Heterologous and efficient expression of a new alkaline pectin lyase in Pichia pastoris

Heterologous and efficient expression of a new alkaline pectin lyase in Pichia pastoris | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Heterologous and efficient expression of a new alkaline pectin lyase in Pichia pastoris Junyi Li, Shuangyan Han This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3846786/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Pectin lyase (PMGL) is an industrially important enzyme with widespread applications in the food, paper, and textile industries, owing to its capacity for direct degradation of highly esterified pectin. In this study, PMGL-Ba derived from Bacillus underwent mining and heterologous expression in P. pastoris. Furthermore, diverse strategies, encompassing the optimization of expression cassette components, elevation of gene dosage, and co-expression of chaperone factors, were employed to augment PMGL-Ba production in P. pastoris . The signaling peptide OST1-pre-α-MF-pro and promoter AOX1 were finally selected as expression elements. By overexpressing the transcription factor Hac1p in conjunction with a two-copy PMGL-Ba setup, a strain yielding high PMGL-Ba production was achieved. In shake flask fermentation lasting 144 hours, the total protein concentration reached 1.81 g/L, and the enzyme activity reached 1821.36 U/mL. For further scale up production, high-density fermentation transpired in a 5 L fermenter for 72 h. Remarkably, the total protein concentration increased to 12.49 g/L, and the enzyme activity reached an impressive 12668.12 U/mL. The successful heterologous and efficient expression of PMGL-Ba not only furnishes a valuable biological enzyme for industrial applications but also contributes to cost reduction in the utilization of biological enzymes in industrial applications. Pectin lyase Efficient expression Pichia pastoris Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Pectin, a structural polysaccharide ubiquitous in plant cell walls, consists of D-galacturonosyl residues linked by α-1,4-glycosidic bonds, imparting both rigidity and flexibility. This polymer serves as a protective agent for plants (Kaur et al. 2023 ; Zheng et al. 2021 ). However, in specific industrial scenarios, altering pectin's structure becomes imperative to curtail energy consumption or enhance product quality. Traditional mechanical and chemical methods pose environmental risks and may harm raw materials. Enzymatic treatments emerge as preferred alternatives due to their efficiency, specificity and environmental friendliness (Haile et al. 2022 ; Kaur et al. 2023 ). The degradation of pectin involves three primary types of pectinases: methyl deesterases (pectin methyl esterases), hydrolases (polygalacturonases), and lyases (pectin lyase) (Suberkropp and Keller 2020). Pectin methyl esterase initiates methoxylation, completing deesterification and producing pectin and methanol. Subsequently, pectin lyase and polygalacturonases preferentially act on highly esterified pectin, catalyzing hydrolytic cleavage of the α-1,4-glycosidic bond in the pectin backbone without methanol production, resulting in 6-methyl-D-galacturonic acid. This crucial step in pectin degradation, involving cutting the pectin skeleton and progressively breaking down the substance, makes pectin lyase vital for industrial environmental protection (Tasgin et al. 2020 ). Pectinase pretreatment finds diverse applications, reducing fruit juice viscosity in food processing (Rahman et al. 2020 ), enhancing cotton fabric absorbency in the textile industry (Aggarwal et al. 2020 ), and improving paper quality while decreasing chemical usage in the paper industry (Ahlawat et al. 2007 ; Nawawi et al. 2021 ; Singh et al. 2020 ). Notably, treating mechanical pulp with pectinase degrades pectin on fibers, reducing the amount of extractives covering the fiber surface. This weakens the bond between lignin and cellulose, refining the pulp further. The enzyme pretreatment exposes more microfibers, facilitating inter-fiber bonding and improving fiber strength, demonstrating the versatility and significance of pectinase in various industrial processes (Ahlawat et al. 2007 ; Li et al. 2010 ; Chen et al. 2020 ). While the application of biological enzymes in industry is highly effective for green manufacturing, elevated production costs present a significant challenge. Hence, improving the production efficiency of enzymes and exploring high-expression strategies are crucial long-term research objectives. P.pastoris , with advantages such as high cell culture density, genetic manipulation, and post-translational modification of proteins, stands as a widely used bacterium for producing exogenous proteins (Ahmad et al. 2014 ; Vijayakumar and Venkataraman 2023 ). Leveraging the secretion mechanism of exogenous proteins in P.pastoris , extensive research has focused on optimizing strategies to enhance protein secretion. These strategies encompass the construction of expression platforms, pathway engineering modification, and optimization of culture conditions, resulting in the efficient expression of diverse exogenous proteins (Liu et al. 2022 ; Raschmanová et al. 2021 ). Specifically, within the expression system, the emphasis lies on targeting promoter strength, selecting secretion signal peptides, and optimizing codons for the target proteins in the expression cassette (Xiao et al. 2023 ; Xu et al. 2023 ). Pathway engineering modification primarily involve using relevant cofactors in the co-expression pathway to promote transcription, translation, processing and secretion of the protein, while averting stress reactions and protein degradation within the cell (Jiang et al. 2023 ; Zahrl et al. 2022 ; Zahrl et al. 2023 ). In the realm of culture conditions, the focus is on optimizing medium composition, pH, temperature, and implementing high-density fermentation (Dixit et al. 2022 ). Additionally, increasing the gene dose through in vitro constructs and in vivo screening is a commonly employed strategy to promote high expression (Peng et al. 2019 ; Chen et al. 2022 ). Although these strategies have shown success in increasing the yield of exogenous proteins, their effects can be unpredictable for different proteins and require customization. However, the effects of these strategies are unpredictable for different proteins, so they can only be "customized" for proteins. Among the studies that have been reported, the highest expression of alkaline pectin lyase in E. coli is currently 147347.6 U/mL, while the expression in P. pastoris is 3620 U/mg (14 L fermenter) (Li et al. 2022 ; Zhou et al. 2017 ; Ma 2023 ; Peng 2020 ). Substantial room for improvement remains in optimizing the expression of PMGL in P. pastoris . In this paper, an unreported PMGL gene derived from Bacillus was selected by homologous sequence comparison and heterologously expressed in P. pastoris . Subsequently, a strain of P. pastoris with high expression of PMGL-Ba was constructed by several strategies, including codon optimization, optimization of gene expression elements, increasing gene dosage and overexpression of cofactors. The objective is to provide an industrial enzyme and reduce the associated costs, addressing a critical aspect in the practical application of these enzymes in various industrial processes. Results 2.1 Gene mining and sequence analysis of PMGL-Ba The gene mining process for PMGL involved using a high enzyme activity pectin lyase gene as the probe sequence, with key residues serving as the screening conditions. The selection criteria included assessing sequence similarity to the probe template, predicting soluble expression of the protein, and considering the type of microorganisms of origin. Ultimately, the identified putative PMGL-Ba (NCBI Reference Sequence: WP_009329358.1) derived from Bacillus licheniformis was finally selected. The protein consists of 494 amino acids with a relative molecular mass of 54693.2. PMGL-Ba was found to be in the same branch as two Bacillus-derived pectin lyases (GenBank: OMI04834.1, QII50179.1) annotated in NCBI, and both are in the polysaccharide lyase 1 family, confirming the reliability of the results(Fig. 1 A). Analysis of the protein structure diagram revealed that PMGL-Ba possesses the typical β-helical structure of pectin lyase proteins, consisting of three parallel β-strands connected through three corners (Zheng et al. 2021 ) (Fig. 1 B). Sequence comparisons indicated that certain sequences of PMGL-Ba overlap with conserved amino acid sequences of the pectin lyase reference sequence or contain key amino acids of the catalytic activity center, suggesting potential functional similarities (Fig. 1 C). Figure 1 Phylogenetic analysis of PMGL-Ba. (A) Selection of signal peptides. (B) The predicted structure of PMGL-Ba. Red represents α-helix, gold represents β-sheet, and green represents random coil structure. (C) Sequence comparison analysis. 2.2 Expression and biochemical characterization of PMGL-Ba The shake flask fermentation of PMGL-Ba recombinant P. pastoris X33 was carried out, with samples taken at 24 h intervals to assess the growth of the recombinant yeast and the enzyme activity of pectin cleavage in the supernatant of the fermentation broth. With an increase in induced expression time, the OD600, fermentation broth supernatant enzyme activity, and protein yield of X33-PMGL-Ba gradually increased. At 144 h of induced expression time, the enzyme activity and protein concentration with esterified pectin as substrate reached 895.69 U/mL and 0.77 g/L, respectively, indicating the successful heterologous secretory expression of recombinant PMGL-Ba using P. pastoris X33 (Fig. 2 A). The purified PMGL-Ba was analyzed by SDS-PAGE, revealing an apparent molecular weight of about 55 kDa, consistent with the theoretical molecular weight of 54.6 kDa༈Fig. 2 B). The enzyme activity of PMGL-Ba exhibited an initial increase followed by a decrease with increasing pH, pinpointing the optimal reaction pH as 8.5 (Fig. 2 C). PMGL-Ba retained more than 80% of its total activity within the pH range of 8.0-9.5. However, the enzyme activity decreased rapidly beyond pH 9.0 or below 8.0. PMGL-Ba has good pH stability, maintaining 60% activity in the pH range of 5.0–11.0(Fig. 2 D). The optimal temperature for PMGL-Ba was 60°C, with relative enzyme activity exceeding 60% at temperatures between 50–70°C༈Fig. 2 E). PMGL-Ba activity decreased with increasing temperature, and relative enzyme activity remained above 60% at temperatures below 70°C after 1 h of exposure. As the temperature increased further, the enzyme activity gradually decreased, with only about 20% of the enzyme activity remained at the temperature of 80 ℃༈Fig. 2 F). PMGL-Ba showed robust enzyme activity and stability over a broad temperature range of 30 to 80°C, indicating its potential advantages for application in medium and high-temperature industries, such as the paper industry. Figure 2 Expression and enzymatic properties of PMGL-Ba. (A) Fermentation of PMGL-Ba. (B) SDS-PAGE analysis. (C-F) Enzymatic properties of PMGL-Ba. 2.3 Effect of expression components on recombinant PMGL-Ba protein expression The secretory expression of heterologous proteins in P. pastoris often