in addition to glucose what other monosaccharide is part of the udp-glucose structure

• Journal Listing
• Front end Bioeng Biotechnol
• v.9; 2021
• PMC8655778

Forepart Bioeng Biotechnol. 2021; 9: 796278.

Label and Heterologous Expression of UDP-Glucose 4-Epimerase From a Hericium erinaceus Mutant with High Polysaccharide Product

Gen Zou, ^{1 , †} Juanbao Ren, ^{1 , 2 , †} Di Wu, ¹ Henan Zhang, ^ane Ming Gong, ⁱ Wen Li, ¹ Jingsong Zhang, ⁱ and Yan Yang ^{1 , *}

Gen Zou

¹ National Engineering Enquiry Center of Edible Fungi, Institute of Edible Fungi, Shanghai University of Agronomical Sciences, Shanghai, Red china,

Juanbao Ren

¹ National Engineering science Enquiry Center of Edible Fungi, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai, Mainland china,

^two College of Food Sciences and Technology, Shanghai Ocean University, Shanghai, Communist china,

Di Wu

^ane National Engineering Research Centre of Edible Fungi, Plant of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai, China,

Observe articles past Di Wu

Henan Zhang

¹ National Applied science Research Centre of Edible Fungi, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai, China,

Ming Gong

¹ National Engineering Enquiry Center of Edible Fungi, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai, China,

Wen Li

¹ National Engineering Research Eye of Edible Fungi, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai, China,

Jingsong Zhang

¹ National Engineering Research Center of Edible Fungi, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai, China,

Yan Yang

¹ National Engineering Research Center of Edible Fungi, Institute of Edible Fungi, Shanghai University of Agronomical Sciences, Shanghai, China,

Received 2021 Oct 16; Accepted 2021 November 3.

Abstract

Hericium erinaceus is an important medicinal mucus in traditional Chinese medicine because of its polysaccharides and other natural products. Compared terpenoids and polyketides, the analysis of synthetic pathway of polysaccharides is more than difficult because of the many genes involved in primal metabolism. In previous studies, A6180, encoding a putative UDP-glucose 4-epimerase (UGE) in an H. erinaceus mutant with loftier production of active polysaccharides, was significantly upregulated. Since there is no reliable genetic manipulation engineering science for H. erinaceus, we employed Escherichia coli and Saccharomyces cerevisiae to study the part and activity of A6180. The recombinant overexpression vector pET22b-A6180 was synthetic for heterologous expression in E. coli. The enzymatic properties of the recombinant protein were investigated. It showed that the recombinant A6180 could strongly convert UDP-α-D-glucose into UDP-α-D-galactose under optimal atmospheric condition (pH 6.0, xxx°C). In add-on, when A6180 was introduced into S. cerevisiae BY4742, xylose was detected in the polysaccharide composition of the yeast transformant. This suggested that the poly peptide coded by A6180 might be a multifunctional enzyme. The generated polysaccharides with a new composition of sugars showed enhanced macrophage activity in vitro. These results betoken that A6180 plays an important office in the structure and activity of polysaccharides. It is a promising strategy for producing polysaccharides with college activity by introducing A6180 into polysaccharide-producing mushrooms.

Keywords: polysaccharide synthesis, heterologous expression, allowed activity, enzymatic properties, polysaccharide, Hericium erinaceus

Introduction

Hericium erinaceus, which grows widely in the mountains of eastern Asia, is a valuable fungus for both medicinal and nutrient utilize and has a long history in the field of medicine in China. Polysaccharides, the main active substance of H. erinaceus, have loftier biological activeness, such as immunomodulation (Wu et al., 2019; Nowak et al., 2020), regulation of glucolipid metabolism (Du et al., 2015; Cai et al., 2020), hypolipidemia (Liang et al., 2013), anti -tumor backdrop (Zeng and Zhu, 2018; Zhang et al., 2021), anti-fatigue properties (Liu et al., 2015), liver protection, and stomach protection (Chen et al., 2020). They have also been widely used in medicine (Li et al., 2018; Jithendra et al., 2020), materials (Low et al., 2015), cosmetics, and other fields. Due to the large number of key enzymes involved in the synthesis of polysaccharides and their unknown characteristics, the synthesis pathway of H. erinaceus polysaccharides has not been clearly antiseptic. In our previous study, two mutant strains with high polysaccharide production of H. erinaceus were bred by atmospheric pressure level room temperature plasma (ARTP) mutagenesis, and polysaccharide production was significantly enhanced, compared with the original strain (Zhu et al., 2019). Through multi-omics analysis, the increased carbohydrate metabolism and the production of glucose-six-phosphate constituted the footing of high polysaccharide yield. The differentially expressed proteins A6180 involved in the mushroom polysaccharide biosynthetic pathways occurred in the mutant strain compared with the original strain, which belonged to GAL10 (UDP-glucose-4-epimerase) involved in the synthesis of polysaccharide echo units, and upregulated mRNA and protein expression based on transcriptome and proteomics information (Gong et al., 2021). Upregulation of the A6180 cistron is known to exist involved in the biosynthesis of polysaccharide repeat units, which is associated with the higher yield of polysaccharides in the mutated strains. However, the function of this gene all the same needs to exist verified through genetic engineering science technology.

Using the method of factor overexpression is an important arroyo to clarify factor function. For example, Jesus et al. constructed recombinant overexpressed strains and homologous overexpressed BL23 gene (encoding UGP) in Lactobacillus casei. In the subsequent enzyme action detection, information technology was found that the enzyme activeness of Fifty. casei increased past approximately seventy times, and the concentration of UDP-Glu increased by 8.v times. Therefore, it is inferred that the BL23 cistron is a key gene in the UDP-glucose synthesis pathway (Rodriguez-Diaz and Yebra, 2011).

