Characterization of the off-flavor from Pichia pastoris GS115 during the overexpression of an α-l-rhamnosidase

Abstract   The off-flavor of Pichia pastoris strains is a negative characteristic of proteins overexpressed with this yeast. In the present study, P. pastoris GS115 overexpressing an α-l-rhamnosidase was taken as the example to characterize the off-flavor via sensory evaluation, gas chromatography–mass spectrometer, gas chromatography–olfaction, and omission test. The result showed that the off-flavor was due to the strong sweaty note, and moderate metallic and plastic notes. Four volatile compounds, that is, tetramethylpyrazine, 2,4-di-tert-butylphenol, isovaleric acid, and 2-methylbutyric acid, were identified to be major contributors to the sweaty note. Dodecanol and 2-acetylbutyrolactone were identified to be contributors to the metallic and plastic notes, respectively. It is the first study on the off-flavor of P. pastoris strains, helping understand metabolites with off-flavor of this yeast. Interestingly, it is the first study illustrating 2-acetylbutyrolactone and dodecanol with plastic and metallic notes, providing new information about the aromatic contributors of biological products. Importance The methylotrophic yeast Pichia pastoris is an important host for the industrial expression of functional proteins. In our previous studies, P. pastoris strains have been sniffed with a strong off-flavor during the overexpression of various functional proteins, limiting the application of these proteins. Although many yeast strains have been reported with off-flavor, no attention has been paid to characterize the off-flavor in P. pastoris so far. Considering that P. pastoris has advantages over other established expression systems of functional proteins, it is of interest to identify the compounds with off-flavor synthesized in the overexpression of functional proteins with P. pastoris strains. In this study, the off-flavor synthesized from P. pastoris GS115 was characterized during the overexpression of an α-l-rhamnosidase, which helps understand the aromatic metabolites with off-flavor of P. pastoris strains. In addition, 2-acetylbutyrolactone and dodecanol were newly revealed with plastic and metallic notes, enriching the aromatic contributors of biological products. Thus, this study is important for understanding the metabolites with off-flavor of P. pastoris strains and other organisms, providing important knowledge to improve the flavor of products yielding with P. pastoris strains and other organisms. One-Sentence Summary Characterize the sensory and chemical profile of the off-flavor produced by one strain of P. pastoris in vitro.


Introduction
The methylotrophic yeast Pichia pastoris is an important strain used as an industrial strain.Pichia pastoris strains can achieve a very high cell density and have a strong, tightly controlled, methanol-inducible promoter.In most cases, the host P. pastoris offers advantages over other established expression systems of functional proteins (Ahmad et al., 2014 ).Hitherto, there are more than 4.4 million international patents involving the use of P. pastoris as a host system to overexpress up to 5000 kinds of proteins (Fischer & Glieder, 2019 ), including αl -rhamnosidase (Li et al., 2019 ), endoxylanase (Wang et al., 2016 ), and tannase (Lebesi & Tzia, 2012 ).
Recently, P. pastoris strains have been observed to synthesize a strong off-flavor during the overexpression of various functional proteins such as αl -rhamnosidase (Li et al., 2020a ), βdglucosidase (Ni et al., 2021 ), and β-xylosidase (Zhang et al., 2020a ), limiting the application of these proteins in food, nutraceutical, and pharmaceutical industries .Although many yeast strains have been reported with off-flavor (Wang et al., 2020 ), little attention has been paid to the off-flavor in P. pastoris so far.Therefore, it is of interest to identify the compounds with off-flavor synthesized in the overexpression of functional proteins with P. pastoris strains.
Currently, various techniques and procedures are available for off-flavor analysis.The analysis using solid-phase microextraction combined with gas chromatography-mass spectrometry-olfactometry (GC-MS-O) shows that short-chain fatty acids, organic acids, higher alcohols, and esters are the potential compounds for the off-odor of Brettanomyces bruxellensis in red wines (Fugelsang & Zoecklein, 2003 ).GC-MS analysis indicates that isobutyric acid, isovaleric acid, and β-phenylethanol contribute to the unpleasant odor of the soy sauce produced by Zygosaccharomyces rouxii (Tomita & Yamamoto, 1997 ).Solventassisted flavor evaporation coupled with GC-MS-O analysis indicates that 4-methylphenol, 3-methylpyridine, 3-methylbutanoic acid, and propionic acid are the main contributors to the offflavors of Angel yeast (Zhang et al., 2017 ).In addition, the aroma extract dilution analysis (AEDA) is the prevailing method for identifying key odor compounds via GC-MS-O analysis (Grosch, 1993 ).These studies provide multimethod references for identifying the off-flavor compounds of P. pastoris strains.
αl -Rhamnosidases that specifically hydrolyze the terminal αl -rhamnosyl-linkages have extensive applications in food and pharmaceutical industries, including improving the aroma of wine (Spagna et al., 2000 ) and juice (Busto et al., 2007 ).In our previous studies, αl -rhamnosidases from various resources were overexpressed with P. pastoris strains such as GS115 and SMD1168 (Li et al., 2019(Li et al., , 2020 b; b;Liao et al., 2019 ).During these studies, P. pastoris strains were observed to synthesize a strong off-flavor.In the context that the compounds with the off-flavor are unclear for P. pastoris during the overexpression of proteins, the present study aims to characterize the off-flavor synthesized in P. pastoris GS115 during the overexpression of the αl -rhamnosidase from Aspergillus tubingensis , which may help understand metabolites with off-flavor in P. pastoris strains and other biological processes.