involves the utilization of the S. cerevisiae α-factor mating pheromone (α-MF) from Saccharomyces cerevisiae. Existing studies have explored the impact of signaling peptides on the secretion of recombinant proteins, revealing varying efficiencies of signaling peptides for different exogenous proteins. Studies on the effect of signal peptides on exogenous protein secretion can be broadly categorized into two groups: modification of existing signal peptides and the discovery of new signal peptides. The commonly used α-MF comprises a two-stage signal, consisting of a 19-amino-acid pre region and a 66-amino-acid pre region at the N-terminal end, demonstrating effective secretion for most exogenous proteins (Barrero et al. 2018 ). Several studies have sought to enhance the secretion efficiency of α-MF various modifications, such as αMF-CC (αM), and αopt obtained after optimization and point mutation of the αMF sequence, resulting in improved protein secretion (Ahn et al. 2016 ; Aza et al. 2021 ; Ito et al. 2022 ). Another approach involves utilizing more efficient sequences to replace parts of the αMF. Juan J et al. designed combined secretion signals, such as the one consisting of the Saccharomyces cerevisiae Ost1 signal sequence and the α-factor PRO region, which has demonstrated superior secretion ability compared to the α-factor signal sequence (Barrero et al. 2018 ). In yeast, secreted proteins are targeted to the endoplasmic reticulum (ER) by either the co-translational pathway or the "post-translational" pathway. The co translational pathway is mediated by the signal recognition particle (SRP), while the "post-translational" pathway, independent of SRP, requires the intact membrane protein Sec62p for ER targeting. However, more hydrophobic signaling sequences utilize the SRP pathway to target the ER in order to increase secretion efficiency (Walter et al. 2011 ). Ost1p, a type I intact membrane protein with a cleavable signal sequence, plays a role in co-translational translocation to the ER (Willer et al. 2008 ). Subsequent bioreactor-scale fermentation has further confirmed that specific recombinant signal peptide promotes protein secretion (Barrero et al. 2021 ). To avoid variability in signal peptide efficiency for different protein secretion, the discovery of new signal peptides is essential (Liang et al. 2012 ; Sastry et al. 2014 ). Systematic analysis of yeast genomes and secretomes has led to the identification of novel signal peptides with more efficient secretion than α-MF, such as the signal peptide derived from the P. pastoris PAS_chr3_0030 gene (PC0) and the signal peptide NCW2 in Saccharomyces cerevisiae (Shen et al. 2022 ; Wang et al. 2015 ; Zou et al. 2022 ). In this study, six distinct types of secreted signaling peptides, namely pPIC-α-MF-PMGL-Ba, pPIC-O-pre-α-pro-PMGL-Ba, pPIC-αopt-PMGL-Ba, pPIC-αM-PMGL-Ba, pPIC-PC0-PMGL-Ba, and pPIC-NCW2-PMGL-Ba, were selected to investigate their effects on PMGL-Ba production. As shown in Fig. 3 , results from shake flask fermentation revealed a significant enhancement in pectinase secretion with O-pre-α-pro and aM compared to α-MF. Conversely, PMGL-Ba secretion by αopt and NCW2 was lower than that of α-MF. The signaling peptide PC0 exhibited secretion levels similar to α-MF. Notably, the protein concentration and enzyme activity of Ba secreted by the signal peptide O-pre-α-pro were 1.40 g/L and 1554.26 U/mL, respectively, representing a 130.71% and 98.13% increase compared to the departure strain pPICZαA-PMGL-Ba. Figure 3 Effects of different signaling peptides on PMGL-Ba expression. (A) Selection of signal peptides. (B) Growth curves of strains corresponding to different signal peptides. (C) Effect of different signal peptides on total protein concentration of PMGL-Ba and SDS-PAGE analysis. (D) Effect of different signaling peptides on PMGL-Ba enzyme activity. P. pastoris , a methylotrophic yeast, tightly regulates the expression of exogenous proteins when utilizing methanol as a carbon source through the methanol utilization pathway (MUT) promoter. The transcription level of exogenous protein genes is intricately linked to the strength of promoter regulation (Püllmann and Weissenborn 2021 ). The commonly employed AOX1 promoter is favored for its robust induction by methanol, although the precise mechanism of its methanol-induced action remains not fully elucidated (Berg et al. 2013 ; Wang et al. 2016 ). Pichia pastoris is a methylotrophic yeast, and the expression of exogenous proteins using methanol as a carbon source is tightly regulated by the methanol utilization pathway (MUT) promoter. The transcription level of exogenous protein genes is closely linked to the strength of promoter regulation. The AOX1 promoter is commonly used and is often the promoter of choice for its strong induction by methanol. However, the mechanism of action of the AOX1 promoter strictly induced by methanol is not fully understood. To unravel the mechanism of AOX1 promoter action, previous studies have utilized mutation screening, followed by sequence analysis, or adjusted regulatory factors of other carbon sources (Hartner et al. 2008 ; Zhan et al. 2017 ). In this study, three MUT strong promoters, reported to be more effective than AOX1 in inducing exogenous proteins, were selected. The first, AOXm, involved deleting a transcription factor sequence associated with glucose repression and copy addition of a positive cis-acting element sequence after AOX1 deletion (Hartner et al. 2008 ; Li et al. 2015 ). The second, from the study by Thomas Vogl et al., utilized genes from the pentose phosphate pathway (PPP) and the defense against reactive oxygen species (ROS) in the MUT pathway to provide strong promoters that somewhat exceed AOX1, such as the promoter of the gene encoding peroxisomal membrane protein (PMP20) (Yurimoto et al. 2000 ; Vogl et al. 2015 ). The third is formaldehyde dehydrogenase 1 (FLD1), a key enzyme in metabolizing methanol as a carbon source (Nakagawa et al. 2004 ; Wang et al. 2019 ). The promoters of their encoding genes show similar yields for FLD1 and AOX1 in exogenous protein expression, with FLD1 even surpassing AOX1 in some proteins (Püllmann and Weissenborn 2021 ; Wang et al. 2016 ). In this study, all four promoters mentioned above were individually employed for PMGL-Ba expression, and the most suitable promoter for PMGL-Ba gene was selected. As depicted in Fig. 4 , AOX1 demonstrated obvious advantages in PMGL-Ba expression. Biomass analysis further indicated a notable decreasing trend for strains PMP20 and FLD1, suggesting that AOX1 remains the most suitable promoter for PMGL-Ba gene expression in this context. Figure 4 Effects of different promoters on PMGL-Ba expression. (A) Selection of promoters. (B) Growth curves of strains corresponding to different promoters. (C) Effect of different promoters on total protein concentration of PMGL-Ba and SDS-PAGE analysis. (D) Effect of different promoters on PMGL-Ba enzyme activity. 2.4 Effect of gene dosage on recombinant PMGL-Ba protein expression In the expression of exogenous proteins in P. pastoris , constructing high-copy strains is often a straightforward and effective approach to enhance protein production. Various methods exist for constructing multicopy strains, with a common strategy involving the in vitro construction of multiple expression cassettes in tandem, subsequently integrated onto the P. pastoris genome. In order to obtain higher copy number strains, different screening markers can be employed for sequential integration into the P. pastoris genome (Wang et al. 2021 ; Yang et al. 2016 ). Another prevalent method involves the gradual increase of antibiotic concentration, with commonly used antibiotics such as zeocin and antibiotic G418; the screening concentration is usually higher than 100 µg/mL, with some reaching up to 2000 µg/mL (Kuo et al. 2015 ; Sha et al. 2013 ; Song et al. 2019 ). However, zeocin is a relatively expensive antibiotic, and its screening process is intricate and time-consuming. Another approach involves the Cre/lox recombination system, using a screening marker for multiple integration, but this method suffers from a prolonged cycle time (Li et al. 2017 ). In addition, Xia et al. constructed a novel double plasmid system using a combined strategy of genomic integration and heterologous expression (Xia et al. 2021 ). In this study, homologous recombination was employed to construct three strains with different screening markers, namely GS115-2PMGL-Ba, GS115-3PMGL-Ba, and GS115-4PMGL-Ba, which were sequentially integrated onto the Picot yeast genome using different screening markers. This method effectively reduced the time required for constructing multiple copies by 2–3 days, streamlining the identification process post-integration into the yeast genome. As illustrated in Fig. 5 , GS115-2PMGL-Ba exhibited improvements over GS115-PMGL-Ba, showcasing increased protein concentration and enzyme activity. However, GS115-3PMGL-Ba demonstrated a significant decrease in yield compared to GS115-2PMGL-Ba. In shake flask fermentation, the protein concentration and enzyme activity of GS115-2PMGL-Ba reached 1.50 g/L and 1658.01 U/mL, respectively, representing a 6.7% and 6.69% increase compared to X33-PMGL-Ba. Figure 5 Effects of gene dosage on PMGL-Ba expression. (A) Schematic of different gene doses. (B) Growth curves of strains corresponding to different gene doses. (C) Effect of different gene doses on total protein concentration of PMGL-Ba and SDS-PAGE analysis. (D) Effect of different gene doses on PMGL-Ba enzyme activity. 