All strains used in this study are listed in Additional file 1: Supplementary Table S1. Escherichia coli were grown at 37°C with constant shaking in LB medium for plasmid propagation. The wild-strain Y. lipolytica 2,021,417 was used as the initial strain for modification. Y. lipolytica cells were cultured at 30°C in YPD medium for strain activation or SC medium for screening transformants. SC medium (g/L): yeast nitrogen base (YNB) 1.7, (NH₄)₂SO₄ 5, glucose 20. Note: add appropriate antibiotics or nutrients (400 μg/ml hygromycin B or 700 μg/ml bleomycin or 0.1 g/l leucine) to the medium before inoculation or plate coating. High glycerol medium (g/L): crude glycerol 500, yeast extract 5. Medium glycerol medium (g/L): crude glycerol 300, yeast extract 5. Erythritol fermentation medium (g/L): crude glycerol 250, yeast extract 1, NH₄Cl 5, KH₂PO₄ 0.25, MgSO₄·7H₂O 0.5. Erythritol fermentation medium (pure glycerol as a carbon source; g/L): pure glycerol 250, yeast extract 1, NH₄Cl 5, KH₂PO₄ 0.25, MgSO₄·7H₂O 0.5. Erythritol fermentation medium (glucose as a carbon source; g/L): glucose 250, yeast extract 1, NH₄Cl 5, KH₂PO₄ 0.25, MgSO₄·7H₂O 0.5. The crude glycerol in this work was obtained from biodiesel waste. The composition of crude glycerol is (v/v): glycerol 80–85%, sodium salt 2.0%, methanol 10–15%, other organic matter 2.5%, and water 2%. 0.1 mol/l lithium acetate: accurately weigh 1.02 g of lithium acetate, dissolve in 90 ml of distilled water, adjust pH 6.0 with acetic acid, then fix the volume to 100 ml, sterilize at 115°C for 20 min and store at −20°C. 40% PEG4000: accurately weigh 20 g of PEG4000, dissolve in 30 ml of 0.1 mol/l lithium acetate (pH 6.0), dissolve fully, fix the volume to 50 ml, sterilize at 115°C for 20 min and store at −20°C.

All plasmids and primers used in this study are listed in Additional file 1: Supplementary Tables S2, Figures S3, respectively. For the knockout of Ku70 and LEU2, two primer pairs (Ku70-sg-1 and LEU2-sg-1) were designed to construct plasmids pCAS1yl-∆Ku70 and pCAS1yl-∆LEU2, respectively. The Ku70-UP-F/R, Ku70-DOWN-F/R, LEU2-UP-F/R, and LEU2-DOWN-F/R primer pairs were used to amplify the upstream and downstream homologous arms of 1,000 bp each from Y. lipolytica 2,021,417 genomic DNA. The hygB gene from Coccidioides posadasii was synthesized in pUC-GW to construct pUC-GW-hygB by Genewiz (Suzhou, China) with codon optimization. The hygB gene cloned into KpnI linearized pINA1269 using the hygB-F/R primer pair to generate plasmid pINA1269-hygB. The fragment of hp4d-hygB-XPR2t from pINA1269-hygB was then amplified using the hp4d-hygB-XPR2t-F/R primer pair. A fusion fragment Ku70 UP-hp4d-hygB-XPR2t-Ku70 DOWN and LEU2 UP-hp4d-hygB-XPR2t- LEU2 DOWN was subsequently generated. The fusion fragments were cloned into PmeI linearized pCAS1yl-∆Ku70 and pCAS1yl-∆LEU2 to generate pCAS2yl-∆Ku70 and pCAS2yl-∆LEU2, respectively. The LEU2 sgRNA expression cassette from pCAS2yl-∆LEU2 was then amplified with LEU2 F/R primer pair and subsequently cascaded with the Ku70 sgRNA expression cassette in pCAS2yl-∆Ku70 to obtain pCAS2yl-∆Ku70∆LEU2 for Y. lipolytica Y01 transformation (Gao et al., 2016). EYD1 was knocked out using bleomycin as a screening marker, and the plasmid construction was the same as the knockout of Ku70 and LEU2.

