Physicochemical characterization of GXDK6
As shown in Fig. 1a, the colony of GXDK6 was milky white, round, convex, and smooth. The SEM results showed that GXDK6 was similar to the typical yeast species of Pichia anomala Y197-13. In addition, the cell size was between 2–12 µm, and the cell morphology was oval or semi-oval with a smooth surface (Fig. 1b).
Multi-stress-tolerant properties of GXDK6
As shown in Fig. 1c, GXDK6 could be incubated continuously in the pH range from 2.5 to 10.0. This result indicated that GXDK6 had a strong resistance to acids and alkalis. However, when GXDK6 was incubated in an acidic condition at pH 2.5 or pH 3.0, the relative biomass GXDK6 was higher than that at pH 9.5 or 10.0, respectively. This result suggested that GXDK6 showed better resistance to an acidic environment.
The salt-tolerance results of GXDK6 indicated that the species was a probiotic with tolerance to high-salt concentrations (Fig. 1d). GXDK6 could be incubated continuously up to 12% NaCl, with the relative biomass of approximately 10% with that in 1% NaCl, and up to 18% KCl or MgCl2, with the relative biomass of near 90% with that with no salt condition. These findings indicated that the growth of GXDK6 decreased drastically with increasing concentration of NaCl up to 12% but showed no significant impact with increasing concentration of KCl or MgCl2 up to 18%. As shown in Fig. 1e, GXDK6 also showed good tolerance to the seven heavy metals (i.e., Cd2+, Cu2+, Mn2+, Co2+, Ni2+, Cr2+, and Zn2+). The maximum tolerated concentration for Ni2+ was 5.8 ppm, while that for Mn2+ was 5494 ppm. The tolerance for Cu2+ was also more than 1000 ppm, but the maximum tolerance for Co2+, Cr2+, and Zn2+ was more than 250 ppm. This characteristic shows the potential of the species for the deodorization and bioremediation in heavy metal environments. Similar results were reported by Fernández et al.  and Bhakta et al. . The temperature sensitivity of GXDK6 was also investigated (Fig. 1f). GXDK6 could be incubated at 25–45 ℃, and the optimal incubation temperature was 30–37 ℃. The relative biomass decreased drastically to 25.22% at 45 ℃ compared with that at 30 ℃. In addition, when the incubation temperature was increased to 50 ℃, GXDK6 stopped to reproduce. A similar result was reported by Ndubuisi et al. , who found that the growth of heat-resistant Pichia is significantly inhibited by increasing the temperature, and the ethanol production efficiency of Pichia is lowered.
Whole genome sequencing analysis of GXDK6
The whole genomic sequencing of GXDK6 was performed using the whole genome shotgun method as reported by Marcel et al. . The number of reads of GXDK6 was 8, 319, 572 items, the total bases were 2, 477, 073, 673 bps, of which the GC content accounted for 38.88% (Additional file 1). The fuzzy bases were only 0.001%, Q20% was 94.60%, and Q30% was 86.73%. Genome of GXDK6 was ~ 15,000 bps. The results showed that the extracted DNA was a clear single band, indicating that the extracted DNA was suitable for subsequent analysis (Fig. 2a). Then, a single-base mass distribution map of the sequencing results was constructed (Fig. 2b). The abscissa is the base position of the reads (5ʹ–3ʹ), while the ordinate is the base Q value statistics of all reads. The red and blue lines represent the median and average value of the reads. The yellow line shows that the reads are in the 25% − 75% interval, and the tentacles represent that the reads are in the 10–90% interval. The results showed that the bases in the middle had higher base quality. The average quality of the data filtered was also reliable (Fig. 2c).
The sequence length distribution of the third-generation sequencing data is also shown in Fig. 2d, which was mainly used to reflect the average mass distribution of the sequencing data . From Fig. 1e, the whole genome sequence length was good, while the ratio of the uncertain bases to the length of the splicing sequence was 0. This result indicated that the sequence could be used for subsequent gene splicing.
Sequencing and analysis of the ITS domain indicated that the strain with higher consistency basically belong to Meyerozyma sp. The evolutionary distance between GXDK6 and other species has been showed in the species evolutionary tree clearly (Fig. 2e). GXDK6 has a high affinity with 88% confidence level with M. guilliermondii KAML05, M. guilliermondii IFM6377, etc., instead of P. guilliermondii ATCC6260. Therefore, GXDK6 was classified as yeast Meyerozyma guilliermondii.
A complete sequence comparison of the genome sequences was conducted by using the online software BUSCO (http://busco.ezlab.org, v3.0.2) to obtain the percentage of single-copy genes in the total single-copy genes . As shown in Fig. 2e, the spliced genome data of the species were relatively complete. According to the alignment results, GXDK6 belonged to the Pichia genus of the Saccharomyces family and showed the closest relation to M. guilliermondii. Therefore, the yeast Pichia was further confirmed as M. guilliermondii GXDK6.