2.5 Effect of overexpression of cofactors on recombinant PMGL-Ba protein expression In the realm of protein expression pathways, alterations in chaperone protein levels can exert influence across multiple stages, encompassing transcription, translation, folding, and secretion. However, the impacts of such alterations are often protein-specific, and determining the singularly most crucial chaperone or the optimal combination remains ambiguous (Samuel et al. 2013 ). In this study, we expanded on GS115-2Ba by overexpressing 11 cofactors previously investigated for their roles in promoting protein expression. A pivotal step in exogenous protein expression in P. pastoris is the translation process, where translation initiation serves as the rate-limiting step in the translation process. Various translation initiation-associated factors have been targeted to study the impact of translation efficiency. Notably, Jennifer Staudacher et al. demonstrated that overexpression of certain translation factors, particularly Pab1 and eIF4G, significantly enhances exogenous protein production in P. pastoris . The synergistic effect was more pronounced when all four loop-closing related factors, eIF4E, eIF4A, eIF4G and Pab1, were simultaneously overexpressed (Staudacher et al. 2022 ). In a previous study in our laboratory, Zheng et al. also proposed a novel yeast transcription factor, tentatively named Fhl1p, implicated in the regulation of rRNA processing genes and genes related to ribosomal small/large subunit biogenesis genes. This factor was found to facilitate the translation efficiency of exogenous proteins (Zheng et al. 2019 ). Correct protein folding is a critical step in P. pastoris , particularly during post-translational translocation in the endoplasmic reticulum (ER). Newly synthesized peptides entering the ER must maintain an unfolded or loosely folded form after release from the ribosome, preventing aggregation before translocation. This process involves binding to cytoplasmic chaperones such as Ssa1 and Ydj1. In contrast, the ATPase activity of the ER luminal chaperone Kar2 powers post-translational translocations, promoting protein folding as well as targeting misfolded proteins to ERAD (Delic et al. 2013 ). In response to endoplasmic reticulum stress induced by the overexpression of exogenous proteins, especially in multicopy strains, a common approach to alleviate these stress effects is the overexpression of cofactors. The HSP30 gene has been shown to play a crucial role in mitigating the repression of target gene transcript levels induced by high-intensity promoters. Besides, HSP30 gene expression is simultaneously regulated by the transcription factors HSF1, MSN2, and MSN4. Notably, the transcription factor HSF1 positively affects alleviating unfolded protein stress (Cui et al. 2023 ). Overexpression of the transcription factor Hac1p is frequently selected during chaperonin assisted secretory protein folding. In multicopy strains, the overexpression of exogenous proteins induces cellular stress responses, such as the common unfolded protein response (UPR), activated by unconventional splicing of HAC1 mRNA in yeast. The spliced HAC1 mRNA encodes an active transcription factor, Hac1p, which binds to UPR response elements in the promoters of UPR target genes, and it induces the expression of other chaperone proteins that assist in protein folding and protein transport (Liu et al. 2020 ; Guerfal et al. 2010 ). In this study, we systematically overexpressed the 11 cofactors previously discussed, building upon the foundation of GS115-2PMGL-Ba. Interestingly, the Fig. 6 indicates that only overexpression of Hac1p increased the protein expression, while overexpression of the other ten cofactors decreased the protein expression. In shake flask fermentation, the protein concentration and enzyme activity of strain GS115-2PMGL-Ba: Hac1p were 1.81 g/L and 1835.53 U/mL, respectively. Compared with GS115-2PMGL-Ba, the protein concentration and enzyme activity increased by 20.85% and 10.7%. Figure 6 Position of different protein cofactors in the protein secretory expression pathway. (A) Schematic of different gene doses. (B) Growth curves of strains corresponding to overexpression of different protein cofactors. (C) Effect of overexpression of different protein cofactors on total protein concentration of PMGL-Ba and SDS-PAGE analysis. (D) Effect of overexpression of different protein cofactors on PMGL-Ba enzyme activity. 2.6 5L fermenter refill batch fermentation After identifying the highest PMGL-Ba yielding strain, GS11-2PMGL-Ba-Hac1p, through the different strategies mentioned above, it was activated and cultured into a seed solution. This seed solution was then introduced into a 5 L fermenter containing 2 L of BSM basal medium to initiate the batch culture phase. The glycerol feed-batch phase commenced after a dissolved oxygen rebound at 28 h. Glycerol replenishment was stopped when the yeast density reached OD600 = about 300, indicating complete glycerol consumption. Subsequently, the methanol feed-batch phase commenced, with samples taken every 8 h, continuing until the completion of 72 h. As shown in the Fig. 7 , the high-density fermentation of GS115-2PMGL-Ba-Hac1p in a 5 L fermenter for 72 h resulted in a substantial increase in protein concentration to 12.49 g/L, representing a remarkable 591.51% improvement compared to shake flask fermentation. Simultaneously, enzyme activity increased to 12668.12 U/mL, reflecting a 595.53% increase compared to shake flask fermentation. Previous experimental findings indicated protein degradation in the late stage of fermentation. To expedite the fermentation process, we increased the starting cell density of the induction stage of methanol flow addition from OD600 = 200 to OD600 = 300, and reduced the fermentation cycle from 144 h in shake flasks to 72 h. Figure 7 PMGL-Ba high density fermentation. (A) 5L type fermenter high density fermentation. (B) Changes in biomass and total protein concentration over time in high-density fermentations. (C) SDS-PAGE analysis of supernatants after 20-fold dilution at different times in high-density fermentation. (D) Changes in PMGL-Ba activity at different times in high-density fermentation. Methods 4.1 Strains, Media, and Reagents The molecular kits used in this experiment were purchased from Magen Biotechnology Co.,Ltd. Restriction endonucleases were purchased from Thermo Fisher Scientific (Shanghai,China). The homologous recombinant enzyme was purchased from Beijing Jinsha Biotechnology Co. Chemical reagents were purchased from Tianjin Damao Chemical Reagent Factory. Pectin was purchased from McLean. Recombinant plasmids and recombinant strains were constructed using Escherichia coli Top10 and P.pastoris as hosts. The medium media and their compositions used in this experiment were LB (0.5% yeast extract, 1% peptone and 1% NaCl), LBL (0.5% yeast extract,1% peptone and 0.5% NaCl), YPD (1% yeast extract, 2% peptone and 2% glucose), MD (1.34% YNB and 2% glucose), BMGY (1% yeast extract, 2% peptone, 1.34% YNB, 0.1 mM PBS pH 6.0 and 1% glycerol) and BMMY (1% yeast extract, 2% peptone, 1.34% YNB, 0.1 mM PBS pH 6.0 and 1% methanol). 4.2 Gene mining for putative xylanase and sequence analysis for target gene Gene mining for PMGL was performed with the help of enzyme resource mining software EnzymeMiner. Two high enzyme activity pectin lyases Q01172 and P94449 of medium alkaline and medium high temperature were used as probe sequences. Additionally, the protein sequences of three PMGLs, Q2TXS4, AY825251, and R9XVW6, were used as auxiliary references for sequence search. The initial set of sequences of potential PMGLs was obtained by performing iterative site-specific homology searches in the NCBI database. The key residues of PMGL were then used as filtering criteria to filter sequences with mismatched key residues in the homology search results. The final selection was based on the sequence similarity and sequence characterization provided by EnzymeMiner, the sequence similarity to the probe template (with a threshold value of 30%-70%), the predicted degree of soluble expression of the protein, and the type of microorganism from which the protein was derived. A phylogenetic tree was constructed between the selected sequences and the probe sequences by neighbor-joining method using MEGA-X software to analyze the homology of the sequences. The protein structure of PMGL-Ba was modeled by SwissModel homology. Sequence comparison analysis was performed using Jalview. The basic physicochemical properties of Bacillus pectin lyase were predicted using ProtParam. 4.3 Heterologous Expression of PMGL-Ba in P. pastoris The PMGL gene (NCBI: WP_009329358.1) derived from Bacillus was mined by homologous sequence comparison. Subsequently, the PMGL gene was codon optimized and constructed in the pPICZαA vector, named pPICZαA-PMGL-Ba. The recombinant plasmid was then transformed into E. coli Top10 receptor cells, coated to solid medium containing zeocin antibiotic in LBL and cultured for 12–14 h. Single colonies, correctly identified by PCR, were transferred to liquid medium of LBL and cultured for 12–14 h. Plasmids were extracted and sequenced according to the kit. The plasmids with correct sequencing were linearized and transformed into P. pastoris by electroporation and selected on YPDZ plates, and incubated at 30°C for 3 d. Yeast single colonies with correct colony morphology were identified by PCR. The recombinant P. pastoris strains were transferred to 10 mL of BMGY medium at 30℃ for 20–24 h, and then transferred to 25 mL of BMMY medium according to the starting OD600 as 1. Samples were taken every 24 h and 1% (v/v) methanol was added. After 144 h of fermentation, the samples were transferred to 50mL centrifuge tubes. After centrifugation, the supernatant was taken for subsequent experiments. The collected fermentation supernatant was centrifuged at high speed (10,000rpm, 4℃, 10 min), and then passed through a 0.22 µm ultrafiltration membrane. Gradient purification of fermentation broth supernatant using Ni-NTA column chromatography, and the eluate was collected. The protein content of the fermentation supernatant and purified PMGL-Ba were determined using the Bradford method, with bovine serum albumin (BSA) as the standard. Finally, the supernatant was diluted 2-fold and 20 µL was taken for SDS-PAGE analysis. 4.4 Enzymatic properties of PMGL-Ba The activity of PMGL-Ba was assessed utilizing the 3,5-dinitrosalicylic acid (DNS) method. Initially, 0.5% pectin was accurately weighed and dissolved in Tris-HCl buffer (50 mM, pH 8.0). The fermentation supernatant was appropriately diluted. Subsequently, 190 µL of the pectin solution was placed in a thermostatic reactor to preheat for 10 min, and 10 µL of sample was added to react for 10 min. 300 µL of DNS solution was added, boiled for 5 min and then cooled down immediately, and 200 µL of the reaction solution was taken to measure the absorbance value at 540 nm. One unit (U) of enzyme activity was defined as the amount of pectin lyase releasing 1 µmol of reducing sugar per minute under the conditions described above. The optimal pH value of PMGL-Ba was measured by determining the activity between pH 5.0–12.0 in 60°C. To determine pH stability of PMGL-Ba, purified enzymes were firstly incubated between pH 5.0–12.0 at 4°C for 12 h. Next, residual activity of PMGL-Ba was further measured using above method. Buffers used were 100 mmol/L PBS buffer (pH 6.0), 50 mmol/L Tris-HCl buffer (pH 7.0–9.0) and Glycine-sodium hydroxide buffer(pH10-11). Optimal temperature of PMGL-Ba was investigated by measuring the activity in a range of 50°C–70°C in pH 8.0. To determine the thermostability of PMGL-Ba, enzymes were pre-incubated at 30°C–90°C for 1 h pH 8.0. 4.5 Construction of highly expressed PMGL-Ba recombinant strains To investigate the expression of PMGL-Ba by secreted signaling peptides, five signaling peptides were selected based on literature research as OST1-pre-αMF-pro(O-pre-α-pro), αopt, αM, PAS_chr3_0030 gene (PC0) and NCW2, respectively. Subsequently, α-MFs were sequentially replaced by homologous recombination, resulting in constructs named pPIC-O-pre-α-pro-PMGL-Ba, pPIC-αopt-PMGL-Ba, pPIC-αM-PMGL-Ba, pPIC-PC0-PMGL-Ba, and pPIC-NCW2-PMGL-Ba. In parallel, three strong promoters, AOXm, FLD1, and PMP20, were also substituted for AOX1 by homologous recombination to explore the expression of the promoters on the proteins, named pPIC-AOXm-PMGL-Ba, pPIC-FLD1-PMGL-Ba, and pPIC-PMP20-PMGL-Ba, respectively. To facilitate in vitro construction of multiple copies, the PMGL-Ba gene expression cassette was constructed on the PHK-PMGL-Ba and pPIC-Cre/lox-PMGL-Ba plasmid vectors. PHK-2PMGL-Ba and pPIC-Cre/lox-2PMGL-Ba were constructed using homologous recombination. Three-copy and four-copy recombinant strains were constructed by screening markers differentially integrated sequentially on the yeast genome. To further promote protein expression, eleven cofactors were overexpressed in multicopy strains as Fhlp, Hsf1, eIF4A, eIF4E, eIF4G, PAB1, Hsp30, Hac1p, MSN2, SSA1, and KAR2, and were named as PHK-2PMGL-Ba-Fhlp, PHK-2PMGL-Ba-Hsf1, PHK-2PMGL-Ba-eIF4A, PHK-2PMGL-Ba-eIF4E, PHK-2PMGL-Ba-eIF4G, PHK-2PMGL-Ba-PAB1, PHK-2PMGL-Ba-Hsp30, PHK-2PMGL-Ba-Hac1p, PHK-2PMGL-Ba-MSN2, PHK-2PMGL-Ba-SSA1, and PHK-2PMGL-Ba-KAR2, respectively. The plasmids, confirmed by correct sequencing, were linearized and transformed into P. pastoris by electroporation and selected on MD plates or YPDZ plates, and incubated at 30°C for 3 d. Yeast single colonies with correct colony morphology were identified by PCR. The recombinant P. pastoris strains were fermented as described above. 