Recombinant expression of Aspergillus niger GH10 endo‐xylanase in Pichia pastoris by constructing a double‐plasmid co‐expression system

Abstract

BACKGROUND: Previous work has shown that GH10 endo-xylanase presented better synergistic cooperation with cellulase to achieve effective hydrolysis. However, most currently reported commercial xylanolytic enzymes from Aspergillus niger belong to GH11 and there have been few studies regarding GH10 xylanase. In addition, to increase overall expression of heterologous endo-xylanase in Pichia pastoris, a double-plasmid co-expression method, including the construction of recombinant P. pastoris using one expression vector followed by inserting another vector which targeted to different locus, was introduced. RESULTS: A GH10 xylanase gene from A. niger BE-2 (XynC) was optimized and constitutively expressed in P. pastoris GS115.Compared with the conventional single-plasmid method, it appeared that the double-plasmid strategy could considerably increase the XynC yield by ∼33%. To further improve enzyme production, cultivation conditions were optimized at shake-flask level and then scaled up to a 5-L bioreactor. Through high cell-density fermentation, the expression level of GH10 XynC reached 1650 U mL −1 . The XynC showed maximum activity at 55 ∘ C and pH 5.0, and exhibited an excellent stability over a wide range of pH from 4.5 to 7.0. The kinetic parameters K m and V max values for beechwood xylan were 3.5 mg mL −1 and 2327 U mg −1 , respectively. CONCLUSION: The double-plasmid co-expression strategy introduced herein could greatly improve the XynC yield in P. pastoris. The GH10 xylanase from A. niger BE-2 shared similar reaction conditions with the existing enzymatic hydrolysis, which might be a good candidate to assist cellulases for industrial application. Biochemical properties of the xylanase XynCIn order to avoid or minimize interference in the assay by media components, XynC expressed from P. pastoris-D was purified wileyonlinelibrary.com/jctb a Shake-flask level. b Fermentor level. J Chem Technol Biotechnol 2020; 95: 535-543

Rational engineering of xylanase hyper-producing system in Trichoderma reesei for efficient biomass degradation

“…Xylanase is a group of xylanolytic enzymes consisting of endo-xylanase, β-xylosidase, α-L-arabinofuranosidase, acetyl xylan esterase, and ferulic acid esterase, which decompose xylan into simple monosaccharide and xylooligosaccharides [ 1 , 8 ], showing great potential in the feed, biorefinery, and pulp paper industry. Generally, the xylanase production is achieved by overexpression of endo-xylanases [ 9 ], which shows high catalyzing performance to the backbone of xylan. However, the unthorough de-branch of the side-chain group also hinders the cleavage efficiency of endo-xylanase [ 1 , 10 ], which then influences the accessibility of cellulose, resulting in lower fermentable sugar yield for downstream biofuel production.…”

Synergism of Recombinant Podospora anserina PaAA9B with Cellulases Containing AA9s Can Boost the Enzymatic Hydrolysis of Cellulosic Substrates

“…The construction of recombinant P. pastoris GS115 by using one expression vector was followed by inserting another vector, which was targeted to a different locus as gene insertions in P. pastoris can occur at either the AOX1 or HIS4 locus (Figure 1B). 14,21 After the electroporation and screening, three recombinant transformants harboring one or both recombinant plasmids (pPIC9K-PaAA9B incorporated into the HIS4 and AOX1 loci and pPICZαA-PaAA9B targeted to the AOX1 locus) were thus generated. As expected, the transformant P. pastoris-D carrying two vectors showed a better expression yield than P. pastoris-S1 and P. pastoris-S2 that each contained one vector only (Table 1).…”

“…The sequence of the PaAA9B gene was analyzed and optimized in its codon sequences using Gene Designer 2.0, according to our earlier reported methods. 20,21 Construction of Recombinant P. pastoris GS115 with Single/Double Plasmid. The recombinant plasmids (pPIC9K-PaAA9BB and pPICZαA-PaAA9B) were linearized using SacI and then electroporated into P. pastoris by a Bio-Rad Micropulser Electroporator using the conditions recommended by the manufacturer.…”

“…To achieve a reasonable expression of PaAA9B at the shake-flask level, the cultivation conditions of three selected transformants, including the initial pH, methanol concentration, inoculum size, and additives, were optimized using a single-factor experiment. 20,21 Additives consisted of 1 g L −1 amino acids (glutamic acid, histidine, tyrosine, and alanine) and nonionic surfactants, including 1 g L −1 Tween-20, 0.5 g L −1 Triton X-100, and 2 g L −1 PEG 2000, PEG 4000, PEG 6000, or PEG 10 000. The transformant P. pastoris-D was incubated in a 1.5 L working volume in a 5 L bioreactor, according to our earlier methods.…”

“…The transformant P. pastoris-D was incubated in a 1.5 L working volume in a 5 L bioreactor, according to our earlier methods. 20,21 Synergetic Role of PaAA9B with Cellulase Acting on Model and "Realistic" Substrates. The enzymatic hydrolysis was carried out by hydrolyzing model substrates, i.e., filter paper (3% solid content), microcrystalline cellulose (MCC, 3% solid content), carboxymethyl cellulose (CMC, 1% solid content), and "realistic" substrates, i.e., al-/ac-AGO-pretreated substrates at a 5% (w/w) solid loading.…”