PEX3 gene knockout influences recombinant xylanase expression by Komagataella phaffii

Abstract

Komagataella phaffii is a methylotrophic yeast harboring a tightly regulated alcohol oxidase promoter (P_AOX1), which is now widely used for recombinant protein production. During P_AOX1 expression phase by methanol induction, a methanol metabolism organelle peroxisome enlarged and occupied 80 % of K. phaffii cell through peroxins functions of matrix protein import and organelle division. Using a K. phaffii expressing xylanase in this study, each of all 23 PEX genes of K. phaffii, encoding peroxin, was knockout to influence the peroxisome size, leading to changes of K. phaffii physiological status and recombinant xylanase expression. It was observed that PEX3 knockout reduced peroxisome size by 54.3 %, increased xylanase expression by 29 %, decreased apoptosis ratio by 70.6 %. Transcriptome analysis revealed that PEX3 gene knockout decreased 18 other PEX genes of all three steps of peroxisome propagation, biogenesis, matrix protein import, and peroxisome fission. PEX3 gene knockout influenced expression of ribosomal subunit-related and protein transportation significantly based on gene function annotation and enrichment analysis. Additionally, Therefore, PEX3 gene knockout promoted xylanase folding correctly via Sec63 complex, and PDI1 significantly. In a summary, PEX3 gene knockout provided a novel strategy to enhance recombinant xylanase by K. phaffii.

Headnote

ARTICLE INFO

Keywords:

Komagataella phaffii

Peroxisome

Peroxin gene

Recombinant protein expression

Transcriptome analysis

ABSTRACT

Komagataella phaffii is a methylotrophic yeast harboring a tightly regulated alcohol oxidase promoter (P_AOX1), which is now widely used for recombinant protein production. During P_AOX1 expression phase by methanol induction, a methanol metabolism organelle peroxisome enlarged and occupied 80 % of K. phaffii cell through peroxins functions of matrix protein import and organelle division. Using a K. phaffii expressing xylanase in this study, each of all 23 PEX genes of K. phaffii, encoding peroxin, was knockout to influence the peroxisome size, leading to changes of K. phaffii physiological status and recombinant xylanase expression. It was observed that PEX3 knockout reduced peroxisome size by 54.3 %, increased xylanase expression by 29 %, decreased apoptosis ratio by 70.6 %. Transcriptome analysis revealed that PEX3 gene knockout decreased 18 other PEX genes of all three steps of peroxisome propagation, biogenesis, matrix protein import, and peroxisome fission. PEX3 gene knockout influenced expression of ribosomal subunit-related and protein transportation significantly based on gene function annotation and enrichment analysis. Additionally, Therefore, PEX3 gene knockout promoted xylanase folding correctly via Sec63 complex, and PDI1 significantly. In a summary, PEX3 gene knockout provided a novel strategy to enhance recombinant xylanase by K. phaffii.

1. Introduction

Komagataella phaffii is a prominent industrial microorganism for recombinant protein and high-value chemical production, owing to its robust alcohol oxidase 1 promoter (Paox1), which drives protein expression using methanol as both an inducer and sole carbon source [1, 2]. Until now, more than 5000 heterologous proteins were expressed by K. phaffii successfully [3]. At the condition of methanol induction in K. phaffii, methanol is oxidized by alcohol oxidase to release hydrogen peroxide and formaldehyde which is divided into fork pathways of dissimilation and assimilation simultaneously [4]. In dissimilation pathway, formaldehyde is oxidized to formate, and carbon dioxide gradually for energy generation. In assimilation pathway, formaldehyde reacts with xylulose-5-phosphate to produce two interchangeable metabolites of dihydroxyacetone and glyceraldehyde-3-phosphate, which enter tricarboxylic acid (TCA) cycle for biomass generation. For the total methanol during metabolic pathway, about 12.0 %-14.9 % of methanol enters dissimilation pathway to provide energy. And 85.1 %-88.0 % methanol enters assimilation pathway for biomass generation [5]. Intracellular accumulation of hydrogen peroxide causes apoptosis which is detrimental to heterologous protein production [6,7]. Intracellular formaldehyde limits K. phaffii not only recombinant protein expression but also cell metabolism [8]. To avoiding the detrimental effects of toxic hydrogen peroxide, formaldehyde, to cell, K. phaffii cell propagates an organelle, named as peroxisome, to embrace the metabolic routes of methanol [9,10].