4.6 Fed-batch fermentation in a 5 L fermenter The screened high yielding strains were first streaked from the preservation tubes to YPD solid medium for 3 d activation. A single colony was picked into 10mL YPD liquid medium and incubated in shaker at 30℃ for 20–24 h to form a primary seed solution. Then the second seed liquid was obtained by transferring to 160mL YPD medium at 4% inoculum volume and culturing in shaker at 30℃ for about 20 h. The resulting seed solution was transferred to a 5 L fermenter containing 2 L of BSM medium, and 8% inoculum was introduced along with 8.7 mL of aseptically added PTM 1 (micronutrient salt). Temperature was controlled at 30°C, and pH was set at 5.5. The glycerol feed-batch phase commenced upon depletion of glycerol in the glycerol batch phase, accompanied by a rebound in dissolved oxygen. Glycerol addition ceased when OD600 = 300, signaling the transition to the methanol fed-batch phase. During this phase, temperature was 25°C, pH = 6.0, and the induction time was 72 h. Cell biomass, protein concentration, and enzyme activity were determined by taking 10 mL samples every 8 h. At the end of fermentation, the broth underwent centrifugation, and the resulting supernatant was utilized for subsequent experiments. Conclusion In this study, we elucidated the amino acid sequence of PMGL-Ba, a novel enzyme derived from Bacillus, previously unreported. This was achieved through homologous sequence comparison and the identification of key amino acids using the EnzymeMiner software. For heterologous expression in P. pastoris , we performed codon optimization on PMGL-Ba. The enzyme exhibited an optimum pH of 8.5 and an optimum temperature of 60 ℃, displaying excellent pH and temperature stability. To enhance PMGL-Ba expression in P. pastoris , we employed a multifaceted approach, encompassing the selection of gene expression components, increased gene dosage, and co-expression of cofactors. Ultimately, a two-copy expression cassette, consisting of AOX1 promoter, OST1-pre-α-MF-pro signal peptide, and AOX1 terminator elements, was selected. This cassette, co-expressed with the transcription factor Hac1p, was utilized for high-density fermentation in a 5 L fermenter for 72 h. The results were impressive, with the total protein concentration reaching 12.49 g/L and the enzyme activity reaching12668.12 U/mL. This represents a remarkable 21-fold increase in total protein expression and a 15-fold increase in enzyme activity compared to the initial bacterial expression. This achievement marks the highest reported expression level of PMGL-Ba to date, providing a yeast strain with substantial PMGL-Ba production potential for future industrial enzyme applications. Declarations Acknowledgments. The authors would like to acknowledge Yian Chen for his assistance in copy editing the manuscript. Author contributions . SH initiated and coordinated the study and reviewed the manuscript. JY designed and performed the research, analysed the data, and draft the manuscript. MY designed and performed the partial research. FZ and YZ reviewed the experimental program and the manuscript. All authors read and approved the final manuscript. Funding. This research was supported by the National Key R&D Program of China (NO. 2021YFC2100400). Availability of data and materials All data generated or analysed during this study are included in this published article. Ethics approval and consent to participate Not applicable. Consent for publication All the coauthors have read and approved the submission to Bioresources and Bioprocessing. Competing interests The authors declare that they have no competing interests Author details 1 Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China. 2 School of Light Industry and Engineering, South China University of Technology, Guangzhou, 510006, China. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3846786","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":266921622,"identity":"60aa2376-1adb-41be-ae15-6be1f73ac89d","order_by":0,"name":"Junyi Li","email":"","orcid":"","institution":"South China University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Junyi","middleName":"","lastName":"Li","suffix":""},{"id":266921623,"identity":"30fb2b5d-a379-496b-9590-8306b1cae1a1","order_by":1,"name":"Shuangyan Han","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYFACHiCuQGITqeUMELORpIWxjRQt8jNyD34unFcnb3C/gfHB2zYGeXNCWgxu5CVLz9x22HDDMQZmw7ltDIY7GwhpkcgxkObddiDB4BgDmzRvG0OCwQGCDssx/s07pw6khf03UVoYbuSYSfM2MINtYSZKi8GZN2bWPMcOG848ltgsOeechOEGgg5rzzG+zVNTJ893+PDBD2/KbOQJO0wgAcZibAASEoTUAwE/QUNHwSgYBaNgxAMARGA67npbVesAAAAASUVORK5CYII=","orcid":"","institution":"South China University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Shuangyan","middleName":"","lastName":"Han","suffix":""}],"badges":[],"createdAt":"2024-01-09 01:07:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3846786/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3846786/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49708572,"identity":"462e94ed-01d5-4b3a-8b3c-a89943d4eee5","added_by":"auto","created_at":"2024-01-16 19:26:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1204347,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of PMGL-Ba. (A) Selection of signal peptides. (B) The predicted structure of PMGL-Ba. Red represents α-helix, gold represents β-sheet, and green represents random coil structure. (C) Sequence comparison analysis.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3846786/v1/83584f84d7d1232f2e62be1f.png"},{"id":49708573,"identity":"cec783bb-0bac-422f-90af-7c7e1d87a638","added_by":"auto","created_at":"2024-01-16 19:26:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":129875,"visible":true,"origin":"","legend":"\u003cp\u003eExpression and enzymatic properties of PMGL-Ba. (A) Fermentation of PMGL-Ba. (B) SDS-PAGE analysis. (C-F) Enzymatic properties of PMGL-Ba.\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3846786/v1/f23c507c3da8aff24e432c02.png"},{"id":49708571,"identity":"e1910a4b-f0e3-42ca-b30c-9b481852377e","added_by":"auto","created_at":"2024-01-16 19:26:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":68313,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different signaling peptides on PMGL-Ba expression. (A) Selection of signal peptides. (B) Growth curves of strains corresponding to different signal peptides. (C) Effect of different signal peptides on total protein concentration of PMGL-Ba and SDS-PAGE analysis. (D) Effect of different signaling peptides on PMGL-Ba enzyme activity.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3846786/v1/7862e30689a6078c1d80c676.png"},{"id":49709214,"identity":"45fb57c8-f549-4541-9a96-8e698bbc54e9","added_by":"auto","created_at":"2024-01-16 19:34:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":58425,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different promoters on PMGL-Ba expression. (A) Selection of promoters. (B) Growth curves of strains corresponding to different promoters. (C) Effect of different promoters on total protein concentration of PMGL-Ba and SDS-PAGE analysis. (D) Effect of different promoters on PMGL-Ba enzyme activity.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3846786/v1/aa3680466d58f253e04a077c.png"},{"id":49709215,"identity":"4314ecab-c11a-4307-b46e-c5bf41c197f1","added_by":"auto","created_at":"2024-01-16 19:34:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":89974,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of gene dosage on PMGL-Ba expression. (A) Schematic of different gene doses. (B) Growth curves of strains corresponding to different gene doses. (C) Effect of different gene doses on total protein concentration of PMGL-Ba and SDS-PAGE analysis. (D) Effect of different gene doses on PMGL-Ba enzyme activity.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3846786/v1/d41ca536850f21f61bebbf57.png"},{"id":49708575,"identity":"9e169783-8d0b-4470-9f60-a95425271109","added_by":"auto","created_at":"2024-01-16 19:26:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":134653,"visible":true,"origin":"","legend":"\u003cp\u003ePosition of different protein cofactors in the protein secretory expression pathway. (A) Schematic of different gene doses. (B) Growth curves of strains corresponding to overexpression of different protein cofactors. (C) Effect of overexpression of different protein cofactors on total protein concentration of PMGL-Ba and SDS-PAGE analysis. (D) Effect of overexpression of different protein cofactors on PMGL-Ba enzyme activity.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-3846786/v1/1fe04e87e3756697108bf90d.png"},{"id":49708578,"identity":"81504f91-573c-436b-a0c6-b1f0df72f136","added_by":"auto","created_at":"2024-01-16 19:26:05","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":93778,"visible":true,"origin":"","legend":"\u003cp\u003ePMGL-Ba high density fermentation. (A) 5L type fermenter high density fermentation. (B) Changes in biomass and total protein concentration over time in high-density fermentations. (C) SDS-PAGE analysis of supernatants after 20-fold dilution at different times in high-density fermentation. (D) Changes in PMGL-Ba activity at different times in high-density fermentation.