The transformation was initiated using S. cerevisiae strain BY4741 (Jia et al., 2018). Escherichia coli Trans1-T1 (Trans Gene Biotech, Beijing, China) was employed for plasmid maintenance and amplification. All yeast strains utilized in this study are listed in Table 1. Yeast strains were selectively inoculated in synthetic complete medium (SC) composed of 6.7 g/L yeast nitrogen base, appropriate amino acid dropout mixture, and approximately 20 g/L glucose. Yeast strains were preserved by being stored in 30% glycerol at −80°C. Unless stated otherwise, chemicals were obtained from Sigma-Aldrich (St. Louis, MO, United States).

All bacterial genes, including alkM (alkane hydroxylase gene) and its coenzyme genes rubA and rubB were codon-optimized and obtained from Kingsley company. Promoter and terminator sequences fragments were amplified from the genome of S. cerevisiae. The recombinant plasmids used in this study were based on modified pRS vector series.

In brief, gene expression units for each enzyme were created in the pRS vector, surrounded by different potent promoters and terminators (Zhou et al., 2021). Unique restriction sites (BamHI/HindIII) were introduced on either side of each gene expression cassette. To enhance recombination efficiency, homologous fragments were utilized to construct plasmids for fusion protein expression. For strain construction, the plasmids were transformed into E. coli and engineered E. coli strains were established through colony PCR verification. Plasmids were isolated from E. coli, and yeast strains were then genetically modified using the LiAc/SS carrier DNA/PEG technique (Agatep et al., 1998). The transformed cells were subsequently selected on appropriate synthetic dropout agar plates (without uracil or leucine) for transformation into S. cerevisiae BY4741.

An isolated yeast strain was cultured in 5 mL of suitable SC medium with 20 g/L glucose for a minimum of 12 h. After that, 50 mL of the corresponding synthetic yeast medium was added to a 250 mL flask, starting with an OD₆₀₀ of 0.2. The fermentation medium was enriched with 10 g/L n-hexadecane. The strain is cultivated in a shaking flask at a temperature of 30°C, pH value of 6, shaking speed of 220 rpm, and a flask volume ratio of 5:1. To assess the hexadecane degradation rate, samples were taken from each flask after 96 h of cultivation. The inoculation ratio of strains is 1:1, with an initial total OD₆₀₀ of 0.2. The microbial consortium was cultivated under single factor-controlled conditions at 35°C, 5% NaCl, and pH = 5.

The n-hexadecane content in each supernatant was extracted using an equal volume (1:1 v/v) of ethyl acetate. Afterwards, 1 mL of the polar phase was analyzed using a Thermo Scientific DSQII single quadrupole gas chromatography mass spectrometry (GC/MS) system with an HP-5MS column (operating at a pulsed split ratio of 1:1 and a split flow rate of 1 mL/min). The oven temperature was first set and maintained at 40°C for 3 min, followed by a gradual temperature increase at a rate of 6°C/min up to 300°C, with a final 2-min hold at that temperature. The quantification of n-hexadecane is achieved by comparing the peak areas of the test sample with those of the standard sample. The amount of n-hexadecane was adjusted by considering its mass spectrometer response factors, which were determined by analyzing dilution series of commercial standards.

We constructed an engineered strain capable of degrading hexadecane based on the S. cerevisiae BY4741. To assess the hexadecane degradation ability of wild strain (S. cerevisiae BY4741), BY4741 and Yarrowia lipolytica were cultured in SC medium until mid-log phase, followed by washing and starvation treatment for 48 h, and subsequently subjected to spot plate experiments. It is known that Y. lipolytica can degrade hexadecane. By comparing the growth of BY4741 and Y. lipolytica, we validated the ability of wild strain to degrade hexadecane. The results showed that wild strain could not utilize hexadecane as the sole carbon source for growth (Figure 1A).

Next, we attempted to introduce the alkane hydroxylase AlkM into wild strain using the single-copy plasmid pRS416, resulting in the strain SAH01 (Figure 1B). To observe the hexadecane degradation ability of this strain, we measured the OD₆₀₀ values of the strain within 96 h and the hexadecane degradation rate after 96 h. The results revealed that SAH01 exhibited better growth in media containing hexadecane and glucose as carbon sources compared to wild strain (Figure 1C), with a hexadecane degradation rate of 10.37% for SAH01 (Figure 1D).

To further enhance the hexadecane degradation ability of the engineered strain, the exogenous gene alkm was subjected to multicopy optimization to generate the strain SAH02. The OD₆₀₀ values of SAH02 approached those of SAH01 after 24 h and this trend continued until 48 h. By 72 h, the OD₆₀₀ value of SAH02 was slightly lower than that of SAH01, but by 96 h, both strains had OD₆₀₀ values exceeding 7.0. SAH02 exhibited growth lag during the logarithmic growth phase (Figure 1C). Degradation rate analysis indicated a significant improvement in the engineered yeast strain SAH02 with multicopy optimization compared to SAH01, reaching 19.68% (Figure 1D). This suggests that the introduction of multiple copies of the alkane hydroxylase gene, while imposing a higher growth burden on the strain, enhances its hexadecane degradation ability. This outcome is analogous to the synthesis of vitamin C in S. cerevisiae (Zhou et al., 2021).

