Enhanced heterologous expression of Trichoderma reesei Cel5A/Cel6A in Pichia pastoris with extracellular co‐expression of Vitreoscilla hemoglobin

Sun, Fubao; Yang, Huimin; Bai, Renhui; Xu, Fang; Wang, Fei; He, Jing; Tu, Maobing

doi:10.1002/jctb.5433

Cited by 9 publications

(12 citation statements)

References 35 publications

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“…Recently, coexpression of independently active genes has been superior to their individual expression for triacylglycerol production in the diatom Phaeodactylum tricornutum (Zulu et al ., ). Furthermore, coexpressing a Vitreoscilla hemoglobin gene with endocellulases ( Cel5A , Cel6A ) in the yeast Pichia pastoris enhanced their carboxymethyl cellulase activity (Sun et al ., ).…”

Section: Discussionmentioning

confidence: 97%

Sequential regiospecific gem‐diprenylation of tetrahydroxyxanthone by prenyltransferases from Hypericum sp.

et al. 2019

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Summary Polyprenylated acylphloroglucinol derivatives, such as xanthones, are natural plant products with interesting pharmacological properties. They are difficult to synthesize chemically. Biotechnological production is desirable but it requires an understanding of the biosynthetic pathways. cDNAs encoding membrane‐bound aromatic prenyltransferase (aPT) enzymes from Hypericum sampsonii seedlings (HsPT8px and HsPTpat) and Hypericum calycinum cell cultures (HcPT8px and HcPTpat) were cloned and expressed in Saccharomyces cerevisiae and Nicotiana benthamiana, respectively. Microsomes and chloroplasts were used for functional analysis. The enzymes catalyzed the prenylation of 1,3,6,7‐tetrahydroxyxanthone (1367THX) and/or 1,3,6,7‐tetrahydroxy‐8‐prenylxanthone (8PX) and discriminated nine additionally tested acylphloroglucinol derivatives. The transient expression of the two aPT genes preceded the accumulation of the products in elicitor‐treated H. calycinum cell cultures. C‐terminal yellow fluorescent protein fusions of the two enzymes were localized to the envelope of chloroplasts in N. benthamiana leaves. Based on the kinetic properties of HsPT8px and HsPTpat, the enzymes catalyze sequential rather than parallel addition of two prenyl groups to the carbon atom 8 of 1367THX, yielding gem‐diprenylated patulone under loss of aromaticity of the gem‐dialkylated ring. Coexpression in yeast significantly increased product formation. The patulone biosynthetic pathway involves multiple subcellular compartments. The aPTs studied here and related enzymes may be promising tools for plant/microbe metabolic pathway engineering.

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Section: Discussionmentioning

confidence: 97%

Sequential regiospecific gem‐diprenylation of tetrahydroxyxanthone by prenyltransferases from Hypericum sp.

et al. 2019

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“…Multiple sequence alignments were carried out using DNAMAN 5.0 (Lynnon Biosoft). 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. , DNA Star software was used to analyze the free energy (Δ G ) of mRNA folding, the average structure, and the sequence around the ATG distribution of G+C and to eliminate AT-rich regions. After codon optimization, the PaAA9B gene was synthesized by Sangon Biotech (Shanghai, China).…”

Section: Methodsmentioning

confidence: 99%

“…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.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

“…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.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

“…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.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

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

Long

Yang

Ren

et al. 2020

ACS Sustainable Chem. Eng.

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To achieve a fast and efficient cellulose hydrolysis with low enzyme loading remains a challenge for an economically feasible biomass biorefinery process. The synergism between cellulase and lytic polysaccharide monooxygenases (LPMOs) has demonstrated great promise, but it appears that such synergistic effects are highly substrate-dependent. In addition, the need for an efficient method of LPMO production has also limited its application. In this study, the production of Podospora anserina AA9B (PaAA9B), one of the most active LPMOs, was optimized by using double-plasmid coexpression in Pichia pastoris, and its hydrolysis-boosting effects on cellulases were assessed on model cellulosic materials and on our in-house-optimized atmospheric glycerol organosolv (AGO)-pretreated lignocellulosic substrates. The results showed that the double-plasmid coexpression technique successfully improved the PaAA9B production, where up to three times more PaAA9B (3.34 g L −1 ) was expressed in the 5 L bioreactor after 4 days of induction. The addition of recombinant PaAA9B to Cellic CTec2 (CTec2) significantly boosted the cellulose hydrolysis of a filter paper and Avicel by 2.3-and 1.4-folds, respectively, while no effects were observed on carboxymethyl cellulose (CMC). When lignocellulosic substrates were assessed, the PaAA9B also successfully enhanced the cellulose hydrolysis of both acid-catalyzed (ac) and alkali-catalyzed (al) AGO-pretreated sugarcane bagasses by 30 and 20%, respectively, at a 5% solid loading (w/v). At industrially relevant high-solid loading (20% w/v) hydrolysis of an al-AGO-pretreated substrate, the combination of 1.0 mg of PaAA9B and 3 FPU of cellulase per gram of substrate achieved 83% cellulose hydrolysis with 105 g L −1 of the corresponding glucose concentration after 72 h. These results indicated that the double-plasmid coexpression strategy is viable for the high-yield PaAA9B production. The mixture of PaAA9B and cellulase enzymes containing other AA9s exhibited a strong cosynergistic interaction and further boosted the enzymatic hydrolysis of both model cellulosic substrates and our optimized AGO-pretreated lignocellulosic biomass with industrially relevant enzyme loading. This study sheds light on the industrially relevant PaAA9B utilization in the enzymatic hydrolysis of lignocellulosic substrates.

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Recent developments in heterologous production of cellulases and xylanases using the Pichia pastoris expression system

Ullah,

Madadi,

Asadollahi

et al. 2023

J of Chemical Tech & Biotech

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The yeast expression system, Pichia pastoris, is one of the most robust and versatile expression systems in biotechnology and molecular biology, generally considered as a safe host for heterologous protein expression, especially for producing cellulases and xylanases at the industrial level. Despite the high recombinant protein expression rate, the potential of the P. pastoris expression system has still not been fully explored. Cultivation of P. pastoris under optimized conditions greatly relies on the strain and is associated with certain problems such as promoter strength, sensitivity to methanol, and oxygen demand. To address these issues, different genetic engineering strategies have been employed. Advancements in promoter engineering, optimization of gene dosage and codon usage, recombinant plasmid engineering using CRISPR/Cas9 system, and directed evolution strategies have proven beneficial to the yield of cellulase expression levels. This study will systemically review recent progress in various genetic engineering strategies to enhance cellulase and xylanase expression in the P. pastoris expression system. The utilization of alcohol oxidase 1 promoter (pAOX1), methanol‐free system, and recombinant plasmid engineering for improved production of these enzymes are highlighted. Additionally, we discuss the recent advancements in the P. pastoris expression system toolbox for improved cellulase and xylanase production, thus providing a deep insight into how P. pastoris is becoming the indispensable platform for heterologous protein production. © 2023 Society of Chemical Industry (SCI).

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Enhanced heterologous expression of Trichoderma reesei Cel5A/Cel6A in Pichia pastoris with extracellular co‐expression of Vitreoscilla hemoglobin

Cited by 9 publications

References 35 publications

Sequential regiospecific gem‐diprenylation of tetrahydroxyxanthone by prenyltransferases from Hypericum sp.

Sequential regiospecific gem‐diprenylation of tetrahydroxyxanthone by prenyltransferases from Hypericum sp.

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

Recent developments in heterologous production of cellulases and xylanases using the Pichia pastoris expression system

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