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Combinatorial mutagenesis and fermentation optimization biotechnologies synergistically enhance monacolin K content
Zhengyan Wu1, Chan Zhang2, Qing Liu1
1Hunan Key Laboratory of Forestry Edible Resources Safety and Processing, Hunan Key Laboratory of Grain-Oil Deep Process and Quality Control, National Engineering Research Center of Rice and Byproduct Deep Processing, School of Food Science and Engineering, Central South University of Forestry and Technology, Changsha, China.
Introduction:
Red yeast rice (RYR) is produced through solid-state fermentation by Monascus genus. Its functional component, Monacolin K (MK), has the same structure as lovastatin and can effectively inhibit HMG-CoA reductase, thereby reducing serum cholesterol.
Methods:
A combinatorial mutagenesis strategy integrating atmospheric room-temperature plasma and heavy-ion radiation was employed to generate mutant strains. The optimizations of substrate components and conditions were carried out during solid-state fermentation (SSF). Subsequently, RYR enriched with MK was produced through scale-up experiments. Additionally, integrated biosynthetic pathway with expression dynamics of MK biosynthetic gene cluster to reveal the efficient biosynthesis of MK in a mutant strain.
Results:
A mutant M. purpureus CSUFT-1, which exhibited a remarkable 1.67-fold increase in MK production during SSF compared to the original strain, was obtained. Through refinement of SSF parameters, specifically, adding optimized corn steep liquor adjuvant solution [60% (v/v) corn steep liquor, pH modulation at 5, 3 g/L of MgSO4·7H2O, 1.5 g/L of KH2PO4] with 20% (v/w) of injection volume, the MK yield was significantly amplified to 32.71 mg/g on day 28. Scale-up experiments confirmed the robustness of the optimized process, with MK production stabilizing approximately at 24.66 mg/g. RT-qPCR results showed that seven key genes, including mokC (6.8-fold upregulation) and mokF (22-fold upregulation), were significantly activated during the early stage of fermentation to drive precursor synthesis, only mokE gene exhibited the sustained overexpression in M. purpureus CSUFT-1 during the entire SSF.
Discussion:
This work successfully demonstrates a synergistic approach combining advanced combinatorial mutagenesis with precise bioprocess optimization to substantially improve MK yield. The overproducing M. purpureus CSUFT-1 and the optimized SSF protocol provide both microbial resource and technical protocol for industrial-scale production of RYR with high MK.
