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.