Because Monascus pigments (MPs) predominantly accumulate in the cytoplasm during submerged fermentation, many biotechnologies are applied to enhance the production of extracellular MPs (exMPs) to reduce the downstream processing costs. In this study, the genes monascus_7017 and monascus_8018, identified as ERG4 genes, were knocked out to disrupt the ergosterol biosynthetic pathway and enhance the production of exMPs in Monascus purpureus LQ-6. Double-deletion of EGR4 in M. purpureus LQ-6 reduced ergosterol concentration by 57.14% and enhanced exMP production 2.06-fold. In addition, integrated transcriptomic and proteomic analyses were performed to elucidate the transmembrane secretion mechanism of exMPs based on the relationship between ergosterol synthesis and membrane permeability, which revealed that several metabolic pathways were noticeably dynamic, including fatty acid degradation, amino acid metabolism, energy metabolism, carbohydrate metabolism, and transport. These findings therefore clarified the secretion mechanism of exMPs and provide a novel strategy for further enhancement of exMP production in submerged fermentation.

BACKGROUND: Monascus pigments (MPs) produced by the genus Monascus, have been utilized for more than 2000 years in the food industry. In the present study, by submerged batch-fermentation (SBF), we were able to obtain a mutant strain with a high tolerance of inhibitory compounds generated from rice husk hydrolysate, allowing the production of MPs.RESULTS: The mutant strain, M. Purpureus M523 with high rice husk hydrolysate tolerance was obtained using the atmospheric and room temperature plasma (ARTP) screening system, producing 39.48 U mL −1 extracellular total MPs (yellow and orange MPs), using non-detoxified rice husk diluted sulfuric acid hydrolysate (RHSAH) as the carbon source in SBF. Extracellular MPs (exMPs) production was enhanced to 72.1 and 80.7 U mL −1 in supplemented SBF (SSBF) and immobilized fermentation (IF) using non-detoxified RHSAH, with productivities of 0.16 and 0.37 U mL −1 h −1 , respectively. In addition, our findings revealed that despite having a high RHSAH tolerance, the mutant strain was unable to degrade phenolic compounds. Furthermore, we discovered that inhibitory compounds, including furfural (Fur) and 5 0 -hydroxymethyl furfural (5 0 -HMF), not only inhibit MP biosynthesis, but also regulate the conversion of pigment components.CONCLUSION: The low-cost agricultural by-product, rice husk, can serve as an efficient substitute for MP production with high productivity via IF by Monascus spp.