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High-level expression of an acidic thermostable xylanase in Pichia pastoris and its application in weaned piglets.

ABSTRACT: An acidic thermostable xylanase (AT-xynA) which was stable at low pH and high temperature was considered to have great potential in animal feed. For large-scale production, AT-xynA activity was enhanced about 1-fold in Pichia pastoris by constructing a double-copy expression strain in this study. Furthermore, impacts of different AT-xynA levels on growth performance, nutrient digestibility, short-chain fatty acids, and bacterial community in weaned piglets were determined. Compared with the control group, ADFI and ADG were higher for the pigs fed 4,000 or 6,000 U/kg AT-xynA (P < 0.05). AT-xynA supplementation also significantly increased the digestibility of OM, GE, and DM (P < 0.05). AT-xynA supplementation increased the concentrations of acetate in ileal (P < 0.01) and cecal digesta (P < 0.05). Isobutyrate (P < 0.05) and valerate (P < 0.05) concentrations in colonic digesta also significantly increased compared with the control group. AT-xynA supplementation increased the abundance of Lactobacillus in the ileal, cecal, and colonic digesta of weaned piglets (P < 0.05). AT-xynA alleviated anti-nutritional effects of nonstarch polysaccharides (NSP) by preventing the growth of Pateurella and Leptotrichia in the ileum (P < 0.05). AT-xynA increased the abundance of NSP-degrading bacteria, such as Ruminococcaceae, Prevotella in the cecum and colon (P < 0.05). In summary, AT-xynA addition could improve the growth performance of weaned piglets by altering gut microbiota.

SUBMITTER: Wang J

PROVIDER: S-EPMC6986428 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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High-level expression of an acidic thermostable xylanase in Pichia pastoris and its application in weaned piglets.

Wang Jian J Liu Yajing Y Yang Yongzhi Y Bao Chengling C Cao Yunhe Y

Journal of animal science 20200101 1

An acidic thermostable xylanase (AT-xynA) which was stable at low pH and high temperature was considered to have great potential in animal feed. For large-scale production, AT-xynA activity was enhanced about 1-fold in Pichia pastoris by constructing a double-copy expression strain in this study. Furthermore, impacts of different AT-xynA levels on growth performance, nutrient digestibility, short-chain fatty acids, and bacterial community in weaned piglets were determined. Compared with the cont ...[more]

PMID: 31778535

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Project description:BackgroundFungal amylase, mainly constitute of fungal α-amylase and glucoamylase, are utilized in a broad range of industries, such as starch hydrolysis, food and brewing. Although various amylases have been found in fungi, the amylases from Aspergillus dominate the commercial application. One of main problems exist with regard to these commercial use of amylases is relatively low thermal and acid stability. In order to maximize the efficiency of starch process, developing fungal amylases with increased thermostability and acid stability has been attracting researchers' interest continually. Besides, synergetic action of glucoamylase and α-amylase could facilitate the degradation of starch. And co-expressing glucoamylase with α-amylase in one host could avoid the need to ferment repeatedly and improves cost-effectiveness of the process.ResultsA novel fungal glucoamylase (RpGla) gene encoding a putative protein of 512 amino acid residues was cloned from Rhizomucor pusillus. BLAST analysis revealed that RpGla shared highest identity of 51% with the Rhizopus oryzae glucoamylase (ABB77799.1). The fungal glucoamylase RpGla was expressed in Pichia pastoris (KM71/9KGla) with maximum activity of 1237 U ml(-1). The optimum pH and temperature of RpGla were pH 4.0 and 70 °C, respectively. Fungal α-amylase (RpAmy) gene was also cloned from R. pusillus and transformed into KM71/9KGla, resulted in recombinant yeast KM71/9KGla-ZαAmy harboring the RpGla and RpAmy genes simultaneously. The maximum saccharogenic activity of KM71/9KGla-ZαAmy was 2218 U ml(-1), which improved 79% compared to KM71/9KGla. Soluble starch hydrolyzed by purified RpGla achieved 43% glucose and 34% maltose. Higher productivity was achieved with a final yield of 48% glucose and 47% maltose catalyzed by purified enzyme preparation produced by KM71/9KGla-ZαAmy.ConclusionsA novel fungal glucoamylase and fungal α-amylase genes were cloned from Rhizomucor pusillus. The two enzymes showed good thermostability and acid stability, and similar biochemical properties facilitated synergetic action of the two enzymes. A dramatic improvement was seen in amylase activity through co-expressing RpGla with RpAmy in Pichia pastoris. This is the first report of improving activity through co-expression glucoamylase with α-amylase in P. pastoris. Besides, fungal glucoamylase and α-amylase from R. pusillus were shown as promising candidates for further application in starch hydrolysis.

Project description:Margatoxin (MgTx) is a high-affinity blocker of voltage-gated potassium (Kv) channels. It inhibits Kv1.1–Kv1.3 ion channels in picomolar concentrations. This toxin is widely used to study physiological function of Kv ion channels in various cell types, including immune cells. Isolation of native MgTx in large quantities from scorpion venom is not affordable. Chemical synthesis and recombinant production in Escherichia coli need in vitro oxidative refolding for proper disulfide bond formation, resulting in a very low yield of peptide production. The Pichia pastoris expression system offers an economical approach to overcome all these limitations and gives a higher yield of correctly refolded recombinant peptides. In this study, improved heterologous expression of recombinant MgTx (rMgTx) in P. pastoris was obtained by using preferential codons, selecting the hyper-resistant clone against Zeocin, and optimizing the culturing conditions. About 36 ± 4 mg/L of >98% pure His-tagged rMgTx (TrMgTx) was produced, which is a threefold higher yield than has been previously reported. Proteolytic digestion of TrMgTx with factor Xa generated untagged rMgTx (UrMgTx). Both TrMgTx and UrMgTx blocked the Kv1.2 and Kv1.3 currents (patch-clamp) (Kd for Kv1.2 were 64 and 14 pM, and for Kv1.3, 86 and 50 pM, respectively) with comparable potency to the native MgTx. The analysis of the binding kinetics showed that TrMgTx had a lower association rate than UrMgTx for both Kv1.2 and Kv1.3. The dissociation rate of both the analogues was the same for Kv1.3. However, in the case of Kv1.2, TrMgTx showed a much higher dissociation rate with full recovery of the block than UrMgTx. Moreover, in a biological functional assay, both peptides significantly downregulated the expression of early activation markers IL2R and CD40L in activated CD4+ TEM lymphocytes whose activation was Kv1.3 dependent. In conclusion, the authors report that the Pichia expression system is a powerful method to produce disulfide-rich peptides, the overexpression of which could be enhanced noticeably through optimization strategies, making it more cost-effective. Since the presence of the His-tag on rMgTx only mildly altered the block equilibrium and binding kinetics, recombinant toxins could be used in ion channel research without removing the tag and could thus reduce the cost and time demand for toxin production.

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Dataset Information

High-level expression of an acidic thermostable xylanase in Pichia pastoris and its application in weaned piglets.

Publications

High-level expression of an acidic thermostable xylanase in Pichia pastoris and its application in weaned piglets.

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