For single gene overexpression, the construction of pINA1269-GUT1 was used as an example. 1,512 bp DNA fragment of gene GUT1 was PCR amplified using genomic DNA of strain Y. lipolytica 2021417 as a template, and primer pair GUT1-F/R, respectively. The purified GUT1 fragment was then digested using KpnI, and cloned at the corresponding sites of plasmid pINA1269 to generate pINA1269-GUT1. For tandem overexpression of two genes, the construction of pINA1269-GUT1-GUT2 was used as an example. The hp4d-GUT2-XPR2t fragment was PCR amplified using pINA1269-GUT2 as a template, and primer pair hp4d-GUT2-XPR2t-F/R, respectively. The GUT1 fragment and hp4d-GUT2-XPR2t fragment were then fused to obtain the GUT1-hp4d-GUT2-XPR2t fragment. The purified GUT1-hp4d-GUT2-XPR2t fragment was digested using KpnI, and cloned at the corresponding sites of plasmid pINA1269 to generate pINA1269-GUT1-GUT2. For tandem overexpression of three genes, the construction of pINA1269-GUT1-GUT2-TKL1 was used as an example. The hp4d-TKL1-XPR2t fragment was PCR amplified using pINA1269-TKL1 as a template, and primer pair hp4d-TKL1-XPR2t-F/R, respectively. The GUT1-hp4d-GUT2-XPR2t fragment and hp4d-TKL1-XPR2t fragment were then fused to obtain the GUT1-hp4d-GUT2-XPR2t-hp4d-TKL1-XPR2t fragment. The purified GUT1-hp4d-GUT2-XPR2t-hp4d-TKL1-XPR2t fragment was digested using KpnI, and cloned at the corresponding sites of plasmid pINA1269 to generate pINA1269-GUT1-GUT2-TKL1. For the construction of pINA1269-GUT1-GUT2-TKL1-TAL1, the hp4d-TAL1-XPR2t fragment was PCR amplified using pINA1269-TAL1 as a template, and primer pair hp4d-TAL1-XPR2t-F/R, respectively. The GUT1-hp4d-GUT2-XPR2t-hp4d-TKL1-XPR2t fragment and hp4d-TAL1-XPR2t fragment were then fused to obtain the GUT1-hp4d-GUT2-XPR2t-hp4d-TKL1-XPR2t-hp4d-TAL1-XPR2t fragment. The purified GUT1-hp4d-GUT2-XPR2t-hp4d-TKL1-XPR2t-hp4d-TAL1-XPR2t fragment was digested using KpnI, and cloned at the corresponding sites of plasmid pINA1269 to generate pINA1269-GUT1-GUT2-TKL1-TAL1.

The wild-strain Y. lipolytica 2,021,417 was cultured in a YPD medium for 24 h, centrifuged at 9000 × g, and washed three times with PBS. Then, the cell suspension was exposed to UV light using Biosan UVC/T-M-AR (Biosan, Latvia) until the cell survival was less than 0.05%, and the cells were inoculated onto YPD plates at 30°C. After the mutagenic strains were cultured, the strains with larger and thicker colonies were selected, numbered, and inserted into a test tube for storage. In contrast, the corresponding strains were inoculated into 250 ml shake flasks (50 ml Erythritol fermentation medium) at 30°C, 220 r/min for 120 h, and the erythritol titers were determined.

