Construction of a Zygosaccharomyces rouxii strain overexpressing the QOR gene for increased HDMF production

Abstract 4‐Hydroxy‐2,5‐dimethyl‐3(2H)‐furanone (HDMF) is a flavor compound widely found in natural products and is used in food as a flavor‐enhancing agent. Quinone oxidoreductase (QOR) was verified as a key enzyme to synthesize HDMF in strawberry, while its impact on HDMF production by Zygosaccharomyces rouxii was still unknown. The QOR gene was cloned and overexpressed in Z. rouxii, and its impact on HDMF production by Z. rouxii was then further analyzed. At the same time, it is expected to obtain engineered strains of Z. rouxii with high HDMF production. The results showed that the engineered strains of Z. rouxii exhibit different levels of QOR gene expression and HDMF production; among them, the QOR6 strain exhibiting the highest gene expression level and HDMF production was named as ZrQOR. The HDMF production of the ZrQOR strain was significantly higher than that of wild‐type Z. rouxii at 3 and 5 days of culture, with 1.41‐fold and 1.08‐fold increases, respectively. At 3 days of fermentation, the highest HDMF yield of ZrQOR strain was obtained (2.75 mg/L), 2 days ahead of the reported highest HDMF production by Z. rouxii. At 3, 5, and 7 days, QOR gene expression was 4.8‐fold, 3.3‐fold, and 5.6‐fold higher in the ZrQOR strain than in the wild‐type Z. rouxii, respectively. Therefore, overexpression of the QOR gene facilitates HDMF synthesis. The genetic stability of the 0–20 generation ZrQOR strain was stable, and there was no significant difference in colony shape, QOR expression, or HDMF production compared to the wild type. In this study, the genetic engineering Z. rouxii strain was used to improve HDMF production. This research has laid the groundwork for further industrial production of HDMF via microbial synthesis.

