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Journal of Industrial Microbiology & Biotechnology logoLink to Journal of Industrial Microbiology & Biotechnology
. 2023 Nov 6;50(1):kuad035. doi: 10.1093/jimb/kuad035

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

YuXuan Yao 1, ShengLan Zheng 2, ShiLin Chi 3, Feng Chen 4, Ning Cai 5, ZhenZhen Cai 6, Zhipeng Li 7,8,9, Hui Ni 10,11,12,13,✉
PMCID: PMC10696632  PMID: 37942557

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.

Keywords: Pichia pastoris, Off-flavor, GC–MS analysis

Graphical Abstract

Graphical Abstract.

Graphical Abstract

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The strong off-flavor (sweaty odor) was identified as a contribution of tetramethylpyrazine, 2,4-di-tert-butylphenol, isovaleric acid, and 2-methylbutyric acid.

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 α-l-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 α-l-rhamnosidase (Li et al., 2020a), β-d-glucosidase (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). Solvent-assisted 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 off-flavors 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.

α-l-Rhamnosidases that specifically hydrolyze the terminal α-l-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, α-l-rhamnosidases from various resources were overexpressed with P. pastoris strains such as GS115 and SMD1168 (Li et al., 2019, 2020b; 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 α-l-rhamnosidase from Aspergillus tubingensis, which may help understand metabolites with off-flavor in P. pastoris strains and other biological processes.

Materials and Methods

Reagents and Chemicals

All chemical standards and internal standards were obtained commercially at high-purity grade (GC grade). Among them, dichloromethane, 2,4,6-trimethylpyridine, benzyl alcohol, decyl aldehyde, dodecyl aldehyde, 2,4-di-tert-butylphenol, and n-alkanes C7–C30 were obtained from Sigma–Aldrich Co., Ltd (Shanghai, China); nonanal, benzaldehyde, limonene, and phenethyl alcohol were obtained from Shanghai Aladdin Co., Ltd (Shanghai, China); ethanol, pantolactone, decanoic acid, dodecanoic acid, 3-methyl-1-butanol, 1-methylpropyl acetate, isobutyric acid, ethyl butyrate, m-xylene, isovaleric acid, o-xylene, 2-methyl butyric acid, 2,5-dimethylpyrazine, 2,6-dimethylpyrazine, δ-valerolactone, 3-methylthiopropanol, trimethylpyrazine, tetramethylpyrazine, methyl phenylacetate, benzothiazole, nonanoic acid, 2-acetylbutyrolactone, geranylacetone, dimethyl phthalate, undecenoic acid, dodecanol, 2-tridecanone, 2,2,4-trimethyl-1,3-pentanediol diisobutyrate, and 2-pentadecanone were obtained from Shanghai Macklin Biochemical Co., Ltd (Shanghai, China). Tryptone, yeast extract powder, and agar were purchased from Huankai Microbial Co., Ltd (Guangdong, China); yeast nitrogen base (YNB) and biotin B were purchased from Beijing Solarbio Co., Ltd (Beijing, China). Anhydrous sodium sulfate (Na2SO4), phosphate buffer (NaH2PO4, Na2HPO4), glycerol, glucose, and methanol were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).

Strain and Fermentation

Pichia pastoris (Komagataella phaffii) GS115 (his4) was purchased from Invitrogen Co., Ltd (Guangzhou, China). The α-l-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.

Table 1.

Volatile Compounds of the Samples Methanol-Added Medium (MAM), Before Methanol Induction (BMI), and After Methanol Induction (AMI).

