Abstract
BackgroundGlucoamylase is one of the most industrially applied enzymes, produced by Aspergillus species, like Aspergillus niger. Compared to the traditional ways of process optimization, the metabolic engineering strategies to improve glucoamylase production are relatively scarce.ResultsIn the previous study combined multi-omics integrative analysis and amino acid supplementation experiment, we predicted four amino acids (alanine, glutamate, glycine and aspartate) as the limited precursors for glucoamylase production in A. niger. To further verify this, five mutants namely OE-ala, OE-glu, OE-gly, OE-asp1 and OE-asp2, derived from the parental strain A. niger CBS 513.88, were constructed respectively for the overexpression of five genes responsible for the biosynthesis of the four kinds of amino acids (An11g02620, An04g00990, An05g00410, An04g06380 and An16g05570). Real-time quantitative PCR revealed that all these genes were successfully overexpressed at the mRNA level while the five mutants exhibited different performance in glucoamylase production in shake flask cultivation. Notably, the results demonstrated that mutant OE-asp2 which was constructed for reinforcing cytosolic aspartate synthetic pathway, exhibited significantly increased glucoamylase activity by 23.5% and 60.3% compared to CBS 513.88 in the cultivation of shake flask and the 5 L fermentor, respectively. Compared to A. niger CBS 513.88, mutant OE-asp2 has a higher intracellular amino acid pool, in particular, alanine, leucine, glycine and glutamine, while the pool of glutamate was decreased.ConclusionOur study combines the target prediction from multi-omics analysis with the experimental validation and proves the possibility of increasing glucoamylase production by enhancing limited amino acid biosynthesis. In short, this systematically conducted study will surely deepen the understanding of resources allocation in cell factory and provide new strategies for the rational design of enzyme production strains.
Highlights
Aspergillus niger, as one of the most efficient cell factories for enzymes, organic acids and food additives production, is widely applied in biochemistry industry
Characterization of the five overexpression mutants Five genes encoding the enzymes for the biosynthesis of four kinds of probable limiting amino acids, namely alanine transaminase, glutamate dehydrogenase, glycine/serine hydroxymethyltransferase, mitochondrial aspartate aminotransferase and cytosolic aspartate aminotransferase were overexpressed in the olvA locus under the control of the constitutive strong promoter PgpdA
Mutant CBS-ΔolvA showed no significant difference in glucoamylase production and biomass growth compared with CBS 513.88 during the fermentation (Additional file 2: Fig. S4a, b), indicating that gene olvA is an ideal locus for the integration of overexpression cassettes
Summary
Aspergillus niger, as one of the most efficient cell factories for enzymes, organic acids and food additives production, is widely applied in biochemistry industry. Oxygen limitation strategy is widely applied because high glucoamylase. Results indicated that under oxygen-limited conditions, cell growth arrest occurs and more precursors such as amino acids may be channeled towards enzyme production, which further increases the enzyme yield. We found that alanine, glutamate, aspartate and glycine, whose intracellular pools decreased sharply in oxygen-limited phases, might form restrictions for further improvement of glucoamylase production. Alanine, aspartate and glycine account for high ratios in amino acid composition of glucoamylase (Additional file 1). Glucoamylase is one of the most industrially applied enzymes, produced by Aspergillus species, like Aspergillus niger. Compared to the traditional ways of process optimization, the metabolic engineering strategies to improve glucoamylase production are relatively scarce
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