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

Abstract

Gene expression is needed to be conducted in an orthogonal manner and controllable independently from the host's native regulatory system. However, there is a shortage of gene expression regulatory toolboxes that function orthogonally from each other and toward the host. Herein, we developed a strategy based on the mutant library to generate orthogonal gene expression toolboxes. A transcription factor, MaR, located in the Monascus azaphilone biosynthetic gene cluster, was taken as a typical example. Nine DNA-binding residues of MaR were identified by molecular simulation and site-directed mutagenesis. We created five MaR multi-site saturation mutagenesis libraries consisting of 10743 MaR variants on the basis of five cognate promoters. A functional analysis revealed that all five tested promoters were orthogonally regulated by five different MaR variants, respectively. Furthermore, fine gene expression tunability and high signal sensitivity of this toolbox are demonstrated by introducing chemically inducible expression modules, designing synthetic promoter elements, and creating protein−protein interaction between MaRs. This study paves the way for a bottom-up approach to build orthogonal gene expression toolboxes.

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

“…As established in our previous work, the saturation induction concentrations of doxycycline and isomaltose were 1.0 μg/mL and 20.0 mg/mL, respectively. 28 Evaluation of the base editing efficiency under various induction conditions revealed that the efficiency of base editing had a positive correlation with the respective inducer concentrations when expressions of ABE8e and AID genes were induced simultaneously (Figure 5A). At saturation inducer concentrations, the mutation rate of the strain reached 3.8 × 10 −7 base p.n.p.g.…”

“…Protein–protein interactions occur specifically between cohesins and dockerins, specifically CTCoh with CTDoc, CCCoh with CCDoc, and RFCoh with RFDoc. − To ascertain which helicase subunit conjugated BE to attain optimal base editing efficiency, MpHLC3, MpHLC4, MpHLC5, and MpHLC6 were individually fused to a cohesin polypeptide (CTCoh–CCCoh–RFCoh); meanwhile, the C-terminal of ABE8e was linked with a dockerin CCDoc (Figure B). Expression of the fused ABE8e protein was regulated by a synthetic MIES promoter, which was induced by doxycycline, as we previously described . 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.…”

“…Controlling the Base Editing Type of MIDBE. To demonstrate the versatility of MIDBE in modulating the type of base editing, we utilized previously established inducible promoters in M. purpureus HJ11 28 to control the expression of the ABE8e and AID genes. Specifically, the gene expression of AID-CCDoc and AID-RFDoc was controlled by a doxycyclineinduced promoter (P min ), and ABE8e-CTDoc gene expression was regulated by an isomaltose-activator-regulated promoter (P amyB ) (Figure 4A).…”

“…Expression of the fused ABE8e protein was regulated by a synthetic MIES promoter, which was induced by doxycycline, as we previously described. 28 To enable rapid screening of mutant variants, we selected the HJ11 strain, which is known for its ability to produce azaphilone pigments, 29 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.…”

“…purpureus HJ11, and the cohesin fragment was chemically synthesized by General Biol Co., Ltd. (Hefei, China). The HYG resistance gene expression cassette was obtained by cloning from plasmid pCB301-PK . These five fragments were subsequently assembled in the following order: HA1-MpHLC4-cohesins-HYG-HA2, with a flexible polypeptide linker (GGGSGGGSGGGS) inserted between the MpHLC4 and cohesin genes, using the Hieff Clone One Step Cloning Kit (YEASEN, China) to generate the artificial hybrid helicase.…”