Strain and Fermentation
Pichia pastoris ( Komagataella phaffii ) GS115 (his4) was purchased from Invitrogen Co., Ltd (Guangzhou, China).The αl -rhamnosidase gene was cloned from A. tubingensis .The methanol-inducible fermentation was conducted following the protocol in a previous study (Li et al., 2018 ).In short, the recombinant P. pastoris was first activated in 30 mL Yeast Extract Peptone Dextrose Medium medium (containing 10 g/L yeast extract powder, 20 g/L peptone, and 20 g/L glucose) for 16 hr, and then was shifted to 100 mL Buffered Glycerol-complex Medium (BMGY) medium (YNB 13.4 g/L, yeast extract powder 14.0 g/L, peptone 28.0 g/L, 0.1 M potassium phosphate, biotin 0.4% w/w, glycerol 1% w/w, pH = 6.0) for 18 hr.Then, the cells were harvested by centrifugation and shifted to Buffered Methanol-complex Medium (BMMY) medium (similar to BMGY except for the substitution of 0.5% MeOH for glycerol) for cell growth and enzyme expression for 7 days.For induction of the protein expression, methanol was injected at a ratio of 0.5% (v/v) every 24 hr.Based on our primary experiments with sensory evaluation and GC-MS analysis, the blank BMMY medium, the medium added with methanol (0.5%) before fermentation (MAM), was almost odorless and contained 22 volatile compounds, including esters, acids, alcohols, and aldehydes.The fermented broth with BMMY medium before methanol induction (BMI) was similarly odorless and contained 23 volatile compounds that are mainly esters, acids, aldehydes, pyrazines, and ketones.The concentration of volatile compounds in both MAM and BMI was very low.The detailed volatile constituents in MAM, BMI, and fermented broth after methanol induction (AMI) are shown in Table 1 .

Sensory Analysis
The samples were sensorially evaluated in random order.According to previous research works (Lin et al., 2014 ;Zhang et al., 2017 ;Zheng et al., 2020 ), eight aroma descriptors, that is, sweaty, roasted, metallic, deep fried/fatty, green, honey-like, floral, and plastic, were sensorially evaluated.A 12-member sensory evaluation panel (3 males and 9 females, 20-30 years old) was trained once per day for 4 weeks before the evaluation.The panelists were asked to determine the intensity of these descriptors by rating scores between 0 and 5, where 0 is unsniffed and 5 is very strong intensity.

