New Promoters for Metabolic Engineering of Ashbya gossypii

Abstract

Ashbya gossypii is a filamentous fungus that is currently exploited for the industrial production of riboflavin. In addition, metabolically engineered strains of A. gossypii have also been described as valuable biocatalysts for the production of different metabolites such as folic acid, nucleosides, and biolipids. Hence, bioproduction in A. gossypii relies on the availability of well-performing gene expression systems both for endogenous and heterologous genes. In this regard, the identification of novel promoters, which are critical elements for gene expression, decisively helps to expand the A. gossypii molecular toolbox. In this work, we present an adaptation of the Dual Luciferase Reporter (DLR) Assay for promoter analysis in A. gossypii using integrative cassettes. We demonstrate the efficiency of the analysis through the identification of 10 new promoters with different features, including carbon source-regulatable abilities, that will highly improve the gene expression platforms used in A. gossypii. Three novel strong promoters (PCCW12, PSED1, and PTSA1) and seven medium/weak promoters (PHSP26, PAGL366C, PTMA10, PCWP1, PAFR038W, PPFS1, and PCDA2) are presented. The functionality of the promoters was further evaluated both for the overexpression and for the underexpression of the A. gossypii MSN2 gene, which induced significant changes in the sporulation ability of the mutant strains.

Metabolic engineering of Ashbya gossypii for limonene production from xylose

“…In addition, A. gossypii has been proposed as an efficient microbial factory for the production of many other metabolites such as nucleosides, folic acid, biolipids, gamma-lactones and recombinant proteins [9][10][11][12]. Thereby, A. gossypii can be considered as a biotechnological chassis owing to (i) the availability of a large molecular toolbox for systems metabolic engineering, including gene-targeting methods, heterologous expression platforms, or CRISPR/Cas9/Cas12 adapted systems [9,[13][14][15][16][17]; (ii) the ability to grow using low-cost oils and industrial residues such as lignocellulosic hydrolysates, molasses, and crude glycerol [8,18,19]; and (iii) and the ease of mycelial harvesting by simple filtration [11], thus making the A. gossypii bioprocessing cost-effective.…”

“…While CltLS1 shows high (99%) selectivity for the production of limonene and can be functionally expressed in different microbial chassis after removal of the plastid targeting signal [37], CltLS2 was reported to be inactive in R. toruloides [38]. Hence, a truncated codon-optimized form of the C. limon LS1 (tLS) (Additional file 1) was synthesized and heterologously overexpressed in A. gossypii under the control of the strong promoter P GPD1 [15]. In addition, the overexpression of three different isoforms of the native Hmg1 was evaluated.…”

“…Consequently, a truncated and codon-optimized form of the NDPS1 gene from Solanum lycopersicum was synthesized (Additional file 1). The tNDPS1 gene was assembled in an integrative expression module and was heterologously overexpressed in the parental strain under the control of the strong promoter P TSA1 , to avoid genomic instability due to the recurrent utilization of P GPD1 [15]. The newly engineered strain was cultured in xylose-based media and produced a limonene titer approximately 30-fold higher than that of the parental strain, reaching 33.6 mg/L (Fig.…”

“…Accordingly, the overexpression of ERG12, ERG8 and ERG19 was assessed in combination with the utilization of the NPP synthase pathway. Both ERG12 and ERG8 were overexpressed using the strong promoter P SED1 , while the overexpression of ERG19 was carried out using the strong promoter P TSA1 [15]. The engineered strains were cultured in xylose-based media and the limonene production was measured.…”

“…Transformation cassettes for genomic integration were used for the overexpression of either endogenous or heterologous genes. For the overexpression of A. gossypii endogenous genes, the transformation cassettes comprised the loxP-KanMX-loxP selectable marker, conferring resistance to G418, followed by the sequence of a strong promoter (P GPD1 , P SED1 or P TSA1 ) [15]. The overexpression cassettes were PCR-amplified using specific primers for each gene (Additional file 5), providing recombinogenic flanks (75-100 bp) for the genomic integration of the cassettes by homologous recombination (Additional file 6).…”