Since genetic manipulation techniques in nigh macro fungi are not sophisticated, it is difficult to investigate the function of a gene by knockout or overexpression of a nativegene. Therefore, genetic verification in heterologous or model microorganisms can more effectively discover the functions of target genes (Bachmann et al., 2014). Escherichia coli and Saccharomyces cerevisiae, as typical eukaryotic model organisms and microbial cell factories, have been widely used in metabolic engineering, organisation biological science, and synthetic biology. For example, S. cerevisiae cannot straight utilize xylan as a carbon source, and strains with high expression can be obtained past integrating xylanase with high enzyme activity into the S. cerevisiae genome by ways of molecular biology (Lu et al., 2017; Wang et al., 2017). Researchers (Biely et al., 2000) expressed the Xyn2 factor of Trichoderma reesei in S. cerevisiae using different promoters, ADH2 and PGK1, and the enzyme activity was 200 and 160 nkat/ml. Besides, the de novo synthesis pathway of g-cresol was constructed from S. cerevisiae glucose past introducing the heterologous pathway of vi-MSA (Hitschler and Boles, 2019). The high yield of emodin reached 528.4 mg/L in Due south. cerevisiae BJ5464-NPGA by heterologous reconstruction of the biosynthesis pathway of endorphin and emodin (Sun et al., 2019).

In this study, E. coli and S. cerevisiae were used every bit heterologously expressing chassis organisms to further verify the biological function of the gene A6180, which is closely related to loftier polysaccharide product in H. erinaceus. The A6180 gene clone and dissimilar recombinant overexpression vector constructs for heterologous expression will be further studied to find out the functional role of this gene in polysaccharide synthesis. The enzyme backdrop of the protein expressed past the A6180 gene were studied to provide a theoretical reference for after development and utilization.

Materials and Methods

Strain and Plasmids

The experimental strain used in this study was H. erinaceus 321, preserved at the Institute of Edible Mushrooms, Shanghai Academy of Agricultural Sciences. Plasmid pET-22b (+) cells were preserved in our laboratory. E. coli strains TOP10 and BL21 (DE3) competent cells were purchased from Weidi Biotechnology (Shanghai, Communist china). S. cerevisiae BY4742 was purchased from Zoman Biotechnology (Beijing, Prc). The pESC-Leu plasmid was purchased from TIANDZ Gene Technology (Beijing, China). To construct the heterologous expression vector, the mycelium of H. erinaceus was scraped and total RNA was extracted using TRIzol RNA Isolation Reagents (TAKARA) kit, followed by Hifair TM II 1st Strand cDNA Synthesis Kit (YEASEN, Shanghai, China). The obtained cDNA was used every bit a template for amplification of the A6180 coding sequence. Yeast genomic DNA was extracted from cultured Saccharomyces cerevisiae BY4742 using the Found Genomic Deoxyribonucleic acid Extraction Kit (TIANGEN, Beijing, People's republic of china). The extracted gDNA was used as a template for amplification of the TDH3 promoter. Primers A6180EC-F and A6180EC-R were designed to amplify the A6180 factor fragment using Primer Premier v.0 based on the results of whole genome sequencing of H. erinaceus 321, and all the primer sequences used in this study are shown in Table 1. The PCR distension procedure for the A6180 target cistron was as follows: 3 min at 98°C; 30 cycles of 98°C for 10 southward, 58°C for 20 s, 72°C for eighty s; and a terminal extension at 72°C for v min. The pET-22b (+) vector was amplified with restriction endonucleases (QuickCut Nde I and Xho I; TAKARA, Dalian, China). Double digestion was performed, followed by the structure of the recombinant vector (pET22b-A6180) using the Hieff Clone^® Plus Multi One Step Cloning Kit (YEASEN) kit.

Table 1

Primer design of the Hericium erinaceus A6180 gene and related functional fragment.

Primer	Sequence (5′-3′)	Descriptions
A6180EC-F	AAC​TTT​AAG​AAG​GAG​ATA​TAC​ATA​TGG​CTG​TTG​CCG​ATA​CCT​C	For full-length A6180
A6180EC-R	TCA​GTG​GTG​GTG​GTG​GTG​GTG​CTC​GAG​CTT​GGA​CTC​GGT​ATC​GTA​GCC​G
A6180SC-F	CAC​ACA​TAA​ACA​AAC​AAA​GCG​GCC​GCA​TGG​CTG​TCG​CTG​ATA​CCT​CTC​T	For full-length A6180
A6180SC-R	CCT​TGT​AAT​CCA​TCG​ATA​CTA​GTT​CAA​TGA​TGA​TGA​TGA​TGA​TGC​TTC​GAC​TCG​GTA​TCG​TAT​CCA​TTC
TDH3-F	AAC​CCT​CAC​TAA​AGG​CAT​ATG​ATA​CTA​GCG​TTG​AAT​GTT​AGC​GTC	For full-length TDH3 promoter
TDH3-R	ATC​AGC​GAC​AGC​Cat​GCG​GCC​GCT​TTG​TTT​GTT​TAT​GTG​TGT​TTA​TTC

Expressing A6180 in E. coli BL21

The constructed vector harboring A6180 (pET22b-A6180) was propagated in TOP10. For heterologous expression of recombinant proteins in Eastward. coli, pET22b-A6180 was transformed into E. coli BL21 (DE3) competent cells. Positive colonies were screened using ampicillin and designated as BL21-A6180. To obtain sufficient recombinant protein, the optimal induction weather were tested at different temperatures (15°C and 37°C) with various doses of isopropyl-β-D-thiogalactopyranoside (IPTG) (ane.0 and 0.2 mmol/50). The recombinant protein was purified using a His-tagged protein purification kit (Beyotime, Shanghai, Mainland china) for assays of enzymatic properties.

