Systematic Metabolic Engineering for the Production of Azaphilones in Monascus purpureus HJ11

Abstract

Fungal azaphilones have attracted considerable interest as they exhibit great potential in food and pharmacological industries. However, there is a severe bottleneck in the low production in wild strains and the ability to genetically engineer azaphilone-producing fungi. Using Monascus azaphilones (MAs) as an example, we demonstrate a systematic metabolic engineering strategy for improving the production of MAs. In this study, Monascus purpureus HJ11 was systematically engineered through a combination of promoter engineering, gene knockout, rate-limiting enzyme overexpression, repression of the competing pathway, enzyme engineering, and metabolic rebalance. The maximum yield and titer of MAs successfully increased to 906 mg/g dry cell weight (DCW) and 14.6 g/L, respectively, 2.6 and 3.7 times higher than those reported in the literature. Our successful model not only offers a practical and efficient way to improve the azaphilone production but also sheds light on the potential of systematic metabolic engineering in nonmodel fungi as a chassis for the production of high-value chemicals.

Versatile Strategy for the Construction of a Transcription Factor-Based Orthogonal Gene Expression Toolbox in Monascus spp

“…In our previous work on the Monascus azaphilone biosynthetic pathway, MaR (also designated as MrPigB) has been found to regulate the transcriptional expression of 14 biosynthetic genes in MABGC. , The expression levels of these genes were significantly different from each other, most likely as a result of the diverse promoter specificity of MaR. It is therefore possible to develop the orthogonal gene expression toolbox by using MaR and its cognate promoters.…”

“…The transcription of the maR gene was set under the control of the constitutive promoter P tef1 based on the prior evidence. 26 In order to achieve the accurate measurement of the conditional expression of the MaR module, a short-lived luciferase, Luc-PEST, was selected as the highly sensitive transcription reporter. 28 These constructed modules were, respectively, inserted into the P-kt strain genome by replacing the genomic region relative to the P mrpigB and mrpigB genes (Figure 2A).…”

“…Using this null background strain, we next turned to assembling the genetic regulation modules using MaR and its cognate promoters, P mrpigE , P mrpigF , and P mrpigG (Figure B). The transcription of the maR gene was set under the control of the constitutive promoter P tef 1 based on the prior evidence . In order to achieve the accurate measurement of the conditional expression of the MaR module, a short-lived luciferase, Luc-PEST, was selected as the highly sensitive transcription reporter …”

“…Gene manipulation in M. purpureus strains was performed as described in our previous work. 26 Replacement of the maR gene with an artificial gene fragment was used as an example. The maR gene and P mrpigG were amplified from M. purpureus HJ11 genomic DNA.…”

Modular and Flexible Molecular Device for Simultaneous Cytosine and Adenine Base Editing at Random Genomic Loci in Filamentous Fungi

“…To enable rapid screening of mutant variants, we selected the HJ11 strain, which is known for its ability to produce azaphilone pigments, as the starting strain. The biosynthesis of these pigments was catalyzed by 16 enzymes encoded within the Monascus pigment biosynthetic gene cluster (MPBGC), culminating in the formation of a deep red colony phenotype . Therefore, disruption of specific genes within the MPBGC or other unknown genes on the chromosome might result in the loss of red pigmentation or a white colony phenotype in the mutant strains.…”