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-3846786/v1/c3c984e49a25d454b8b84b98.png"},{"id":49710359,"identity":"530565a3-107b-494c-b7ac-aa28dbaa657f","added_by":"auto","created_at":"2024-01-16 19:42:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2233935,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3846786/v1/62e05427-09f8-4b9e-a4ea-c3eb3e6ce934.pdf"},{"id":49708577,"identity":"db4ffb52-23e4-4357-a629-b23b02996965","added_by":"auto","created_at":"2024-01-16 19:26:05","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3010765,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical Abstract\u003c/p\u003e","description":"","filename":"renamed132e6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3846786/v1/2d27bcb116951f9c58d0a5b7.jpg"}],"financialInterests":"","formattedTitle":"Heterologous and efficient expression of a new alkaline pectin lyase in Pichia pastoris","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePectin, a structural polysaccharide ubiquitous in plant cell walls, consists of D-galacturonosyl residues linked by α-1,4-glycosidic bonds, imparting both rigidity and flexibility. This polymer serves as a protective agent for plants (Kaur et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zheng et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, in specific industrial scenarios, altering pectin's structure becomes imperative to curtail energy consumption or enhance product quality. Traditional mechanical and chemical methods pose environmental risks and may harm raw materials. Enzymatic treatments emerge as preferred alternatives due to their efficiency, specificity and environmental friendliness (Haile et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kaur et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The degradation of pectin involves three primary types of pectinases: methyl deesterases (pectin methyl esterases), hydrolases (polygalacturonases), and lyases (pectin lyase) (Suberkropp and Keller 2020). Pectin methyl esterase initiates methoxylation, completing deesterification and producing pectin and methanol. Subsequently, pectin lyase and polygalacturonases preferentially act on highly esterified pectin, catalyzing hydrolytic cleavage of the α-1,4-glycosidic bond in the pectin backbone without methanol production, resulting in 6-methyl-D-galacturonic acid. This crucial step in pectin degradation, involving cutting the pectin skeleton and progressively breaking down the substance, makes pectin lyase vital for industrial environmental protection (Tasgin et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Pectinase pretreatment finds diverse applications, reducing fruit juice viscosity in food processing (Rahman et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), enhancing cotton fabric absorbency in the textile industry (Aggarwal et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and improving paper quality while decreasing chemical usage in the paper industry (Ahlawat et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Nawawi et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Singh et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Notably, treating mechanical pulp with pectinase degrades pectin on fibers, reducing the amount of extractives covering the fiber surface. This weakens the bond between lignin and cellulose, refining the pulp further. The enzyme pretreatment exposes more microfibers, facilitating inter-fiber bonding and improving fiber strength, demonstrating the versatility and significance of pectinase in various industrial processes (Ahlawat et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile the application of biological enzymes in industry is highly effective for green manufacturing, elevated production costs present a significant challenge. Hence, improving the production efficiency of enzymes and exploring high-expression strategies are crucial long-term research objectives. \u003cem\u003eP.pastoris\u003c/em\u003e, with advantages such as high cell culture density, genetic manipulation, and post-translational modification of proteins, stands as a widely used bacterium for producing exogenous proteins (Ahmad et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Vijayakumar and Venkataraman \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Leveraging the secretion mechanism of exogenous proteins in \u003cem\u003eP.pastoris\u003c/em\u003e, extensive research has focused on optimizing strategies to enhance protein secretion. These strategies encompass the construction of expression platforms, pathway engineering modification, and optimization of culture conditions, resulting in the efficient expression of diverse exogenous proteins (Liu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Raschmanov\u0026aacute; et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Specifically, within the expression system, the emphasis lies on targeting promoter strength, selecting secretion signal peptides, and optimizing codons for the target proteins in the expression cassette (Xiao et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Pathway engineering modification primarily involve using relevant cofactors in the co-expression pathway to promote transcription, translation, processing and secretion of the protein, while averting stress reactions and protein degradation within the cell (Jiang et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zahrl et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zahrl et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In the realm of culture conditions, the focus is on optimizing medium composition, pH, temperature, and implementing high-density fermentation (Dixit et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Additionally, increasing the gene dose through in vitro constructs and in vivo screening is a commonly employed strategy to promote high expression (Peng et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Although these strategies have shown success in increasing the yield of exogenous proteins, their effects can be unpredictable for different proteins and require customization. However, the effects of these strategies are unpredictable for different proteins, so they can only be \"customized\" for proteins. Among the studies that have been reported, the highest expression of alkaline pectin lyase in \u003cem\u003eE. coli\u003c/em\u003e is currently 147347.6 U/mL, while the expression in \u003cem\u003eP. pastoris\u003c/em\u003e is 3620 U/mg (14 L fermenter) (Li et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Ma \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Peng \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Substantial room for improvement remains in optimizing the expression of PMGL in \u003cem\u003eP. pastoris\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn this paper, an unreported PMGL gene derived from \u003cem\u003eBacillus\u003c/em\u003e was selected by homologous sequence comparison and heterologously expressed in \u003cem\u003eP. pastoris\u003c/em\u003e. Subsequently, a strain of \u003cem\u003eP. pastoris\u003c/em\u003e with high expression of PMGL-Ba was constructed by several strategies, including codon optimization, optimization of gene expression elements, increasing gene dosage and overexpression of cofactors. The objective is to provide an industrial enzyme and reduce the associated costs, addressing a critical aspect in the practical application of these enzymes in various industrial processes.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Gene mining and sequence analysis of PMGL-Ba\u003c/h2\u003e \u003cp\u003eThe gene mining process for PMGL involved using a high enzyme activity pectin lyase gene as the probe sequence, with key residues serving as the screening conditions. The selection criteria included assessing sequence similarity to the probe template, predicting soluble expression of the protein, and considering the type of microorganisms of origin. Ultimately, the identified putative PMGL-Ba (NCBI Reference Sequence: WP_009329358.1) derived from Bacillus licheniformis was finally selected. The protein consists of 494 amino acids with a relative molecular mass of 54693.2. PMGL-Ba was found to be in the same branch as two Bacillus-derived pectin lyases (GenBank: OMI04834.1, QII50179.1) annotated in NCBI, and both are in the polysaccharide lyase 1 family, confirming the reliability of the results(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Analysis of the protein structure diagram revealed that PMGL-Ba possesses the typical β-helical structure of pectin lyase proteins, consisting of three parallel β-strands connected through three corners (Zheng et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Sequence comparisons indicated that certain sequences of PMGL-Ba overlap with conserved amino acid sequences of the pectin lyase reference sequence or contain key amino acids of the catalytic activity center, suggesting potential functional similarities (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e Phylogenetic analysis of PMGL-Ba. (A) Selection of signal peptides. (B) The predicted structure of PMGL-Ba. Red represents α-helix, gold represents β-sheet, and green represents random coil structure. (C) Sequence comparison analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Expression and biochemical characterization of PMGL-Ba\u003c/h2\u003e \u003cp\u003eThe shake flask fermentation of PMGL-Ba recombinant \u003cem\u003eP. pastoris X33\u003c/em\u003e was carried out, with samples taken at 24 h intervals to assess the growth of the recombinant yeast and the enzyme activity of pectin cleavage in the supernatant of the fermentation broth. With an increase in induced expression time, the OD600, fermentation broth supernatant enzyme activity, and protein yield of X33-PMGL-Ba gradually increased. At 144 h of induced expression time, the enzyme activity and protein concentration with esterified pectin as substrate reached 895.69 U/mL and 0.77 g/L, respectively, indicating the successful heterologous secretory expression of recombinant PMGL-Ba using \u003cem\u003eP. pastoris X33\u003c/em\u003e(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The purified PMGL-Ba was analyzed by SDS-PAGE, revealing an apparent molecular weight of about 55 kDa, consistent with the theoretical molecular weight of 54.6 kDa༈Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eThe enzyme activity of PMGL-Ba exhibited an initial increase followed by a decrease with increasing pH, pinpointing the optimal reaction pH as 8.5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). PMGL-Ba retained more than 80% of its total activity within the pH range of 8.0-9.5. However, the enzyme activity decreased rapidly beyond pH 9.0 or below 8.0. PMGL-Ba has good pH stability, maintaining 60% activity in the pH range of 5.0\u0026ndash;11.0(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The optimal temperature for PMGL-Ba was 60\u0026deg;C, with relative enzyme activity exceeding 60% at temperatures between 50\u0026ndash;70\u0026deg;C༈Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). PMGL-Ba activity decreased with increasing temperature, and relative enzyme activity remained above 60% at temperatures below 70\u0026deg;C after 1 h of exposure. As the temperature increased further, the enzyme activity gradually decreased, with only about 20% of the enzyme activity remained at the temperature of 80 ℃༈Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). PMGL-Ba showed robust enzyme activity and stability over a broad temperature range of 30 to 80\u0026deg;C, indicating its potential advantages for application in medium and high-temperature industries, such as the paper industry.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e Expression and enzymatic properties of PMGL-Ba. (A) Fermentation of PMGL-Ba. (B) SDS-PAGE analysis. (C-F) Enzymatic properties of PMGL-Ba.