Determination of Enzymatic Backdrop of Recombinant Protein

High performance liquid chromatography (HPLC) was employed to evaluate the recombinant putative UGE by detecting the conversion charge per unit of UDP-α-D-glucose (UDP-Glu) into UDP-α-D-galactose (UDP-Gal) (Goulard et al., 2001). Thereafter, to study the enzymatic backdrop of recombinant UGE proteins, such every bit optimum pH and temperature. In optimal pH analysis, different buffers including 10 mM citric acid-sodium citrate buffer (pH 3.0, 4.0, 5.0, and 6.0), x mM Tris-HCl buffer (pH 7.0, 8.0 and ix.0), 10 mM sodium carbonate-sodium hydroxide buffer (pH x.0, and 11.0) were prepared for reaction at 35°C for 1 h. In optimal temperature assay, the enzyme reaction mixture was incubated at dissimilar temperatures (15°C, 20°C, 25°C, thirty°C, 35°C, 40°C, 45°C, 50°C, and 55°C) for 1 h at optimal pH. In guild to make up one's mind the effect of different ions on UGE activity, 1.0 mM ions were respectively added into the reaction system, including Thousand⁺, Ni²⁺, Mg^two+, Ba²⁺, Caⁱⁱ⁺, Cu²⁺, Co²⁺, and Fe³⁺. The optimal separation conditions were: Athena NH₂ (250 mm × 4.half dozen mm, 5 µm); mobile phase: KH₂PO₄ buffer (0.125 mol/Fifty, pH three.6): ethanol = forty:lx (v:v); cavalcade temperature: 30°C; menstruum rate: one.0 ml/min; detector: UV absorption detector; detection wavelength: 254 nm; injection volume: xx µL. One unit of enzyme activity is defined as the amount of enzyme required to catechumen 1 µmol of substrate UDP-Glu in 1 min.

Transforming A6180 Into South. cerevisiae

Saccharomyces cerevisiae competent cells were prepared using the Super Yeast Transformation Kit (Coolaber). The recombinant vector pESC-Leu-A6180 was transformed into S. cerevisiae co-ordinate to the manufacturer's instructions. Positive transformants were screened by PCR and designated as BY4742-A6180. Western blotting was used to further verify the right expression of the protein encoded by A6180 (Supplementary Data S1). Yeast cells were harvested after 24 h incubation in yeast extract peptone dextrose medium (YPD) (1% yeast excerpt, 2% peptone, and ii% dextrose) at 28°C and 160 r/min. The nerveless cells were frozen in liquid nitrogen and footing in a mortar with a pestle. The intracellular protein was extracted using RIPA lysis buffer (Yeasen) for western blotting. In the fermentation analysis, all the S. cerevisiae strains were inoculated in 4 ml YPD for 18 h at 28°C and 160 r/min. One milliliter of civilization pause was used as a seed to transfer into 100 ml YPD for scale-up fermentation on different civilisation days (28°C 160 r/min).

Yeast Polysaccharide Extraction and in vitro Immune Activity Evaluation

Yeast cells were collected by centrifuging day 1–9 of fermentation. The precipitates were washed with distilled h2o and freeze-stale to obtain dried cell debris. The polysaccharides of yeast cells were extracted and adamant co-ordinate to a previously reported method (Zhang et al., 2021). The β-glucan content of polysaccharides was determined using β-glucan analysis kits provided by Megazyme International Republic of ireland Limited (yeast and mushroom). The polysaccharide samples were hydrolyzed and analyzed past HPAEC system (Dionex ICS-2500, Dionex, Sunnyvale, CA, United States) equipped with a CarboPac™ PA20 cavalcade (Dionex, Sunnyvale, CA, The states) for monosaccharide limerick testing co-ordinate to the reference (Jiang et al., 2016). The generated polysaccharide solution was dialyzed (with a molecular weight of iii.0 kDa intercepted) by distilled h2o for 48 h then freeze-dried. The freeze-dried sample was prepared into a v mg/ml original liquor with PBS (phosphate-buffered saline, pH 7.4) solution and centrifuged at 12,000 × k for 30 min. The supernatant was diluted to 0.five, 2.0, and 5.0 mg/ml for jail cell testing (concluding concentrations in cell culture medium were 50, 200, and 500 µg/ml, respectively). The in vitro immune activity of the dialyzed polysaccharide samples was studied by measuring the NO production of RAW264.7, co-ordinate to the previous report (Wu et al., 2019), and the in vitro immunoactivity was assessed based on the amount of NO released.

Statistical Analysis

Statistical analysis was carried out using SPSS 26.0 software (SPSS Inc., Chicago, United States). A probability level of p < 0.05 was set as statistical significance. Data of the NO production of RAW264.7were presented as mean ± standard deviation (SD) of at least three independent experiments.