Ability for single-organic matter fermentation
As shown in Fig. 3a, 21 organic matters (i.e., glucose, sucrose, fructose, xylose, xylan, sorbitol, raffinose, mannose, trehalose, cellulose, maltose, arabic candy, inulin, mannitol, sorbose, D-galactose, cellobiose, wheat bran, ethanol, succinic, and L-rhamnose) as the sole carbon source could be fermented by GXDK6. This result indicated that GXDK6 showed a strong ability to utilize organic matter, such as pentose and hexose.
Figure 3b showed that the GXDK6 grew best when used with sorbitol as the sole carbon source, with a dry cell weight of ~ 1.499 g/L, but grew slowest with L-rhamnose, with a dry cell weight of ~ 0.437 g/L. Therefore, the growth rate results showed significant differences in the rate by which GXDK6 utilized diverse organic matters. The order of utilization could be summarized as follows: sorbitol༞xylan༞raffinose༞mannose༞sucrose༞fructose༞trehalose༞inulin༞maltose༞arabic candy༞mannitol༞glucose༞sorbose༞D-galactose༞cellobiose༞xylose༞cellulose༞ethanol༞wheat bran༞succinic༞L-rhamnose.
Many types of aroma-producing yeasts, such as Hansenula, Candida, S. cerevisiae, Pichia pastoris, and Sphaeropsis sphaeroides, had been reported . The different yeasts also presented diverse aroma-producing characteristics, such as floral fragrance, fruity, delicateness, sweetness, and wine aroma. However, an aroma-producing yeast that can ferment various organic matters, produce abundant aromatic beneficial metabolites, and possess a strong multi-stress tolerance to various environments has not been reported yet. Therefore, M. guilliermondii GXDK6 will be a potential probiotic with important application value.
The metabolites produced by GXDK6 with glucose, sucrose, fructose, or xylose as sole carbon sources was detected with GC–MS method, results showed that 14, 20, 16, and 26 peaks were detected in the samples (Fig. 3a, 3b, 3c, and 3d). Among them, when GXDK6 was fermented with six carbon sugars (glucose, sucrose) as a single carbon source for 72 h, the top five substances with peak areas were 3,5-ditert-butylphenol, sec-butyl cyclohexyl sulfide, isoamyl alcohol, 3,5-dimethylbenzaldehyde, and ethanol, respectively. However, when GXDK6 was fermented for 72 h with pentose (fructose, xylose) as a single carbon source, the top five substances in peak area were 3,5- ditert-butylphenol, sec-butyl cyclohexyl sulfide, 3,5-dimethylbenzaldehyde, nerol, and isoamyl alcohol (or 2-ethyl hexanol), respectively. Thus, GXDK6 could ferment with different kinds of organic matter as the sole carbon source to produce aromatic metabolites. However, the distinct metabolic regulation networks and mechanisms remain unknown [19, 20], which still need further study.
Metabolomic analysis of GXDK6
As shown in Fig. 4, the metabolites of GXDK6 produced from the selected carbon sources can be classified as alcohols, esters, acids, hydrocarbons, aldehydes, ketones, sulfide, phenolics, and other organic substance (Fig. 4a). However, alcohols, lipids and organic acids are usually defined as the main aromatic metabolites. Based on this, the main aromatic metabolites produced by GXDK6 fermentation of glucose and sucrose are 9 and 13 types, respectively (Table 1), and the aromatic metabolites produced by fermentation of fructose and xylose are 8 and 14 types, respectively (Table 1).
Aromatic metabolites of GXDK6 fermented with inconsistent organic matter.
Percentage of total metabolites (%)
Isopentanol, Ethanol, Nerol, Phenylethanol, Isobutanol, 2-ethylhexyl alcohol, propionic acid, formic acid, 1-methoxy-2-propanol.
Isopentanol, Ethanol, Nerol, 2-ethy-hexanol, Isobutanol, Phenylethanol, propionic acid, formic acid, Dibutyl phthalate, α - terpineol, 1-methoxy-2-propanol, Cyclopentyl 4-ethylbenzoate, Farnesol.
Nerol, 2-ethylhexanol, Isopentanol, Phenylethanol, propionic acid, formic acid, α - terpineol, Dibutyl phthalate.
Nerol, Isopentanol, Phenylethanol, propionic acid, formic acid, 2-ethylhexyl alcohol, α - terpineol, Diisobutyl phthalate, acetic acid, Dimethylsiloxanediol, Methyl 4-ethylbenzoate, Farnesol, Butyl isobutyl phthalate, 3-ethoxypropionic acid.
