Fig.3-18 Expression levels of growth and metabolism genes ofMonascus C100, ΔMareA and OE-MareA in solid-state
fermentation under different nitrogen sources
This study used M. ruber C100 as the starting strain and amplified theMare A gene through PCR. MareA gene knockout and
overexpression vectors were constructed. The study then used
Agrobacterium-mediated transformation to infect Monascus purpureus with
the vectors, and selected the correct ΔMareA and OE-MareA strains based on the principle of homologous recombination. The study
then compared the changes in colony morphology, spore production, and
mycelial morphology among the three strains to investigate the impact of
the MareA gene on the mycelial morphology of Monascus
purpureus.We conducted a preliminary study on the impact of theMareA gene on growth and reproduction in monascus. The
regulation of the MareA gene on monascus (a secondary metabolite)
in monascus was examined through solid-state fermentation by
analyzing expression differences in red currant pigment and genes
related to growth and development. This analysis aimed to understand the
role played by the MareA gene in monascus’ metabolic process. The
research results of this thesis are as follows:
The DNA of M. ruber C100 was used as a template, and theMareA gene homologous sequence C5.761 in the genome of M.
purpureus YY-1 was used as a reference to design primers. The full
length of the MareA gene fragment, 2746 bp, was obtained through
PCR amplification and sequencing. The sequencing results of C100 were
compared with those of C5.761 in the YY-1 genome using DNAMAN software
for analysis, resulting in a 99.15% similarity. This fragment contained
889 amino acids and exhibited the conserved GATA-type zinc finger
structure (C-X(2)-C-X(17)-C-X(2)-C).
Using p59 as the original vector, the homologous arms of the MareA gene
on both ends were amplified by PCR and then inserted into the two ends
of the p59 vector with the selection marker gene Hyg. The successfully
reconstructed vector p59ss was screened and verified. Using p1301 as the
original vector, a fragment of the MareA gene was amplified by PCR and
then inserted into the CaMV 35S strong promoter and CaMV 35S terminator
between the p1301 vector. The correct overexpression recombinant plasmid
p1301s was screened and verified. The obtained recombinant vectors p59ss
and p1301s were introduced into Agrobacterium rhizogenes separately, followed by co-culturing with monascus throughAgrobacterium-mediated transformation to screen and validate
ΔMareA knockout strain and OE-MareA overexpression strain.
The monascus were cultivated through solid-state fermentation under
different nitrogen source conditions, and the yield of MPs was tested
after 13 days of fermentation. The results showed that the ΔMareA strain reduced the color values of red, orange, and yellow pigments,
while OE-MareA increased the color values of these pigments.
Meanwhile, the MareA gene does not affect the species of the six
major pigments but influences their contents. Specifically, it has
little effect on Y1 and Y2 contents but mainly affects the percentage of
peak area occupied by R1, R2, O1, and O2. Comparing fermentations under
different nitrogen source conditions revealed that the ΔMareA strain
could preferentially utilize Gln and
(NH4)2SO4 but had
limited utilization ability for NaNO3 and Urea.
Additionally, it had varying effects on pigment color values. In
summary, these results demonstrate that knocking down the MareA gene decreases total pigment color value in red yeast while
overexpressing this gene increases it. This indicates that metabolism of
red yeast pigment is regulated by MareA gene expression levels.
Furthermore, different nitrogen sources are utilized differently byMareA gene. Gln can counteract its effects on growth and
development in monascus whereas NaNO3 most prominently
reflects its impact during fermentation.
RT-qPCR was used to analyze the expression of four MPs genes and four
growth-regulated genes. From the expression of the four key genes
involved in MPs biosynthesis, it can be observed that after deleting theMareA gene, the gene expression of mppE was significantly
up-regulated, while the relative expressions of MpPKS5, mppG , andmppD genes were down-regulated to varying degrees. On the other
hand, overexpressing the MareA gene suppressed the expression of
all four key pigmentation genes. Regarding growth regulation, deletion
of MareA gene notably reduced VosA and LaeA gene
expressions, whereas overexpression promoted GprD gene
significantly. In presence of Gln (glutamine), deletion of MareA did not affect monascus growth, however, in presence
NaNO3 (sodium nitrate), its deletion had a clear
inhibitory effect on both growth and reproduction. These results
indicate that MareA gene influences sexual and asexual
development as well as metabolism in monascus.
Discussion
In this study, overgene knockout and overexpression techniques were used
to achieve the knockout and overexpression of MAreA gene, the
global transcriptional regulatory factor of monascus, and theMAreA knockout strain ΔMAreA and the overexpression strain
OE-MAreA were successfully obtained through
agrobacterium-mediated transformation technology. Through solid state
fermentation, it was found that overexpression of MAreA gene
would increase the yield of Mps, and different types of Mps would be
obtained due to different utilization of nitrogen source by MAreA gene. Especially, the yield of red pigment R1 and R2 increased
significantly after urea was added. The effects of MAreA gene on
the sexual and asexual development and pigment metabolism of monascus
were studied. The study results provided a reference for determining the
functional characteristics of AreA gene, theoretical guidance and
technical support for monascus large-scale fermentation of Mps in the
future, and provided a basis for the production of Mps strains for
industrial use. However, due to time constraints, only RT-qPCR was used
to achieve the difference analysis of gene expression in the functional
prediction of MAreA gene, and the number of genes for difference
analysis was small. In subsequent studies, high-throughput sequencing
can be used to analyze the function of MAreA gene on the
transcript data. In addition, due to the hygromycin antibiotic gene in
the strain genome, it can not be used for industrial scale fermentation
of Mps. After removing the hygromycin resistance gene, it should be
considered as a high-yield pigment engineering strain. Previous studies
have shown that there is no secondary metabolite citrinin in Monascus
rubrus C100 or the content is very small. Therefore, the effect of MAreA
gene on the yield of citrinin was not studied in this paper. In future
studies, it is possible to select monascus strains with high yield of
citrinin, such as monascus purple M8, for MAreA gene knockout and
overexpression, so as to study the effect of MAreA gene on citrinin
metabolism. Finally, the optimal process of industrial production and
preparation of Mps can be achieved by cultivation under different
nitrogen source conditions, so as to achieve large-scale production of
Mps and improve its application in various industries.
Conflict of Interest Statement
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.
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