Dataset Information

Overexpression of a modified 6-phosphofructo-1-kinase results in an increased itaconic acid productivity in Aspergillus niger.

ABSTRACT: A modified 6-phosphofructo-1-kinase was expressed in a citrate producing Aspergillus niger strain in combination with cis-aconitate decarboxylase from Aspergillus terreus to study the effect on the production of itaconic acid. The modified pfkA gene was also expressed in combination with the itaconic acid biosynthetic cluster from A. terreus, which consists of cis-aconitate decarboxylase cadA, a putative mitochondrial transporter mttA and a putative plasmamembrane transporter mfsA. The combined expression of pfkA and cadA resulted in increased citrate levels, but did not show increased itaconic acid levels. The combined expression of pfkA with the itaconic acid biosynthetic cluster resulted in significantly increased itaconic acid production at earlier time points. Also the itaconic acid productivity increased significantly. The maximum itaconic acid productivity that was reached under these conditions was 0.15 g/L/h, which is only a factor 17 lower than the 2.5 g/L/h that according to the US Department of Energy should be achieved to have an economically feasible production process.

SUBMITTER: van der Straat L

PROVIDER: S-EPMC4016035 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Overexpression of a modified 6-phosphofructo-1-kinase results in an increased itaconic acid productivity in Aspergillus niger.

van der Straat Laura L Tamayo-Ramos Juan A JA Schonewille Tom T de Graaff Leo H LH

AMB Express 20130913 1

A modified 6-phosphofructo-1-kinase was expressed in a citrate producing Aspergillus niger strain in combination with cis-aconitate decarboxylase from Aspergillus terreus to study the effect on the production of itaconic acid. The modified pfkA gene was also expressed in combination with the itaconic acid biosynthetic cluster from A. terreus, which consists of cis-aconitate decarboxylase cadA, a putative mitochondrial transporter mttA and a putative plasmamembrane transporter mfsA. The combined ...[more]

PMID: 24034235

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Project description:Background:Bio-based production of organic acids promises to be an attractive alternative for the chemicals industry to substitute petrochemicals as building-block chemicals. In recent years, itaconic acid (IA, methylenesuccinic acid) has been established as a sustainable building-block chemical for the manufacture of various products such as synthetic resins, coatings, and biofuels. The natural IA producer Aspergillus terreus is currently used for industrial IA production; however, the filamentous fungus Aspergillus niger has been suggested to be a more suitable host for this purpose. In our previous report, we communicated the overexpression of a putative cytosolic citrate synthase citB in an A. niger strain carrying the full IA biosynthesis gene cluster from A. terreus, which resulted in the highest final titer reported for A. niger (26.2 g/L IA). In this research, we have attempted to improve this pathway by increasing the cytosolic acetyl-CoA pool. Additionally, we have also performed fermentation optimization by varying the nitrogen source and concentration. Results:To increase the cytosolic acetyl-CoA pool, we have overexpressed genes acl1 and acl2 that together encode for ATP-citrate lyase (ACL). Metabolic engineering of ACL resulted in improved IA production through an apparent increase in glycolytic flux. Strains that overexpress acl12 show an increased yield, titer and productivity in comparison with parental strain CitB#99. Furthermore, IA fermentation conditions were improved by nitrogen supplementation, which resulted in alkalization of the medium and thereby reducing IA-induced weak-acid stress. In turn, the alkalizing effect of nitrogen supplementation enabled an elongated idiophase and allowed final titers up to 42.7 g/L to be reached at a productivity of 0.18 g/L/h and yield of 0.26 g/g in 10-L bioreactors. Conclusion:Ultimately, this study shows that metabolic engineering of ACL in our rewired IA biosynthesis pathway leads to improved IA production in A. niger due to an increase in glycolytic flux. Furthermore, IA fermentation conditions were improved by nitrogen supplementation that alleviates IA induced weak-acid stress and extends the idiophase.

Project description:BackgroundAs an attractive platform chemical, malic acid has been commonly used in the food, feed and pharmaceutical field. Microbial fermentation of biobased sources to produce malic acid has attracted great attention because it is sustainable and environment-friendly. However, most studies mainly focus on improving yield and ignore shortening fermentation time. A long fermentation period means high cost, and hinders the industrial applications of microbial fermentation. Stresses, especially oxidative stress generated during fermentation, inhibit microbial growth and production, and prolong fermentation period. Previous studies have shown that polypeptides could effectively relieve stresses, but the underlying mechanisms were poorly understood.ResultsIn this study, polypeptides (especially elastin peptide) addition improves the productivity of malic acid in A. niger, resulting in shortening of fermentation time from 120 to 108 h. Transcriptome and biochemical analyses demonstrated that both antioxidant enzyme-mediated oxidative stress defense system, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), and nonenzymatic antioxidant system, such as glutathione, were enhanced in the presence of elastin peptide, suggesting elastin peptide relieving oxidative stresses is involved in many pathways. In order to further investigate the relationship between oxidative stress defense and malic acid productivity, we overexpressed three enzymes (Sod1, CAT, Tps1) related to oxidation resistance in A. niger, respectively, and these resulting strains display varying degree of improvement in malic acid productivity. Especially, the strain overexpressing the Sod1 gene achieved a malate titer of 91.85 ± 2.58 g/L in 96 h, corresponding to a productivity of 0.96 g/L/h, which performs better than elastin peptide addition.ConclusionsOur investigation provides an excellent reference for alleviating the stress of the fungal fermentation process and improving fermentation efficiency.

Dataset Information

Overexpression of a modified 6-phosphofructo-1-kinase results in an increased itaconic acid productivity in Aspergillus niger.

Publications

Overexpression of a modified 6-phosphofructo-1-kinase results in an increased itaconic acid productivity in Aspergillus niger.

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