The lithium acetate method was used to transform Y. lipolytica as described previously (Gietz and Schiestl, 2007). In brief, a single colony of Y. lipolytica was inoculated into 10 ml of YPD and incubated with shaking at 30°C, 220 r/min for 8–10 h (OD₆₀₀ ≈ 0.3 ~ 0.5). Cells were harvested by centrifugation at 6000 × g for 5 min. The cells were then washed with sterile water, resuspended the cells with 1 ml 0.1 mol/l lithium acetate and incubated for 10 min at room temperature for transformation. The transformation mix was added to the cells in the following order: 10 μl 10 mg/ml ssDNA, 1 μg linearized plasmid DNA, 700 μl 40% PEG4000. The centrifuge tube was vortexed until the cell pellet was completely mixed. Cells were incubated at 30°C for 30 min and then heat shocked in a water bath at 42°C for 30 min. Cells were centrifuged to remove the transformation mix and resuspended in 100 μl of sterile water. Cells were then plated on the appropriate selection agar plates. Colonies were verified by PCR and then selected for erythritol production.

Seed culture was carried out in a 500 ml flask containing 50 ml of YPD medium on a rotary shaker at 30°C and 220 r/min for 20 h. An inoculum of 20% was introduced into a shake flask containing 30 ml of the Erythritol fermentation medium. Shake flasks were performed on a rotary shaker at 30°C and 220 r/min for 120 h. An inoculum of 20% was introduced into a bioreactor containing 1.6 l of the Erythritol fermentation medium. Batch cultivations were carried out in a 5-L fermenter (Baoxing Co., China) at 30°C with a working volume of 2 l. The aeration rate was fixed at 1.0 l/min. The stirrer speed was adjusted to 800 rpm and the dissolved oxygen concentration was maintained at 20–30%. pH was maintained automatically at 3.0 by the addition of 20% (w/v) of NaOH solution. All cultures were carried out in three replications.

The concentrations of erythritol, mannitol, arabinitol, and glycerol were determined by HPLC (Agilent 1,200 series; Agilent Technologies). The sample volume was 20 μl using an amino column 70 Å NH₂ (250 × 4.6 mm; 5 μm) eluted with 80% acetonitrile as the mobile phase at a flow rate of 1.0 ml/min. The detector was a RID detector coupled to a detector temperature of 35°C and a column temperature of 40°C. The mass yield (Y_ERY) and volumetric productivity (Q_ERY) of erythritol and glycerol consumption were calculated based on previous studies (Mawire et al., 2020; Wang et al., 2020).

Total RNA was extracted from Y. lipolytica according to the manufacturer’s instructions using the Bead-Beat Total RNA Mini kit (A&A Biotechnology, Gdynia, Poland). The TranScriba kit (A&A Biotechnology, Gdynia, Poland) was used to synthesize the cDNA strands.

Quantitative PCR (qPCR) was performed in a 7,500 real-time PCR thermal cycler (Applied Biosystems, Waltham, MA, USA) using an SYBR®Green B PCR MasterMix (A&A Biotechnology, Gdynia, Poland). Each reaction contained 0.5 μl of cDNA template, 5 μl of SYBR®Green B PCR MasterMix, and 0.5 μl of forwarding and reversed primers, which were made up to 10 μl using ddH₂O. Reaction conditions were as follows:95°C for 3 min, 95°C for 15 s, 60°C for 30 s and 72°C for 30 s, 2–4 steps of 40 cycles, and melting curve phases: 94°C for 15 s, 60°C for 60 s, 95°C for 30 s, 60°C for 15 s. Each qPCR reaction was performed in technical replicates.

According to recent studies, highly osmotolerant yeast strains isolated from the daily activities of honey bees were the major source of erythritol-producing strains (Moon et al., 2010). To obtain effective strains for erythritol production, we screened surviving strains from honey with high glycerol medium and inoculated them into medium glycerol medium for fermentation. The erythritol-producing strain 2,021,417 was screened and analyzed by HPLC. It was sequenced and identified as Y. lipolytica, producing 10 ± 0.07 g/l erythritol in shake flask fermentation (Figure 1A). To further enhance the erythritol production capacity of Y. lipolytica 2,021,417, UV mutagenesis was first performed (Figure 1B). The strains obtained by UV mutagenesis were fermented in a shake flask. The fermentation results are shown in Figure 1C. The erythritol production of some strains decreased after UV mutageneses, such as UV-2 and UV-9. Meanwhile, the erythritol production was also increased in most strains, such as UV-3, UV-5, UV-6, UV-11, UV-12, UV-16, UV-17, UV-21, and UV-22 reached more than 20 g/l, among which UV-16 had the highest erythritol titer of 27 ± 0.03 g/l. Compared to the initial strain 2,021,417, the erythritol titer of UV-16 increased by 170%. Therefore, UV-16 was renamed Y. lipolytica Y01 for further metabolic engineering.

