Cloning, Expression, Characterization and Application of the Pectin Esterase MtCE12-1 from Myceliophthora thermophila

doi:10.13560/j.cnki.biotech.bull.1985.2024-0336

Abstract

Abstract:

【Objective】 To explore a novel pectin esterase enzyme, a homologous pectin esterase significantly up-regulated in tobacco biomass induction was highly expressed in Myceliophthora thermophila. The enzymatic properties of this pectin esterase were investigated, along with its role in assisting the degradation of tobacco biomass. 【Method】 RT-qPCR was used to analyze the expression of the pectin esterase gene Mtce12-1 in M. thermophila under the condition of tobacco biomass. Using a viral 2A peptide-mediated expression screening system, the pectin esterase gene Mtce12-1 was highly expressed in the M. thermophila wild-type strain ATCC 42462. The positive transformants overexpressing MtCE12-1-His-2A-GFP were subjected to recombinant enzyme production and protein purification, and the enzymatic properties of the pectin esterase MtCE12-1 were characterized. 【Result】 The transcription level of the pectin esterase gene Mtce12-1 was significantly up-regulated by about 109-110 folds under tobacco biomass conditions as compared to glucose condition. The sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, copy number determination and Western blotting indicated that the recombinant proteins were successfully expressed and secreted in M. thermophila, with expressions reaching as the 464.08 U/mL. The MtCE12-1 presented the highest activity at 75℃ and pH 8.0. It had good enzymatic activity in the range of 50-80℃ and pH 7.0-9.0 and demonstrated excellent thermal stability. The addition of 100-300 µg of MtCE12-1 to the degradation system resulted in an increase in cellulose degradation efficiency in tobacco bar and tobacco stem by 18.5%-30.7% and 14.6%-30.5%, respectively. 【Conclusion】 The use of the 2A peptide-mediated expression system in M. thermophila facilitates efficient target protein expression and purification. The alkaline pectin esterase MtCE12-1 not only shows good temperature stability but also demonstrates exceptional effectiveness in tobacco biomass degradation, providing potential high-quality enzyme resources for tobacco industry applications.

Key words: Myceliophthora thermophila, pectin esterase, gene expression, enzymatic properties, tobacco biomass

ZHANG Man-yu, DONG Jia-cheng, GOU Fu-fan, GONG Chao-hui, LIU Qian, SUN Wen-liang, KONG zhen, HAO Jie, WANG Min, TIAN Chao-guang. Cloning, Expression, Characterization and Application of the Pectin Esterase MtCE12-1 from Myceliophthora thermophila[J]. Biotechnology Bulletin, 2024, 40(9): 291-300.

Figures/Tables 6

Table 1 Primers for amplifying the target sequence

名称 Primer	序列 DNA sequence（5'-3'）	用途 Application
Mtce12-1-RT-F	GACGACATTGTAGTGATC	基因表达分析
Mtce12-1-RT-R	TAGTGGTTGAAGGTGTAG	基因表达分析
actin-RT-F	AACGCTCCTGCCTTCTAC	基因表达分析
actin-RT-R	GTAACACCATCACCAGAGTC	基因表达分析
Mtce12-1-F	GCCAGTTTCGTTCTTCAGAACTAGTATGCGACCGTGGTCGACTCT	基因克隆
Mtce12-1-R	GTGGTGGTGGTGGTGGTGGATATCGAACACCTGAGGCACCGGGG	基因克隆
Ptef1-F	CTTCGACCCCTCCTCAAATCTTCTT	基因鉴定
TtrpC-R	GAGCTATTAAATCACTAGAAGGCAC	基因鉴定
Mtalp1-ko-F	TTCTGGCCTGCCCTTTTCTTTCAAC	基因鉴定
Mtalp1-ko-R	GCCCCTTCTTCCGAAAGGGGAGGTA	基因鉴定

Fig. 1 Expression of the Mtce12-1 gene for pectin esterase under conditions of the glucose and tobacco biomass

Fig. 2 Recombinant expressed pectin esterase MtCE12-1 in M. thermophile A: Schematic illustration of the expressed vector and recombinant expression of MtCE12-1. B: Microscopic fluorescence imaging of conidia and mycelia of the overexpressing strain OE-MtCE12-1-GFP. Scale bar 10 μm

