Synergistic effects of multiple enzymes from industrial Aspergillus niger strain O1 on starch saccharification

Abstract

Background Starch is one of the most important renewable polysaccharides in nature for production of bio-ethanol. The starch saccharification step facilitates the depolymerization of starch to yield glucose for biofuels production. The filamentous fungus Aspergillus niger (A. niger) is the most used microbial cell factory for production of the commercial glucoamylase. However, the role of each component in glucoamylases cocktail of A. niger O1 for starch saccharification remains unclear except glucoamylase. Results In this study, we identified the key enzymes contributing to the starch saccharification process are glucoamylase, α-amylase and acid α-amylase out of 29 glycoside hydrolases from the 6-day fermentation products of A. niger O1. Through the synergistic study of the multienzymes for the starch saccharification in vitro, we found that increasing the amount of α-amylase by 5-10 times enhanced the efficiency of starch saccharification by 14.2-23.2%. Overexpression of acid α-amylase in strain O1 in vivo increased the total glucoamylase activity of O1 cultures by 15.0%. Conclusions Our study clarifies the synergistic effects among the components of glucoamylases cocktail, and provides an effective approach to optimize the profile of saccharifying enzymes of strain O1 for improving the total glucoamylase activity.

Development of Genetic Tools in Glucoamylase-Hyperproducing Industrial Aspergillus niger Strains

“…While this manuscript was in revision, two papers regarding the strain engineering of A. niger N1 [ 71 ] and O1 [ 72 ] using the protoplast transformation procedure described in M. thermophila were published. In A. niger O1, Guo et al described that the four overexpression and four knockout transformants of the acid α-amylase gene were simply obtained after protoplast transformation, while four overexpression and two knockout transformants of the neutral α-amylase gene were generated [ 72 ]. In A. niger N1, 6 out of 52 transformants were triple-gene mutants, with a gene editing efficiency of 11.5% [ 71 ].…”

“…In A. niger N1, 6 out of 52 transformants were triple-gene mutants, with a gene editing efficiency of 11.5% [ 71 ]. Compared to previous studies [ 71 , 72 ], the PMT method we developed here is relatively simple. First, in previous studies, the young mycelia were washed twice with 1 mol/L MgSO 4 before lysing with enzymatic hydrolysate [ 71 , 72 ], while in our study, enough protoplasts could be obtained without carrying out these washing steps; therefore, the PMT method that we developed is relatively simple and saves time.…”

“…Compared to previous studies [ 71 , 72 ], the PMT method we developed here is relatively simple. First, in previous studies, the young mycelia were washed twice with 1 mol/L MgSO 4 before lysing with enzymatic hydrolysate [ 71 , 72 ], while in our study, enough protoplasts could be obtained without carrying out these washing steps; therefore, the PMT method that we developed is relatively simple and saves time. Second, previous studies washed the protoplasts with STC solution twice after the young mycelia had been lysed with enzymatic hydrolysate [ 71 , 72 ]; however, the protoplasts were only washed with solution B once in our study, so the method that we developed can shorten the time required for the protoplasts to remain in the osmotic stabilizer solution, thus reducing the damage to the protoplasts obtained by centrifuging twice.…”

“…Third, there are some differences between the PMT method that we established in this manuscript and the method used in previous studies. For example, in the transformation procedure, the protoplast mixture was heated at 60 °C for 2 min after the DNA mixture was added into the protoplast STC solution [ 71 , 72 ]; however, no heat treatment is used in our method. Finally, although the flow cytometry-based plating-free system developed by Yang et al [ 71 ] was more convenient than the PMT method used in our manuscript, the selection marker gene was still contained in the transformants generated by Yang et al, while an efficient marker-free gene deletion system was constructed in this manuscript.…”

Manipulation of an α-glucosidase in the industrial glucoamylase-producing Aspergillus niger strain O1 to decrease non-fermentable sugars production and increase glucoamylase activity

“…* * P < 0.01 (Student's t-test), * P < 0.05 (Student's t-test), n.s., not significant. most abundant protein in the secretomes of 2-day, 4-day, 6day cultures (Guo et al, 2021). Peptide search analyses revealed that A2QAC1 (UniProt accession) was the most abundant αglucosidase, ranked fifth among all detected proteins in the O1 secretome.…”