A mutant Y. lipolytica Y01 was obtained by UV mutagenesis. Next, metabolic engineering will be combined to improve erythritol production further. First, the antibiotic markers were screened for Y. lipolytica Y01 knockout to create a chassis strain that could be genetically modified (The screening of antibiotics is listed in Additional file 1: Supplementary Figure S1). Non-homologous end joining (NHEJ) and homologous recombination (HR) are the main genome engineering approaches used by Y. lipolytica. Due to non-specific NHEJ, HR in Y. lipolytica is limited in terms of integration efficiency and length (Abdel-Mawgoud and Stephanopoulos, 2020). The HR efficiency of short-length flanking fragments was enhanced when the Ku70 gene responsible for repairing DNA double-strand breaks (DSBs) in the NHEJ pathway was absent (Verbeke et al., 2013). Therefore, the gene Ku70 was knocked out to improve integration efficiency. Furthermore, since the integration plasmid used in this work was leucine back-complemented, the auxotrophic strain for leucine was constructed by knocking out gene LEU2. Hygromycin B was used as a screening marker to knockout genes Ku70 and LEU2, laying the foundation for later molecular manipulation (Figure 2A). The Y. lipolytica Y01-ΔKu70ΔLEU2 and Y. lipolytica Y01 were incubated in Erythritol fermentation medium for 120 h, and their growth curve was determined. The results showed that the growth trends of Y. lipolytica Y01-ΔKu70ΔLEU2 and Y. lipolytica Y01 were similar, which indicated that the knockout of Ku70 and LEU2 had no negative effects on fitness (Figure 2B). In addition, the conversion rate of positive transformants was 2% (1/50) when the 7.2 kb DNA fragment was integrated into the Y. lipolytica Y01 genome, whereas the conversion rate reached 80% (40/50) for Y. lipolytica Y01-ΔKu70ΔLEU2 (Figure 2C). Next, we will construct and optimize the erythritol-producing strains based on Y. lipolytica Y01-ΔKu70ΔLEU2.

Glycerol kinase and glycerol-3-P dehydrogenase are encoded by GUT1 (YALI0F00484g) and GUT2 (YALI0B13970g), respectively. These two enzymes are mainly involved in glycerol assimilation in the erythritol synthesis pathway (Figure 3; Mirończuk et al., 2015). To improve the glycerol assimilation of Y. lipolytica Y01-ΔKu70ΔLEU2, we obtained strains Y-02, Y-03 and Y-04 by overexpressing GUT1 and GUT2 separately and in tandem. We first evaluated the expression levels of GUT1 and GUT2 by RT-qPCR of total RNA. 18sRNA was used as the reference gene. According to the analysis, the Y-02, Y-03, and Y-04 strains had higher expression levels of GUT1 and GUT2 (Figure 4A). Notably, both GUT1 and GUT2 expression levels were raised by overexpressing either GUT1 or GUT2, with an 11-fold increase in GUT1 expression in strain Y-02 (pINA1269-GUT1). However, the expression levels of GUT1 and GUT2 were only slightly up-regulated in the co-expression strain, which may be due to the mutual coordination between genes to maintain the dynamic balance of the cells. The results were similar to those obtained in previous studies (Mirończuk et al., 2016; Martău et al., 2020). Based on these results, the erythritol titer, Y_ERY, Q_ERY, and glycerol assimilation of the engineered strains were determined by shaking the flask. The results of the shake flask experiments are summarized in Figures 4B–D. The results indicated that there was no significant different in crude glycerol consumption before 48 h. After 48 h, the crude glycerol consumption of all engineered strains increased rapidly. In strain Y-04 (pINA1269-GUT1-GUT2), crude glycerol consumption was increased by 33% compared to the control strain (Y. lipolytica Y01-ΔKu70ΔLEU2; Figure 4B). Mirończuk et al. (2016) also found that glycerol consumption was higher in Y. lipolytica strain Y101 with tandem overexpression of GUT1 and GUT2. Carly et al. (2017) overexpressed GUT1 and GUT2 separately or in tandem for Y. lipolytica strain Po1d and found that the engineered strain overexpressing GUT1 had a higher specific glycerol consumption rate. This may be due to the different expression levels of the same gene in different hosts. Furthermore, we found that crude glycerol consumption of the engineered strains increased rapidly after 48 h. As the synthesis of erythritol was regulated by growth, the cells were induced to produce erythritol after the culture reached a stationary phase at about 48 h. The rapid increase in crude glycerol consumption indicated that crude glycerol consumption and erythritol synthesis were interrelated.