Aroma Characteristic ion Dentification Standard Rang MAM BMI AMI
No. RTa compound RIb RIc fragment basisd curves R 2 (mg/L) (mg/L) (mg/L) (mg/L)
Alcohols
 1 4.678 3-Methyl-1-butanol 743 743 42 70 84 MS, Std, RI Y = 0.2957X−0.0081 0.9999 0.03–10 - - 6.22 ± 0.12
 2 11.801 Benzyl alcohol 1039 1038 79 108 51 MS, Std, RI Y = 0.4606X−0.2043 0.9970 0.03–30 - - 36.62 ± 0.19
 3 13.507 Phenethyl alcohol 1116 1116 91 122 65 MS, Std, RI Y = 0.8985X−0.5400 0.9976 0.03–30 4.32 ± 0.06 - 65.16 ± 0.28
 4 20.679 Dodecanol 1490 1490 41 69 93 MS, Std, RI Y = 0.8452X−0.1138 0.9992 0.03–80 - - 2146.29 ± 7.22
Esters
 1 5.146 1-Methylpropyl acetate 763 - 43 87 61 MS, Std Y = 1.3443X−0.0037 0.9999 0.03–10 0.09 ± 0.05 - 0.44 ± 0.01
 2 6.116 Ethylbutyrate 805 805 43 71 88 MS, Std, RI Y = 0.4014X−0.0044 0.9999 0.03–10 - - 3.32 ± 0.33
 3 9.964 δ-Valerolactone 961 961 42 100 73 MS, Std, RI Y = 0.2952X−0.0127 0.9997 0.03–10 - 2.37 ± 0.26 6.67 ± 0.77
 4 11.857 Pantolactone 1042 1032 71 43 85 MS, Std, RI Y = 0.3612X−0.2335 0.9995 0.03–80 6.06 ± 0.93 - -
 5 14.814 Methyl phenylacetate 1178 1178 91 150 39 MS, Std, RI Y = 1.4740X−0.0316 0.9999 0.03–10 - - 8.13 ± 0.2
 6 17.471 2-Acetylbutyrolactone 1312 1312 43 86 60 MS, Std, RI Y = 0.1051X−0.0133 0.9943 0.03–10 - 89.62 ± 2.69 95.82 ± 1.72
 7 20.041 Dimethyl phthalate 1454 1454 163 77 133 MS, Std, RI Y = 1.2893X−0.0244 0.9999 0.03–10 0.81 ± 0.56 2.91 ± 0.08 6.45 ± 0.17
 8 22.355 2,2,4-Trimethyl-1,3-pentanediol diisobutyrate 1591 1588 55 69 82 MS, Std, RI Y = 1.6715X−0.0323 0.9997 0.03–10 - 1.46 ± 0.01 -
Acids
 1 5.578 Isobutyric acid 765 765 43 73 88 MS, Std, RI Y = 0.2883X−0.0582 0.9991 0.03–30 11.49 ± 0.07 - 55.35 ± 1.06
 2 6.295 Butanoic acid 812 812 60 41 88 MS, RI SCIS - - 4.34 ± 0.16 - -
 3 8.181 Isovaleric acid 888 888 60 43 87 MS, Std, RI Y = 0.8888X−0.0256 0.9999 0.03–80 6.72 ± 0.03 - 303.39 ± 1.72
 4 8.470 2-Methylbutyric acid 899 898 74 57 87 MS, Std, RI Y = 0.9277X−0.0931 0.9991 0.03–80 2.98 ± 0.12 - 187.6 ± 1.4
 5 12.550 Levulinic acid 1073 1063 43 85 73 MS, RI SCIS - - 10.29 ± 0.16 - -
 6 16.521 Phenylacetic acid 1263 1263 91 136 65 MS, RI SCIS - - 2.94 ± 0.71 4.19 ± 2.46 50.2 ± 1.7
 7 16.735 Nonanoic acid 1274 1274 60 57 115 MS, Std, RI Y = 0.4465X−0.3004 0.9951 0.03–30 4.81 ± 0.13 - 36.23 ± 0.75
 8 18.070 3-Phenylpropionic acid 1344 1344 91 150 78 MS, Std, RI Y = 0.2351X−0.0247 0.9991 0.03–80 0.79 ± 0.07 - 1.24 ± 0.9
 9 18.437 3-Decanoic acid 1364 1364 60 41 91 MS, Std, RI Y = 0.2003X−0.7213 0.9999 0.03–80 21.23 ± 0.25 - -
 10 20.548 Undecenoic acid 1483 1484 56 69 98 MS, Std, RI Y = 0.6127X−0.1023 0.9995 0.03–80 - 1029.71 ± 11.6 -
 11 21.825 Dodecanoic acid 1559 1559 73 43 85 MS, Std,RI Y = 0.1708X−0.9701 0.9999 0.03–80 29.02 ± 0.73 - -
