Improved biomass and protein production in solid-state cultures of an Aspergillus sojae strain harboring the Vitreoscilla hemoglobin

Abstract

The biotechnological value of Aspergillus sojae ATCC 20235 (A. sojae) for production of pectinases in solid-state fermentation (SSF) has been demonstrated recently. However, a common drawback of fungal solid-state cultures is the poor diffusion of oxygen into the fungi that limits its growth and biological productivity. The bacterial Vitreoscilla hemoglobin (VHb) has favored the metabolism and productivities of various bacterial and yeast strains besides alleviating hypoxic conditions of its native host, but the use of VHb in filamentous fungi still remains poor explored. Based on the known effects of VHb, this study assessed its applicability to improve A. sojae performance in SSF. The VHb gene (vgb) under control of the constitutive Aspergillus nidulants gpdA promoter was introduced into the genome of A. sojae by Agrobacterium-mediated transformation. Successful fungal transformants were identified by fluorescence microscopy and polymerase chain reaction (PCR) analyses. In solid-state cultures, the content of protease, exo-polygalacturonase (exo-PG), and exo-polymethylgalacturonase (exo-PMG) of the transformed fungus (A. sojae vgb+) improved were 26, 60, and 44 % higher, respectively, in comparison to its parental strain (A. sojae wt). Similarly, biomass content was also 1.3 times higher in the transformant strain. No significant difference was observed in endo-polygalacturonase (endo-PG) content between both fungal strains, suggesting dissimilar effects of VHb towards different enzymatic productions. Overall, our results show that biomass, protease, and exo-pectinase content of A. sojae in SSF can be improved by transformation with VHb.

Keywords: Agrobacterium tumefaciens-mediated transformation; Aspergillus sojae; Biomass production; Pectinases and protease; Solid-state fermentation; Vitreoscilla hemoglobin.

Fig. 1

a The vector pRM-vgb was used for ATMT of A. sojae and contains the kanamycin resistance gene (kanR), trans-acting factor A gene (trfA), replication origin (oriV), and transfer DNA region (T-DNA). b Enlargement of the T-DNA region that is delimited by the right and left border (RB and LB, respectively) and contains the Vitreoscilla hemoglobin gene (vgb) and the reporter enhanced green fluorescent protein gene (egfp) under control of the constitutive Aspergillus nidulants gpdA promoter (PgpdA), the phleomycin resistance gene from Stretoalloteichus hindustanus (Sh ble) under control of the A. nidulants trpC promoter (PtrpC) and trpC terminator (TtrpC). The red arrows indicate the target sites for the oligonucleotide primers BLE-F/R, VHb-F/R. Target sites for restriction enzymes are also shown

Fig. 2

a Verification of putative A. sojae transformants by PCR analysis. The amplified PCR products at the expected size (highlighted with arrows) confirm the presence of the ble gene and the vgb gene in the genomic DNA samples of the fungal transformants. Genomic DNA of A. sojae wt was used as negative (W) and purified pRM-vgb vector as positive control (C); molecular size marker (M). b Verification of egfp expression in the selected A. sojae vgb + transformant by fluorescence microscopy analysis. A. sojae wt was used as negative control

Fig. 3

Typical growth of A. sojae wt and A. sojae vgb + on solid substrate during the 10-day fermentation period (only selected days are shown). The column on the left shows a representative flask of the inoculation day (0), and the column on the right shows the non-inoculated control media after 10 days of incubation under the same conditions (C)

Fig. 4

Protein production of A. sojae wt and A. sojae vgb + in SSF. Exo-PG (a), exo-PMG (b), endo-PG (c), and protease (d) content were determined from enzymatic extracts collected every 24 h during the 10-day incubation period. Solid lines indicate the enzymatic yield, and dashed lines indicate specific activity. Each data point represents the average ± SD from fermentations carried out in triplicates

Fig. 5

Biomass content of A. sojae wt and A. sojae vgb + in SSF. The values plotted are expressed as milligram of glucosamine (mg_GlcN) per gram of dried fermented substrate (dfs), which were collected every 24 h during the 10-day incubation time. Each data point represents the average ± SD from fermentations carried out in triplicates

Fig. 6

Relative yields of A. sojae wt and A. sojae vgb + in SSF. The maximum yields of the parental A. sojae wt strain were adjusted to 1.0 and compared with the normalized maximum titers of the transformed A. sojae vgb + strain