For strains Y-02 (pINA1269-GUT1) and Y-03(pINA1269-GUT2), the erythritol titers were 35 g/l and 31 g/l, which increased by 29.6 and 14.8% compared to the control strain, achieving Y_ERY 0.39 g/g and 0.35 g/g, Q_ERY 0.292 g/l/h and 0.258 g/l/h, respectively (Figures 4C,D). Previous research discovered that erythritol synthesis and the conversion of erythritol to glycerol were significantly affected by the overexpression of GUT1. Still, overexpression of GUT2 only affected glycerol consumption of the engineered strain and did not significantly improve erythritol synthesis, which was verified by our results (Mirończuk et al., 2016; Carly et al., 2017). The highest erythritol titer, Y_ERY, and Q_ERY were achieved in strain Y-04 (pINA1269-GUT1-GUT2). For strain Y-04, the erythritol titer, Y_ERY, and Q_ERY were 51.9, 56.7, and 52% higher than the control strain (41 and 27 g/l, 0.47 and 0.30 g/g, 0.342 and 0.225 g/l/h, respectively; Figures 4C,D). A significant increase in all process parameters could be found for strain Y-04, which further demonstrated that the tandem overexpression of GUT1 and GUT2 not only enhanced crude glycerol consumption but also improved erythritol synthesis.

Transketolase and transaldolase, which are primarily involved in the supply of precursors in the erythritol synthesis pathway, are encoded by the genes TKL1 (YALI0E06479g) and TAL1 (YALI0F15587g), respectively (Figure 3; Mirończuk et al., 2017). Thus, to further enhance precursors supply, TKL1 and TAL1 were overexpressed in Y. lipolytica Y01-ΔKu70ΔLEU2, and engineered strains Y-05, Y-06, and Y-07 were obtained. The RT-qPCR results of total RNA showed that the relative expression levels of GUT1, GUT2, TKL1, and TAL1 were increased in all engineered strains, which was consistent with the overexpression results of GUT1 and GUT2 (Figure 4A).

Based on these results, we evaluated the effect of TKL1 and TAL1 overexpression on erythritol production. For strain Y-05 (pINA1269-TKL1), the erythritol titer, Y_ERY and Q_ERY increased to 40 g/l, 0.45 g/g and 0.333 g/l/h, which were increased by 48.1, 50 and 48% compared to the control strain, respectively (Figure 4C, D). This suggested that transketolase (TKL1) is more important in promoting erythritol synthesis. Carly et al. (2017) found that overexpression of TKL1 obtained the best results in terms of reduced fermentation time and improved erythritol production compared to overexpression of other genes. In addition, Mirończuk et al. (2017) discovered that transketolase was a key enzyme for erythritol synthesis in Y. lipolytica, and overexpression of TKL1 resulted in a 2-fold improvement in erythritol synthesis. In light of these findings, we further investigated the effect of tandem overexpression of multiple genes on erythritol synthesis.