Results

A6180 Contains a Typical UGE Domain

To empathize the functions of the poly peptide encoded by A6180, a neighbour-joining phylogenetic tree was established to clarify the evolutionary relationships of fungi, including Ascomycetes and Basidiomycetes. The results showed that H. erinaceus was close to the fungus H. alpestre (Figure 1A). Intriguingly, the ortholog in Dentipellis fragilis was also closely related to A6180, although it was incorporated with an Northward-terminal THO circuitous subunit 1 transcription elongation factor domain and a C-concluding epimrase domain (Effigy 1B). In South. cerevisiae, the ortholog protein Gal10P contains a galactose mutarotase domain (Figure 1B). Thus, the orthologs of UDP-glucose-4-epimerases in fungi are classified into three forms with distinct poly peptide structures (Figure 1B). Based on sequence alignment in the SWISS-MODLE server (https://swissmodel.expasy.org/), the crystal structure of UDP-glucose-4-epimerases of Burkholderia pseudomallei (PDB ID: 3enk.1) was called every bit the template, and the tertiary structure of A6180 was modeled using the SWISS-MODLE server (Figure 2). Combined with the prediction of conserved domains on NCBI, A6180 was predicted to be a homodimeric UDP-glucose-4-epimerases catalyzing the NAD-dependent interconversion of UDP-galactose and UDP-glucose ( Effigy 2A ). It has an N-form catalytic tetrad composed of residues N126, S150, Y174, and K178 (Effigy 2B). 20-one residues (G16, A18, G19, Y20, I21, C80, D81, L82, V106, A107, A108, K110, N125, S148, S149, S150, Y174, K178, Y203, F204, and P206) were predicted every bit NAD binding sites (Figure 2C) and eighteen residues were substrate bounden sites (S150, A151, T152, Y174, Y203, F204, N205, G222, N225, L226, L243, K244, V245, F246, C257, R259, Y260, and V307) ( Effigy 2nd ). These regions were and so close that some of the residues overlapped, such every bit S150, Y174, and Y178. In the intermediate region of the homodimer, polypeptide binding motif (constituted by T116, I118, P119, V120, Y123, H124, V127, S128, I131, F132, L134, Q135, D138, P173, K176, M180, and T183. I184, D186. D187, and L188) were located in the α-helix (Figure 2E). This indicated that the monomers interacted to class homodimers.

Phylogenetic and structural analysis of UDP-glucose-4-epimerases in fungi. (A) Phylogenetic analysis of UDP-glucose-4-epimerases of fungi including: Hericium erinaceus (Data S1), H. alpestre (accession no. {"type":"entrez-protein","attrs":{"text":"TFY78463.1","term_id":"1608096701","term_text":"TFY78463.1"}}TFY78463.one), Dentipellis_fragilis (accretion no. {"type":"entrez-protein","attrs":{"text":"TFY62748.i","term_id":"1608057673","term_text":"TFY62748.i"}}TFY62748.one), Punctularia_strigosozonata (accession no. {"type":"entrez-poly peptide","attrs":{"text":"XP_009540529.1","term_id":"695531083","term_text":"XP_009540529.ane"}}XP_009540529.1), Xerocomus_badius (accretion no. {"type":"entrez-protein","attrs":{"text":"KAF8559888.ane","term_id":"1929005596","term_text":"KAF8559888.ane"}}KAF8559888.1), Postia_placenta (accretion no. {"blazon":"entrez-protein","attrs":{"text":"KAF8559888.1","term_id":"1929005596","term_text":"KAF8559888.1"}}KAF8559888.1), Ganoderma_sinensis_ZZ0214-i (accession no. {"type":"entrez-protein","attrs":{"text":"PIL31081.one","term_id":"1275222715","term_text":"PIL31081.ane"}}PIL31081.one), Trametes_cinnabarina (accretion no. {"type":"entrez-protein","attrs":{"text":"CDO77294.1","term_id":"691785697","term_text":"CDO77294.one"}}CDO77294.one), Lentinus_tigrinus_(Aga) (accretion no. {"type":"entrez-protein","attrs":{"text":"RPD64555.1","term_id":"1519171871","term_text":"RPD64555.1"}}RPD64555.ane), Lentinus_tigrinus_(Sec) (accretion no. {"type":"entrez-protein","attrs":{"text":"RPD82942.i","term_id":"1519190468","term_text":"RPD82942.1"}}RPD82942.1), Volvariella_volvacea_(accession no. {"type":"entrez-protein","attrs":{"text":"KAF8665304.ane","term_id":"1930081519","term_text":"KAF8665304.1"}}KAF8665304.1) Pleurotus_eryngii_(accession no. {"type":"entrez-protein","attrs":{"text":"KDQ31371.ane","term_id":"646310228","term_text":"KDQ31371.ane"}}KDQ31371.one), Hypsizygus_marmoreus (accretion no. {"type":"entrez-protein","attrs":{"text":"RDB19506.i","term_id":"1434138030","term_text":"RDB19506.1"}}RDB19506.i), Agaricus_bisporus (accession no. {"type":"entrez-poly peptide","attrs":{"text":"XP_006454268.ane","term_id":"568435366","term_text":"XP_006454268.one"}}XP_006454268.ane), Lentinula_edodes (accession no. {"type":"entrez-protein","attrs":{"text":"GAW00910.1","term_id":"1139916063","term_text":"GAW00910.1"}}GAW00910.1), Saccharomyces_cerevisiae (accession no. {"type":"entrez-poly peptide","attrs":{"text":"AJQ11874.1","term_id":"762200587","term_text":"AJQ11874.1"}}AJQ11874.1:), and Cordyceps_militaris (accession no. {"type":"entrez-protein","attrs":{"text":"XP_006672787.1","term_id":"573989733","term_text":"XP_006672787.1"}}XP_006672787.i). A neighbour-joining tree was built using MEGA5.0 and the bootstrap method with 1000 replicates. The superscript numbers represent iii types of orthologs shown in B. (B) The structural functional domain analysis of UDP-glucose-four-epimerases. 1) Typical UGE with unique functional domain. 2) A yeast UGE containing N-terminal epimerase domain and a C-terminal mutarotase domain. iii) An exclusive UGE to basidiomycetes containing N-terminal THO circuitous subunit one transcription elongation factor domain and C-terminal epimerase domain.