 12 21.871 Cis-5-Dodecenoic acid 1562 1562 55 69 82 MS, RI SCIS - - - 6.76 ± 0.28 -
 13 25.242 Myristic acid 1765 1765 70 97 41 MS, RI SCIS - - - - 103.97 ± 0.49
 14 30.525 Palmitic acid 1980 1980 73 43 129 MS, RI SCIS - - - - 1112.75 ± 8.85
Aldehydes
 1 8.659 3-(Methylthio)propionaldehyde 907 907 48 104 76 MS, RI SCIS - - 3.01 ± 1.44 - -
 2 10.055 Benzaldehyde 965 965 77 106 51 MS, Std, RI Y = 0.4718X−0.0124 0.9999 0.03–10 2.33 ± 0.41 7.35 ± 0.29 10.08 ± 0.26
 3 13.286 Nonanal 1106 1106 49 84 70 MS, Std, RI Y = 0.3961X−0.0073 0.9999 0.03–10 1.93 ± 0.07 4.73 ± 0.16 7.06 ± 0.38
 4 15.425 Decyl aldehyde 1207 1207 57 82 68 MS, Std, RI Y = 0.1529X−0.0040 0.9998 0.03–10 2.77 ± 0.21 4.98 ± 0.63 15.73 ± 2.22
 5 18.970 Dodecyl aldehyde 1393 1392 45 82 70 MS, Std, RI Y = 0.1876X−0.0058 0.9999 0.03–10 - 9.42 ± 0.54 -
Pyrazines
 1 8.813 2,5-Dimethylpyrazine 913 913 108 42 81 MS, Std, RI Y = 0.6836X−0.0159 0.9999 0.03–10 - 32.92 ± 0.1 -
 2 8.868 2,6-Dimethylpyrazine 916 916 108 42 81 MS, Std, RI Y = 0.5896X−0.0157 0.9999 0.03–10 - - 43.6 ± 1.41
 3 10.993 Trimethylpyrazine 1004 1004 42 122 81 MS, Std, RI Y = 0.8209X−0.0136 0.9990 0.03–10 - 18.67 ± 0.4 32.28 ± 0.5
 4 12.864 Tetramethylpyrazine 1086 1086 136 54 95 MS, Std, RI Y = 0.3783X−0.0015 0.9999 0.03–10 - 4.98 ± 0.53 33.44 ± 0.65
Ketones
 1 19.975 Geranylacetone 1450 1450 43 69 107 MS, Std, RI Y = 0.4218X−0.0128 0.9998 0.03–10 - 2.52 ± 0.36 -
 2 20.775 2-Tridecanone 1496 1496 58 71 85 MS, Std, RI Y = 0.8079X−0.0204 0.9997 0.03–10 - 0.9 ± 0.01 -
 3 24.110 2-Pentadecanone 1701 1702 58 71 85 MS, Std, RI Y = 0.5843X−0.0150 0.9998 0.03–10 - 7.68 ± 0.11 39.07 ± 0.94
Sulfur compounds
 1 10.496 3-Methylthiopropanol 983 983 106 281 61 MS, Std, RI Y = 0.2957X−0.0081 0.9977 0.03–30 - - 38.1 ± 1.37
 2 15.976 Benzothiazole 1235 1236 135 108 69 MS, Std, RI Y = 0.7465X−0.0230 0.9998 0.03–10 - 1.18 ± 0.05 3.16 ± 0.06
Amides
 1 9.063 Isobutyramide 924 923 44 72 87 MS, RI SCIS - - - - 5.23 ± 0.45
 2 19.208 2-Phenylacetamide 1406 1411 92 135 65 MS, RI SCIS - - - 2.38 ± 0.22 -
Benzodiazepines
 1 7.772 m-Xylene 871 870 91 106 77 MS, Std, RI Y = 0.9455X−0.0025 0.9999 0.03–10 - 1.51 ± 0.09 -
 2 8.315 o-Xylene 893 893 91 106 51 MS, Std, RI Y = 1.0967X−0.0034 0.9998 0.03–10 0.36 ± 0.13 0.83 ± 0.12 -
Phenol
 1 13.409 Maltol 1111 1111 126 43 71 MS, RI SCIS - - 1.82 ± 0.99 - -
 2 21.082 2,4-Di-tert-butylphenol 1514 1513 191 57 206 MS, Std, RI Y = 1.4646X−0.0717 0.9998 0.03–30 6.93 ± 0.06 16.96 ± 0.34 82.26 ± 2.68
Olefin
 1 11.643 Limonene 1032 1032 68 93 41 MS, Std, RI Y = 0.2852X−0.0001 0.9999 0.03–10 1.21 ± 0.04 12.35 ± 0.12 5.54 ± 0.31
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aLinear retention time calculated on Rtx-5 MS column.