GC-MS Analysis
The 70 mL suspensions were fetched from the fermented broth AMI with centrifuge, and were extracted for 12 h with 105 mL dichloromethane.The organic phase (lower phase) was separated into a separatory funnel, followed by concentration with a termovap sample concentrator.Anhydrous sodium sulfate was added into the concentrated extract to remove water.The final Table 1.Volatile Compounds of the Samples Methanol-Added Medium (MAM), Before Methanol Induction (BMI), and After Methanol Induction (AMI).extract was condensed to 1 mL.For the analysis using GC-MS, 200 μL of the condensed extract was mixed with 300 μL dichloromethane and 1 μL 1000 ppm 2,4,6-trimethylpyridine (dissolved in ethanol) as internal standards.A QP2020 GC-MS (Shimadzu, Kyoto, Japan) was utilized for both qualitative and quantitative analyses.Full-scan mode was utilized for qualitative analysis, while SIM mode was utilized for quantitative analysis.One microliter of sample was injected in the splitless mode at 250°C.Chromatographic separation was achieved using a 30 m × 0.25 mm × 0.25 μm Rtx-5MS column (Bellefonte, PA, USA) at a flow rate of 3.13 mL/min with the carrier gas helium (99.999% purity).The GC oven temperature was maintained at 35°C for 3 min, ramped to 200°C at a rate of 8°C/min, maintained for 8 min, ramped again to 270°C at a rate of 5°C per minute, and then maintained for 10 min.The MS analysis was operated in electron impact mode at 70 eV with the scanning range from 35 to 500 m/z.The solvent delay was 3 min.The transfer line and ion source temperatures were 250 and 230°C, respectively.

GC-MS-O Analysis
A QP 2020 GC-MS (Shimadzu, Kyoto, Japan) equipped with an OP 275 Olfactory Detector Port (GL Sciences Inc., Kyoto, Japan) was used.The sample was separated on a 30 m × 0.25 mm × 0.25 μm Rtx-5 MS fused silica capillary column (Bellefonte, PA, USA).The operating conditions for GC-MS-O were the same as for the GC-MS.The volatile extract was split between the olfactory detection port and MS with 16:9 proportions.The transfer line to the GC-O sniffing port was held at 200°C; humidified air was added to the sniffing port at 50 mL/min to maintain olfactory sensitivity by reducing dehydration of mucous membranes in the nasal cavity (Ni et al., 2021 ).AEDA analysis was conducted by the dilution of 1, 2, 4, 8, 16, 64, and 1024 folds.The aroma compounds were identified by comparing their MS fragments and RIs with those of authentic standards.The flavor dilution (FD) factor was defined as the maximum dilution where the aroma compound could be detected (Fan et al., 2015 ).Each diluted sample was analyzed consecutively three times by three panelists.

Aroma Recombination and Omission Tests
The recombination and omission experiments were conducted according to the method mentioned in the previous study (Neugebauer et al., 2020 ).Based on aromatic compounds in the GC-MS-O analysis, 13 key volatile compounds, that is, isovaleric acid, 2-methylbutyric acid, 2,6-dimethylpyrazine, trimethylpyrazine, tetramethylpyrazine, nonanal, phenethyl alcohol, methyl phenylacetate, 2-acetylbutyrolactone, 3phenylpropionic acid, dimethyl phthalate, dodecanol, and 2,4-di-tert -butylphenol were chosen to simulate the AMI sample with the sterilized BMMY medium as the substrate.All the compounds were tested in the concentration detected in the samples.To further analyze the contribution of each volatile, single-omission models were constructed by omitting the volatile compounds one by one from the AMI model.

Statistical Analysis
Each sample was prepared and analyzed three times.The average and standard deviation were calculated with the Office 2019 software.The radar chart was drawn with the Office 2019 software.Significant analysis (Least Significant Difference) was conducted by the IBM SPSS 26.0 software.

Descriptive Sensory Evaluation of the Off-Flavor
To preliminarily evaluate the off-flavor, the fermented broth AMI was taken for the sensory evaluation.Methanol induction is a critical approach for promoting the heterologous expression of proteins.As shown in Fig. 1 , the AMI sample was sniffed to have a strong sweaty note (score = 4.80), a moderate metallic note (score = 2.24), and a moderate plastic note (score = 2.13).A researcher has reported that yeast extract was dominated by fermented, caramel, and roasted notes (Alim et al., 2018 ).Another researcher regarded "sweaty" note as similar to "rancid" and "sour" notes in the case of describing the off-flavor of foods (Rudman et al., 2018 ).Therefore, the off-flavor might be attributed to the strong sweaty note and moderate metallic and plastic notes.