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Effect of expression components on recombinant PMGL-Ba protein expression\u003c/h2\u003e \u003cp\u003eThe secretory expression of heterologous proteins in \u003cem\u003eP. pastoris\u003c/em\u003e often involves the utilization of the \u003cem\u003eS. cerevisiae\u003c/em\u003e α-factor mating pheromone (α-MF) from Saccharomyces cerevisiae. Existing studies have explored the impact of signaling peptides on the secretion of recombinant proteins, revealing varying efficiencies of signaling peptides for different exogenous proteins. Studies on the effect of signal peptides on exogenous protein secretion can be broadly categorized into two groups: modification of existing signal peptides and the discovery of new signal peptides. The commonly used α-MF comprises a two-stage signal, consisting of a 19-amino-acid pre region and a 66-amino-acid pre region at the N-terminal end, demonstrating effective secretion for most exogenous proteins (Barrero et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Several studies have sought to enhance the secretion efficiency of α-MF various modifications, such as αMF-CC (αM), and αopt obtained after optimization and point mutation of the αMF sequence, resulting in improved protein secretion (Ahn et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Aza et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ito et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Another approach involves utilizing more efficient sequences to replace parts of the αMF. Juan J et al. designed combined secretion signals, such as the one consisting of the Saccharomyces cerevisiae Ost1 signal sequence and the α-factor PRO region, which has demonstrated superior secretion ability compared to the α-factor signal sequence (Barrero et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In yeast, secreted proteins are targeted to the endoplasmic reticulum (ER) by either the co-translational pathway or the \"post-translational\" pathway. The co translational pathway is mediated by the signal recognition particle (SRP), while the \"post-translational\" pathway, independent of SRP, requires the intact membrane protein Sec62p for ER targeting. However, more hydrophobic signaling sequences utilize the SRP pathway to target the ER in order to increase secretion efficiency (Walter et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Ost1p, a type I intact membrane protein with a cleavable signal sequence, plays a role in co-translational translocation to the ER (Willer et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Subsequent bioreactor-scale fermentation has further confirmed that specific recombinant signal peptide promotes protein secretion (Barrero et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To avoid variability in signal peptide efficiency for different protein secretion, the discovery of new signal peptides is essential (Liang et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sastry et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Systematic analysis of yeast genomes and secretomes has led to the identification of novel signal peptides with more efficient secretion than α-MF, such as the signal peptide derived from the \u003cem\u003eP. pastoris\u003c/em\u003e PAS_chr3_0030 gene (PC0) and the signal peptide NCW2 in Saccharomyces cerevisiae (Shen et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zou et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, six distinct types of secreted signaling peptides, namely pPIC-α-MF-PMGL-Ba, pPIC-O-pre-α-pro-PMGL-Ba, pPIC-αopt-PMGL-Ba, pPIC-αM-PMGL-Ba, pPIC-PC0-PMGL-Ba, and pPIC-NCW2-PMGL-Ba, were selected to investigate their effects on PMGL-Ba production. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, results from shake flask fermentation revealed a significant enhancement in pectinase secretion with O-pre-α-pro and aM compared to α-MF. Conversely, PMGL-Ba secretion by αopt and NCW2 was lower than that of α-MF. The signaling peptide PC0 exhibited secretion levels similar to α-MF. Notably, the protein concentration and enzyme activity of Ba secreted by the signal peptide O-pre-α-pro were 1.40 g/L and 1554.26 U/mL, respectively, representing a 130.71% and 98.13% increase compared to the departure strain pPICZαA-PMGL-Ba.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e Effects of different signaling peptides on PMGL-Ba expression. (A) Selection of signal peptides. (B) Growth curves of strains corresponding to different signal peptides. (C) Effect of different signal peptides on total protein concentration of PMGL-Ba and SDS-PAGE analysis. (D) Effect of different signaling peptides on PMGL-Ba enzyme activity.\u003c/p\u003e \u003cp\u003e \u003cem\u003eP. pastoris\u003c/em\u003e, a methylotrophic yeast, tightly regulates the expression of exogenous proteins when utilizing methanol as a carbon source through the methanol utilization pathway (MUT) promoter. The transcription level of exogenous protein genes is intricately linked to the strength of promoter regulation (P\u0026uuml;llmann and Weissenborn \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The commonly employed AOX1 promoter is favored for its robust induction by methanol, although the precise mechanism of its methanol-induced action remains not fully elucidated (Berg et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePichia pastoris is a methylotrophic yeast, and the expression of exogenous proteins using methanol as a carbon source is tightly regulated by the methanol utilization pathway (MUT) promoter. The transcription level of exogenous protein genes is closely linked to the strength of promoter regulation. The AOX1 promoter is commonly used and is often the promoter of choice for its strong induction by methanol. However, the mechanism of action of the AOX1 promoter strictly induced by methanol is not fully understood.\u003c/p\u003e \u003cp\u003eTo unravel the mechanism of AOX1 promoter action, previous studies have utilized mutation screening, followed by sequence analysis, or adjusted regulatory factors of other carbon sources (Hartner et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Zhan et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In this study, three MUT strong promoters, reported to be more effective than AOX1 in inducing exogenous proteins, were selected. The first, AOXm, involved deleting a transcription factor sequence associated with glucose repression and copy addition of a positive cis-acting element sequence after AOX1 deletion (Hartner et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The second, from the study by Thomas Vogl et al., utilized genes from the pentose phosphate pathway (PPP) and the defense against reactive oxygen species (ROS) in the MUT pathway to provide strong promoters that somewhat exceed AOX1, such as the promoter of the gene encoding peroxisomal membrane protein (PMP20) (Yurimoto et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Vogl et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The third is formaldehyde dehydrogenase 1 (FLD1), a key enzyme in metabolizing methanol as a carbon source (Nakagawa et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The promoters of their encoding genes show similar yields for FLD1 and AOX1 in exogenous protein expression, with FLD1 even surpassing AOX1 in some proteins (P\u0026uuml;llmann and Weissenborn \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, all four promoters mentioned above were individually employed for PMGL-Ba expression, and the most suitable promoter for PMGL-Ba gene was selected. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, AOX1 demonstrated obvious advantages in PMGL-Ba expression. Biomass analysis further indicated a notable decreasing trend for strains PMP20 and FLD1, suggesting that AOX1 remains the most suitable promoter for PMGL-Ba gene expression in this context.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e Effects of different promoters on PMGL-Ba expression. (A) Selection of promoters. (B) Growth curves of strains corresponding to different promoters. (C) Effect of different promoters on total protein concentration of PMGL-Ba and SDS-PAGE analysis. (D) Effect of different promoters on PMGL-Ba enzyme activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Effect of gene dosage on recombinant PMGL-Ba protein expression\u003c/h2\u003e \u003cp\u003eIn the expression of exogenous proteins in \u003cem\u003eP. pastoris\u003c/em\u003e, constructing high-copy strains is often a straightforward and effective approach to enhance protein production. Various methods exist for constructing multicopy strains, with a common strategy involving the in vitro construction of multiple expression cassettes in tandem, subsequently integrated onto the \u003cem\u003eP. pastoris\u003c/em\u003e genome. In order to obtain higher copy number strains, different screening markers can be employed for sequential integration into the \u003cem\u003eP. pastoris\u003c/em\u003e genome (Wang et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Another prevalent method involves the gradual increase of antibiotic concentration, with commonly used antibiotics such as zeocin and antibiotic G418; the screening concentration is usually higher than 100 \u0026micro;g/mL, with some reaching up to 2000 \u0026micro;g/mL (Kuo et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Sha et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Song et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, zeocin is a relatively expensive antibiotic, and its screening process is intricate and time-consuming. Another approach involves the Cre/lox recombination system, using a screening marker for multiple integration, but this method suffers from a prolonged cycle time (Li et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In addition, Xia et al. constructed a novel double plasmid system using a combined strategy of genomic integration and heterologous expression (Xia et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, homologous recombination was employed to construct three strains with different screening markers, namely GS115-2PMGL-Ba, GS115-3PMGL-Ba, and GS115-4PMGL-Ba, which were sequentially integrated onto the Picot yeast genome using different screening markers. This method effectively reduced the time required for constructing multiple copies by 2\u0026ndash;3 days, streamlining the identification process post-integration into the yeast genome. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, GS115-2PMGL-Ba exhibited improvements over GS115-PMGL-Ba, showcasing increased protein concentration and enzyme activity. However, GS115-3PMGL-Ba demonstrated a significant decrease in yield compared to GS115-2PMGL-Ba. In shake flask fermentation, the protein concentration and enzyme activity of GS115-2PMGL-Ba reached 1.50 g/L and 1658.01 U/mL, respectively, representing a 6.7% and 6.69% increase compared to X33-PMGL-Ba.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e Effects of gene dosage on PMGL-Ba expression. (A) Schematic of different gene doses. (B) Growth curves of strains corresponding to different gene doses. (C) Effect of different gene doses on total protein concentration of PMGL-Ba and SDS-PAGE analysis. (D) Effect of different gene doses on PMGL-Ba enzyme activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Effect of overexpression of cofactors on recombinant PMGL-Ba protein expression\u003c/h2\u003e \u003cp\u003eIn the realm of protein expression pathways, alterations in chaperone protein levels can exert influence across multiple stages, encompassing transcription, translation, folding, and secretion. However, the impacts of such alterations are often protein-specific, and determining the singularly most crucial chaperone or the optimal combination remains ambiguous (Samuel et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In this study, we expanded on GS115-2Ba by overexpressing 11 cofactors previously investigated for their roles in promoting protein expression.