K. phaffii peroxisomes biogenesis fell into two ways, de novo synthesis, division and proliferation. The protein associated with peroxisome biogenesis and proliferation was called peroxin which was coded by PEX gene [11]. Currently, it was discovered that peroxins were involved in all steps of biogenesis, matrix protein import, growth, and regulation of peroxisome abundance and size [12]. In case of peroxisomes de novo synthesis, peroxisomal vesicle precursors (pre-peroxisomal vesicles, ppVs) were formed on the ER firstly, then peroxisomal matrix proteins imported into ER through a joint action of peroxin protein Pex3 and Pex19 [13]. The peroxisome transported matrix proteins into the peroxisome via the receptor to grow and divide, which was influenced by several PEX genes in the terms of peroxisomes size and number [14]. The knockout of PEX genes in K. phaffii significantly impacts peroxisome biogenesis and function. For instance, deletion of PEX1 or PEX6 causes defective matrix protein import and impaired methanol utilization due to incomplete peroxisome maturation [15,16]. Knockout of PEX3 or PEX19 prevents peroxisome membrane formation, leading to mislocalisation of peroxisomal matrix proteins and growth defects on methanol [17,18]. Additionally, PEX5 or PEX7 deletions block peroxisomal targeting of PTS1/PTS2-dependent proteins, disrupting methanol metabolism and increasing reactive oxygen species (ROS) accumulation [19,20]. These genetic modifications often reduce cell fitness under peroxisome-dependent conditions while showing minimal effects on glucose-based growth [8,21].

Under methanol incubation, peroxisomes occupy as high as 40 % volume of whole K. phaffii cell extremely because a large amount of alcohol oxidase is needed as the results of low affinity of alcohol oxidase to oxygen for methanol oxidase [22,23]. Considering the high total occupy ratio of peroxisome in K. phaffii cell, it was assumed that compressed endoplasmic reticulum (ER) was too narrow for nascent polypeptide chains to fold correctly because the crosstalk among organelles network [24]. K. phaffii cells of the stress state led unfolded protein response (UPR) which were reported by several researchers [25,26]. For example, UPR triggered up-regulated expression of chaperones, such as immunoglobulin-binding protein (BiP) [27,28]. Therefore, chaperone co-expression became an effective approach to assist the folding step of nascent polypeptide chains [29]. Therefore, it was estimated to alleviate the cellular stress through adjustment of peroxisome abundance and size, benefiting heterologous protein expression.

In this study, we knocked out 23 PEX genes in an engineered К. phaffii strain expressing xylanase-an industrially relevant enzyme for lignin degradation. Xylanase was the second largest enzyme which was required by biomass degradation. Effects of PEX knockout on cell growth, viability, peroxisome morphology, and xylanase production were evaluated. Transcriptomic analysis of selected PEX knockout strains was performed to elucidate global gene expression changes.

2. Materials and method

2.1. Microbial strains, vectors and chemicals

K. phaffii GS115 and vectors pPIC3.5K, pPIC9K, pPICHA were provided from Invitrogen (Now part of Thermo Fisher Scientific, Waltham, USA). The recombinant KpxynB expressing xylanase was constructed in our laboratory, known as K. phaffii-xynB previously [30]. Chemicals were purchased from Sinopharm chemical reagent Co., Ltd. (Shanghai, China). All biological reagents were purchased from Takara Biomedical Technology (Beijing) Co., Ltd. (Beijing, China). Primers (Table S1) and DNA sequencing were performed by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). Yeast extract peptone dextrose (YPD) medium consisted of 10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose, and 20 g/L agar only for plate preparation. Minimal dextrose (MD) medium consisted of 13.4 g/L yeast nitrogen base (YNB), 0.4 mg/L biotin, 20 g/L glucose. Buffered minimal glycerol complex (BMGY) medium consisted of 10 g/L yeast extract, 20 g/L peptone, 20 g/L glycerol, 100 mmol/L phosphate buffered saline (PBS, pH 6.0), 13.4 g/L YNB, 0.4 mg/L biotin. All electrophoresis gel kits, DNA recovery kit, and plasmid extraction kit were provided by Nanjing Vazyme Biotech Co., Ltd. (Nanjing, China).