Three-dimensional structures of UDP-glucose-4-epimerases in H. erinaceus. (A) Homodimer was developed by homology modeling. (B) Active sites (N126, S150, Y174, K178) were illustrated in colored sticks. (C) NAD bounden sites of H. erinaceus UGE were shown in colored sticks (G16, A18, G19, Y20, I21, C80, D81, L82, V106, A107, A108, K110, N125, S148, S149, S150, Y174, K178, Y203, F204, and P206). (D) Homology modeling of substrate binding sites (S150, A151,T152, Y174, Y203, F204, N205, G222, N225, L226, L243, K244, V245, F246, C257, R259, Y260, and V307). (E) Monomer interactions caused by peptide binding sites in homodimer interface (T116, I118, P119, V120, Y123, H124, V127, S128, I131, F132, L134, Q135, D138, P173, K176, M180, T183. I184, D186. D187, and L188). All models were generated by PyMOL.

A6180 Is Highly Expressed in E. coli

The Due east. coli heterologous expression system is a reliable tool for characterizing protein function. Thus, A6180 fragments were ligated into the pET22b (+) vector for protein expression. A schematic of the recombinant overexpression vector structure is shown in Figure 3A. The selected positive clones were cultured under diverse civilisation conditions. Rough extracts of cultured prison cell debris were verified by SDS-Page. The full-length recombinant putative UGE had a predicted molecular weight of 41.7 kD with 379 amino acids. SDS–Folio assay indicated that recombinant UGE was expressed under all the test conditions. Among these, the highest yield of recombinant protein was observed in precipitates and supernatants when the clones were induced by one.0 mmol/L IPTG at 37°C (Effigy 3B). Although near of the recombinant proteins existed every bit inclusion bodies in the precipitate under these weather condition, the recombinant proteins in the supernatant were also the highest of all the tested conditions. Thus, E. coli cells were harvested from 2 50 of culture pause after 4 h of induction with 1.0 mmol/Fifty IPTG at 37°C. Finally, the SDS-Folio purified samples of recombinant UGE poly peptide were analyzed using Quantity One gel analysis software, which showed that the target protein reached 95% purity (Effigy 3C).

Heterologous expression of A6180 in E. coli. (A) Schematic diagram of the construction of recombinant overexpression vector. (B) SDS-Folio analysis of the poly peptide expression of positive transformants under the four inductive expression weather condition. 1000: protein marking; 1: 37°C 1.0 mmol/L IPTG induced precipitation; ii: 37°C ane.0 mmol/50 IPTG induced supernatant sample; iii: 37°C 0.2 mmol/L IPTG induced precipitation sample; four: 37°C 0.2 mmol/L IPTG induced supernatant sample; 5: xv°C i.0 mmol/L IPTG induced precipitation sample; 6: 15°C one.0 supernatant sample after consecration of mmol/L IPTG; vii: precipitation sample afterwards induction of 0.two mmol/L IPTG at 15°C; viii: sample of supernatant after induction of 0.ii mmol/L IPTG at 15°C. (C) Purified recombinant poly peptide expressed in Eastward. coli. M: marker; South: Purified poly peptide.

Enzyme Activity Characteristics Prove A6180 Is a Real UGE

The optimum chromatographic conditions were selected for the detection and separation of UDP-Glu and UDP-Gal past screening the mobile phase ratio and flow rate conditions of HPLC. As shown in Figure 4A, the absorption peaks of UDP-Glu and UDP-Gal standards appeared at eighteen.90 min (UDP-Glu) and 20.10 min (UDP-Gal). This indicated that the 2 standards could be finer separated under the tested conditions (Supplementary Figure S1). The enzymatic reaction charge per unit of UGE was measured for different concentrations of UDP-Glu substrate. The double inverse equation (Figure 4B) showed that the V _grand of the target protein to UDP-Glu is 11.86 mmol/min and K _m is 0.34 mM. This suggests that A6180 encodes a real UGE with the action of converting UDP-Glu into UDP-Gal.

Activities of recombinant protein expressed in E. coli. (A) High performance liquid chromatography detection separation UDP-Glu and UDP-Gal. Peak 1: UDP-Glu; Peak ii: UDP-Gal. (B) Double reciprocal graph. (C) Optimal pH for action. (D) Optimal temperature for activity. (E) The result of metal ions on enzyme activity. (F) The effect of chemical reagents on enzyme activity.

Under the reaction weather of T = 35°C, the enzymatic activity of UGE poly peptide showed an initial increasing trend and then a decreasing tendency with the increase in pH, and had the highest enzymatic activity at pH vi, while the enzymatic action was basically lost when the pH was ten.0 and 11.0, respectively (Figure 4C) (Supplementary Effigy S2). This indicates that the change in pH affects the rate of enzymatic reaction by affecting the dissociation of the enzyme agile center and the substrate of UGE protein, while at pH 10.0 and xi.0, the hyperalkaline state denatures the enzyme protein and thus loses its enzymatic activity.

Nether the optimum pH condition for enzymatic hydrolysis, the enzyme activity beginning increased so decreased with increasing temperature, and the UGE protein had the highest enzyme activeness at a temperature of xxx°C, just the enzyme activeness decreased rapidly in the range of 35–fifty°C, and reached 0 at 50°C. Nevertheless, when the temperature was college than the optimum temperature, the protein gradually denatured and inactivated the enzyme, resulting in a significant decrease in enzyme activeness (Effigy 4D) (Supplementary Figure S3).

The general culture conditions for the mycelium of H. erinaceus were 26°C and natural pH medium (pH 5.8), and the results of the written report also showed that the optimum pH and optimum temperature of UGE were closer to the culture conditions.