and HDMF production; among them, the QOR6 strain exhibiting the highest gene expression level and HDMF production was named as ZrQOR.The HDMF production of the ZrQOR strain was significantly higher than that of wild-type Z.rouxii at 3 and 5 days of culture, with 1.41-fold and 1.08-fold increases, respectively.At 3 days of fermentation, the highest HDMF yield of ZrQOR strain was obtained (2.75 mg/L), 2 days ahead of the reported highest HDMF production by Z. rouxii.At 3, 5, and 7 days, QOR gene expression was 4.8-fold, 3.3-fold, and 5.6-fold higher in the ZrQOR strain than in the wild-type Z.rouxii, respectively.Therefore, overexpression of the QOR gene facilitates HDMF synthesis.The genetic stability of the 0-20 generation ZrQOR strain was stable, and there was no significant difference in colony shape, QOR expression, or HDMF production compared to the wild type.In this study, the genetic engineering Z. rouxii strain was used to improve HDMF production.This research has laid the groundwork for further industrial production of HDMF via microbial synthesis.
Accompanied by the improvement of people's living standards, the demand for HDMF in the food industry is increasing.However, the production of HDMF using Z. rouxii involves a high cost of the precursor substance fructose 1,6-diphosphate (FDP) (Wang et al., 2009).
Therefore, cost reduction and yield improvement are topics of intense interest in current research (Raab et al., 2006).
Quinone oxidoreductase (QOR) exhibits selectivity toward quinone substrates and serves as a crucial catalyst during the synthesis of HDMF through FDP metabolism in yeast (Hecquet et al., 1996;Landry et al., 2021;Zhang et al., 2018).QOR was discovered in the biosynthetic pathway of HDMF and its derivatives in strawberries (Yamada et al., 2018).QOR was found to be the last enzyme involved in the HDMF synthesis pathway and is functionally expressed in Escherichia coli; thus, it was hypothesized that QOR can catalyze HDMF production via 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone (HMMF) (Raab et al., 2006).Further studies revealed the role of QOR in HMMF for the synthesis of HDMF by hydrogenation of a previously unknown HMMF or 4-hydroxy5-methyl-3(2H)-furanone (HMF) derivatives substituted at the methylene functional group, rather than reduction of the double bond of straight-chain 2-alkenals or 2-alkenones, and this pathway is operative in tomato fruit (Klein et al., 2007).The exogenous nutrients and their intermediate metabolites, such as those FDP involved in EMP pathway and ribulose 5-phosphate (R5P) in PP pathway, were also verified as precursors (Li et al., 2020).Thus, QOR and other enzymes involved play important roles in regulating the synthesis of HDMF, as shown in Figure 1.
The construction of engineered yeast mainly occurs through the regulation of metabolic pathways and the construction of synthetic pathways for the target product in the host cell.The construction of genetically engineered yeast with the desired characteristics is efficient and environmentally friendly and might be produced in large quantities (Rahmat & Kang, 2020).To date, the use of Saccharomyces cerevisiae to build engineered strains with high production of hydroxytyrosol (Liu et al., 2022) and p-coumaric acid (Zhang et al., 2020) and the use of Pichia pastoris to produce keratinase (Xu et al., 2019) have achieved good results.However, studies on the use of Z. rouxii to construct genetically engineered yeast for high yields of HDMF have not been reported.Some modern biotechnologies, such as genetic engineering technology, can be used to construct genetically engineered strains with high HDMF production.This can achieve the characteristics of economy, efficiency, and environmental friendliness in the large-scale production of HDMF using microbial factories.
The aim of this study was to clone the QOR gene and overexpress it in Z. rouxii.The genetically engineered strain with higher production of HDMF was then screened through HDMF production and gene expression.Furthermore, the genetic stability and HDMFproduced function of this engineering strain were analyzed, which will provide a theoretical basis for enterprises to further utilize engineering strains on a large scale to produce natural HDMF.vector was constructed by Wuhan Transduction Biological Laboratory (Wuhan, China).Yeast peptone dextrose medium (YPD, containing 20.0 g/L peptone, 20.0 g/L glucose, 10.0 g/L yeast extract, and 180 g/L NaCl) was obtained from Qingdao Hope Bio-Technology Co., Ltd.(Qingdao, China).The standard for HDMF was obtained from Sigma Aldrich (St. Louis, MO, purity ≥97%).

| Cultivation of Z. rouxii
According to the method of Fu et al. (2022), with slight modifications, the freeze-dried powder of native Z. rouxii was activated.In a biological clean room, a sample was added to 100 mL of sterilized YPD broth and incubated for 3-4 days at 28°C and 180 rpm in a temperaturecontrolled shaking incubator.The culture was stored when cell counts were measured with a hemacytometer at 2 × 10 8 CFU/mL.The activated seed was inoculated at 5% (approximately 2 × 10 8 CFU in 1 mL of solution) into a new 100 mL of sterile YPD and incubated at 28°C and 180 rpm for 30-35 h.The seeded fermentation broth was obtained when the total number of cells reached 2 × 10 8 CFU/mL.Seed broths in triplicate were added to the sterilized medium at 5%, then each of them was incubated at 0, 3, 5, and 7 days, and then those were subsequently added with an equal volume of 70% glycerol into the strains, mixed well, and stored at −80°C in the fridge.Biomass calculation was carried out according to the method of Li et al. (2020).

