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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