Qualitative and Quantitative Analysis of Volatile Content
For identifying the volatile compounds contributing to the offflavor, the AMI was submitted to GC-MS analysis.According to Table 1 , a total of 30 volatile compounds were detected in the AMI, including eight acids, six esters, one ketone, four alcohols, three aldehydes, three pyrazines, two sulfur compounds, one olefin, one amide, and one phenol.The quantitative analysis showed that the AMI had a noticeable content of alcohol and acid compounds, including dodecanol (2146.29 mg/L), isovaleric acid (303.39 mg/L), 2-methylbutyric acid (187.6 mg/L), palmitic acid (1112.75mg/L), and myristic acid (103.97mg/L) (Table 2 ).A previous study has shown that Saccharomyces cerevisiae can synthesize various aromatic volatiles, including higher alcohols, fatty acids, acetates, ethyl esters, ketones, and aldehydes (Chen & Xu, 2010 ).In general, the results of the present study seem to be similar to those of the previous study in that P. pastoris can synthesize aromatic higher alcohols, fatty acids, acetates, ethyl esters, ketones, and aldehydes.

Recombination and Omission Tests
According to the AEDA, 13 key odorants that were detected to have FD factor ≥2 were submitted to simulate the aroma of AMI using a recombination test with their measured concentration.As shown in Fig. 1 , the AMI and recombination models were similar in aroma profiles, and no significant difference ( p > .05)was observed in the eight sensory attributes between the AMI and recombination models (Fig. 1 ).The aroma of AMI was well reconstructed, indicating that these odorants have dominated contributions to the off-flavor.Furthermore, the result of the omission test showed that six compounds had noticeable effects on the sweaty, metallic, and plastic notes (Table 3 ).In short, the omission of tetramethylpyrazine, 2,4-di-tert -butylphenol, isovaleric acid, and 2-methylbutyric acid led to a significant decrease in the sweaty note.The omission of dodecanol and 2-acetylbutyrolactone led to notable decreases in the intensities of metallic and plastic notes, respectively.Taking all together, tetramethylpyrazine, 2,4-di-tertbutylphenol, isovaleric acid, and 2-methylbutyric acid were the key contributors to the sweaty notes; 2-acetylbutyrolactone and dodecanol were the contributors to the plastic and metal notes.
In this study, the off-flavor was determined as an outstanding sweaty note and moderate metallic and plastic notes by the sensory evaluation.Furthermore, the GC-MS analysis, GC-O analysis, and omission test showed that the key sweaty odorants of P. pastoris were identified to be tetramethylpyrazine, 2,4-di-tert -butylphenol, isovaleric acid, and 2-methylbutyric acid.
The key contributors to the plastic and metallic notes were identified to be 2-acetylbutyrolactone and dodecanol, respectively.Tetramethylpyrazine is reported in yeast to generate the fermented soybean-like notes (Raza et al., 2019 ).Isovaleric acid, which is the classical sweaty malodorant (Gross, 2007 ), brings the sweaty flavor to fish products (Fukami et al., 2006 ), mushrooms (Zhang et al., 2020 b), alcoholic drinks (Lee et al., 2000 ), and milks (Fricke & Schieberle, 2020 ).Tetramethylpyrazine has a significant contribution to the overall aroma profile of food systems (Adams et al., 2008 ;Müller & Rappert, 2010 ).By comparison, it is clear that the sweaty-note contributors of P. pastoris , that is, tetramethylpyrazine, 2,4-di-tert -butylphenol, isovaleric acid, and 2-methylbutyric acid, were similar to those identified in other biological products.Interestingly, 2-acetylbutyrolactone and dodecanol were first identified to be the contributors to the plastic and metallic notes .Thus, this research will helppeople to understand the off-flavor of P. pastoris and other biological products.