Effects of Metallic Ions and Organic Reagents on UGE Enzyme Activity

The addition of the same concentration of several metal ions at pH 6 and 30°C produced different furnishings on enzyme action. Among them, K⁺ and Mg²⁺ had 2.8 and 4% enhancement effects on enzyme activity, while Ni²⁺, Cu²⁺, Co²⁺, and Feⁱⁱⁱ⁺ showed dissimilar degrees of inhibition of enzyme activity, with Cu²⁺ having the most inhibitory result, reducing the enzyme activity to 58% of the original activity. In contrast, Baⁱⁱ⁺ and Caⁱⁱ⁺ had no effect on enzyme activity. This could be attributed to the metallic ions combining with the sparse group, sulfur grouping, or amino group in the target enzyme protein molecule, thus affecting the structure of the active center of the enzyme protein molecule and leading to a reduction in enzyme action (Figure 4E) (Supplementary Effigy S4).

The different chemic reagents added to the enzyme reaction system at pH 6 and 30°C produced dissimilar degrees of inhibition of enzyme activity, with isopropanol, n-butanol, ethyl acetate, and trichloromethane, which showed the strongest inhibition of enzyme action. The reaction enhanced the contact between organic solvents and h2o molecules through oscillation, resulting in the removal of the hydrophilic residues surrounding the surface of the enzyme protein molecules, causing changes in the spatial configuration of the protein thus reducing the enzyme activity to dissimilar degrees. The strong electrostatic interaction between SDS every bit an anionic surfactant and the enzyme molecule caused a change in enzyme conformation, which led to a pregnant decrease in enzyme activity (Guo et al., 2006) (Effigy 4F).

A6180 Expressed in South. cerevisiae Constitutively

A schematic diagram of the recombinant overexpression vector is shown in Figure 5A. Since the GAL1, GAL10 promoter of the pESC-Leu overexpression vector was repressed in the presence of glucose in the culture medium, the TDH3 strong promoter of South. cerevisiae BY4742, which is non repressed by glucose, was added between the GAL1 and GAL10 promoters and the A6180 target gene, thus enabling stable and efficient expression of the A6180 target gene in South. cerevisiae BY4742. Moreover, a 6×His protein tag coding sequence was fused to the 3′ concluding of A6180 for Western blot analysis using the responding monoclonal antibody.

Expression of A6180 in yeast. (A) Schematic diagram of the construction of recombinant overexpression vector pESC-Leu-A6180. (B) Verification for positive transformants of yeast. Grand: D2000 Marker; 1–3: blank controls; 4–9: transformants ane–6. (C) Western blot results of positive transformants. Grand: Prestained poly peptide Marker 10–180 kDa; i: Parent strain. 2–three: Positive transformants.

PCR was performed on nine randomly selected unmarried colonies, and the results are shown in Figure 5B. According to the previous primer design, it tin be seen that the results of lanes iv, 5, and 6 are consistent with the expected size and brightness, so the single colonies corresponding to lanes 4, 5, and 6 can be identified as positive transformant single colonies BY4742-A6180.

The total poly peptide of the positive transformant BY4742-A6180 was verified by western blotting, and the results are shown in Effigy 5C. A clear band at the size of 42 kDa consisted with the size of the target protein expressed by the A6180 gene predicted past Expasy (https://web.expasy.org/protparam/), which can be determined that the target protein, was normally induced to exist expressed in the positive transformant BY4742-A6180 induction group.

Effect of Gene A6180 Transformation on Physicochemical Properties of Southward. cerevisiae Polysaccharides

The total dextran of yeast polysaccharides in the original and transformed strains is shown in Figure 6A, with the bluish line for the original strain (command group) and the black line for the transformed strain. The dextran content of the control group showed an increasing trend in the first 4 days, while there was a decreasing trend on the fifth twenty-four hour period, but it slowly increased and stabilized during the sixth to ninth days. The total dextran content in the transformed and control groups was like after cultivation for 5 days, but there was an obvious decrease in the transformed strain on the third and fourth days. This indicated that the A6180 gene became functional on the third and fourth days, and was consistent with the bioinformatics analysis of its part as a UDP-glucose-4-epimerase (EC:5.1.iii.2). The A6180 gene was transformed into yeast by converting UDP-Glu to UDP-Gal, which in turn led to the reduction in the corporeality of glucan synthesis precursor substance (UDP-Glu) and consequently to a reduction in the full amount of glucan on days 3 and iv in the transformed group compared to the command group.

Effect on polysaccharide later on expressing A6180 in yeast. (A) The trend of glucan in Southward. cerevisiae control group and transformation group. The effect of the control group is a bluish curve and the upshot of the transformation grouping is a black curve. (B) Monosaccharide composition results of S. cerevisiae control group and transformation grouping on day 3. The result of the control group can be seen in the blueish bend, and the consequence of the transformation group is present in the black curve. (C) Immune activity of polysaccharides from S. cerevisiae command grouping and transformation group. PBS (phosphate-buffered saline): bare control; SCT: polysaccharides from S. cerevisiae control group; SCA: polysaccharides from S. cerevisiae transformation group; LPS (Lipopolysaccharides): positive control.

Cells were collected to determine the monosaccharide limerick of polysaccharide after 1–ix days of civilization. After comparing the monosaccharide composition of yeast polysaccharide produced on each solar day, merely the monosaccharide composition of the third day changed to a larger extent, and the monosaccharide limerick of yeast polysaccharide on the other days remained the aforementioned (Figure 6B). The results of the monosaccharide composition data of the third solar day are shown in Table ii, which shows that a variety of new monosaccharides appeared in the transformation group compared with the command group. Yeast polysaccharides, usually, contain merely glucose and mannose and a minor amount of fructose, every bit shown in Table two, and the monosaccharide composition of the polysaccharides produced by the yeast transformed with the A6180 gene contained four new monosaccharides (rhamnose, galactose, xylose, and galacturonic acrid) which accounted for 0.55, nine.24, 11.83, and 1.54% of the full sugars, respectively. The per centum of arabinose increased past iii.1% and the per centum of glucosamine, glucose, mannose, and fructose decreased past 0.29, seven.88, 16.87, and i.23%, respectively.