The genes rubA and rubB encode for the coenzymes rubredoxin and rubredoxin reductase of the alkane hydroxylase AlkM, respectively (Ratajczak and Hillen, 1998), which play crucial roles in electron transfer during the enzymatic process. To enhance the catalytic effect of alkane hydroxylase and improve degradation efficiency in engineered yeast, the alkane hydroxylase was fused with its coenzymes. To ensure that the structure of the alkane hydroxylase and its coenzymes would not negatively impact each other due to proximity, a linker sequence was inserted between the two genes while constructing the plasmid containing the target genes. Subsequently, the alkM gene fused with different coenzyme genes was separately ligated to single-copy and multicopy vectors, obtaining four strains: SAH03, SAH04, SAH05, and SAH06. Results from growth conditions and degradation rate determination experiments indicated that among the strains using the single-copy vector, three strains exhibited similar trends in OD₆₀₀ values, with the order of SAH01 > SAH03 > SAH05. The degradation rates of these strains were found to be SAH03 > SAH01 > SAH05. Notably, the strain SAH03, which was introduced with a single coenzyme, achieved the highest degradation rate of 20.48%, which was 1.97 times higher than the strain without coenzyme introduction. In contrast, among the strains using a multi-copy vector, a distinct difference in growth was observed after 24 h, with OD₆₀₀ values being SAH02 > SAH04 > SAH06. Moreover, the introduction of coenzymes resulted in a decrease in degradation rates of the strains. Specifically, compared to the control strain SAH02, the degradation rates of SAH04 and SAH06, which introduced one and two coenzymes respectively, decreased by 5.21% and 2.04%. Interestingly, we found that the growth of strains weakened with the introduction of coenzymes, but the corresponding degradation rates did not show a complete positive correlation with the growth conditions.

The GSH synthesis and cycling process were divided into four modules: SOD module, GSH synthesis module, GSH oxidation module, and GSH reduction module (Pastore et al., 2003). The principal reaction in the SOD module involves the clearance of superoxide anions, catalyzed by the SOD enzyme (Dubois-Randé et al., 1994). The GSH synthesis module is composed of two reactions, each catalyzed by different GSH synthesizing enzymes. The GSH oxidation module primarily encompasses the oxidation of GSH to oxidized glutathione (GSSG), catalyzed by glutathione peroxidase (GPx) and glutaredoxin (Grx) (Grant et al., 1996). This oxidation process simultaneously reduces H₂O₂, phospholipid hydroperoxides, and protects thermosensitive thiol proteins. The GSH reduction module bifurcates, first catalyzing the conversion of NADH to NADPH through NADH kinase to generate reducing power, and then reducing GSSG to GSH via glutathione oxidoreductase. Kluyveromyces marxianus exhibits elevated thermotolerance in yeasts (Cui et al., 2023), Whereas mellicola (Sun et al., 2019) is an acid-resistant marine bacterium, and Thermus thermophilus HB27 (Gallo et al., 2021) is an extremophilic thermophile. These microbes demonstrate substantial tolerance under various conditions. Consequently, the twelve genes selected for this study are derived from the three microorganisms. After undergoing codon optimization provided by a commissioned company, these genes were introduced into the S. cerevisiae BY4741 (Figure 2A).

To validate the strain tolerance during growth at 40°C, yeast strains YGSH01-YGSH12, along with BY4741, were inoculated into YPD liquid medium at a final OD₆₀₀ of 0.2 and cultured under 40°C at 220 rpm for 48 h. As shown in Figure 2B, strain YGSH10 exhibited notably superior heat resistance compared to other strains, characterized by a brief lag phase, prolonged log phase, high cell concentration, swift recovery from heat-induced damage, and sustained, enhanced cell vitality at 40°C. Although strain YGSH11 exhibited slower recovery of cell activity from high temperatures, its log phase was shorter than that of YGSH10, indicating higher cell vitality under heat, and its final OD₆₀₀ was second only to YGSH10. Both YGSH10 and YGSH11 express the GSH reduction module, suggesting a pivotal role of GSH reduction module in yeast’s defense against heat damage. Additionally, strains YGSH12, YGSH09, YGSH01, YGSH06, and YGSH04 (order from fast to slow) could recover cell vitality at a relatively quick pace under high-temperature stress, reaching cell concentrations comparable to wild strain (S. cerevisiae BY4741). Compared to BY4741, the remaining strains YGSH03, YGSH07, YGSH08, YGSH02, and YGSH05 (order from strong to weak) showed noticeably enhanced sensitivity to heat. Moreover, no significant secondary growth phenomena were observed for all twelve yeast strains and BY4741 at 40°C.