2.2. PEX gene knockout

PEX gene knockout in K. phaffii using homologous recombinant was stated as following using PEX1 knockout as an example. Primers PEX1UP-F/R, PEX1-DOWN-F/R were used to amplify upstream and downstream fragments of PEX1 from the K. phaffii GS115 genome. The hygromycin B resistance cassette was amplified from plasmid pPICHA using primers hyg-F/R. These fragments were ligated using primers PEX1-UP-F and PEX1-DOWN-R to generate the PEX1 knockout cassette. The cassette was electroporated into 90 µL of KpxynB cells using a Gene Pulser (Bio-Rad, Hercules, CA, USA) at 1.1 kV, 200 Q, and 25 mF. Transformed cells were incubated at 30 °C for 2 h in 0.5 mL of 1 M sorbitol and 0.5 mL YPD medium. Transformants were selected on YPD agar containing 0.2 mg/mL hygromycin B at 30 °C for 3-5 days. Successful knockouts were confirmed by PCR using primers PEX1-F-Y/ PEX1-R-Y (for PEX1) and F-hyg-Y/R-hyg-R (for the hygromycin resistance gene), followed by sequencing. All 23 PEX genes were sequentially knocked out in KpxynB, and knockout strains were verified via PCR with primers listed in Table S1. Molecular procedures followed standard protocols from Molecular Cloning [31].

2.3. Recombinant K. phaffii cultivation

A single colony was inoculated into 5 mL YPD medium and cultured at 30 °C with shaking (200 rpm) for 24 h. A 600 µL aliquot was transferred to 15 mL BMGY medium and incubated under the same conditions for 48 h. Methanol (1 % v/v) was added every 24 h to induce protein expression. After 72 h of induction, cultures were harvested for xylanase analysis.

2.4. Cell density measurement

Cell densities were measured using a microplate reader (SuPerMax 2700 MF, Shanghai Flash Spectrum Biological Technology Co. Ltd., Shanghai, China) at 600 nm after appropriate dilutions.

2.5. Xylanase activity measurement

The xylanase activity was determined by reducing sugar method using the cell fermentation supernatant as a crude xylanase solution after dilution. For detail, 500 µL 1 % xylan solution in 50 mmol/L NazHPO4-citric acid buffer (pH 6.0) was mixed with 200 µL xylanase solution to incubated at 50 °C for 5 min. Then, 200 µL DNS solution (18.1992 g sodium potassium tartrate tetrahydrate, 0.6313 g 3',5-dinitrosalicylic acid, 0.5128 g phenol, 0.5064 g anhydrous sodium sulfite, and 2.096 g sodium hydroxide in 100 mL) was added for 100 °C for 10 min, which followed by water addition to 2 mL. The absorbance of the reaction solution was determined at OD540. One unit of xylanase activity was defined as the amount of xylanase that produced reducing sugar equivalent to 1 pmol of xylose per minute.

