Cloning of a novel gene from Penicillium oxalicum I 1 which in Escherichia coli enhances the secretion of acetic acid

(1) Chinese Academy of Agricultural Sciences. Institute of Environment and Sustainable Development in Agriculture. Beijing 100081 (P.R. China). (2) Chinese Academy of Agricultural Sciences. Key Laboratory of Microbial Resources. Ministry of Agriculture / Institute of Agricultural Resources and Regional Planning. Beijing 100081 (P.R. China). E-mail: gongcaas@163.com (3) Université de Liège Gembloux Agro-Bio Tech. Microbial Processes and Interactions(MiPI). Passage des Déportés, 2. BE-5030 Gembloux (Belgium).


INTRODUCTION
Many soil microorganisms can secrete organic acids, such as oxalic, lactic, acetic, propionic, malic, tartaric, citric, butyric, malonic, succinic, gluconic, and fumaric acids (Banik & Dey, 1982;Altomare et al., 1999;Fomina et al., 2005;Khan et al., 2007;Bianco & Defez, 2010;Gulati et al., 2010).These acids play important roles in agriculture by improving for instance phosphate solubilization and releasing metal ions to improve the concentration of plant essential nutrients (Dessureault-Rompré et al., 2007;Singh et al., 2007).Organic acids can chelate Fe 3+ , Fe 2+ , Ca 2+ , and Al 3+ , thereby converting insoluble forms of nutrients to soluble ones (Walpola & Yoon, 2013).These acids could decrease fertilizer requirement and improve fertilizer utilization.However, they are scarcely found and the effects are limited in soil.Therefore, highly efficient expression of genes related to exogenous organic acid secretion for organic acid production is an optimal strategy for improving fertilizer utilization.In this strategy, the critical step is obtaining genes that can improve organic acid secretion and expressing these genes heterologously.Numerous studies on genes that can enhance organic acid secretion have been conducted.Genes in bacteria are primarily cloned to improve gluconic acid secretion (Goldstein et al., 1999).For example, pyrroloquinoline quinone genes from Enterobacter intermedium, Klebsiella pneumoniae, and Rahnella aquatilis have been expressed in Escherichia coli (Meulenberg et al., 1992;Kim et al., 1998;Kim et al., 2003).Few fungal genes demonstrate this function as well.Fungal genes have been expressed in E. coli to improve various organic acid secretions.Lü et al. (2012) cloned mitochondrial malate dehydrogenase from Penicillium oxalicum and expressed the gene in E. coli to improve malate, lactate, acetate, citrate, and oxalate secretions.Gong et al. (2014a) also cloned delta-1-pyrroline-5-carboxylate dehydrogenase from P. oxalicum and expressed the gene in E. coli to enhance the secretion of acetic acid and α-ketoglutarate.
Penicillium oxalicum I1 is a fungus that secretes oxalic acid.In the current study, we constructed a primary cDNA library of P. oxalicum and screened clones that can solubilize phosphate in tricalcium phosphate (TCP) medium by clear halos.We aimed to obtain the gene expressed in E. coli that can enhance organic acid secretion.

Construction of cDNA library and screening for genes enhancing secretion of organic acid
A cDNA library of P. oxalicum I1 was constructed using the SMART method.The cDNA sequences were linked to pBluescript II SK(+) and recombinant plasmids were transformed into competent E. coli DH5α™ cells.Colonies diluted to 100-fold were spread on TCP medium agar plates containing 100 μg .ml -1 ampicillin.Clear halos appeared after three days.These halos resulted from the solubilization of TCP by the clones secreting organic acid, and the clones were subcultured to confirm stability.The cDNA sequences were blasted in the GenBank database.
The PCR mix contained 1 µM of each primer, 10 µM deoxynucleotide, 10× buffer (NEB, USA), and 1 unit of Taq Polymerase (NEB).This process was conducted under the following thermocycling conditions: initial denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C for 60 s, 60 °C for 60 s, 72 °C for 60 s, and a final elongation at 72 °C for 10 min (ABI 9700).The ORF sequence and pBluescript II SK(+) vector were digested with EcoRI and XhoI enzymes at 37 °C for 4 h.Digested products were linked to pBluescript II SK(+) by T4 DNA ligase.Plasmids containing the ORF sequence were transformed into E. coli DH5α™ competent cells.Transformants showing clear halos were selected from the TCP plates.

