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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). [ABSTRACT FROM AUTHOR]

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 Km and Vmax 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. © 2019 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

BACKGROUND The deficiency of family 5 endoglucanase ( Cel5A) and family 6 cellobiohydrolase ( Cel6A) has become a key limiting factor on cellulase enzymatic hydrolysis in bioprocessing of cellulosic biomass. To improve the production of Trichoderma reesei Cel5A / Cel6A, a Vitreoscilla hemoglobin (VHb) gene was tried to co-express extracellularly for the first time with Cel5A / Cel6A in Pichia pastoris GS115. RESULTS Newly constructed recombinant of co-expressing Cel5A / Cel6A extracellularly with VHb was consistent with the single expression at some key variables of culture condition, i.e. inoculum size, initial pH, culture temperature and methanol concentration. Comparing their single expression, the CMCase activity of co-expressed Cel5A and Cel6A enzymes enhanced by 40% and 30%, respectively. With high-cell-density fed-batch (HCDFB) fermentation, the co-expressed Cel5A enzyme activity was 366.8 U mL-1 with 4.3 g L-1 protein content and the Cel6A enzyme activity reached 1.3 U mL-1 with 2.23 g L-1 protein content. The two co-expressed enzyme activities were enhanced by 35% and 20%, respectively, compared with the single expression. CONCLUSION VHb protein capable of binding oxygen can be successfully co-expressed extracellularly with other target proteins. The co-expression of VHb with recombinant Cel5A / Cel6A is efficient at improving oxygen-limited condition and thus enzyme production in both shake-flask and HCDFB fermentation. © 2017 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

BACKGROUND Trans-4-hydroxy-L-proline (Hyp) is a value-added amino acid that is an applicable chiral building block in some industries, such as the pharmaceutical, cosmetic and healthcare industries. Given that glycerol is a good carbon source for microbial fermentation and a large amount of glycerol is produced by the biodiesel industry, this study investigated a value-added Hyp fermentation from glycerol using an engineered Escherichia coli strain. RESULTS By optimizing the proline-4-hydroxylase ( P4H) gene codons and vectors, a constructed recombinant Escherichia coli BL21 strain successfully converted L-proline to Hyp. With a chemo-physical combination mutagenesis, a high Hyp production strain that can grow with glycerol as a sole carbon source, NA45 was isolated, yielding 1.24 g L−1 from 20 g L−1 glycerol at 12 h in a 30 mL/250 mL conical flask fermentation. With further systemic optimization of nutritional elements, the output was enhanced to 1.62 g L−1. In a 5 L fermentator, the strain achieved a surprisingly high output of 25.4 g L−1 at 48 h in fed batch mode. CONCLUSION A high Hyp production strain growing well on glycerol as carbon source was successfully engineered, which is a promising candidate for the industrial Hyp production. © 2016 Society of Chemical Industry [ABSTRACT FROM AUTHOR]