| PCR amplification and construction of the overexpression vector
The QOR gene sequence (AC: XM_002494472.1)from native Z. rouxii was first obtained from the NCBI database, and primers were designed using Primer Premier 5.0 Software (Premier Biosoft Co. Ltd., Palo Alto, California, USA) (Table 1).The genomic DNA was extracted using the yeast genomic DNA extraction kit (Qualityard Biotechnology Co., Ltd.), and the specific process was carried out according to its instructions.Using genomic DNA as a template, the QOR gene was then amplified by PCR using the 2 × PCR MiX enzyme (a high-fidelity enzyme) and finally observed by 1% agarose gel electrophoresis.The PCR program was set to predenaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min for a total of 35 cycles.The ZrQOR gene was purified by the gel recovery method, connected to the vector pESC-MCS2 with a strong GAL promoter and Amp + resistance, and inserted between the polyclonal sites 5′ BamHI-SalI 3′.The ligated product was transferred into E. coli DH5 alpha, the plasmid was extracted, and the construction of the target gene recombinant, named pESC-URA-QOR, was identified by 1% agarose gel electrophoresis.

| Acquisition of the positive transformants
First, the recombinant plasmids in E. coli were extracted using an OMEGA Plasmid Mini Kit I. Overexpression of the QOR gene in a yeast strain was constructed according to the method of Wang et al. ( 2022) with minor modifications.A total of 1-5 μg of purified plasmid and 100 μL of receptor cells were mixed, transferred to an electrotransfer cup, and electroshocked for 5 milliseconds at 2.1 kV.
After shocking, 1 mL of YPD was quickly added to the electrotransfer cup, and the cells were gently suspended before being transferred to a 1.5 mL centrifuge tube and resuscitated for 2 h at 28°C and 180 rpm.Finally, the cell was cultured for 2-3 days in solid YPD medium until a single colony was formed.The positive clones were screened through colony PCR using multiple pairs of primers.The positive transformants with QOR gene overexpression were subsequently referred to the engineered yeast strain.

| RNA extraction, cDNA synthesis, and qRT-PCR analysis
Total RNA from native Z. rouxii and engineered yeast strain samples was obtained using TRIzol reagent (Invitrogen, Carlsbad, CA, USA).
The quality of the extracted RNA was determined using agarose gel electrophoresis.A Nanodrop 2000 (Thermo Scientific, Waltham, MA, USA) was used to determine the concentration of extracted RNA samples.Then, ReverTra Ace qPCR RT Master Mix with a gDNA Remover kit (TOYOBO, Dalian, China) was used for cDNA synthesis.
The diluted cDNA was taken, and qRT-PCR was performed using THUNDERBIRD SYBR® qPCR Mix (TOYOBO).The qRT-PCR program was set to predenaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s, for a total of 30 cycles, and NCBI/Primer-BLAST was used to design the primers.The gene expression levels were measured after 0, 3, 5, and 7 days of fermentation, and the gene expression in the original group was set as the control group.After being fermented for 0, 3, 5, and 7 days, the QOR gene expression levels were TA B L E 1 Primers sequence design.

GAPDH-R CCTTAGCAGCACCGGTAGAG
Note: Underlines in primer sequences mean the cleavage site of the enzyme.
measured in Z. rouxii and the transformed yeast, and the GAPDH gene was used as an internal reference gene.Three biological and technical replicates were measured, and the relative gene expression was calculated using the 2 −∆Ct method (Elcik et al., 2021).

| Determination of the HDMF content
According to the modified method (Li et al., 2020), the HDMF content was determined on a High Performance Liquid Chromatography (HPLC) system equipped with an Agilent 1260 Infinity II UV-VIS Diode Array Detector (DAD, Agilent Technologies, CA, USA).The fermentation broth was centrifuged at 8000 rpm for 10 min, and the supernatant was transferred to a freshly sterilized centrifuge tube and filtered through a 0.22 μm membrane before loading.The column was a ZORBAX Eclipse XDB-C18 column (5 μm, 250 × 4.6 mm, Agilent) with a mobile phase of 0.5% formic acid and an injection volume of 10 μL.A gradient starting at 95% A (0.5% formic acid in water) and 5% B (acetonitrile) to 80% A within 10 min and then to 0% A in 15 min was used at a flow rate of 1 mL/min and recorded at a wavelength of 287 nm.HDMF standard chromatography is shown in Figure 1S.The linearity of a standard curve using an external standard was assessed over the range of 5-90.0 mg/mL for HDMF.The linear regression equation for HDMF is y = 32.397×+ 3.904 (Figure 2S, R 2 > 0.9997, 98.5%-103.5% accuracy).