Among these aromatic metabolites, no matter which carbon source is used for fermentation, there are five aromatic metabolites which are the same (Fig. 4b), namely isoamyl alcohol, nerol, phenethyl alcohol, formic acid, and propionic acid, respectively. These results indicate that GXDK6 has the same metabolic pathway when fermenting five-carbon sugar and six-carbon sugar. In order to further studying the difference of aroma production from inconsistent carbon sources of GXDK6 fermentation, the top five aromatic metabolites were selected according to the percentage of content. Among them, isopentanol (15.24%, 17.60%), ethanol (9.83%, 9.13%), nerol (6.60%, 6.46%), isobutanol (9.83%, 9.13%), and 2- ethylhexanol (9.83%, 9.13%) are the top five aromatic metabolites of six-carbon sugar (glucose and sucrose) fermented by GXDK6. Among the aromatic metabolites of five-carbon sugars (fructose and xylose) fermented by GXDK6, nerol (9.17%, 10.15%), isopentanol (2.29%, 5.39%), phenethyl alcohol (1.77%, 1.62%), formic acid (1.09%, 1.23%), 2- ethylhexanol (1.09%, Fig. 4e) or propionic acid (1.49%) ranked in the top five (Fig. 4f). These results suggested that GXDK6 showed distinct fermentation abilities with different carbon sources as substrates. However, this species could also produce partial similar beneficial aromatic metabolites, which could be due to its diverse mechanisms in fermenting organic matters [21, 22].
Metabolic pathways of nerol and its biosynthesis mechanism
In order to further studying the molecular mechanism of aroma production by GXDK6 fermentation, nerol (Additional file 2) was taken as a representative novel aromatic metabolite from M. guilliermondii, which was further compared and analyzed with the whole genome data of GXDK6 (Additional file 3), and the metabolic pathway was further elucidated. As shown in Fig. 5, the upstream sources of glycolysis or citric acid cycle 1-deoxy-d-xylulose5-phosphate geranyl diphosphate or linalool nerol, was firstly converted into final geranic acid or 8-Oxogeranial. In this process, the proteins involved in nerol synthesis were geranyldi phosphotase, monoterphenyl diphosphatase, and geraniol isomerase. The proteins involved in nerol metabolic transformation were geraniol 8-hydroxyase, alcohol dehydrogenase, and geraniol dehydrogenase. As reported by Zong et al. , nerol was biosynthesized in the metabolic engineered Escherichia coli from glucose, and the biosynthesis mechanism had also been revealed. The truncated neryl diphosphate synthase gene tNDPS1 was expressed that catalyzed isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) form neryl diphosphate (NPP), and then the nerol synthase gene GmNES was co-expressed to synthesize the final product nerol from NPP. However, the biosynthesis of nerol in native Meyerozyma guilliermondii has not been reported yet.
The structure and function of the six proteins were further investigated (Fig. 5), results showed that geranyl diphosphatase was a dimer protein with ~ 65 kDa, and its corresponding ligand was found as Mn2+ (Fig. 5b), which suggested that geranyl diphosphatase could be bound and interacted with Mn2+, and promoted the catalytic reaction to produce more nerol . Monoterpenyl diphosphatase was also a dimer protein with a molecular weight ~ 68 kDa, but its corresponding ligand had not been found yet (Fig. 5c), this indicated it should be a non-allosteric enzyme with auxiliary catalysis . Geraniol isomerase was a pentameric protein with ~ 44 kDa (Fig. 5d), and its corresponding ligand was found as geraniol (ligand for nonmetallic ion not shown), indicating that the existence of geraniol was beneficial to the catalytic reaction . In summary, these proteins were indispensable and directly participate in the regulation and biosynthesis of nerol.
The subsequent metabolism or transformation of nerol was catalyzed by geraniol 8-hydroxyase, alcohol dehydrogenase, and geraniol dehydrogenase. Among them, geraniol 8-hydroxyase was a monomeric protein of ~ 55 kDa , and its corresponding ligand had not been found yet (Fig. 5e). Alcohol dehydrogenase was also a monomeric protein with a molecular weight of ~ 39 kDa , its corresponding ligand was found as Zn2+ (Fig. 5f), indicating alcohol dehydrogenase could be bound and interacted with Zn2+, it would contribute to the formation of nerol. Geraniol dehydrogenase was a dimer protein with ~ 41 kDa , and its corresponding ligand had not been found (Fig. 5g), suggesting that it could be a non-allosteric enzyme with auxiliary catalysis. These evidences indicated that the generation of nerol was mainly attributed to the existence of corresponding enzymatic system and metabolic pathway in GXDK6. Furthermore, nerol was classified as a typical example of aromatic metabolites in GXDK6, why GXDK6 can maintain a long-term aroma production should be the contribution of various aromatic metabolites. Therefore, the biosynthesis mechanism of nerol will contribute to better understand the aroma-producing mechanism of GXDK6.