\u003c/p\u003e \u003cp\u003eA pivotal step in exogenous protein expression in \u003cem\u003eP. pastoris\u003c/em\u003e is the translation process, where translation initiation serves as the rate-limiting step in the translation process. Various translation initiation-associated factors have been targeted to study the impact of translation efficiency. Notably, Jennifer Staudacher et al. demonstrated that overexpression of certain translation factors, particularly Pab1 and eIF4G, significantly enhances exogenous protein production in \u003cem\u003eP. pastoris\u003c/em\u003e. The synergistic effect was more pronounced when all four loop-closing related factors, eIF4E, eIF4A, eIF4G and Pab1, were simultaneously overexpressed (Staudacher et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In a previous study in our laboratory, Zheng et al. also proposed a novel yeast transcription factor, tentatively named Fhl1p, implicated in the regulation of rRNA processing genes and genes related to ribosomal small/large subunit biogenesis genes. This factor was found to facilitate the translation efficiency of exogenous proteins (Zheng et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCorrect protein folding is a critical step in \u003cem\u003eP. pastoris\u003c/em\u003e, particularly during post-translational translocation in the endoplasmic reticulum (ER). Newly synthesized peptides entering the ER must maintain an unfolded or loosely folded form after release from the ribosome, preventing aggregation before translocation. This process involves binding to cytoplasmic chaperones such as Ssa1 and Ydj1. In contrast, the ATPase activity of the ER luminal chaperone Kar2 powers post-translational translocations, promoting protein folding as well as targeting misfolded proteins to ERAD (Delic et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn response to endoplasmic reticulum stress induced by the overexpression of exogenous proteins, especially in multicopy strains, a common approach to alleviate these stress effects is the overexpression of cofactors. The HSP30 gene has been shown to play a crucial role in mitigating the repression of target gene transcript levels induced by high-intensity promoters. Besides, HSP30 gene expression is simultaneously regulated by the transcription factors HSF1, MSN2, and MSN4. Notably, the transcription factor HSF1 positively affects alleviating unfolded protein stress (Cui et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Overexpression of the transcription factor Hac1p is frequently selected during chaperonin assisted secretory protein folding. In multicopy strains, the overexpression of exogenous proteins induces cellular stress responses, such as the common unfolded protein response (UPR), activated by unconventional splicing of HAC1 mRNA in yeast. The spliced HAC1 mRNA encodes an active transcription factor, Hac1p, which binds to UPR response elements in the promoters of UPR target genes, and it induces the expression of other chaperone proteins that assist in protein folding and protein transport (Liu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Guerfal et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we systematically overexpressed the 11 cofactors previously discussed, building upon the foundation of GS115-2PMGL-Ba. Interestingly, the Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e indicates that only overexpression of Hac1p increased the protein expression, while overexpression of the other ten cofactors decreased the protein expression. In shake flask fermentation, the protein concentration and enzyme activity of strain GS115-2PMGL-Ba: Hac1p were 1.81 g/L and 1835.53 U/mL, respectively. Compared with GS115-2PMGL-Ba, the protein concentration and enzyme activity increased by 20.85% and 10.7%.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e Position of different protein cofactors in the protein secretory expression pathway. (A) Schematic of different gene doses. (B) Growth curves of strains corresponding to overexpression of different protein cofactors. (C) Effect of overexpression of different protein cofactors on total protein concentration of PMGL-Ba and SDS-PAGE analysis. (D) Effect of overexpression of different protein cofactors on PMGL-Ba enzyme activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 5L fermenter refill batch fermentation\u003c/h2\u003e \u003cp\u003eAfter identifying the highest PMGL-Ba yielding strain, GS11-2PMGL-Ba-Hac1p, through the different strategies mentioned above, it was activated and cultured into a seed solution. This seed solution was then introduced into a 5 L fermenter containing 2 L of BSM basal medium to initiate the batch culture phase. The glycerol feed-batch phase commenced after a dissolved oxygen rebound at 28 h. Glycerol replenishment was stopped when the yeast density reached OD600\u0026thinsp;=\u0026thinsp;about 300, indicating complete glycerol consumption. Subsequently, the methanol feed-batch phase commenced, with samples taken every 8 h, continuing until the completion of 72 h.\u003c/p\u003e \u003cp\u003eAs shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, the high-density fermentation of GS115-2PMGL-Ba-Hac1p in a 5 L fermenter for 72 h resulted in a substantial increase in protein concentration to 12.49 g/L, representing a remarkable 591.51% improvement compared to shake flask fermentation. Simultaneously, enzyme activity increased to 12668.12 U/mL, reflecting a 595.53% increase compared to shake flask fermentation. Previous experimental findings indicated protein degradation in the late stage of fermentation. To expedite the fermentation process, we increased the starting cell density of the induction stage of methanol flow addition from OD600\u0026thinsp;=\u0026thinsp;200 to OD600\u0026thinsp;=\u0026thinsp;300, and reduced the fermentation cycle from 144 h in shake flasks to 72 h.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e PMGL-Ba high density fermentation. (A) 5L type fermenter high density fermentation. (B) Changes in biomass and total protein concentration over time in high-density fermentations. (C) SDS-PAGE analysis of supernatants after 20-fold dilution at different times in high-density fermentation. (D) Changes in PMGL-Ba activity at different times in high-density fermentation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Strains, Media, and Reagents\u003c/h2\u003e \u003cp\u003eThe molecular kits used in this experiment were purchased from Magen Biotechnology Co.,Ltd. Restriction endonucleases were purchased from Thermo Fisher Scientific (Shanghai,China). The homologous recombinant enzyme was purchased from Beijing Jinsha Biotechnology Co. Chemical reagents were purchased from Tianjin Damao Chemical Reagent Factory. Pectin was purchased from McLean. Recombinant plasmids and recombinant strains were constructed using \u003cem\u003eEscherichia coli Top10\u003c/em\u003e and \u003cem\u003eP.pastoris\u003c/em\u003e as hosts. The medium media and their compositions used in this experiment were LB (0.5% yeast extract, 1% peptone and 1% NaCl), LBL (0.5% yeast extract,1% peptone and 0.5% NaCl), YPD (1% yeast extract, 2% peptone and 2% glucose), MD (1.34% YNB and 2% glucose), BMGY (1% yeast extract, 2% peptone, 1.34% YNB, 0.1 mM PBS pH 6.0 and 1% glycerol) and BMMY (1% yeast extract, 2% peptone, 1.34% YNB, 0.1 mM PBS pH 6.0 and 1% methanol).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Gene mining for putative xylanase and sequence analysis for target gene\u003c/h2\u003e \u003cp\u003eGene mining for PMGL was performed with the help of enzyme resource mining software EnzymeMiner. Two high enzyme activity pectin lyases Q01172 and P94449 of medium alkaline and medium high temperature were used as probe sequences. Additionally, the protein sequences of three PMGLs, Q2TXS4, AY825251, and R9XVW6, were used as auxiliary references for sequence search. The initial set of sequences of potential PMGLs was obtained by performing iterative site-specific homology searches in the NCBI database. The key residues of PMGL were then used as filtering criteria to filter sequences with mismatched key residues in the homology search results. The final selection was based on the sequence similarity and sequence characterization provided by EnzymeMiner, the sequence similarity to the probe template (with a threshold value of 30%-70%), the predicted degree of soluble expression of the protein, and the type of microorganism from which the protein was derived. A phylogenetic tree was constructed between the selected sequences and the probe sequences by neighbor-joining method using MEGA-X software to analyze the homology of the sequences. The protein structure of PMGL-Ba was modeled by SwissModel homology. Sequence comparison analysis was performed using Jalview. The basic physicochemical properties of Bacillus pectin lyase were predicted using ProtParam.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Heterologous Expression of PMGL-Ba in \u003cem\u003eP. pastoris\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe PMGL gene (NCBI: WP_009329358.1) derived from Bacillus was mined by homologous sequence comparison. Subsequently, the PMGL gene was codon optimized and constructed in the pPICZαA vector, named pPICZαA-PMGL-Ba. The recombinant plasmid was then transformed into \u003cem\u003eE. coli Top10\u003c/em\u003e receptor cells, coated to solid medium containing zeocin antibiotic in LBL and cultured for 12\u0026ndash;14 h. Single colonies, correctly identified by PCR, were transferred to liquid medium of LBL and cultured for 12\u0026ndash;14 h. Plasmids were extracted and sequenced according to the kit. The plasmids with correct sequencing were linearized and transformed into \u003cem\u003eP. pastoris\u003c/em\u003e by electroporation and selected on YPDZ plates, and incubated at 30\u0026deg;C for 3 d. Yeast single colonies with correct colony morphology were identified by PCR.\u003c/p\u003e \u003cp\u003eThe recombinant \u003cem\u003eP. pastoris\u003c/em\u003e strains were transferred to 10 mL of BMGY medium at 30℃ for 20\u0026ndash;24 h, and then transferred to 25 mL of BMMY medium according to the starting OD600 as 1. Samples were taken every 24 h and 1% (v/v) methanol was added. After 144 h of fermentation, the samples were transferred to 50mL centrifuge tubes. After centrifugation, the supernatant was taken for subsequent experiments.