Systems metabolic engineering for citric acid production by Aspergillus niger in the post-genomic era.
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SUBMITTER: Tong Z
PROVIDER: S-EPMC6362574 | biostudies-literature | 2019 Feb
REPOSITORIES: biostudies-literature
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Systems metabolic engineering for citric acid production by Aspergillus niger in the post-genomic era.
Tong Zhenyu Z Zheng Xiaomei X Tong Yi Y Shi Yong-Cheng YC Sun Jibin J
Microbial cell factories 20190204 1
Citric acid is the world's largest consumed organic acid and is widely used in beverage, food and pharmaceutical industries. Aspergillus niger is the main industrial workhorse for citric acid production. Since the release of the genome sequence, extensive multi-omic data are being rapidly obtained, which greatly boost our understanding of the citric acid accumulation mechanism in A. niger to a molecular and system level. Most recently, the rapid development of CRISPR/Cas9 system facilitates high ...[more]
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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.
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Project description:Agricultural waste is as an alternative low-cost carbon source or beneficial additives which catch most people's eyes. In addition, methanol and sweet potato vine hydrolysate (SVH) have been reported as the efficient enhancers of fermentation according to some reports. The objective of the present study was to confirm SVH as an efficient additive in CA production and explore the synergistic effects of methanol and SVH in fermentation reactions.The optimal fermentation conditions resulted in a maximum citric acid concentration of 3.729 g/L. The final citric acid concentration under the optimized conditions was increased by 3.6-fold over the original conditions, 0.49-fold over the optimized conditions without methanol, and 1.8-fold over the optimized conditions in the absence of SVH. Kinetic analysis showed that Qp, Yp/s, and Yx/s in the optimized systems were significantly improved compared with those obtained in the absence of methanol or SVH. Further, scanning electron microscopy (SEM) revealed that methanol stress promoted the formation of conidiophores, while SVH could neutralize the effect and prolong Aspergillus niger vegetative growth. Cell viability analysis also showed that SVH might eliminate the harmful effects of methanol and enhance cell membrane integrity.SVH was a superior additive for organic acid fermentation, and the combination of methanol and SVH displayed a significant synergistic effect. The research provides a preliminary theoretical basis for SVH practical application in the fermentation industry.
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Project description:NADP-specific isocitrate dehydrogenase [threo-DS-isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42] was purified 200-300-fold from the citric acid-accumulating fungus Aspergillus niger. The enzyme consists of a single polypeptide chain with a molecular mass of 60 +/- 4 kDa and has a pI of 5.9 +/- 0.2. Only a single enzyme protein was found, although the enzyme appears to occur both in the mitochondrion and in the cytoplasm. Growth on organic acids as carbon sources or on NO3- as nitrogen source led to increased activities, whereas the presence of amino acids led to lower activities. The enzyme exhibits hyperbolic kinetics with respect to its substrates isocitrate and NADP+. Mn2+ and Mg2+ are obligatory for enzyme activity. The enzyme is inhibited by its products alpha-oxoglutarate and NADPH. Among various metabolites, ATP and citrate appear to inhibit the enzyme at a concentration considered to occur intracellularly. In both cases, however, the mechanism is a removal of the metal ion cofactor from the substrates. It is concluded that under physiological conditions, where the Mg2+ content is around 10 mM, the observed inhibition by ATP or citrate is of poor regulatory significance.
| S-EPMC1146875 | biostudies-other
Project description:BackgroundThis work investigates the production of citric acid from cashew apple juice, an abundant waste in the processing of cashew, using a local strain of Aspergillus niger and the application of the citric acid as a coagulant for the production of soy cheese. Fungal isolates were obtained from a cashew plantation in Ogbomoso, Nigeria, using potato dextrose agar. Further screening was undertaken to determine the qualitative strength of acid production by the fungi on Czapek-Dox agar supplemented with bromocresol green, with the development of yellow zone taken as an indication of citric acid production. Thereafter, the best producing strain was cultivated in a cashew apple juice medium.ResultsOut of 150 fungal isolates generated from the cashew plantation, 92 (61.3%), 44 (29.3%) and 14 (9.3%) were obtained from cashew fruits, soil and cashew tree surfaces, respectively. Different strains of fungi isolated include Aspergillus niger, A. flavus, A. foetidus, A. heteromorphus, A. nidulans and A. viridinutans. The isolates produced yellow zonation of 0.4-5.5 cm on modified Czapek-Dox agar; the highest was observed for a strain of A. niger LCFS 5, which was identified using molecular tools. In the formulated cashew apple juice medium, the citric acid yield of LCFS 5 ranged 16.0-92.8 g/l with the peak obtained on the 10th day of fermentation. The citric acid produced was recovered using the double precipitation method with Ca(OH)2 and H2SO4 having ≈ 70% purity of citric acid on HPLC. The citric acid acted as a coagulant to produce soy cheese with 66.67% acceptability by panelists.ConclusionThis work has extended the frontiers of valorization of cashew waste by a strain of A. niger to produce citric acid in high yield, with potential application in food industries.
| S-EPMC8448800 | biostudies-literature