TABLE two

The proportion of monosaccharide composition in S. cerevisiae and its transformant on twenty-four hours 3.

Monosaccharide type	Monosaccharide in control group	Monosaccharide in transformed group (%)	Increased monosaccharides in transformed grouping	Reduced monosaccharides in transformed group
Rhamnose	N.Da	0.55	0.55%	N.A b
Arabinose	0.15%	3.25	3.10%	N.A.
Glucosamine	0.82%	0.53	Northward.A.	0.29%
Galactose	N.D	9.24	9.24%	Northward.A.
Glucose	33.fifty%	25.62	Due north.A.	7.88%
Xylose	North.D.	11.83	11.83%	N.A.
Mannose	61.81%	44.94	N.A.	xvi.87%
Fructose	3.72%	2.49	N.A.	1.23%
GalA	North.D.	ane.54	1.54%	N.A.

Due to the overexpression of the A6180 gene in the transformed group of S. cerevisiae BY4742-A6180, a new target protein was produced to participate in the polysaccharide synthesis pathway in S. cerevisiae cells. In plough, a new conversion pathway from glucose to galactose emerged, resulting in the production of galactose products that did not exist in the transformant strain and deemed for ix.24% of the total sugars. The increase in galactose content in the transformed group led to the production of galacturonic acid which besides appeared in the transformed grouping. However, the target protein did not have an efficient catalytic role in the galactose-to-galacturonic acid pathway, resulting in an increase of galacturonic acid by 1.54%. Protein role prediction of the A6180 gene by the Poly peptide Family Data Banking company (http://pfam.xfam.org/) Pfam showed that it too functions equally a GDP-mannose four,6 dehydratase (PF16363) (EC: 4.2.1.47), which converts Gross domestic product-α-D-mannose to Gross domestic product-iv-dehydro-α-D-rhamnose, corroborating the appearance of rhamnose in the monosaccharide limerick of the transformed yeast polysaccharide. The results also showed a small amount of rhamnose produced in polysaccharides of the Brewer'southward yeast BY4742-A6180 transformation group, which did not appear in the Brewer'south yeast BY4742 command group, although not as significant every bit the elevation of galactose. Moreover, PF16363 domain is also contained in UDP-xylose synthase which converts UDP-glucuronic acid into UDP-xylose (Borg et al., 2021).

Compared with the control group, the composition of glucose, mannose, fructose, and glucosamine was reduced to different degrees in the transformed group. This could be attributed to the protein expressed by the A6180 cistron promoting the conversion of glucose to galactose and galacturonic acid, leading to a meaning subtract in the conversion of glucose to mannose, fructose, and glucosamine. Overexpression of the A6180 gene promoted the simultaneous conversion of glucose every bit a reaction substrate to multiple monosaccharide conversions, leading to an increase in the consumption of glucose, which in turn led to a seven.88% decrease in the total sugar percentage of glucose.

In vitro Bioactivity of Polysaccharides From the S. cerevisiae BY4742-A6180

Enhancing macrophage activity in vitro is one way to evaluate the allowed activity of polysaccharide fractions (Hitschler and Boles, 2019). Later the pre-experiment of the in vitro immunoreactivity by determining the NO product of RAW264.7 cells treated with the S. cerevisiae polysaccharide on the 3rd day at different concentrations, the highest activity of the sample was establish at 500 μg/ml concentration. The 500 μg/ml concentration, therefore, was chosen to go along the immunoreactivity assay of the polysaccharide obtained from S. cerevisiae BY4742-A6180 transformation strain and control strain cultivated on days 2–5. The results showed that the polysaccharide activity in the transformed group increased by 71.8% on the 3rd twenty-four hour period compared to the control group, and there was no significant change after 4 days incubation (Figure 6C). This is also consistent with the previous results for monosaccharide composition, in which only the third twenty-four hour period of the transformation group showed a significant change in monosaccharide limerick. This indicates that the activity of yeast polysaccharides is closely related to their structure, and is especially related to the composition of its monosaccharide. The transformation of A6180 into yeast inverse the construction of the yeast polysaccharides and further changed the activity of its polysaccharide.

In this written report, heterologous expression of A6180 in E. coli and yeast confirmed that UGE encoded by A6180 is involved in polysaccharide production past H. erinaceus (Effigy vii). In particular, the expression results in yeast indicated that UGE derived from H. erinaceus could change the limerick of fungal polysaccharides and increase their activity. In the future, it is promising to use it to engineer strains for producing polysaccharides with high activity.

Schematic diagram of this study.

Discussion

About of the current research on UDP-glucose-4-epimerases is express to model species, including E. coli (Zhu et al., 2019), Aspergillus (Lee et al., 2014; Park et al., 2014), and Arabidopsis thaliana (Hairdresser et al., 2006), rather than macrofungi. In our previous study, a predicted UDP-glucose-four-epimerases could be involved in the high yield of high-bioactivity polysaccharides in an H. erinaceus mutant. To date, there have been limited reports on the role of UDP-glucose-iv-epimerases in macrofungi. In the present report, the enzymatic characteristics of the purified UGE (heterologously expressed in Eastward. coli) showed that the optimum atmospheric condition were consistent with the cultivation conditions of H. erinaceus. Moreover, the heterologous expression of UGE in Southward. cerevisiae also indicates that UGE participates in the synthesis of polysaccharides.