bLinear retention index calculated on Rtx-5 MS column.

cLinear retention index reported from http://webbook.nist.gov/chemistry/.

dMethod 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.

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 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.

The reference databases (NIST11, NIST11s, and FFNSC1.3) were used to search and screen similar substances with mass spectrometry matching greater than 80%. Moreover, retention index (RI) was calculated from each compound, based on analyses of n-alkane (C7–C30) following Vandendool and Kratz RI for using a temperature ramp (Vandendool & Kratz, 1963). Thereafter, most of the volatile compounds were identified by comparing their detected mass spectra and RI with those of standard compounds. The 3-methyl-1-butanol, 1-methylpropyl acetate, isobutyric acid, ethyl butyrate, m-xylene, isovaleric acid, o-xylene, 2-methylbutyric acid, 2,5-dimethylpyrazine, 2,6-dimethylpyrazine, δ-valerolactarone, benzaldehyde, 3-methylthiopropanol, trimethylpyrazine, limonene, benzyl alcohol, tetramethylpyrazine, nonanal, phenethyl alcohol, methyl phenylacetate, decyl aldehyde, benzothiazole, nonanoic acid, 2-acetylbutyrolactone, dodecyl aldehyde, geranylacetone, dimethyl phthalate, undecenoic acid, dodecanol, 2-tridecanone, 2,4-di-tert-butylphenol, 2,2,4-trimethyl-1,3-pentanediol diisobutyrate, pantolactone, decanoic acid, dodecanoic acid, 3-phenylpropionic acid, and 2-pentadecanone were quantitatively analyzed in the SIM model by an external standard method according to their respective calibration curves. The content of volatiles without matched standards, including isobutyramide, phenylacetic acid, 2-phenylacetamide, cis-5-dodecenoic acid, myristic acid, butanoic acid, 3-(methylthio) propionaldehyde, levulinic acid, maltol, and palmitic acid, was tentatively analyzed using the internal standard method with 2,4,6-trimethylpyridine as the internal standard.

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, 3-phenylpropionic 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.

Results

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.

Fig. 1.

Fig. 1.

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

Qualitative and Quantitative Analysis of Volatile Content

For identifying the volatile compounds contributing to the off-flavor, 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 (1112.75 mg/L), and myristic acid (103.97 mg/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.

Table 2.

Aroma Extract Dilution Analysis of Before Methanol Induction (BMI) and After Methanol Induction (AMI) Samples

No. Odorants Odor characteristica RIb FD factorc
Sweaty
 1 3-Methyl-1-butanol Sour, herb 743 1
 2 Isobutyric acid Sour, leather-like, gasoline 781 1
 3 Ethyl butyrate Sour, bitter 804 1
 4 Isovaleric acid Sweaty, rancid, sour 887 128
 5 2-Methylbutyric acid Sweaty, rancid, sour 898 128
 6 Benzaldehyde Sour, paint 964 1
 7 3-Methylthiopropanol Sour, paint 981 1
 8 Benzyl alcohol Sour, bitter 1038 1
 9 Tetramethylpyrazine Rancid, moldy, fermented 1085 256
 10 Nonanal Sweaty, sour 1104 4
 11 Methyl phenylacetate Sour, leather-like 1176 2
 12 3-Phenylpropionic acid Sour 1344 512
 13 3-Decenoic acid Sour 1363 1
 14 2,4-Di-tert-butylphenol Sour, phenol 1510 256
Metallic
 1 Dimethyl phthalate Metallic, rust, bitter 1451 2
 2 Dodecanol Metallic, rust, bitter 1489 256
Plastic
 1 2-Acetylbutyrolactone Plastic, rubbery 1312 2
Roasted
 1 Trimethylpyrazine Roasted, moldy 1002 4
Floral
 1 Phenethyl alcohol Rosy, fermented 1116 16
Deep-fried, fatty
 1 2,6-Dimethylpyrazine Fatty, salty 915 128
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aOdor descriptions perceived by panelists through the sniffing port.

bLinear retention index calculated on Rtx-5 MS column.

cFlavor dilution perceived through the sniffing port.

