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Deletion of glucose oxidase changes the pattern of organic acid production in Aspergillus carbonarius.

Yang L, Lübeck M, Lübeck PS - AMB Express (2014)

Bottom Line: Aspergillus carbonarius has potential as a cell factory for the production of different organic acids.The effect of genetic engineering was examined by testing glucose oxidase deficient (Δgox) mutants for the production of different organic acids in a defined production medium.The results obtained showed that the gluconic acid accumulation was completely inhibited and increased amounts of citric acid, oxalic acid and malic acid were observed in the Δgox mutants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Section for Sustainable Biotechnology, Aalborg University Copenhagen, A. C. Meyers Vænge 15, Copenhagen, DK-2450, SV, Denmark.

ABSTRACT
Aspergillus carbonarius has potential as a cell factory for the production of different organic acids. At pH 5.5, A.carbonarius accumulates high amounts of gluconic acid when it grows on glucose based medium whereas at low pH, it produces citric acid. The conversion of glucose to gluconic acid is carried out by secretion of the enzyme, glucose oxidase. In this work, the gene encoding glucose oxidase was identified and deleted from A. carbonarius with the aim of changing the carbon flux towards other organic acids. The effect of genetic engineering was examined by testing glucose oxidase deficient (Δgox) mutants for the production of different organic acids in a defined production medium. The results obtained showed that the gluconic acid accumulation was completely inhibited and increased amounts of citric acid, oxalic acid and malic acid were observed in the Δgox mutants.

No MeSH data available.


Related in: MedlinePlus

Verification of deletion of thegoxgene in transformant 4 and 5 (a) Disruption ofgoxgene and primer binding sites (b) Amplification of internal sequence with primer Gox Fw1-Rv1 and Fw2-Rv2. Lane 1–2, Δgox transformant 4; lane 3–4, Δgox transformant 5; lane 5–6, ITS sequence amplified from Δgox transformant 4 and 5; lane 7–8, internal fragments of the gox gene from the wildtype strain (~1,5 kb and 0,6 kb) (c) Amplification of the gox gene containing region with the external primers Gox Fw3 and Rv3. Lane 1–2, amplified fragments from gox transformants 4 and 5. (~6.2 kb). Lane 3, amplified fragment from wild type (~7.7 kb).
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Figure 1: Verification of deletion of thegoxgene in transformant 4 and 5 (a) Disruption ofgoxgene and primer binding sites (b) Amplification of internal sequence with primer Gox Fw1-Rv1 and Fw2-Rv2. Lane 1–2, Δgox transformant 4; lane 3–4, Δgox transformant 5; lane 5–6, ITS sequence amplified from Δgox transformant 4 and 5; lane 7–8, internal fragments of the gox gene from the wildtype strain (~1,5 kb and 0,6 kb) (c) Amplification of the gox gene containing region with the external primers Gox Fw3 and Rv3. Lane 1–2, amplified fragments from gox transformants 4 and 5. (~6.2 kb). Lane 3, amplified fragment from wild type (~7.7 kb).

Mentions: Protoplast transformation was carried out with circular pSB414gox plasmids. Two Δgox transformants were obtained and checked for the deletion of the putative gox gene by 3 different pairs of primers (Table 1). Two pairs of primers (Gox Fw1-Rv1 and Fw2-Rv2) amplified the internal sequence of the putative gox gene, and the third pair of primers (Gox Fw3-Rv3) was designed to amplify the putative gox gene together with the flanking regions in order to check the replacement of the gox gene (Figure 1a). As shown in Figure 1b, no fragments could be amplified by the two pairs of internal primers in PCR, indicating that the gox gene was deleted. In PCR with external primers, a fragment at approx. 6.2 kb was amplified from both of the transformants, which indicated that the putative gox gene had been successfully replaced by the marker gene, since the length of the original sequence containing the gox gene was approx.7.7 kb (Figure 1c). A PCR amplifying the ITS region (Bulat et al. [2000]) confirmed the quality of the genomic DNA (Figure 1b). The size of all the amplified DNA fragments were estimated by comparing with 1 kb DNA ladder (Figure 1b).