Organic acid secretion
The organic acid secretion ability of E. coli DH5α™ with overexpressed P. oxalicum I1 gene was tested in liquid TCP medium.Escherichia coli DH5α™ containing pBluescript II SK(+) and ORF sequence (pPos) or pBluescript II SK(+) (pBlu) were grown in TCP.An aliquot (100 µl) of each bacterial culture (10 8 cfu .ml -1 ) was grown in 50 ml of broth.The culture was shaken at 120 rpm at 37 °C.Changes in pH of the medium and organic acid concentrations were measured in the culture filtrates at 0, 8, 16, 24, 36, and 48 h.The pH of the medium was measured using a pH meter equipped with a glass electrode.The cell density of E. coli DH5α™ was tested by plate counting at 24 h and 48 h.Organic acids produced were determined at 48 h by an anion chromatographic system (ICS-3000, Dionex, USA).The experiment was performed three times.

Substrate utilization
Pyruvic acid, citric acid, α-ketoglutaric acid, succinic acid, fumaric acid, and malic acid were used as sole carbon source substitutes for glucose in the TCP medium whose pH was adjusted to 7.0.An aliquot (100 µl) of E. coli DH5α™ containing pPos culture (10 8 cfu .ml -1 ) was grown in 50 ml of broth.The culture was shaken at 120 rev .min -1 at 37 °C.Organic acids produced were determined at 48 h by an anion chromatography system (ICS-3000, Dionex, USA).The experiment was performed three times.

Statistical analysis
Statistical analysis was conducted by using Analysis of Variance (ANOVA) statistical package for social sciences (SPSS) software, version 21.0 followed by comparison of multiple treatment levels with the control, using the significant difference (LSD) at p ≤ 0.05.

Screening for genes enhancing of secretion of organic acids
Selection of the E. coli transformants with cDNA from P. oxalicum were tested on their ability to produce larger amounts of organic acid by checking the clear halos they induce on TCP agar plates because of the phosphate solubilization by the clones after 3 d of incubation at 37 °C.The titer of the primary cDNA library was 5.65 × 10 6 cfu .ml -1 with a recombination rate of 99.15%.A total of 106 positive clones were obtained, and the diameters of the clear halos ranged from 1.3 mm to 4.2 mm.The diameter of the clear halo of the clone I-2 was 3.6 mm.The full-length cDNA of the clone I-2 was 1,151 bp, and this clone contained an ORF of 702 bp (pPos).This ORF encoded a 25.41 kDa polypeptide comprising 233 amino acids.The cDNA sequence was deposited to the GenBank under accession number JF419552.
The cDNA sequence was analyzed through the GenBank database.Pennicillium oxalicum I1 cDNA showed 68% identity and 73% query cover with Penicillium chrysogenum Wisconsin 54-1255 partial mRNA (accession number XM_002569279.1).The sequence from P. oxalicum I1 also showed < 66% identity and 53% query cover with other fungi.The deduced amino acid sequence of P. oxalicum I1 was analyzed using the Blast program (DNAMAN 6.0) via the GenBank database.Multiple alignment results revealed 100% identity between the amino acid sequences of P. oxalicum I1 and P. oxalicum 114-2 (EPS25113.1),which has an unknown gene function.In addition, the sequence from P. oxalicum I1 can be aligned with hypothetical proteins of other closely related fungi (Figure 1).Even though for some the identity is below 63% identity, the alignment clearly shows that these hypothetical proteins belong to a same group that should share the same function.

Organic acid secretion
The subcloned ORF of the cDNA clone was inserted into pBluescript II SK(+), and the resulting recombinant plasmid pPos was transformed into E. coli DH5α™.Clear halos resulting from TCP solubilization appeared in vitro.Finally, E. coli DH5α™ containing the target gene was grown in TCP liquid medium to demonstrate the organic acid secretion ability of the recombinant.The pH values of the TCP liquid medium were more quickly reduced in E. coli bearing the cloned pPos (Figure 2a).The pH level decreased from pH 7.0 to pH 3.57 by pPos after 48 h.Under the same condition, pH level decreased from pH 7.0 to pH 5.41 by pBlu (control E. coli DH5α™ transformed with the pBluescript SK vector without any insert).However, the cell density of E. coli DH5α™ was similar.The cell density of E. coli DH5α™ with pPos was 37.67 x 107,93.33x 10 7 CFU .
We tested whether the decrease in pH in the presence of E. coli was correlated with organic acid secretion.Escherichia coli DH5α™ (pPos and pBlu) secreted acetic acid, and the acetic acid concentration was 168.6 mg .l -1 after 48 h.However, the acetic acid concentration reached 567.3 mg .l -1 by E. coli DH5α™ (pPos), which was significantly higher than those from E. coli DH5α™ (pBlu) (Figure 2b).