GC-O Analysis

The GC-O analysis showed that 14 compounds, that is, 3-methyl-1-butanol, isobutyric acid, ethyl butyrate, isovaleric acid, 2-methylbutyric acid, benzaldehyde, 3-methylthiopropanol, benzyl alcohol, tetramethylpyrazine, nonanal, methyl phenylacetate, 3-phenylpropionic acid, 3-decenoic acid, and 2,4-di-tert-butylphenol, were sniffed with a sweaty/rancid/sour note. Dimethyl phthalate and dodecanol were sniffed with metallic notes. Limonene and 2-pyrrolidinone were sniffed with fragrant notes. The phenethyl alcohol, 2-acetylbutyrolactone, trimethylpyrazine, and 2,6-dimethylpyrazine were sniffed with floral, plastic, roasted, and deep fried/fatty notes, respectively. It has been reported that yeasts generally synthesized δ-valerolactone, dimethyl phthalate, phenylacetic acid, benzaldehyde, nonanal, decyl aldehyde, trimethylpyrazine, tetramethylpyrazine, benzothiazole, 2,4-di-tert-butylphenol, and limonene as the basic metabolic products with “generalist” in aroma (Ames & Leod, 2006; Comuzzo et al., 2006). It seems that the P. pastoris strain and other yeasts can similarly synthesize sniffed benzaldehyde, nonanal, tetramethylpyrazine, 2,4-di-tert-butylphenol, etc. However, the P. pastoris strain synthesized high FD of isovaleric acid, 2-methyl butyric acid, tetramethylpyrazine, 3-phenylpropionic acid, 2,4-di-tert-butylphenol, dodecanol, and 2,6-dimethyl pyrazine, whereas other yeast had not been reported to synthesize these compounds.

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-tert-butylphenol, 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.

Table 3.

Omission Tests of After Methanol Induction (AMI)

No. Omission compound from AMIR Sweaty Deep-fried, Fatty Metallic Green Roasted Honey-like Plastic Floral
AMIR 4.10 ± 0.52 1.89 ± 1.21 2.47 ± 1.38 1.10 ± 1.61 0.77 ± 0.73 0.30 ± 0.18 2.44 ± 1.25 0.35 ± 0.20
 1 3-Phenylpropionic acid 3.70 ± 0.29 1.90 ± 1.1 2.40 ± 0.43 1.00 ± 0.39 0.50 ± 0.38 0.40 ± 0.20 2.30 ± 0.33 0.30 ± 0.22
 2 Nonanal 3.80 ± 0.24 1.90 ± 0.65 2.40 ± 0.42 0.90 ± 0.75 0.70 ± 0.35 0.40 ± 0.22 2.30 ± 0.73 0.40 ± 0.28
 3 Methyl phenylacetate 4.00 ± 0.47 1.90 ± 1.00 2.30 ± 1.05 1.00 ± 0.40 0.70 ± 0.24 0.30 ± 0.16 2.30 ± 0.55 0.30 ± 0.22
 4 Dodecanol 4.20 ± 0.20 2.00 ± 0.51 1.40 ± 0.32* 0.90 ± 0.56 0.60 ± 0.55 0.40 ± 0.25 2.20 ± 0.53 0.40 ± 0.33
 5 Dimethyl phthalate 4.00 ± 0.28 1.90 ± 0.98 2.30 ± 0.50 0.90 ± 0.66 0.60 ± 0.30 0.40 ± 0.19 2.30 ± 0.32 0.40 ± 0.33
 6 2-Acetylbutyrolactone 4.10 ± 0.24 1.90 ± 0.63 2.30 ± 0.84 1.00 ± 0.56 0.60 ± 0.44 0.50 ± 0.25 1.30 ± 0.56* 0.40 ± 0.30
 7 Trimethylpyrazine 4.00 ± 0.38 1.90 ± 1.12 2.40 ± 0.62 0.90 ± 0.43 0.41 ± 0.23 0.60 ± 0.28 2.30 ± 0.56 0.20 ± 0.11
 8 Phenethyl alcohol 4.20 ± 0.31 1.90 ± 0.90 2.40 ± 0.72 1.00 ± 0.48 0.60 ± 0.32 0.50 ± 0.21 2.30 ± 0.38 0.30 ± 0.24
 9 2,6-Dimethylpyrazine 4.20 ± 0.33 1.90 ± 0.50 2.40 ± 0.78 0.90 ± 0.29 0.60 ± 0.30 0.40 ± 0.22 2.40 ± 0.62 0.30 ± 0.2
 10 Tetramethypyrazine 3.30 ± 1.43* 2.00 ± 0.6 2.30 ± 0.72 0.90 ± 0.49 0.80 ± 0.30 0.60 ± 0.37* 2.20 ± 0.41 0.50 ± 0.23
 11 2,4-Di-tert-butylphenol 3.00 ± 1.21* 1.90 ± 0.75 2.30 ± 0.49 0.90 ± 0.46 0.70 ± 0.16 0.40 ± 0.24 2.20 ± 0.50 0.30 ± 0.22
 12 Isovaleric acid 2.80 ± 0.39* 2.00 ± 0.82 2.30 ± 0.67 0.80 ± 0.42 0.60 ± 0.35 0.60 ± 0.32 2.30 ± 0.78 0.30 ± 0.23
 13 2-Methylbutyric acid 3.30 ± 1.15* 1.90 ± 0.93 2.30 ± 0.80 0.90 ± 0.48 0.80 ± 0.64 0.60 ± 0.34 2.00 ± 0.97 0.40 ± 0.25
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*Significant (α ≤ 0.05).