2.6. Transmission electron microscopy (TEM) test

One mililiter K. phafii fermentation broth were subjected to centrifuge at 4 °C and 4000 rpm for 5 min in a 2.0 mL Eppendorf tube to obtain cell pellets which was washed twice by 10 mol/L PBS (pH 7.4). Then, cell pellets were mixed with 1 mL 2.5 % glutaraldehyde slowly, and stored at 4°C overnight. After glutaraldehyde removal, К. phaffii cell pellets were mixed with 1 mL 100 mmol/L PBS (pH 7.0) for 15 min for washing three times. K. phaffii cell pellets were mixed with 1 % osmium tetroxide solution for 2 h, and then were mixed with 1 mL 10 mmol/L PBS (pH 7.4) for 15 min, and was washed for three times by 1 mL 10 mmol/L PBS (pH 7.4) after osmium tetroxide solution removal. K. phaffii cells were then subjected to dehydration process using 30 %, 50 %, 70 %, 80 %, 90 %, 95 % ethanol for 15 min each in sequence, and was mixed with absolute ethanol for 20 min twice, then mixed with mixture (1:1) of ethanol and isoamyl acetate for 30 min, isoamyl acetate mixing for 1 h respectively. Finally, K. phaffii was suffered to TEM analysis after critical point drying and coating. Eight photos of each recombinant K. phaffii strain were obtained for peroxisome area evaluation. The peroxisome occupancy in K. phaffii was calculated through total peroxisome area which was divided by total cell area.

2.7. Apoptosis assay by flow cytometry

One mililiter K. phafii fermentation broth was centrifuged at 4 °C and 4000 rpm for 5 min in a 2.0 mL Eppendorf tube, and washed twice by 10 mmol/L PBS (pH 7.4) for 50-fold dilution. Then, 100 µL diluted K. phafii solution was centrifuged at 4000 rpm for 5 min at 4 °C, which was added by 100 µL of annexin V binding buffer, 4 µL of FITC-annexin V; dye, and 5 µL of propidium iodide (PI) dye after the supernatant removal. The solution was incubated on ice for 30 min, and detected by flow cytometry (FACSCalibur, BD, Franklin Lakes, USA). The data obtained were analyzed using FlowJo.

2.8. Hydrogen peroxide content measurement

One mililiter К. phafii fermentation broth (about 10% cells) was centrifuged at 4 °C and 4000 rpm for 5 min in a 2.0 mL EP tube, and washed twice by 10 mmol/L PBS (pH 7.4). The cell pellets were mixed with 1 mL acetone for sonication using 40 cycles of 3 s work and 10 s interval at the condition of 20 W, and followed by centrifugation of 8000xg, 4 °C for 10 min to obtain supernatant for hydrogen peroxide content measurement. The hydrogen peroxide content in supernatant (250 µL) was measured using the hydrogen peroxide content assay kit (D799774-0100, Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China). The principle was that hydrogen peroxide and titanium sulfate formed a yellow complex with absorption at 415 nm. The 250 µL 2 pmol/mL hydrogen peroxide solution and 250 µL acetone were used as control respectively.

2.9. Transcriptome analysis

After recombinant K. phaffii cultivation in BMGY medium at 30 °C and 200 rpm for 48 h, 1.0 % methanol was added per 24 h to induce xylanase expression. Recombinant K. phaffii was used for transcriptome analysis after 72 h induction. RNA-seq was performed by Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China) using hiseq 2500 (Illumina, California, USA). The data was analyzed through Majorbio cloud platform (www.majorbio.com). In detail, the DNA library was constructed through trusegTM RNA prep kit (Illumina, California, USA). The DNA sequences in fastq files were evaluated by software fastx_toolkit_0.0.14. and screened by software Sickle to obtain clean reads. Clean reads were transferred to transcripts by software Trinity. Then, transcripts were used to blast with K. phaffii GS115 genome (PRJNA1207999, https://dataview.ncbi.nlm.nih.gov/object/ PRINA1207999?reviewer=-1tidc7264o4adijdjg4oicgm51) by software TopHat2. The transcriptional levels of transcripts were quantitated and analyzed by software Salmon using fragments per kilobase per million reads (FPKM) as the parameter of transcriptional level. The differentially expressed genes (DEGs) were analyzed using DESeq2 with the default parameters (Padjust < 0.05 & |1log2FC| = > 1). These transcripts were compared with GO (gene ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes) databases with software BLAST2GO, KOBAS2.1 respectively. The enrichment analysis of transcripts was carried out using software Goatools and R script into GO and KEGG metabolic pathways (Padjust <0.05). Raw data was deposited in National Center for Biotechnology Information (NCBI) database.