Fig. 3 Detecting the expression of recombinant protein MtCE12-1 by using the SDS-PAGE electrophoresis, Western blotting, and copy number A: SDS-PAGE analysis of secreted proteins from the CK and OE-MtCE12-1-GFP strains after 4-day of growth. M: the protein molecular weight markers. CK is used as the negative control for the transformation of the empty vector pAN52-1N into the host wild-type strain. B: Western blotting of culture supernatants from the strains and probed with anti-His antibody. C: Assay of Mtce12-1 copy number in overexpressing strains by RT-qPCR. D: SDS-PAGE analysis of the purified MtCE12-1. M: the protein molecular weight markers; Lane 1: the culture supernatants of the OE-MtCE12-1-GFP strain T8; Lane 2: the purified enzyme of MtCE12-1

Fig. 4 Enzymatic properties of pectin esterase MtCE12-1 A: Effect of pH on the enzyme activity of MtCE12-1. B: Effect of temperature on the enzyme activity of MtCE12-1. C: Effect of pH on the enzyme activity of MtCE12-1. D: Effect of temperature on the stability of MtCE12-1. E: Substrate specificity of MtCE12-1

Fig. 5 Synergistic degradation of tobacco waste biomass by pectin esterase MtCE12-1 and cellulase A: Synergistic degradation of tobacco bar by MtCE12-1 and cellulase. B: Synergistic degradation of tobacco stem by MtCE12-1 and cellulase. 1: Control（no enzyme）; 2: only cellulase; 3: 100 μg MtCE12-1 and cellulase; 4: 200 μg MtCE12-1 and cellulase; 5: 300 μg MtCE12-1 and cellulase. Different letters are statistically significant（Tukey’s HSD, P < 0.05）