Deletion of glucose oxidase changes the pattern of organic acid production in Aspergillus carbonarius.

Yang L, Lübeck M, Lübeck PS - AMB Express (2014)

Verification of deletion of thegoxgene in transformant 4 and 5 (a) Disruption ofgoxgene and primer binding sites (b) Amplification of internal sequence with primer Gox Fw1-Rv1 and Fw2-Rv2. Lane 1–2, Δgox transformant 4; lane 3–4, Δgox transformant 5; lane 5–6, ITS sequence amplified from Δgox transformant 4 and 5; lane 7–8, internal fragments of the gox gene from the wildtype strain (~1,5 kb and 0,6 kb) (c) Amplification of the gox gene containing region with the external primers Gox Fw3 and Rv3. Lane 1–2, amplified fragments from gox transformants 4 and 5. (~6.2 kb). Lane 3, amplified fragment from wild type (~7.7 kb).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230901&req=5

Figure 1: Verification of deletion of thegoxgene in transformant 4 and 5 (a) Disruption ofgoxgene and primer binding sites (b) Amplification of internal sequence with primer Gox Fw1-Rv1 and Fw2-Rv2. Lane 1–2, Δgox transformant 4; lane 3–4, Δgox transformant 5; lane 5–6, ITS sequence amplified from Δgox transformant 4 and 5; lane 7–8, internal fragments of the gox gene from the wildtype strain (~1,5 kb and 0,6 kb) (c) Amplification of the gox gene containing region with the external primers Gox Fw3 and Rv3. Lane 1–2, amplified fragments from gox transformants 4 and 5. (~6.2 kb). Lane 3, amplified fragment from wild type (~7.7 kb).
Mentions: Protoplast transformation was carried out with circular pSB414gox plasmids. Two Δgox transformants were obtained and checked for the deletion of the putative gox gene by 3 different pairs of primers (Table 1). Two pairs of primers (Gox Fw1-Rv1 and Fw2-Rv2) amplified the internal sequence of the putative gox gene, and the third pair of primers (Gox Fw3-Rv3) was designed to amplify the putative gox gene together with the flanking regions in order to check the replacement of the gox gene (Figure 1a). As shown in Figure 1b, no fragments could be amplified by the two pairs of internal primers in PCR, indicating that the gox gene was deleted. In PCR with external primers, a fragment at approx. 6.2 kb was amplified from both of the transformants, which indicated that the putative gox gene had been successfully replaced by the marker gene, since the length of the original sequence containing the gox gene was approx.7.7 kb (Figure 1c). A PCR amplifying the ITS region (Bulat et al. [2000]) confirmed the quality of the genomic DNA (Figure 1b). The size of all the amplified DNA fragments were estimated by comparing with 1 kb DNA ladder (Figure 1b).

Bottom Line: Aspergillus carbonarius has potential as a cell factory for the production of different organic acids.The effect of genetic engineering was examined by testing glucose oxidase deficient (Δgox) mutants for the production of different organic acids in a defined production medium.The results obtained showed that the gluconic acid accumulation was completely inhibited and increased amounts of citric acid, oxalic acid and malic acid were observed in the Δgox mutants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Section for Sustainable Biotechnology, Aalborg University Copenhagen, A. C. Meyers Vænge 15, Copenhagen, DK-2450, SV, Denmark.

ABSTRACT
Aspergillus carbonarius has potential as a cell factory for the production of different organic acids. At pH 5.5, A.carbonarius accumulates high amounts of gluconic acid when it grows on glucose based medium whereas at low pH, it produces citric acid. The conversion of glucose to gluconic acid is carried out by secretion of the enzyme, glucose oxidase. In this work, the gene encoding glucose oxidase was identified and deleted from A. carbonarius with the aim of changing the carbon flux towards other organic acids. The effect of genetic engineering was examined by testing glucose oxidase deficient (Δgox) mutants for the production of different organic acids in a defined production medium. The results obtained showed that the gluconic acid accumulation was completely inhibited and increased amounts of citric acid, oxalic acid and malic acid were observed in the Δgox mutants.

No MeSH data available.


Related in: MedlinePlus