Fig.3-11 Red, orange and yellow values of C100, ΔMareA and OE-MareA under different nitrogen sources
3.4 HPLC analysis of pigments produced by solid state fermentation of C100, ΔMareA and OE-MareA strains
As shown in Figure 3-12.In the rice fermentation medium without additional nitrogen source, the pigment species of all three strains were dominated by Y1, O1 and O2, among which, O2 was the most abundant. The contents of yellow pigments of the three strains did not differ much, and they contained more Y1 than Y2.The contents of C100 red pigments R1 and R2 were significantly lower relative to orange and yellow pigments. The content comparison of ΔMareA red pigments R1 and R2 were lower than that of C100.Peak area comparison of OE-MareA in R1 was higher than that of C100 and R2 peak area was lower than that of C100.As shown in Tables 3-1
As shown in Figure 3-13.After the addition of Gln to the rice fermentation medium, there was not much difference in the peak area share of the six pigments C100, ΔMareA , and OE-MareA . The contents of all three strains were dominated by Y1, O1, and O2. Among them, O1 and O2 were the most abundant while Y2 was the least abundant. Compared with the control group without an added nitrogen source, there was an increase in the peak area share of R1 and R2 while a decrease in the peak area share of O1 and O2. This indicates that after adding Gln, it promotes the transformation from orange pigment amination to red pigment during red pigment synthesis. As shown in Tables 3-1.
As shown in Figure 3-14.After the addition of (NH4)2SO4, there was no significant difference in pigment type and content between the original strain C100 and the knockout strain ΔMareA . However, the red pigment content of the overexpression strain OE-MareA increased significantly. There was no significant difference in Y1 and Y2 contents between OE-MareA and C100. Compared to the control group without nitrogen source, there was an exponential increase in peak area occupied by R1 and R2 while orange pigment decreased; however, Y1 and Y2 contents did not change much. The only strain that showed a significant change towards red pigment synthesis with this addition was OE-MareA. This indicates that (NH4)2SO4 promotes amination of orange pigment to red during red pigment synthesis. As shown in Tables 3-2.
As shown in Figure 3-15. Using NaNO3 as the nitrogen source, there was no significant difference in pigment types and contents between the original strain C100 and the overexpression strain OE-MareA . The red pigments R1 and R2 were very low in C100, while the knockout strain ΔMareA had slightly higher red pigment content compared to C100. The percentage of yellow pigments Y1 and Y2 in the peak area of ΔMareA was reduced compared to that of C100, with a relatively large reduction observed for Y1. The orange pigment remained most abundant in C100, with a slightly increased O1 content observed in ΔMareA compared to the original strain, while O2 content did not differ significantly. As shown in Tables 3-3.
As shown in Figure 3-16. The addition of Urea as a nitrogen source resulted in the most noticeable change, which was the undetectable levels of orange pigments O1 and O2 in the original strain C100, knockout strain ΔMareA , and overexpression strain OE-MareA due to their extremely low levels. The other four major alcohol-soluble pigments in these three strains had the highest percentage of R1 and R2 and the lowest percentage of Y2. As shown in Tables 3-4.
In summary, knockdown and overexpression of the MareA gene do not affect the types of pigments but only their contents. Among them, the most noticeable changes were observed in orange and red pigments, while the content of yellow pigment remained relatively unchanged. Knocking down the MareA gene resulted in a decrease in red pigment content, particularly R2. Conversely, overexpressing the MareA gene led to an increase in orange pigment content, especially O2. It can be hypothesized that the MareA gene may influence the conversion of orange pigment to red pigment through amination in the synthesis pathway. Deletion of this gene inhibits this process, whereas its overexpression promotes it. After adding Gln and (NH4)2SO4, even without presence of the MareA gene, preferred nitrogen sources are still utilized resulting in similar pigmentation as observed in original strain. These two preferred nitrogen sources facilitate ammonification process. The addition of NaNO3 did not promote or inhibit the conversion from orange to red pigment mediated byMareA gene. When Urea is added and heated for decomposition into NH3 with high concentration of (NH4)2+ present in medium, sufficient ammonification occurs leading to substantial conversion from orange to red pigment and consequently significant increase in red pigment content.