Saccharomyces cerevisiae could generate higher alcohols such as isoamylol and amyl alcohol during wine and beer brewing (Ma et al., 2017 ) and could transform in downstream pathways to synthesize organic acids with unpleasant aroma, including isovaleric acid (Thierry et al., 2002 ) and 2-methyl butyric acid (Dickinson et al., 2000 ).It has been reported that higher alcohols and acids could be synthesized via the Ehrlich pathway from the degradation of amino acids, and the Harris pathway from glycometabolism (Avalos et al., 2013 ).The 2,4-di-tert -butylphenol is a common metabolite that has been reported in yeast and other organisms (Zhao et al., 2020 ) to provide a phenol note (Pang et al., 2021 ).In addition, it has been reported that S. cerevisiae synthesizes tetramethylpyrazine via valine metabolism and spontaneous reaction (Heidlas & Tressl, 1990 ;González et al., 2010 ).Potentially, tetramethylpyrazine, 2,4-di-tert -butylphenol, isovaleric acid, 2-methylbutyric acid, 2-acetylbutyrolactone, and dodecanol likely originate from a similar pathway of other microorganisms such as S. cerevisiae , but further studies are needed to confirm the putative hypothesis.It was found that P. pastoris cells have different metabolic regulation in the case of different carbon sources such as glycerol and methanol (Ren et al., 2003 ;Van Der Klei et al., 2006 ).In the glycerol growth phase, the intracellular metabolic pathways are mainly concentrated in oxidative phosphorylation, glycolysis, tricarboxylic acid cycle cycle, and electronic respiratory chain.During the methanol induction period, the intracellular metabolic pathway is mainly concentrated in the methanol metabolic pathway (Ren et al., 2003 ).In the glycerol growth phase, P. pastoris grows in a medium containing glycerol as a carbon source that represses the expression of methanol promoter AOX1 through transcription repressor factors such as Mig1, Mig2, and Nrg1, and hexose transporters such as Hxt1 and Hxt2.At this stage, the related induction of exogenous protein was strongly inhibited due to the carbon source metabolic repression (Orman et al., 2009 ;Vogl et al., 2018 ).In the methanol induction stage, methanol activates AOX1 transcriptional expression through transcriptional activators Mxr1 and Mitl, and initiates the downstream metabolic pathway (Vanz et al., 2012 ;Vogl & Glieder, 2013 ).In comparison to the AMI, the medium added with MAM and the fermented broth BMI have a much lower content of off-flavor contributors, that is, tetramethylpyrazine, 2,4-di-tert -butylphenol, isovaleric acid, 2-methylbutyric acid, 2-acetylbutyrolactone, and dodecanol (Table 1 ), indicating that these compounds were synthesized after the methanol induction.Thus, the synthesis of volatiles with the off-flavor might be related to the methanol induction pathway in relation to the AOX1 activations, providing a fundamental basis for regulating the biosynthetic pathway to control the offflavor.However, further research is needed to investigate how the off-flavor contributors are synthesized after methanol induction.Additionally, the gene-editing technologies such as CRISPR-Cas9 can facilitate blocking the synthetic pathway of off-flavor contributors, based on the illustration of the off-flavor synthetic pathway in the P. pastoris strains.
calculated on Rtx-5 MS column.b Linear retention index calculated on Rtx-5 MS column.cLinear retention index reported from http://webbook.nist.gov/chemistry/.d Method of identification: MS, compounds were identified by matching mass spectrometry spectra in the data library (NIST11, NIST11s, and FNSSC 1.3); Std, compound confirmed by matching standard reference; and when only MS or RI is available for the identification of a compound, it must be considered as an attempt of identification.-=not available; SCIS = concentration of compound estimated by internal standard.

Fig. 1 .
Fig. 1.Aroma profiles and aroma recombination model of the sample after methanol induction (AMI).

Table 2 .
Aroma Extract Dilution Analysis of Before Methanol Induction (BMI) and After Methanol Induction (AMI) Samples a Odor descriptions perceived by panelists through the sniffing port.b Linear retention index calculated on Rtx-5 MS column.c Flavor dilution perceived through the sniffing port.

Table 3 .
Omission Tests of After Methanol Induction (AMI)