2.10. Data statistics

All K. phaffii cultivation experiments were repeated three replicates. And Microsoft Office 2016 (Microsoft, Seattle, USA) was used to analyze data.

3. Results and discussion

3.1. PEX gene knockout from K. phaffii strain KpxynB

PEX genes were found in various biological species, such as mammalian cell, plant cell, and fungi and yeast cell [32]. According to K. phaffii GS115 genome, 23 PEX genes were summarized according to gene homology using Blast [33] in Table 1. Using homologous recombination, all 23 PEX genes were knocked out in the KpxynB strain, generating 23 knockout mutants. Fig. 1 showed the PEX genes knockout verification through PCR amplification. PCR products were sequenced for confirmation.

3.2. Effects of PEX gene knockout on peroxisome morphology and recombinant xylanase expression of K. phaffii

After methanol induction, the area values of peroxisome in K. phaffii cells were showed in Fig. 2A which were extracted from TEM photos in Fig. S1 partly. The percentages of peroxisome in K. phaffii after PEX gene knockout were varied from 8.97 % to 20.96 % which was lower than that of 21.02 % by KpxynB (Fig. 2A). The ΔPEX6, ΔPEX2, and ΔPEX3 had the three lowest percentages of peroxisomes, 8.75 %, 9.16 %, and 9.62 %, respectively, which were 41.60 %, 43.55 %, and 45.74 % of that of KpxynB. In contrast, ΔPEX29, ΔPEX20 and ΔPEX8 had the peroxisome percentages of 20.96 %, 19.68 % and 19.54 %, respectively, which were 99.69 %, 93.62 % and 92.94 % of that of KpxynB. Therefore, different PEX genes knockout brought fluctuant influences on peroxisome size, which was attributed to various functions of peroxins. Pex6 was an ATPases associated with various cellular activities (AAA-ATPase), which was involved in receptor recirculation of peroxisome. Motley et al. showed that Pex6 and Pex1, another AAA-ATPase, participated directly in the import of matrix proteins [16]. Pex2 and Pex12 formed a complex which was required for peroxisomal matrix protein import also. Platta et al. reported that Pex2 and Pex12 as E3 proteins were required for ubiquitination of the import receptor Pex5 [35]. Pex3 was a key peroxin in de novo pathways, growth, and division process of peroxisome biogenesis [40]. Therefore, deletion of these key peroxins resulted in a significant reduction in peroxisome size. The insignificant influence of PEX genes knockout on peroxisome size indicated that these PEX genes encoded peroxins of other functions in peroxisome. Also, peroxins interacted with each other to functionalize in peroxisome. For example, Farré et al. found that Pex36 was essential for peroxisome proliferation at the condition of PEX25 knockout [40]. PEX knockout also increased expression of other proteins to maintain peroxisome function. Liu.et al. demonstrated that the presence of redundant ubiquitin-conjugating enzyme E2s (UBCs) proteins functioned in the absence of Pex4, and that the associated ubiquitination was delayed or slowed due to the low efficiency of UBCs proteins [53]. Therefore, it was reasonable that knockout of some PEX genes, such as PEX29, in this study did not reduce peroxisome size significantly.