References 31

[1]	Zhang GT, Zhan JJ, Fu HQ. Trends in smoking prevalence and intensity between 2010 and 2018: implications for tobacco control in China[J]. Int J Environ Res Public Health, 2022, 19(2): 670.
[2]	Zhang Y, Li RD, Shang GL, et al. Effects of multiscale-mechanical fragmentation on techno-functional properties of industrial tobacco waste[J]. Powder Technol, 2022, 402: 117327.
[3]	Tao JM, Chen QS, Chen SY, et al. Metagenomic insight into the microbial degradation of organic compounds in fermented plant leaves[J]. Environ Res, 2022, 214(Pt 1): 113902.
[4]	Lin YN, Wang C, Yu GF, et al. Transformation of tobacco biomass into value-added carbohydrate, aromatics, and biochar[J]. Biomass Convers Biorefin, 2024, 14(10): 11697-11705.
[5]	黄申, 芦尧, 刘强, 等. 生物酶在烟草工业中的应用研究进展[J]. 轻工学报, 2023, 38(5): 112-118.
	Huang S, Lu Y, Liu Q, et al. Review on application of biological enzymes in tobacco industry[J]. J Light Ind, 2023, 38(5): 112-118.
[6]	郝捷, 李选文, 张宝, 等. 纤维素酶在烟草中的应用进展[J]. 生物技术进展, 2023, 13(2): 166-173. doi: 10.19586/j.2095-2341.2022.0127
	Hao J, Li XW, Zhang B, et al. Application progress of cellulase in tobacco[J]. Curr Biotechnol, 2023, 13(2): 166-173. doi: 10.19586/j.2095-2341.2022.0127
[7]	Zou G, Shi SH, Jiang YP, et al. Construction of a cellulase hyper-expression system in Trichoderma reesei by promoter and enzyme engineering[J]. Microb Cell Fact, 2012, 11: 21.
[8]	Daly P, Cai F, Kubicek CP, et al. From lignocellulose to plastics: knowledge transfer on the degradation approaches by fungi[J]. Biotechnol Adv, 2021, 50: 107770.
[9]	Meng JL, Mäkelä MR, de Vries RP. Molecular engineering to improve lignocellulosic biomass based applications using filamentous fungi[J]. Adv Appl Microbiol, 2021, 114: 73-109. doi: 10.1016/bs.aambs.2020.09.001 pmid: 33934853
[10]	Kun RS, Gomes ACS, Hildén KS, et al. Developments and opportunities in fungal strain engineering for the production of novel enzymes and enzyme cocktails for plant biomass degradation[J]. Biotechnol Adv, 2019, 37(6): 107361.
[11]	Berka RM, Grigoriev IV, Otillar R, et al. Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris[J]. Nat Biotechnol, 2011, 29: 922-927.
[12]	Kolbusz MA, Di Falco M, Ishmael N, et al. Transcriptome and exoproteome analysis of utilization of plant-derived biomass by Myceliophthora thermophila[J]. Fungal Genet Biol, 2014, 72: 10-20.
[13]	Liu Q, Li JG, Gao RR, et al. CLR-4, a novel conserved transcription factor for cellulase gene expression in ascomycete fungi[J]. Mol Microbiol, 2019, 111(2): 373-394. doi: 10.1111/mmi.14160 pmid: 30474279
[14]	Li N, Liu Y, Liu DF, et al. MtTRC-1, a novel transcription factor, regulates cellulase production via directly modulating the genes expression of the Mthac-1 and Mtcbh-1 in Myceliophthora thermophila[J]. Appl Environ Microbiol, 2022, 88(19): e0126322.
[15]	Zhu ZJ, Zhang MY, Liu DD, et al. Development of the thermophilic fungus Myceliophthora thermophila into glucoamylase hyperproduction system via the metabolic engineering using improved AsCas12a variants[J]. Microb Cell Fact, 2023, 22(1): 150.
[16]	Li JG, Lin LC, Sun T, et al. Direct production of commodity chemicals from lignocellulose using Myceliophthora thermophila[J]. Metab Eng, 2020, 61: 416-426.
[17]	Liu J, Chen MX, Gu SY, et al. Independent metabolism of oligosaccharides is the keystone of synchronous utilization of cellulose and hemicellulose in Myceliophthora[J]. PNAS Nexus, 2024, 3(2): pgae053.
[18]	Gu SY, Wu TJ, Zhao JQ, et al. Rewiring metabolic flux to simultaneously improve malate production and eliminate by-product succinate accumulation by Myceliophthora thermophila[J]. Microb Biotechnol, 2024, 17(2): e14410.
[19]	Kashyap DR, Vohra PK, Chopra S, et al. Applications of pectinases in the commercial sector: a review[J]. Bioresour Technol, 2001, 77(3): 215-227.
[20]	Hoondal G, Tiwari R, Tewari R, et al. Microbial alkaline pectinases and their industrial applications: a review[J]. Appl Microbiol Biotechnol, 2002, 59(4): 409-418.
[21]	Lara-Márquez A, Zavala-Páramo MG, López-Romero E, et al. Biotechnological potential of pectinolytic complexes of fungi[J]. Biotechnol Lett, 2011, 33(5): 859-868. doi: 10.1007/s10529-011-0520-0 pmid: 21246254
[22]	Sista Kameshwar AK, Qin WS. Structural and functional properties of pectin and lignin-carbohydrate complexes de-esterases: a review[J]. Bioresour Bioprocess, 2018, 5(1): 43.
[23]	Schmitz K, Protzko R, Zhang LS, et al. Spotlight on fungal pectin utilization-from phytopathogenicity to molecular recognition and industrial applications[J]. Appl Microbiol Biotechnol, 2019, 103(6): 2507-2524. doi: 10.1007/s00253-019-09622-4 pmid: 30694345
[24]	Benz JP, Chau BH, Zheng DA, et al. A comparative systems analysis of polysaccharide-elicited responses in Neurospora crassa reveals carbon source-specific cellular adaptations[J]. Mol Microbiol, 2014, 91(2): 275-299.
[25]	Liu Q, Zhang YL, Li FY, et al. Upgrading of efficient and scalable CRISPR-Cas-mediated technology for genetic engineering in thermophilic fungus Myceliophthora thermophila[J]. Biotechnol Biofuels, 2019, 12: 293.
[26]	Yang YJ, Liu Y, Liu DD, et al. Development of a flow cytometry-based plating-free system for strain engineering in industrial fungi[J]. Appl Microbiol Biotechnol, 2022, 106(2): 713-727.
[27]	Liu Q, Gao RR, Li JG, et al. Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase production strain engineering[J]. Biotechnol Biofuels, 2017, 10: 1.
[28]	Gao LW, Liu GD, Zhao QQ, et al. Customized optimization of lignocellulolytic enzyme cocktails for efficient conversion of pectin-rich biomass residues[J]. Carbohydr Polym, 2022, 297: 120025.
[29]	Sakai T, Sakamoto T, Hallaert J, et al. Pectin, pectinase and protopectinase: production, properties, and applications[J]. Adv Appl Microbiol, 1993, 39: 213-294. pmid: 8213306
[30]	华婷, 李雅楠, 王凯凯, 等. 蓝状菌(Talaromyces leycettanus JCM12802)高温果胶甲酯酶PmeT在毕赤酵母中的高效表达及酶学性质[J]. 微生物学报, 2018, 58(1): 122-130.
	Hua T, Li YN, Wang KK, et al. High-level expression and characterization of pectin methylesterase Pme T from Talaromyces leycettanus JCM12802 in Pichia pastoris[J]. Acta Microbiol Sin, 2018, 58(1): 122-130.
[31]	Kumar R, Meghwanshi GK, Marcianò D, et al. Sequence, structure and functionality of pectin methylesterases and their use in sustainable carbohydrate bioproducts: a review[J]. Int J Biol Macromol, 2023, 244: 125385.