| Genetic stability analysis
The ZrQOR strains were inoculated on YPD plates and incubated at 28°C for 3 days to check the morphology of the colonies.The cell morphology was also examined using an electron microscope (Chongqing Optics & Electronics Instrument Co., Ltd., China).The HDMF production and QOR gene expression in engineered yeast at 0, 5, 10, 15, and 20 generations were determined by referring to the above method at 3 days; each 10-15 h was regarded as one generation.

| Statistical analysis
All tests were performed in triplicate, and expressed as the mean ± SD deviation.The data were analyzed using SPSS 20.0 (SPSS, Inc., Chicago, IL, USA), and an independent sample t test was used to compare the data for the two groups.p < .05,p < .01,or p < .001indicates that the data were statistically significant.The figures were prepared with GraphPad Prism 8.0 (GraphPad Software, Inc., San Diego, CA, USA).

| Determination of QOR gene expression
Cloning and overexpression vector construction of the QOR gene (Supplementary Material 1-Data S1, Figure 3S) and screening of 7 positive clones of the ZrQOR strain (Supplementary Material 2-Data S1, Figure 4S) were prepared.To confirm whether the QOR gene was overexpressed in the 7 positive clones, QOR gene expression was measured by qRT-PCR at 3 days of fermentation using GAPDH as the internal reference gene.According to the results in Figure 2, the QOR gene's relative expression of QOR6 and QOR11 clones was significantly higher than those of the native Z. rouxii, with 3.2-fold (p < .001)and 2.1-fold (p < .01)higher expression, respectively.Therefore, QOR6 and QOR11 were identified as effective engineered yeasts overexpressing QOR.QOR6 showed the highest expression, so it was used in the subsequent experiments.

| HDMF production and relative expression of QOR6 in different culture periods
The HDMF content was measured at different fermentation times using HPLC.The results are shown in Figure 3a.At 3 and 5 days, QOR6 produced significantly more HDMF than native Z. rouxii (1.41-fold and 1.08-fold increases, respectively) (p < .01& p < .05).
At 5 days of fermentation, the highest HDMF yield of native Z.
rouxii was 2.35 mg/L, while the highest HDMF yield of QOR6 was 2.75 mg/L at 3 days of fermentation.Therefore, almost 2 days of fermentation time reduction were conducted at the level of similar HDMF production between native Z. rouxii and genetic strains.
Regardless of the presence or absence of D-fructose in YPD culture medium, the highest HDMF production was observed in Z. rouxii on the fifth day of fermentation (Li et al., 2020;Zhou, 2018).It is evident that overexpression of the QOR gene in Z. rouxii leads to an earlier production decreased beginning at 7 days due to substrate depletion in the medium, which is consistent with previously reported results (Li et al., 2020;Li, Zhao, et al., 2018;Li, Zhou, et al., 2018).However, compared with the addition of FDP, the amount of HDMF produced by the ZrQOR strain was lower (Li et al., 2020).Pichia guilliermondii HDMF production was up to 92.5 mg/L in 100 g/L FDP medium (Zhang, 2009).After 5 days of complete fermentation in YPD supplemented with 120 g/L D-fructose and 180 g/L NaCl, HDMF production by native Z. rouxii reached 6.77 mg/L (Zhou, 2018).This result indicates that external supplementation with a carbon source is essential for HDMF production in native Z. rouxii (Thomas et al., 2001).
For dynamic analysis of QOR expression in engineered yeast (Figure 3b), QOR6 and native Z. rouxii were inoculated into YPD for simultaneous cultivation.Expression in both yeasts at 0, 3, 5, and 7 days was determined by qRT-PCR.Expression in QOR6 was found to be significantly higher than that in native Z. rouxii at 3, 5, and 7 days of fermentation (p < .001),with 4.8-fold, 3.3-fold, and 5.6-fold higher expression, respectively.The overall trend in relative QOR gene expression followed the same trend as HDMF production, first increasing and then decreasing.And biomass between the QOR6 and ZrQOR strains indicated a similar tendency (Figure 3c).Based on the above results, QOR gene expression and HDMF yield were positively correlated.This finding indicates that QOR, as a key catalyst for the biosynthesis of HDMF in Z. rouxii, can promote the production and accumulation of HDMF via the genetically engineered yeast, thus increasing the yield of HDMF.

