β-Glucan Synthase Gene Overexpression and β-Glucans Overproduction in Pleurotus ostreatus Using Promoter Swapping

Abstract

Mushroom β-glucans are potent immunological stimulators in medicine, but their productivities are very low. In this study, we successfully improved its production by promoter engineering in Pleurotus ostreatus. The promoter for β-1,3-glucan synthase gene (GLS) was replaced by the promoter of glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans. The homologous recombination fragment for swapping GLS promoter comprised five segments, which were fused by two rounds of combined touchdown PCR and overlap extension PCR (TD-OE PCR), and was introduced into P. ostreatus through PEG/CaCl2-mediated protoplast transformation. The transformants exhibited one to three fold higher transcription of GLS gene and produced 32% to 131% higher yield of β-glucans than the wild type. The polysaccharide yields had a significant positive correlation to the GLS gene expression. The infrared spectra of the polysaccharides all displayed the typical absorption peaks of β-glucans. This is the first report of successful swapping of promoters in filamentous fungi.

Biocatalytic Synthesis of Fungal β-Glucans

“…In S. cerevisiae, the most well-studied microorganism in respect to GLS molecular biology, there are two fks genes, as well as in L. edodes, Auricularia auricula-judae and P. ferulae [10], while in the strains C. albicans, C. neoformans, A. fumigatus, Ustilago maydis, P. ostreatus and P. eryngii, a single gene copy has been found [10,11,38,41,70,71]. According to a recent classification from Yang et al [72], Ascomycetes gls genes are widespread mostly in the subphylum Pezizomycotina, while for Basidiomycetes, gls genes are distributed to four subphyla, with the majority of them belonging to subphylum Agaricomycotina, class Agaricomycetes.…”

“…Both these attributes are damaging for most host systems. However, Chai et al [11] succeeded in overexpressing the relevant genes of P. ostreatus by swapping the native promoter with the constitutive promoter of glyceraldehyde-3-phosphate dehydrogenase (gpd) from A. nidulans. Thus, they obtained from 32 to 131% higher β-glucan yield in the supernatants of six transformants.…”

“…Alternative attempts to achieve high-yields of β- (1,3)-glucan synthesis include the in vitro synthesis of these polymers [6], or the adjustment of the culture medium that is used for the microorganism growth [7][8][9]. Along with the above, the increase of β- (1,3)-glucan synthase (GLS) expression which is the key enzyme in the β- (1,3)-glucan synthesis [10], as well as GLS gene overexpression using promoter swapping [11] seem to be promising alternatives for higher productivities. Therefore, the main objective of this review article is the relevant research on GLS, summarizing previously published studies, since it is regarded as a largely unexplored biotechnological tool.…”

β-(1→3),(1→6)-Glucans: medicinal activities, characterization, biosynthesis and new horizons

“…Source: Adapted from work of Reverberi et al (2004) Pleurotus ostreatus was performed by polyethyleneglycol (PEG)-mediated protoplast and CaCl 2 . Due to being wild in type, glucan production increased from 32 to 131 %, enhancing from two to four times the gene expression of the GLS (Chai et al 2013). Malavazi et al (2014) found that nutrient changes or exposure to substances which cause toxicity and stress to filamentous fungi can interfere directly in the cell wall biosynthesis, as well as in growth the cell, showing that the cell wall is a first form of defense.…”

“…In an attempt to obtain β-(1→3)-glucans or β-(1→3),(1→ 6)-glucans with less interfering and higher yields, some advances have shown that the in vitro synthesis of these polysaccharides (Tomazett et al 2010) as well as modifications of the culture medium Papaspyridi et al 2010;Bzducha-Wróbel et al 2015), the increase of the protein expression β-(1→3)-glucan synthase (GLS) that realizes the synthesis of β-(1→3)-glucan (Reverberi et al 2004), and the genetic manipulation (Chai et al 2013) may be described as alternative methods.…”