\u003c/p\u003e \u003cp\u003eThe collected fermentation supernatant was centrifuged at high speed (10,000rpm, 4℃, 10 min), and then passed through a 0.22 \u0026micro;m ultrafiltration membrane. Gradient purification of fermentation broth supernatant using Ni-NTA column chromatography, and the eluate was collected. The protein content of the fermentation supernatant and purified PMGL-Ba were determined using the Bradford method, with bovine serum albumin (BSA) as the standard. Finally, the supernatant was diluted 2-fold and 20 \u0026micro;L was taken for SDS-PAGE analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Enzymatic properties of PMGL-Ba\u003c/h2\u003e \u003cp\u003eThe activity of PMGL-Ba was assessed utilizing the 3,5-dinitrosalicylic acid (DNS) method. Initially, 0.5% pectin was accurately weighed and dissolved in Tris-HCl buffer (50 mM, pH 8.0). The fermentation supernatant was appropriately diluted. Subsequently, 190 \u0026micro;L of the pectin solution was placed in a thermostatic reactor to preheat for 10 min, and 10 \u0026micro;L of sample was added to react for 10 min. 300 \u0026micro;L of DNS solution was added, boiled for 5 min and then cooled down immediately, and 200 \u0026micro;L of the reaction solution was taken to measure the absorbance value at 540 nm. One unit (U) of enzyme activity was defined as the amount of pectin lyase releasing 1 \u0026micro;mol of reducing sugar per minute under the conditions described above.\u003c/p\u003e \u003cp\u003eThe optimal pH value of PMGL-Ba was measured by determining the activity between pH 5.0\u0026ndash;12.0 in 60\u0026deg;C. To determine pH stability of PMGL-Ba, purified enzymes were firstly incubated between pH 5.0\u0026ndash;12.0 at 4\u0026deg;C for 12 h. Next, residual activity of PMGL-Ba was further measured using above method. Buffers used were 100 mmol/L PBS buffer (pH 6.0), 50 mmol/L Tris-HCl buffer (pH 7.0\u0026ndash;9.0) and Glycine-sodium hydroxide buffer(pH10-11). Optimal temperature of PMGL-Ba was investigated by measuring the activity in a range of 50\u0026deg;C\u0026ndash;70\u0026deg;C in pH 8.0. To determine the thermostability of PMGL-Ba, enzymes were pre-incubated at 30\u0026deg;C\u0026ndash;90\u0026deg;C for 1 h pH 8.0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.5 Construction of highly expressed PMGL-Ba recombinant strains\u003c/h2\u003e \u003cp\u003eTo investigate the expression of PMGL-Ba by secreted signaling peptides, five signaling peptides were selected based on literature research as OST1-pre-αMF-pro(O-pre-α-pro), αopt, αM, PAS_chr3_0030 gene (PC0) and NCW2, respectively. Subsequently, α-MFs were sequentially replaced by homologous recombination, resulting in constructs named pPIC-O-pre-α-pro-PMGL-Ba, pPIC-αopt-PMGL-Ba, pPIC-αM-PMGL-Ba, pPIC-PC0-PMGL-Ba, and pPIC-NCW2-PMGL-Ba. In parallel, three strong promoters, AOXm, FLD1, and PMP20, were also substituted for AOX1 by homologous recombination to explore the expression of the promoters on the proteins, named pPIC-AOXm-PMGL-Ba, pPIC-FLD1-PMGL-Ba, and pPIC-PMP20-PMGL-Ba, respectively. To facilitate in vitro construction of multiple copies, the PMGL-Ba gene expression cassette was constructed on the PHK-PMGL-Ba and pPIC-Cre/lox-PMGL-Ba plasmid vectors. PHK-2PMGL-Ba and pPIC-Cre/lox-2PMGL-Ba were constructed using homologous recombination. Three-copy and four-copy recombinant strains were constructed by screening markers differentially integrated sequentially on the yeast genome. To further promote protein expression, eleven cofactors were overexpressed in multicopy strains as Fhlp, Hsf1, eIF4A, eIF4E, eIF4G, PAB1, Hsp30, Hac1p, MSN2, SSA1, and KAR2, and were named as PHK-2PMGL-Ba-Fhlp, PHK-2PMGL-Ba-Hsf1, PHK-2PMGL-Ba-eIF4A, PHK-2PMGL-Ba-eIF4E, PHK-2PMGL-Ba-eIF4G, PHK-2PMGL-Ba-PAB1, PHK-2PMGL-Ba-Hsp30, PHK-2PMGL-Ba-Hac1p, PHK-2PMGL-Ba-MSN2, PHK-2PMGL-Ba-SSA1, and PHK-2PMGL-Ba-KAR2, respectively. The plasmids, confirmed by correct sequencing, were linearized and transformed into \u003cem\u003eP. pastoris\u003c/em\u003e by electroporation and selected on MD plates or YPDZ plates, and incubated at 30\u0026deg;C for 3 d. Yeast single colonies with correct colony morphology were identified by PCR. The recombinant \u003cem\u003eP. pastoris\u003c/em\u003e strains were fermented as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.6 Fed-batch fermentation in a 5 L fermenter\u003c/h2\u003e \u003cp\u003eThe screened high yielding strains were first streaked from the preservation tubes to YPD solid medium for 3 d activation. A single colony was picked into 10mL YPD liquid medium and incubated in shaker at 30℃ for 20\u0026ndash;24 h to form a primary seed solution. Then the second seed liquid was obtained by transferring to 160mL YPD medium at 4% inoculum volume and culturing in shaker at 30℃ for about 20 h. The resulting seed solution was transferred to a 5 L fermenter containing 2 L of BSM medium, and 8% inoculum was introduced along with 8.7 mL of aseptically added PTM\u003csub\u003e1\u003c/sub\u003e (micronutrient salt). Temperature was controlled at 30\u0026deg;C, and pH was set at 5.5. The glycerol feed-batch phase commenced upon depletion of glycerol in the glycerol batch phase, accompanied by a rebound in dissolved oxygen. Glycerol addition ceased when OD600\u0026thinsp;=\u0026thinsp;300, signaling the transition to the methanol fed-batch phase. During this phase, temperature was 25\u0026deg;C, pH\u0026thinsp;=\u0026thinsp;6.0, and the induction time was 72 h. Cell biomass, protein concentration, and enzyme activity were determined by taking 10 mL samples every 8 h. At the end of fermentation, the broth underwent centrifugation, and the resulting supernatant was utilized for subsequent experiments.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we elucidated the amino acid sequence of PMGL-Ba, a novel enzyme derived from Bacillus, previously unreported. This was achieved through homologous sequence comparison and the identification of key amino acids using the EnzymeMiner software. For heterologous expression in \u003cem\u003eP. pastoris\u003c/em\u003e, we performed codon optimization on PMGL-Ba. The enzyme exhibited an optimum pH of 8.5 and an optimum temperature of 60 ℃, displaying excellent pH and temperature stability. To enhance PMGL-Ba expression in \u003cem\u003eP. pastoris\u003c/em\u003e, we employed a multifaceted approach, encompassing the selection of gene expression components, increased gene dosage, and co-expression of cofactors. Ultimately, a two-copy expression cassette, consisting of AOX1 promoter, OST1-pre-α-MF-pro signal peptide, and AOX1 terminator elements, was selected. This cassette, co-expressed with the transcription factor Hac1p, was utilized for high-density fermentation in a 5 L fermenter for 72 h. The results were impressive, with the total protein concentration reaching 12.49 g/L and the enzyme activity reaching12668.12 U/mL. This represents a remarkable 21-fold increase in total protein expression and a 15-fold increase in enzyme activity compared to the initial bacterial expression. This achievement marks the highest reported expression level of PMGL-Ba to date, providing a yeast strain with substantial PMGL-Ba production potential for future industrial enzyme applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe authors would like to acknowledge Yian Chen for his assistance in copy editing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003cstrong\u003e. \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSH initiated and coordinated the study and reviewed the manuscript. JY designed and performed the research, analysed the data, and draft the manuscript. MY designed and performed the partial research. FZ and YZ reviewed the experimental program and the manuscript.\u0026nbsp;All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Key R\u0026amp;D Program of China (NO. 2021YFC2100400).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the coauthors have read and approved the submission to Bioresources and\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBioprocessing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eGuangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China. \u003csup\u003e2\u003c/sup\u003eSchool of Light Industry and Engineering, South China University of Technology, Guangzhou, 510006, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAggarwal R, Dutta T, Sheikh J (2020) Extraction of pectinase from Candida isolated from textile mill effluent and its application in bio-scouring of cotton. 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Biotechnol Biofuels Bioprod 15(1)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bioresources-and-bioprocessing","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biob","sideBox":"Learn more about [Bioresources and Bioprocessing](http://bioresourcesbioprocessing.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/biob/default.aspx","title":"Bioresources and Bioprocessing","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Pectin lyase, Efficient expression, Pichia pastoris","lastPublishedDoi":"10.21203/rs.3.rs-3846786/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3846786/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePectin lyase (PMGL) is an industrially important enzyme with widespread applications in the food, paper, and textile industries, owing to its capacity for direct degradation of highly esterified pectin. In this study, PMGL-Ba derived from Bacillus underwent mining and heterologous expression in \u003cem\u003eP. pastoris.\u003c/em\u003e Furthermore, diverse strategies, encompassing the optimization of expression cassette components, elevation of gene dosage, and co-expression of chaperone factors, were employed to augment PMGL-Ba production in \u003cem\u003eP. pastoris\u003c/em\u003e. The signaling peptide OST1-pre-α-MF-pro and promoter AOX1 were finally selected as expression elements. By overexpressing the transcription factor Hac1p in conjunction with a two-copy PMGL-Ba setup, a strain yielding high PMGL-Ba production was achieved. In shake flask fermentation lasting 144 hours, the total protein concentration reached 1.81 g/L, and the enzyme activity reached 1821.36 U/mL. For further scale up production, high-density fermentation transpired in a 5 L fermenter for 72 h. Remarkably, the total protein concentration increased to 12.49 g/L, and the enzyme activity reached an impressive 12668.12 U/mL. The successful heterologous and efficient expression of PMGL-Ba not only furnishes a valuable biological enzyme for industrial applications but also contributes to cost reduction in the utilization of biological enzymes in industrial applications.\u003c/p\u003e","manuscriptTitle":"Heterologous and efficient expression of a new alkaline pectin lyase in Pichia pastoris","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-16 19:26:00","doi":"10.21203/rs.3.rs-3846786/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-01-12T21:39:09+00:00","index":0,"fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-12T01:58:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bioresources and Bioprocessing","date":"2024-01-08T06:27:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bioresources-and-bioprocessing","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biob","sideBox":"Learn more about [Bioresources and Bioprocessing](http://bioresourcesbioprocessing.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/biob/default.aspx","title":"Bioresources and Bioprocessing","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e5b84052-df3e-4564-b8e5-1c96ae777a9f","owner":[],"postedDate":"January 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-03-17T09:53:39+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-16 19:26:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3846786","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3846786","identity":"rs-3846786","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}