Fig. 2B and C showed cell densities, xylanase activities of ΔPEX strains respectively. The cell densities fluctuated from 6.9 OD3600 to 9.0 OD600, which was 78.4 %-102.3 % of cell density of KpxynB (8.8 OD600). It was observed that most ΔPEX strains showed lower cell density than that of KpxynB, except ΔPEX25. This phenomenon was also found in the recombinant xylanase expression by K. phaffii strains. The ΔΔPEX3 showed the highest xylanase activity of 895.3 U/mL. And ΔPEX14 showed the lowest xylanase activity of 432.3 U/mL. To clarify the influence on PEX gene knockout on capability of xylanase expression by K. phaffii, we deduced specific xylanase activities of K. phaffii strains in Fig. 2D. ΔPEX3, ΔPEX11C, ΔPEX19 had top three specific xylanase activity of 104.9 U/(mL-OD600), 101.9 U/(mL-OD600) and 98.7 U/ (mL-OD600) which was 28.7 %, 25 % and 21.1 % higher than that of KpxynB (81.5 U/(mL-OD600)) respectively. Also, several ΔPEX strains had lower specific xylanase activities than that of KpxynB. For example, ΔPEX14 had the lowest specific xylanase activity of 60.1 U/(mL-OD600) which was 73.7 % of that of KpxynB.

3.3. Hydrogen peroxide contents and apoptosis ratios of ΔPEX strains

Hydrogen peroxide generation during methanol metabolism located in peroxisome, was an important factor influencing K. phaffii physiological status, such as apoptosis. Therefore, hydrogen peroxide contents and apoptosis ratios of ΔPEX strains were analyzed with results in Fig. 3. Nineteen of 23 ΔPEX strains showed higher hydrogen peroxide contents than that of KpxynB. Hydrogen peroxide contents of ΔPEX strains were in a range of 0.19 pmol/L - 0.42 pmol/L (Fig. ЗА) which was 82.6 %-182.6 % of that of KpxynB (0.23 pmol/L). ΔPEX3 and ΔPEXS provided the lowest and the highest hydrogen peroxides content respectively. Apoptosis of ΔPEX strains were analyzed by flow cytometry (Fig. S2) with results in Fig. 3B. Apoptosis ratios of ΔPEX strains were detected in range of 0.32 %-1.86 % which was 22.4 %-130.1 % of KpxynB. The ΔPEX25, ΔPEX3, ΔPEX30 had the three lowest apoptosis ratios in sequence as 0.32 %, 0.42 %, and 0.66 % respectively. Therefore, the hydrogen peroxide contents and apoptosis ratios were not consistent to peroxisome size. In yeast, peroxisomes played important roles in cell metabolism, especially in different catabolic processes including fatty acid p-oxidation, the glyoxylic shunt and methanol metabolism, as well as some biosynthetic processes [11]. Therefore, the diverse effects of PEX genes knockout on recombinant xylanase expression was estimated. Therefore, maintenance of peroxisome homeostasis represented an effective method for reduction of oxidative stress and heterologous gene expression, which was consistent to Bonekamp et al. [20]. In this study, only ΔPEX3 increased xylanase expression, and decreased hydrogen peroxide content and apoptosis ratio. ΔPEX3 was used for transcriptome analysis disclosing different genes expression, especially of recombinant xylanase expression related.

3.4. DEGs of ΔPEX3 strain based on transcriptome analysis

As ΔPEX3 brought relative higher specific xylanase activity, transcriptome analysis was carried out using ΔPEX3 and KpxynB with aim of DEGs finding. Raw data of RNA-seq was deposited in the NCBI database with SRA Study accession number SRR31929893, SRR31929894). Both of the Q30 reads (percentage of DNA fragments of over 30 bp) of ΔPEX3 and KpxynB were 94.2 %. Finally, 5039 genes were assembled, which were used to analyze the correlation by Pearson's correlation coefficient (r = 0.84) (Table S2). Fig. 4 showed the DEGs distribution of ΔPEX3 using criterion of p-value< 0.05 & fold change >2. Comparison with KpxynB, ΔPEX3 showed 1371 DEGs including 230 up-regulated DEGs and 1141 down-regulated DEGs. Considering the total genes of 5313 in K. phaffii [54], PEX3 gene knockout influenced 25.8 % gene expression in K. phaffii which indicated PEX3 gene knockout influenced gene expression in K. phaffii drastically.