To further validate the heat tolerance of strains at 55°C, secondary seed cultures were obtained and diluted to an OD₆₀₀ of 1 using sterile water. For the experimental group, the cultures underwent heat shock at 55°C in a metal bath for 15 min while control groups remained untreated. Three biological replicates per group were conducted. Yeast cell counts under both conditions were obtained through gradient dilution plating, and the cell mortality induced by 55°C heat shock was calculated. As shown in Figure 2C, except for strain YGSH09, the remaining eleven strains exhibited lower cell mortality rates compared to wild strain under 55°C heat shock, with more cells surviving. The poorer heat tolerance of YGSH09 may be attributed to the gene grx35095 sourced from W. mellicola, which has lower homology with yeast. Among YGSH01-YGSH12, YGSH05 exhibited the strongest heat tolerance at 55°C, followed by YGSH04 and YGSH01, while the mortality rate of the remaining strains was around 50%. Divided per module, strains from the SOD and GSH reduction modules displayed higher cell survival rates under 55°C heat stress compared to others. It is inferred that high-temperature heat shock may necessitate increased intracellular O^2- clearance speed and maintaining GSH in a reduced state to protect mitochondria from oxidative damage, thereby sustaining normal life activities.

To ascertain the NaCl concentration that induces osmotic stress and preliminarily understand the strains’ osmotic tolerance, the study cultivated YGSH01-YGSH12, along with wild strain expressing pRS416, on SC-ura solid plates with varying NaCl concentrations. Figure 2D displays the outcomes: the twelve yeast strains seemingly exhibited no significant variance in their defense against osmotic stress caused by NaCl. However, the growth conditions of three strains, YGSH07, YGSH08, and YGSH10, were superior to BY4741 (pRS416) on solid plates containing 5% and 7.5% NaCl. For a more accurate characterization of strain tolerance, YGSH01-YGSH12, along with BY4741, were inoculated in YPD liquid medium containing 5% NaCl and cultured for 48 h. The results, as shown in Figure 2E, categorized the twelve yeast strains into two groups based on salt tolerance: YGSH12, YGSH08, YGSH09, YGSH06, YGSH10, YGSH07, and YGSH04 (order from strong to weak) exhibited enhanced salt tolerance compared to the wild-type strain; whereas YGSH02, YGSH11, YGSH03, YGSH05, and YGSH01 (order from strong to weak) showed salt tolerance slightly higher than or equivalent to BY4741. Furthermore, compared to H₂O₂ and thermal stress, 5% NaCl had a milder impact on yeast growth, characterized by a short lag phase and higher final OD₆₀₀. The influence of 5% NaCl on yeast growth predominantly occurred during the log phase, slowing the growth rate. Moreover, according to module classification, the three strains from the GSH reduction module and two from the GSH oxidation module exhibited higher osmotic tolerance. This suggests that upregulation of exogenous gene expression related to GSH cycling positively affected yeast growth under salt stress induced by NaCl.

To validate the strains’ tolerance to inorganic acid, yeast strains YGSH01-YGSH12, along with BY4741, were inoculated to a final OD₆₀₀ of 0.2 and cultivated in SC-ura liquid medium (acidified with HCl) at pH 3.0 for 48 h. YGSH10 uniquely exhibited a secondary growth phase among the twelve strains, recovering cellular vitality rapidly from the inorganic acid stress (Figure 2F). Following YGSH10, strains YGSH08, YGSH12, YGSH07, and YGSH04 demonstrated resilience under inorganic acid stress, with a final OD₆₀₀ around 4.0. The remaining strains displayed a sensitivity to inorganic acid similar to that of BY4741. Categorized by module, strains from the GSH reduction module exhibited superior inorganic acid tolerance. This study infers that maintaining a reduced state of GSH in yeast cells under inorganic acid stress is crucial for cellular protection, especially in preventing mitochondrial oxidative damage and sustaining normal intracellular life processes (Outten and Culotta, 2004; Outten et al., 2005).

The intracellular and extracellular GSH levels were measured in the most tolerant strain YGSH10 among the 12 engineered strains. The results showed that YGSH10 could secrete more glutathione compared to the wild strain BY4741. At 24 h and 48 h, the intracellular GSH content in YGSH10 was higher than that in BY4741. However, at 72 h and 96 h, the intracellular GSH content in YGSH10 was lower than that in BY4741 (Figure 2G). Meanwhile, the extracellular GSH content of YGSH10 was higher than that of the yeast BY4741. The highest increase in GSH content for YGSH10 was observed at 24–48 h, reaching 0.218 mM. From 48 h to 72 h, the increase in GSH content for YGSH10 was 2.13 times higher than that of BY4741 (Figure 2H). These findings indicate that between the 48–72 h period, YGSH10 secretes a significant quantity of intracellular GSH into the extracellular environment, resulting in a reduction of intracellular GSH and an elevation of extracellular GSH.