A novel transcriptional activation mechanism of inulinase gene in Kluyveromyces marxianus involving a glycolysis regulator KmGcr1p with unique and functional Q‐rich repeats

Abstract

Kluyveromyces marxianus is the most suitable fungus for inulinase industrial production. However, the underlying transcriptional activation mechanism of the inulinase gene (INU1) is hitherto unclear. Here, we undertook genetic and biochemical analyses to elucidate that a glycolysis regulator KmGcr1p with unique Q‐rich repeats is the key transcriptional activator of INU1. We determined that INU1 and glycolytic genes share similar transcriptional activation patterns and that inulinase activity is induced by fermentable carbon sources including the hydrolysis products of inulin (fructose and glucose), which suggests a novel model of product feedback activation. Furthermore, all four CT‐boxes in the INU1 promoter are important for KmGcr1p DNA‐binding in vitro, but the most downstream CT‐box 1 primarily confers upstream activating sequence activity in vivo. More intriguingly, the use of artificial and natural GCR1 mutants suggests that the Q‐rich repeats act as a functional module to maintain KmGcr1p transcriptional activity by contributing to its solubility and DNA‐binding affinity. Altogether, this study uncovers a novel transcriptional activation mechanism for the inulinase gene, that is different from the previous understanding for filamentous fungi, but might have universal significance among inulinase‐producing yeasts, thereby leading to a better understanding of the regulation mechanism of yeast inulinase genes.

Customizable and stable multilocus chromosomal integration: a novel glucose-dependent selection system in Aureobasidium spp.

“…The fluorescence intensity was measured using Varioskan ™ LUX Multimode Microplate Reader (Thermo Fisher, USA) with an excitation wavelength of 480 nm and emission wavelength of 520 nm according to our previous method [ 49 ]. The yeast cells were resuspended in the sterile PBS and diluted until the OD 600nm value of the suspension reached 0.8.…”

“…Purification of total RNA, quantification of synthesized cDNAs by qPCR, and data analysis were carried out following the protocol as detailed in [ 49 ]. The β-actin gene was used as an internal reference.…”

“…The gene copy numbers of GFP in different yeast strains were analyzed by Quantitative Real-time PCR using genomic DNA as templates according to a previous study [ 49 ]. The glyceraldehyde-3-phosphate dehydrogenase gene ( GAPDH ) was used as a reference gene.…”

Bioconversion of non-food corn biomass to polyol esters of fatty acid and single-cell oils

“…RNA samples were isolated from P4R5 cells grown in the PEFA production medium at 28 °C for 72 h with different monosaccharides of d -glucose, d -xylose, d -fructose, d -arabinose, or l -arabinose as the carbon source. Purification of total RNA, quantification of synthesized cDNAs by qPCR (Additional file 3 : Table S3), and data analysis were conducted essentially as described [ 46 ].…”