3.5. Gene annotation and gene set enrichment analysis based on transcriptome

DEGs were divided into groups of gene functions in gene ontology (GO) database, and metabolic pathways according to KEGG with results in Fig. 5. Using GO, there were 187 up-regulated DEGs and 833 downregulated DEGs were annotated in Fig. 5A and B respectively. Among 187 up-regulated DEGs, biological process, cellular component, and molecular function occupied 151 up-regulated DEGs, 148 up-regulated DEGs, 148 up-regulated DEGs respectively. Cell part, cell process, and metabolic process were the top three catalogs which consisted in 123 upregulated DEGs, 116 up-regulated DEGs, 118 up-regulated DEGs. For 833 down-regulated DEGs, there were 622 down-regulated DEGs, 625 down-regulated DEGs, 530 down-regulated DEGs involved in biological process, cellular component, and molecular function respectively. And the cell part, cell process, and metabolic process occupied top three down-regulated DEGs as 526, 464, 394 respectively.

DEGs were also was annotated into metabolic pathways according to KEGG database with results in Fig. 5C and D. There were 121 upregulated DEGs and 337 down-regulated DEGs which were distributed into five groups of metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems according to the KEGG database. Impressively, translation occupied 55 up-regulated DEGs which was significantly higher than that of other metabolic pathways (Fig. 5C). The most down-regulated DEGs also fell into transport and catabolism (61 down-regulated DEGs), folding, sorting and degradation (53 down-regulated DEGs), and translation (49 down-regulated DEGs) (Fig. 5D). Therefore, it was necessary to enrich these DEGs to specific functions and metabolic pathways.

GO enrichment and KEGG enrichment were performed with the results in Fig. 6. For GO enrichment, there were 164 up-regulated DEGs and 553 down-regulated DEGs at the condition of Padjust <0.05. Within 164 enriched up-regulated DEGs, the highest value of rich factor was observed as 0.64 at three GO term of cytosolic small ribosomal subunit, cell wall, and external encapsulating structure which contained 9 enriched up-regulated DEGs each (Fig. 6A). Then, the subsequent high rich factors as 0.50 and 0.41 were obtained at two GO terms of small ribosomal subunit occupying 12 enriched up-regulated DEGs and ribosomal subunit occupying 22 enriched up-regulated DEGs. Within 553 enriched down-regulated DEGs, the highest value of rich factor was observed as 0.83 at three GO term of exocyst containing 5 enriched down-regulated DEGs (Fig. 6B). Then, the subsequent high rich factors as 0.71 and 0.55 were obtained at two GO terms of ribosomal small subunit biogenesis occupying 5 enriched down-regulated DEGs and endonucleolytic cleavage involved in rRNA processing occupying 12 enriched down-regulated DEGs. Therefore, PEX3 gene knockout influenced gene expression of ribosomal subunit-related and protein transportation according to the GO enrichment.

For KEGG enrichment, there were 121 up-regulated DEGs and 337 down-regulated DEGs were obtain at the condition of Padjust <0.05. Within 121 enriched up-regulated DEGs, the highest value of rich factor was observed as 0.43 at ribosome pathway which contained the highest value of enriched up-regulated DEGs each (50 enriched up-regulated DEGs, Fig. 6C). Within 337 down-regulated DEGs, diterpenoid biosynthesis pathway showed the highest rich factor of 1 because it only had one gene (PAS_chr4_0006, high affinity nicotinic acid plasma membrane permease) which benefited protein secretion [55]. The second highest rich factor was obtained at the pathway of non-homologous end-joining containing 4 down-regulated DEGs (Fig. 6D). Therefore, PEX3 gene knockout influenced genes expression of ribosomal subunit-related and protein transportation according to the KEGG enrichment also.

