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Semirational Directed Evolution of Loop Regions in Aspergillus japonicus ?-Fructofuranosidase for Improved Fructooligosaccharide Production.

ABSTRACT: The Aspergillus japonicus ?-fructofuranosidase catalyzes the industrially important biotransformation of sucrose to fructooligosaccharides. Operating at high substrate loading and temperatures between 50 and 60°C, the enzyme activity is negatively influenced by glucose product inhibition and thermal instability. To address these limitations, the solvent-exposed loop regions of the ?-fructofuranosidase were engineered using a combined crystal structure- and evolutionary-guided approach. This semirational approach yielded a functionally enriched first-round library of 36 single-amino-acid-substitution variants with 58% retaining activity, and of these, 71% displayed improved activities compared to the parent. The substitutions yielding the five most improved variants subsequently were exhaustively combined and evaluated. A four-substitution combination variant was identified as the most improved and reduced the time to completion of an efficient industrial-like reaction by 22%. Characterization of the top five combination variants by isothermal denaturation assays indicated that these variants displayed improved thermostability, with the most thermostable variant displaying a 5.7°C increased melting temperature. The variants displayed uniquely altered, concentration-dependent substrate and product binding as determined by differential scanning fluorimetry. The altered catalytic activity was evidenced by increased specific activities of all five variants, with the most improved variant doubling that of the parent. Variant homology modeling and computational analyses were used to rationalize the effects of amino acid changes lacking direct interaction with substrates. Data indicated that targeting substitutions to loop regions resulted in improved enzyme thermostability, specific activity, and relief from product inhibition.

SUBMITTER: Trollope KM

PROVIDER: S-EPMC4579456 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Semirational Directed Evolution of Loop Regions in Aspergillus japonicus β-Fructofuranosidase for Improved Fructooligosaccharide Production.

Trollope K M KM Görgens J F JF Volschenk H H

Applied and environmental microbiology 20150807 20

The Aspergillus japonicus β-fructofuranosidase catalyzes the industrially important biotransformation of sucrose to fructooligosaccharides. Operating at high substrate loading and temperatures between 50 and 60°C, the enzyme activity is negatively influenced by glucose product inhibition and thermal instability. To address these limitations, the solvent-exposed loop regions of the β-fructofuranosidase were engineered using a combined crystal structure- and evolutionary-guided approach. This semi ...[more]

PMID: 26253664

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Project description:Research backgroundMicrobial β-fructofuranosidases are widely employed in food industry to produce inverted sugar or fructooligosaccharides. In this study, a newly isolated Aspergillus carbonarius PC-4 strain was used to optimize the β-fructofuranosidase production in a cost-effective process and the sucrose hydrolysis was evaluated to produce inverted sugars.Experimental approachOptimization of nutritional components of culture medium was carried out using simplex lattice mixture design for 72 and 120 h at 28 °C. One-factor-at-a-time methodology was used to optimize the physicochemical parameters. Crude enzyme was used for sucrose hydrolysis at different concentrations.Results and conclusionsThe optimized conditions of enzyme production were achieved from cultivations containing pineapple crown waste (1.3%, m/V) and yeast extract (0.3%, m/V) after 72 h with an enzyme activity of 9.4 U/mL, obtaining R2=91.85%, R2 adjusted=85.06%, highest F-value (13.52) and low p-value (0.003). One-factor-at-a-time used for optimizing the physicochemical conditions showed optimum temperature (20 °C), pH (5.5), agitation (180 rpm) and time course (72 h) with a 3-fold increase of enzyme production. The invertase-induced sucrose hydrolysis showed the maximum yield (3.45 mmol of reducing sugars) using 10% of initial sucrose concentration. Higher sucrose concentrations caused the inhibition of invertase activity, possibly due to the saturation of substrate or formation of sucrose aggregates, making it difficult for the enzyme to access sucrose molecules within the created clusters. Therefore, a cost-effective method was developed for the invertase production using agroindustrial waste and the produced enzyme can be used efficiently for inverted sugar production at high sucrose concentration.Novelty and scientific contributionThis study presents an efficient utilization of pineapple crown waste to produce invertase by a newly isolated Aspergillus carbonarius PC-4 strain. This enzyme exhibited a good potential for inverted sugar production at high initial sucrose concentration, which is interesting for industrial applications.

Project description:BACKGROUND:Nicotinamide adenine dinucleotide phosphate (NADPH) is an important cofactor ensuring intracellular redox balance, anabolism and cell growth in all living systems. Our recent multi-omics analyses of glucoamylase (GlaA) biosynthesis in the filamentous fungal cell factory Aspergillus niger indicated that low availability of NADPH might be a limiting factor for GlaA overproduction. RESULTS:We thus employed the Design-Build-Test-Learn cycle for metabolic engineering to identify and prioritize effective cofactor engineering strategies for GlaA overproduction. Based on available metabolomics and 13C metabolic flux analysis data, we individually overexpressed seven predicted genes encoding NADPH generation enzymes under the control of the Tet-on gene switch in two A. niger recipient strains, one carrying a single and one carrying seven glaA gene copies, respectively, to test their individual effects on GlaA and total protein overproduction. Both strains were selected to understand if a strong pull towards glaA biosynthesis (seven gene copies) mandates a higher NADPH supply compared to the native condition (one gene copy). Detailed analysis of all 14 strains cultivated in shake flask cultures uncovered that overexpression of the gsdA gene (glucose 6-phosphate dehydrogenase), gndA gene (6-phosphogluconate dehydrogenase) and maeA gene (NADP-dependent malic enzyme) supported GlaA production on a subtle (10%) but significant level in the background strain carrying seven glaA gene copies. We thus performed maltose-limited chemostat cultures combining metabolome analysis for these three isolates to characterize metabolic-level fluctuations caused by cofactor engineering. In these cultures, overexpression of either the gndA or maeA gene increased the intracellular NADPH pool by 45% and 66%, and the yield of GlaA by 65% and 30%, respectively. In contrast, overexpression of the gsdA gene had a negative effect on both total protein and glucoamylase production. CONCLUSIONS:This data suggests for the first time that increased NADPH availability can indeed underpin protein and especially GlaA production in strains where a strong pull towards GlaA biosynthesis exists. This data also indicates that the highest impact on GlaA production can be engineered on a genetic level by increasing the flux through the pentose phosphate pathway (gndA gene) followed by engineering the flux through the reverse TCA cycle (maeA gene). We thus propose that NADPH cofactor engineering is indeed a valid strategy for metabolic engineering of A. niger to improve GlaA production, a strategy which is certainly also applicable to the rational design of other microbial cell factories.

Dataset Information

Semirational Directed Evolution of Loop Regions in Aspergillus japonicus ?-Fructofuranosidase for Improved Fructooligosaccharide Production.

Publications

Semirational Directed Evolution of Loop Regions in Aspergillus japonicus β-Fructofuranosidase for Improved Fructooligosaccharide Production.

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