Disruption of a putative mitochondrial oxaloacetate shuttle protein in Aspergillus carbonarius results in secretion of malic acid at the expense of citric acid production

FWN – Publicaties zonder aanstelling Universiteit Leide

Metabolic engineering with ATP-citrate lyase and nitrogen source supplementation improves itaconic acid production in Aspergillus niger

FWN – Publicaties zonder aanstelling Universiteit Leide

Itaconic acid degradation in Aspergillus niger: the role of unexpected bioconversion pathways

Abstract Background Itaconic acid (IA), a C5-dicarboxylic acid, has previously been identified as one of the top twelve biochemicals that can be produced by biotechnological means. IA is naturally produced by Aspergillus terreus, however, heterologous production in the related species Aspergillus niger has been proposed earlier. Remarkably, we observed that during high producing conditions and elevated titers A. niger detoxifies the extracellular medium of IA. In order to determine the genes responsible for this decline in IA titers a transcriptome analysis was performed. Results Transcriptome analysis has led to the identification of two novel and previously unknown IA bioconversion pathways in A. niger. One pathway is proposed to convert IA into pyruvate and acetyl-CoA through the action of itaconyl-CoA transferase (IctA), itaconyl-CoA hydratase (IchA) and citramalyl-CoA lyase, similar to the pathway identified in A. terreus. Another pathway putatively converts IA into 1-methyl itaconate through the action of trans-aconitate methyltransferase (TmtA). Upon deleting the key genes ictA and ichA we have observed increased IA production and titers and cessation of IA bioconversion. Surprisingly, deletion of tmtA lead to strong reduction of heterologous IA production. Conclusion Heterologous IA production in A. niger induces the expression of IA bioconversion pathways. These pathways can be inhibited by deleting the key genes ictA, ichA and tmtA. Deletion of ictA and ichA resulted in increased IA production. Deletion of tmtA, however, resulted in almost complete cessation of IA production

Microbial itaconic acid production from starchy food waste by newly isolated thermotolerant Aspergillus terreus strain

In the present study, we have explored the potential of newly isolated Aspergillus terreus BD strain, which can accumulate itaconic acid (IA) at higher temperature. The shake flask cultivation of thermotolerant strain with medium optimized using Box-Behnken Design at 45 °C resulted in IA accumulation of 28.9 g/L with yield of 0.27 g/g. The enzymatic saccharification of the synthetic food waste (SFW) consisting of potatoes, rice & noodles were optimized using Taguchi method of orthogonal array to maximize the release of fermentable sugar. The maximum glucose release of 0.60 g/g was achieved with 10% biomass loading, 5% enzyme concentration, pH 5.5 and temperature 60 0C. The sugars obtained from SFW was integrated with IA production and maximum IA titer achieved with SFW hydrolysate during bioreactor cultivation was 41.1 g/L with conversion yield of 0.27 g/g while with pure glucose IA titer and yield were 44.7 g/L and 0.30 g/g, respectively

In Pursuit of Protein Targets: Proteomic Characterization of Bacterial Spore Outer Layers

<i>Bacillus cereus</i>, responsible for food poisoning, and <i>Clostridium difficile</i>, the causative agent of <i>Clostridium difficile</i>-associated diarrhea (CDAD), are both spore-forming pathogens involved in food spoilage, food intoxication, and other infections in humans and animals. The proteinaceous coat and the exosporium layers from spores are important for their resistance and pathogenicity characteristics. The exosporium additionally provides an ability to adhere to surfaces eventually leading to spore survival in food. Thus, studying these layers and identifying suitable protein targets for rapid detection and removal of spores is of the utmost importance. In this study, we identified 100 proteins from <i>B. cereus</i> spore coat, exosporium and 54 proteins from the <i>C. difficile</i> coat insoluble protein fraction. In an attempt to define a universal set of spore outer layer proteins, we identified 11 superfamily domains common to the identified proteins from two <i>Bacilli</i> and one <i>Clostridium</i> species. The evaluated orthologue relationships of identified proteins across different spore formers resulted in a set of 13 coat proteins conserved across the spore formers and 12 exosporium proteins conserved in the <i>B. cereus</i> group, which could be tested for quick and easy detection or targeted in strategies aimed at removal of spores from surfaces

MOESM1 of Metabolic engineering with ATP-citrate lyase and nitrogen source supplementation improves itaconic acid production in Aspergillus niger