3.6. Gene analysis of PEX genes and UPR-related genes

ΔPEX3 showed 18 down-regulated PEXs and 4 up-regulated PEXs. Eighteen down-regulated PEXs distributed all three steps of peroxisome propagation, biogenesis and matrix protein import, and peroxisome fission (Fig. 7). Pex3 was an essential component of peroxisome biogenesis, helping Pex19 location to peroxisome [17] as Pex3 formed nascent preperoxisomal vesicles with Pex19, Pex36, and Pex30 together. Although 4 up-regulated PEXs were also involved in all 3 steps of peroxisome biogenesis, it was reasonable to conclude that significant decreasing of Pex events led to lower percentage of peroxisome in K. phaffii cell [56]. We also analyzed the xylanase secretion pathway including UPR event in Fig. 8. Only three secretion associated proteins, Sec63, PDI1 [26], and Hacl, increased their expression levels by 30.2 %, 104.7 %, and 68.3 % respectively. Overexpression of HAC1 improved recombinant protein production, such as recombinant a-amylase expression enhancement in K. phaffii by Huang et al. [57]. All other UPR-related proteins decreased their expression levels at different extents. Therefore, more proportion of xylanase nascent polypeptides folded via Sec63 complex and PDI1 correctly. This result was consistent to that overexpression of PDI1 was one of the approaches to alleviate UPR [58]. Less proportion of xylanase nascent polypeptides folded improperly as Kar2 and Ypt6 decreased their expression levels by 38.2 %, and 72.3 % respectively. Less Kar2 expression level indicated alleviated UPR in K. phaffii cell because Kar2 was an ER-resident chaperone that recognized misfolded /unfolded proteins and assisted proper protein folding [25]. ΔPEX3 also showed 51.8 % lower Aox1 expression than that of KpxynB, indicating lower expression of xylanase nascent polypeptides. Therefore, it was reasonable to notify that most of chaperonins decreased their expression levels. Singh et al. also summarized the relationship between recombinant protein expression levels and phenotypes of K. phaffii with the result that seven out of ten studies reported higher recombinant protein expression levels by K. phaffii Muts strains [59].

4. Conclusion

In this study, we investigated the impact of PEX gene knockout on peroxisome proliferation and recombinant xylanase expression in K. phaffii using a strain expressing xylanase under the PAOX1 promoter. Among the 23 PEX knockouts, the ΔPEX3 strain exhibited a 54.3 % reduction in peroxisome occupancy, 1.25-fold higher xylanase activity, and 1.29-fold increase in specific activity compared to the parent strain (KpxynB). Transcriptomic analysis of ΔPEX3 revealed 187 upregulated and 833 downregulated differentially expressed genes (DEGs), with enrichment in ribosomal subunit biogenesis and protein trafficking pathways (GO/KEGG). Notably, ΔPEX3 downregulated 18 PEX genes spanning peroxisome biogenesis, matrix protein import, and fission. Upregulation of Sec63 and PDI1 expression increased the transporter efficiency of protein synthesis and increased folding correctness, thereby increasing xylanase production. These findings demonstrate that PEX3 deletion optimizes recombinant xylanase production in K. phaffii by modulating peroxisome homeostasis and protein transport pathways.

CRediT authorship contribution statement

Ziwei Zhou: Writing - review & editing, Writing - original draft, Investigation, Formal analysis, Data curation. Wenjie Cong: Writing - review & editing, Supervision, Project administration. Mingxuan Wang: Writing - review & editing, Supervision, Project administration. Hualan Zhou: Writing - review & editing, Supervision, Methodology, Formal analysis. Jianguo Zhang: Writing - review & editing, Writing - original draft, Supervision, Project administration, Funding acquisition, Formal analysis, Conceptualization.

Data availability statement

The datasets generated during the current study are available from the corresponding author up on request.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

This work was supported by the Science and Technology Commission of Shanghai Municipality (No. 22dz1205800), National Natural Science Foundation of China (No. 31870045), and Open Funding Project of the State Key Laboratory of Bioreactor Engineering.

Sidebar

Peer review under the responsibility of Editorial Board of Synthetic and Systems Biotechnology.

Received 9 January 2025; Received in revised form 28 February 2025; Accepted 27 March 2025

Available online 28 March 2025

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Footnote

* Corresponding author. 516 Jungong Road, Shanghai, 200093, China.

E-mail address: [email protected] (J. Zhang).