Discussion

The host system of P. pastoris has been developed to heterologously produce proteins (Yang & Zhang, 2018; Fischer & Glieder, 2019). In our recent research works, the P. pastoris GS115 strain has been used to express various heterologous proteins (Li et al., 2020a; Zhang et al., 2020a; Ni et al., 2021). In these processes, the fermented broth of P. pastoris GS115 was sniffed with a strong off-flavor that has negative effects on the aromatic quality of the proteins. Various compounds with off-flavor have been illustrated in yeast products by researchers. For instance, sulfide compounds [e.g. hydrogen sulfide (Dequin, 2001) and thiophene (Lee et al., 2000)], and phenolic substances (e.g., 2-/4-methoxyphenol) are commonly presented in beer and wine fermented by S. cerevisiae. Yeast extracts have been determined to contain various aromatic volatiles, including 2-/3-methylbutanoic acid, 2-/3-methylbutanal, 3-mercapto-2-pentanone, 4-hydroxy-2,5-dimethyl-3(2H)-furanone, 2,3-butanedione, 3-methylpyridine, 2-acetyl-1-pyrroline, phenylacetaldehyde, and 2-acetyl-2-thiazoline (Münch et al., 1997; Münch & Schieberle, 1998; Zhang et al., 2017). However, it has not been illustrated which compounds contribute to the off-flavor of the fermented broth with P. pastoris strains. 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., 2020b), 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 off-flavor. 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.

Contributor Information

YuXuan Yao, College of Ocean Food and Biological Engineering, Jimei University, Xiamen 361021, People's Republic of China.

ShengLan Zheng, College of Ocean Food and Biological Engineering, Jimei University, Xiamen 361021, People's Republic of China.

ShiLin Chi, College of Ocean Food and Biological Engineering, Jimei University, Xiamen 361021, People's Republic of China.

Feng Chen, Department of Food, Nutrition and Packaging Sciences, Clemson University, Clemson, SC 29634, USA.

Ning Cai, Xiamen Ocean Vocational College, Xiamen, Fujian 361021, People's Republic of China.

ZhenZhen Cai, Xiamen Ocean Vocational College, Xiamen, Fujian 361021, People's Republic of China.

Zhipeng Li, College of Ocean Food and Biological Engineering, Jimei University, Xiamen 361021, People's Republic of China; Key Laboratory of Food Microbiology and Enzyme Engineering Technology of Fujian Province, Xiamen, Fujian 361021, People's Republic of China; Research Center of Food Biotechnology of Xiamen City, Xiamen, Fujian 361021, People's Republic of China.

Hui Ni, College of Ocean Food and Biological Engineering, Jimei University, Xiamen 361021, People's Republic of China; Xiamen Ocean Vocational College, Xiamen, Fujian 361021, People's Republic of China; Key Laboratory of Food Microbiology and Enzyme Engineering Technology of Fujian Province, Xiamen, Fujian 361021, People's Republic of China; Research Center of Food Biotechnology of Xiamen City, Xiamen, Fujian 361021, People's Republic of China.

Author Contributions

Y.Y.: methodology, experiment preparation, and data analysis; S.Z.: data curation, software, and validation; S.C.: investigation; F.C.: resources; N.C.: visualization; Z.C.: methodology; Z.L.: conceptualization, project administration, and supervision; H.N.: resources, methodology, funding acquisition, project administration, and supervision. All authors read and approved the manuscript.

Funding

This study was financially supported by the Program for the National Natural Science Foundation of China (32172339).

Conflict of Interest

There are no financial conflicts of interest to disclose.

Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval

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

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval

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

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