Impact Of NADPH Supply On Protein Synthesis In Aspergillus Niger

Aspergillus niger has the advantages of extreme environmental tolerance,food safety(GRAS),strong protein secretion ability,and high production economy.It is the main engineering strain for producing homologous or heterologous proteins.The imbalance of regeneration and consumption of NADH and the low availability of NADPH are one of the limiting factors for efficient protein synthesis in A.niger.NADPH regeneration enzyme(malic enzyme Mae A)can effectively convert NADP+ to NADPH and improve the availability of intracellular NADPH;NAD kinase can directly convert NAD+/H to NADP+/H,there are three kinds of NAD kinases in A.niger,two kinds of localization in the cytoplasm(AN03,AN14),one is located in the mitochondria(AN17).In this study,A.niger SH-2 was used as the host,and glucoamylase was used as the protein production model to study the impacts of recombinant expression of NAD kinase and malic enzyme on protein synthesis in A.niger.Using CRISPR/Cas9 editing technology combined with marker-free gene integration to improve the multiple expression efficiency of NAD kinase and malic enzyme,and explore the effect of their combination on glucoamylase production in.The main research contents are as follows:(1)A universal expression vector and Afpyr G-labeled cas9 plasmid p FC330-amy A were constructed,and the coding sequences of three NAD kinases(AN03,AN14,AN17)and malic enzyme(Mae A)were cloned into the universal expression vector.In the host A.niger SH-2,recombinant expression of NAD kinase and malic enzyme under the control of the Pgpd A promoter was achieved by CRISPR/Cas9 gene-editing technology.The overexpression strains of NAD kinases(AN03,AN14)increased NAD kinase activity in the cytoplasm by 80%,NADP+ content increased by 40% and 68%,and NADPH content increased by 60% and 40%,respectively.The overexpression strain of malic enzyme(Mae A)increased the content of NADPH in the cytoplasm by 24% and the ratio of NADPH/NADP+ by 2.2-fold.The overexpression strain of NAD kinase(AN17)increased the transcription of the glucoamylaseencoding gene gla A by 2-fold.Compared with the control,the overexpression recombinant strains of the three NAD kinases(AN03,AN14,AN17)and malic enzyme(Mae A)increased the glucoamylase activity by about 70%,50%,90%,and 70%,respectively,and the total secreted protein increased by about 25%,22%,52% and 26%,respectively.(2)Single-gene overexpression of NAD kinase and malic enzyme was achieved by CRISPR/Cas9 editing technology combined with marker-free gene integration technology.And then the episomal cas9 plasmid p FC330-amy A in the recombinant strain OEmae A overexpressing malic enzyme(Mae A)can be recycled.Rapidly realize marker-free gene integration events,obtain secondary transformation host OEmae A-R,improve the efficiency of multiple genetic transformation of NAD kinase and malic enzyme,and facilitate further study the cumulative effect of multiple genetic manipulations of NAD kinase and malic enzyme on the production of glucoamylase in A.niger.(3)The candidate genes AN03,AN17 and mae A were cloned into an expression vector under the control of the Ptef promoter.The linearized fragment and the hyg B-labeled cas9 plasmid p FC332-aam A were co-transformed into the OEmae A-R strain to obtain the doublegene expression strain.Compared with the control strain,in the strain OEmm with an additional malic enzyme-encoding gene mae A,although the transcription level of mae A was increased,the activity of glucoamylase did not change significantly.The combination of AN03 and mae A strain OEm03 increased NADP+ and NADPH by 20% and 40%,respectively,but decreased glucoamylase activity and total protein level by about 12% and 10%,respectively.The combination of AN17 and mae A strain OEm17 further increased the activity of glucoamylase by about 19%,and the total protein level did not change significantly.