Substrate utilization
Glucose, pyruvic acid, citric acid, α-ketoglutaric acid, succinic acid, fumaric acid, and malic acid participate in the tricarboxylic acid cycle of E. coli DH5α™.Escherichia coli DH5α™ containing the target gene was grown in TCP liquid medium with one of the six organic acids as sole carbon sources instead of glucose to demonstrate the function of the cloned pPos sequence.The pH value significantly decreased and the acetic acid concentration increased with E. coli DH5α™ bearing pPos compared to E. coli DH5α™ (pBlu) in the TCP liquid medium after 48 h with glucose, pyruvic acid, α-ketoglutaric acid, and malic acid.The acetic acid concentration reached 567.3, 326.4,213.2, and 225.3 mg .l -1 , respectively.No difference was found in the pH values and acetic acid contents between E. coli DH5α™ (pPos) and E. coli DH5α™ (pBlu) in TCP liquid medium with citric acid, succinic acid, and fumaric acid (Figure 3a and Figure 3b).The hypothetical protein could improve glucose, pyruvic acid, α-ketoglutaric acid, and malic acid utilization in E. coli.

DISCUSSION
In this study, P. oxalicum I1 secreted a high amount of oxalic acid that reached 593.9 μg .ml -1 at 30 h in culture, which decreased the pH of culture from 6.90 to 1.65, and showed a strong ability to convert a wide range of insoluble phosphate into soluble forms (Gong et al., 2014b).Genes that can induce secretion of organic acids are present in the P. oxalicum I1 genome.Therefore P. oxalicum I1 was as a suitable organism from which to clone gene that may enhance organic acids secretion.From P. oxalicum I1, a total of 106 clones were obtained as candidates, and the cDNA sequence I-2 we obtained showed only 68% identity and 73% query cover with fungi.The deduced amino acid sequence showed 100% identity with a hypothetical protein in P. oxalicum 114-2, for which no gene function had been reported.The protein sequence from P. oxalicum I1 could be aligned with another hypothetical protein of the Penicillium genus.
Even though for some alignments the identity is less than 63%, it clearly forms a group of proteins that most probably share the same function.In this study, we proved that the protein can improve acetic acid secretion in E. coli DH5α™.In addition, the E. coli DH5α™ containing the target gene secreted up to 567 mg .l -1 acetic acid within 48 h.Lü et al. (2012) cloned a full-length gene encoding mitochondrial malate dehydrogenase from P. oxalicum, which was expressed in E. coli to secrete malic, lactic, acetic, and citric acids, and the obtained amount of organic acids was < 60 mg .l -1 .Gong et al. (2014a) cloned the delta-1-pyrroline-5-carboxylate dehydrogenase gene that secreted acetic acid and α-ketoglutaric acids, and the obtained the acetic acid concentration was 389.81 μg .ml -1 .Many organic acid metabolic pathways, such as glycometabolism and tricarboxylic acid cycle, are also present in E. coli.Glucose, pyruvic acid, citric acid, α-ketoglutaric acid, succinic acid, fumaric acid, and malic acid are the main substrates in the tricarboxylic acid cycle of E. coli DH5α™.Escherichia coli DH5α™ containing the target gene showed improved acetic acid secretion with the use of glucose, pyruvic acid, α-ketoglutaric acid, and malic acid.By contrast, acetic acid secretion was not improved when citric acid, succinic acid, and fumaric acid were used.This result indicated that the hypothetical protein was related to the utilization of glucose, pyruvic acid, α-ketoglutaric acid, and malic acid in E. coli.

CONCLUSIONS
We obtained a novel gene from Penicillium oxalicum I1 whose overexpression in E. coli DH5α™ increased the secretion of acetic acid.Alignment of the putative amino acid sequence of the protein with other comparable sequences of the Penicillium genus suggests that this is a protein group sharing similar functions, nothing however is known about their role in the other organisms.The results reported here may help to understand the function of these genes and the corresponding protein group.