]. Biotechnology Bulletin, 2020, 36(5): 159-168.

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