The general culture conditions for the mycelium of H. erinaceus were 26°C and natural pH medium (approximately 5.8), and the results of the study showed that the optimum pH and optimum temperature of UGE were closer to the culture conditions. The optimum pH and temperature of UGE from oyster (Vocal et al., 2018), E. coli (Guo et al., 2006) were 8.v and 8, 16°C and 37°C, respectively. This indicates that the reaction atmospheric condition of UGE from H. erinaceus are shut to the optimum conditions for fungal growth, which is more suitable for practical product applications of UDP-Gal. This suggests that this UGE encoding gene could be used to engineer other fungi to produce highly agile polysaccharides under optimal conditions with the highest activity.

The heterologous expression and polysaccharide backdrop of the A6180 cistron in S. cerevisiae yielded iii results. Starting time, the overall trend of glucan content in the experimental group was like to that of the control grouping, with a meaning decrease in glucan content on days 3 and 4, which tentatively demonstrated that the A6180 cistron functions on days iii and iv; and was found to have the power to catechumen glucose into other monosaccharides. UDP-glucose-4-epimerase (EC:5.one.3.2) converts UDP-α-D-glucose to UDP-α-D-galactose, corroborating the appearance of galactose in the monosaccharide composition results. It also functions equally GDP-mannose 4,6 dehydratase (EC: four.2.1.47), which converts GDP-α-D-mannose to Gross domestic product-4-dehydro-α-D-rhamnose, corroborating the appearance of rhamnose in the monosaccharide composition results. As shown past the monosaccharide composition results, it can likewise promote the production of xylose, and it accounts for a larger proportion of monosaccharide conversion (xi.83%). The polysaccharide composition of Due south. cerevisiae is generally considered to exist mainly composed of glucan and mannan. The analysis of the results of our command group is consistent with the previous report (Free, 2013). The A6180 gene is the only difference between the transformed group and the command group. Therefore, nosotros speculate that A6180 coded a poly peptide with multi-role as well the isomerization between different hexoses. This speculation is similar to that of a previously reported bifunctional UGE. Information technology catalyzes the isomerization betwixt a variety of UDP-sugars, including UDP-hexose and UDP-pentose (Schäper et al., 2019). In addition, the possible activities of the conserved functional domains contained in A6180 include GDP-mannose four,six-dehydratase (Li et al., 2021), UDP-glucuronate 4-epimerase (Gauttam et al., 2021), and UDP-glucuronate decarboxylase (Woo et al., 2019), and so on. This versatile A6180 that may cause the dramatic alter in the composition and proportions of yeast polysaccharides.

In the budding yeast Southward. cerevisiae, Gal10p contains both galactose mutarotase (mutarotase) and UDP-galactose-4-epimerase (referred to every bit epimerase) (Majumdar et al., 2004). This dual activeness appears to exist unique to Southward. cerevisiae and other yeasts such as Kluyveromyces fragilis, K. lactis, and Pachysolen tannophilus (Brahma and Bhattacharyya, 2004). It is not usual to meet two not-sequential enzymatic activities encoded in the aforementioned poly peptide, and it is not clear why the two activities are linked this manner in yeasts. Previous reports take indicated that this biofunctional protein would have the advantage of sequestering galactose 1-phosphate, which is toxic to both yeasts and mammals (Tsakiris et al., 2002; Scott and Timson, 2007). Nevertheless, this report showed that the expression of UGE with a unmarried part did not cause toxicity in yeast (Scott and Timson, 2007). Moreover, large amounts of galactose were detected in the polysaccharides. Therefore, we hypothesize that the bifunctional GAL10P in yeast is responsible for the absence of galactose in wild-type yeast polysaccharides (Lozančić et al., 2021). Still, it is difficult to explain the special structure of basidiomycetes. Research is yet to exist carried out on the N-terminal THO complex subunit 1 transcription elongation factor domain in fungi. In humans, it functions in the cotranscriptional recruitment of mRNA to export proteins to the nascent transcript (Luo et al., 2012).

In our previous study, A6180 was speculated to exist related to polysaccharide product. However, we found that heterologous expression in yeast was not significantly related to polysaccharide yield. This may be related to the fact that nosotros are simply heterologously expressed instead of replacing the endogenous UGE gene in yeast. However, in some found UGE functional studies, it is related to the production of polysaccharides. In Brassica rapa, BrUGE1 was cloned and introduced into the genome of wild blazon rice (Gopum) using the Agrobacterium-mediated transformation method. Agronomic trait evaluation of the transgenic plants under optimal field weather revealed enriched biomass product, particularly in panicle length, number of productive tillers, number of spikelets per panicle, filled spikelets, and polysaccharide content (Guevara et al., 2014; Abdula et al., 2016). In add-on, our results reveal that UGE is not only related to polysaccharide production, just also to the structure and activity of polysaccharides. It is important to acquit in-depth research on the functions of UGE.

Information Availability Statement

The original contributions presented in the study are included in the article/Supplementary Fabric, further inquiries tin can be directed to the respective writer.

Author Contributions

YY and GZ designed the experiments. All experiment data were acquired by JR, GZ, DW, MG, HZ, WL, and JZ, YY, JR, and GZ wrote the newspaper. JR and GZ analyzed the data. YY and GZ edited the article. All authors read and approved the article.

Funding

This work was supported financially by Shanghai Agriculture Applied Technology Development Programme, China (Grant No. Z 20180101), Shanghai leading Talent Projection (2018).

Conflict of Involvement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed every bit a potential conflict of interest.

Publisher'southward Note

All claims expressed in this article are solely those of the authors and practise non necessarily stand for those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Whatsoever product that may be evaluated in this article, or merits that may exist made by its manufacturer, is not guaranteed or endorsed past the publisher.

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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8655778/

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