Additional file 1: Figure S1. Design of acl1 and acl2 expression cassette. Figure S2. Shake flask cultivation of CitB#99 ACL G1 Z. Table S1. Relative copy numbers of introduced acl12 and IA titers achieved in shake flask cultivations. Figure S3. Southern-Blot results of selected CitB#99-acl transformants. Table S2. Carbon balance of 10L fed-batch bioreactor cultivation of ACL G1 Z. Figure S4. Dissolved oxygen profile and pH profile during 10 L controlled fed-batch cultivation of strain CitB#99-ACL G1 Z. Figure S5. Total nitrogen concentration during 10 L controlled fed-batch cultivation of ACL G1 Z. Figure S6. Repeat fed-batch cultivation of ACL G1 Z performed in 5 L BioFlo 320 (Eppendorf) controlled bioreactors. Table S3. Carbon balance of repeat fed-batch 5 L bioreactor cultivation of ACL G1 Z

MOESM1 of Disruption of a putative mitochondrial oxaloacetate shuttle protein in Aspergillus carbonarius results in secretion of malic acid at the expense of citric acid production

Additional file 1: Figure S1. Transcriptional analysis of the mtpA gene

Disruption of a putative mitochondrial oxaloacetate shuttle protein in Aspergillus carbonarius results in secretion of malic acid at the expense of citric acid production

Abstract Background In filamentous fungi, transport of organic acids across the mitochondrial membrane is facilitated by active transport via shuttle proteins. These transporters may transfer different organic acids across the membrane while taking others the opposite direction. In Aspergillus niger, accumulation of malate in the cytosol can trigger production of citric acid via the exchange of malate and citrate across the mitochondrial membrane. Several mitochondrial organic acid transporters were recently studied in A. niger showing their effects on organic acid production. Results In this work, we studied another citric acid producing fungus, Aspergillus carbonarius, and identified by genome-mining a putative mitochondrial transporter MtpA, which was not previously studied, that might be involved in production of citric acid. This gene named mtpA encoding a putative oxaloacetate transport protein was expressed constitutively in A. carbonarius based on transcription analysis. To study its role in organic acid production, we disrupted the gene and analyzed its effects on production of citric acid and other organic acids, such as malic acid. In total, 6 transformants with gene mtpA disrupted were obtained and they showed secretion of malic acid at the expense of citric acid production. Conclusion A putative oxaloacetate transporter gene which is potentially involved in organic acid production by A. carbonarius was identified and further investigated on its effects on production of citric acid and malic acid. The mtpA knockout strains obtained produced less citric acid and more malic acid than the wild type, in agreement with our original hypothesis. More extensive studies should be conducted in order to further reveal the mechanism of organic acid transport as mediated by the MtpA transporter

Disruption of a putative mitochondrial oxaloacetate shuttle protein in Aspergillus carbonarius results in secretion of malic acid at the expense of citric acid production

Abstract Background In filamentous fungi, transport of organic acids across the mitochondrial membrane is facilitated by active transport via shuttle proteins. These transporters may transfer different organic acids across the membrane while taking others the opposite direction. In Aspergillus niger, accumulation of malate in the cytosol can trigger production of citric acid via the exchange of malate and citrate across the mitochondrial membrane. Several mitochondrial organic acid transporters were recently studied in A. niger showing their effects on organic acid production. Results In this work, we studied another citric acid producing fungus, Aspergillus carbonarius, and identified by genome-mining a putative mitochondrial transporter MtpA, which was not previously studied, that might be involved in production of citric acid. This gene named mtpA encoding a putative oxaloacetate transport protein was expressed constitutively in A. carbonarius based on transcription analysis. To study its role in organic acid production, we disrupted the gene and analyzed its effects on production of citric acid and other organic acids, such as malic acid. In total, 6 transformants with gene mtpA disrupted were obtained and they showed secretion of malic acid at the expense of citric acid production. Conclusion A putative oxaloacetate transporter gene which is potentially involved in organic acid production by A. carbonarius was identified and further investigated on its effects on production of citric acid and malic acid. The mtpA knockout strains obtained produced less citric acid and more malic acid than the wild type, in agreement with our original hypothesis. More extensive studies should be conducted in order to further reveal the mechanism of organic acid transport as mediated by the MtpA transporter