Abstract
BackgroundMonascus azaphilone pigments (MonAzPs), which were produced by Monascus species, have been used as important food colorant and food supplements for more than one billion people during their daily life. Moreover, MonAzPs recently have received more attention because of their diverse physiological activities. However, the high microbial production of MonAzPs is still not always guaranteed. Herein, the aim of this study was to develop an efficient biotechnological process for MonAzPs production.ResultsIn this study, exogenous cyclic adenosine monophosphate (cAMP) treatment not only induced MonAzPs production, but also stimulated the expression of a cAMP phosphodiesterase gene, named as mrPDE, in M. purpureus HJ11. Subsequently, MrPDE was identified as a cAMP phosphodiesterase by in vitro enzymatic reaction with purified enzyme. Further, a gene knockout mutant of mrPDE was constructed to verify the activation of cAMP signalling pathway. Deletion of mrPDE in M. purpureus HJ11 improved cAMP concentration by 378% and enhanced PKA kinase activity 1.5-fold, indicating that activation of cAMP signalling pathway was achieved. The ΔmrPDE strain produced MonAzPs at 8563 U/g, with a 2.3-fold increase compared with the WT strain. Moreover, the NAPDH/NADP+ ratio of the ΔmrPDE strain was obviously higher than that of the wild type strain, which led to a higher proportion of yellow MonAzPs. With fed-batch fermentation of the ΔmrPDE strain, the production and yield of MonAzPs achieved 332.1 U/mL and 8739 U/g.ConclusionsA engineered M. purpureus strain for high MonAzPs production was successfully developed by activating the cAMP signalling pathway. This study not only describes a novel strategy for development of MonAzPs-producing strain, but also provides a roadmap for engineering efforts towards the production of secondary metabolism in other filamentous fungi.
Highlights
Monascus azaphilone pigments (MonAzPs), which were produced by Monascus species, have been used as important food colorant and food supplements for more than one billion people during their daily life
M. purpureus HJ11 strain was co-incubated with Agrobacterium tumefaciens EHA105, containing recombinant pCAMBIA3300, to obtain deletion mutants. pCAMBIA3300 vector was used as deletion vector and pET30a(+) was used as heterologous gene expression in E. coli BL21 (DE3)
The MonAzPs production was determined under different concentrations (0, 1.0, 2.0, 3.0 and 4.0 mM) of cyclic adenosine monophosphate (cAMP) in glucose mineral salt (GM) medium
Summary
Monascus azaphilone pigments (MonAzPs), which were produced by Monascus species, have been used as important food colorant and food supplements for more than one billion people during their daily life. MonAzPs have been extensively used as natural food coloring agents and food supplements for more than one billion people [5, 6]. The highest MonAzPs production was achieved with 211 U/mL using a mutant M. purpureus strain M183 in liquid-state (submerged) fermentation [15]. These microbial productions of MonAzPs still do not fully meet the demand for cost-effective production
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