| Observation of colony morphology and cell morphology
Due to the existence of repair mechanisms in yeast strains, not all transformed strains are able to stably maintain their beneficial traits (Hülter et al., 2020).Therefore, genetic stability analysis of strains is needed.
The cell morphology of each generation was observed by electron microscopy, as shown in Figure 4a.Generations 1-20 were all oval-shaped, and there was no significant difference in the morphology compared to the original generation of the engineered yeast.
The morphology of the colonies of the original and 20th generations of the ZrQOR strain is shown in Figure 4b, with a white, round, and smooth appearance, typical of yeast colonies.There was no significant difference between the generations, the morphology was the same as that of the original generation, and there were no aberrant colonies.

| Detection of target fragments
During successive iterations of engineered strains, plasmid replication is often delayed compared to cell division, resulting in the loss of the target plasmids in the engineered strain (Zheng et al., 2021).
Therefore, it is crucial to verify the stability of the plasmids carried by recombinant engineered strains to cultivate high-quality recombinant engineered strains.In this experiment, PCR was performed to verify the presence of plasmids in the 1st, 5th, 10th, 15th, and 20th generations of QOR6.This finding shows that the genotype of the ZrQOR strain remained unchanged after 20 generations and has high genetic stability.

| Analysis of HDMF production and stability of QOR gene expression by generations of QOR6
The production of HDMF is directly correlated with the growth of Z. rouxii (Thomas et al., 2001).The HDMF yield was measured after 3 days of incubation for each QOR6 generation from 0, 5, 10, 15, and 20.As shown in Figure 5a, the yield of HDMF was relatively stable at approximately 2.6 mg/L among all generations of QOR6.There was no significant difference in yield among generations, indicating that the HDMF yield was relatively stable at the 20th generation of inheritance.Therefore, the QOR gene can be stably inherited, and the yield of HDMF by the ZrQOR strain is significantly higher than that  Although FDP is the most favorable precursor substance found to promote HDMF production, it is not an ideal precursor substance for industrial production because of its high cost.Therefore, it is a new trend in development to combine fermentation engineering with genetic engineering to construct engineered yeast for producing target products.In this study, we successfully transferred the gene for the key enzyme involved in HDMF synthesis into native Z. rouxii.A genetically stable ZrQOR strain was developed with a brief cultivation period, producing a high HDMF yield.The objectives of time reduction and yield enhancement were successfully attained.However, the yield of HDMF was still lower in comparison with an exogenously added carbon source.Therefore, finding a suitable carbon source is necessary to increase the yield of HDMF.It is therefore necessary to continue studies on improving HDMF yield in the ZrQOR strain itself and its metabolic mechanism.Further pilot scale production is possible to produce HDMF in large quantities by the microbiological method in factories.
peak in HDMF synthesis by 2 days.HDMF production by QOR6 initially increased and then decreased, which was the same as the trend of HDMF production by native Z. rouxii.It is possible that HDMF F I G U R E 2 QOR gene expression of positive clones and native Zygosaccharomyces rouxii, based on the qRT-PCR results.*Representing p < .05,**representing p < .01,and ***representing p < .001are considered as statistically significant, respectively, the same as below.QOR, quinone oxidoreductase.