As mentioned above, tandem overexpression of GUT1 and GUT2 enhanced crude glycerol assimilation and improved erythritol synthesis in the engineered strain. Overexpression of TKL1 significantly increased erythritol titer, Y_ERY, and Q_ERY. Co-expression of multiple key genes has been shown to improve cellular metabolic performance. Therefore, to further enhance erythritol production, GUT1, GUT2, and TKL1 or GUT1, GUT2, and TAL1 were overexpressed in tandem in strains Y-08 and Y-09. For strain Y-08, the erythritol titer, Y_ERY, and Q_ERY reached 65 g/l, 0.56 g/g, and 0.541 g/l/h, which were increased by 58.5, 19.2, and 58.2% compared to the control strain Y-04, respectively (Figures 4C,D). This suggested that the primary rate-limiting processes in erythritol synthesis were glycerol assimilation and precursor supply, and enhancing these reactions might assist increase erythritol production. To further develop this push and pull strategy, GUT1, GUT2, TKL1, and TAL1 were overexpressed in tandem to obtain the engineered strain Y-10. For strain Y-10 (pINA1269-GUT1-GUT2-TKL1-TAL1), erythritol titer, Y_ERY, and Q_ERY were reduced by 15.3, 9.1, and 15.3% compared to the strain Y-08 (pINA1269-GUT1-GUT2-TKL1; Figures 4C,D). Overexpression of TAL1 with GUT1, GUT2 and TKL1 in tandem could not further enhance erythritol production. Therefore, overexpression of GUT1, GUT2, and TKL1 in tandem was the combination that maintained the highest erythritol titer, Y_ERY, and Q_ERY.

Although Y. lipolytica can produce high levels of erythritol, it is also capable of consuming erythritol as a carbon source. This ability has a negative impact on erythritol productivity and is a serious drawback for the development of high erythritol-producing strains. The EYD1 (YALI0F01650g) gene encoding erythritol dehydrogenase is involved in the catabolic pathway of erythritol (Carly et al., 2018). To prevent the catabolism of erythritol, the EYD1 gene was knocked out and strain Y-11 was obtained. As expected, strain Y-11 was unable to use erythritol in a crude glycerol-deficient medium, whereas strain Y-08 was able to begin consuming erythritol after crude glycerol depletion (Figure 5A). As a result, it can be inferred that EYD1 is involved in the catabolic pathway of erythritol, and the knockdown of EYD1 effectively inhibited the catabolism of erythritol. In addition, strain Y-11 had better erythritol production performance. Compared to strain Y-08, the erythritol titer, yield, and productivity were increased by 9.4, 3.6, and 9.2%, respectively (Figure 5B).

Previous experiments have shown that engineered strain Y-11 was the most capable of producing erythritol from crude glycerol. Next, we investigated whether the Y-11 producer capabilities could be fully utilized for large-scale production. In the 5-L bioreactor, the capacity of Y-11 and control strain (Y. lipolytica Y01-ΔKu70ΔLEU2) to produce erythritol was evaluated. Cell growth, crude glycerol consumption, erythritol titer, Y_ERY, and Q_ERY of the fermentation process were monitored (Figure 6). There was no significant difference in cell growth between engineered strain Y-11 and the control strain. For strain Y-11, it rapidly grew to a maximum OD₆₀₀ of 109 and reached a stable phase at 48 h. The crude glycerol consumption of strain Y-11 was similar to that of the control strain before 48 h. After 48 h, the crude glycerol consumption of Y-11 increased rapidly, which was consistent with the results of the shake flask experiments. The erythritol titer, Y_ERY, and Q_ERY of Y-11 reached 150 g/l, 0.62 g/g, and 1.25 g/l/h, which increased by 172.7, 31.9, and 179.4% compared to the control strain, respectively, indicating that appropriate metabolic modification greatly improved crude glycerol consumption, erythritol titer, Y_ERY and Q_ERY of Y. lipolytica (Figure 6). In addition, the results demonstrated that the erythritol titer, Y_ERY, and Q_ERY in the 5-L bioreactor were increased by 111, 7, and 111% compared to that obtained in shake flasks, respectively. It was identified that erythritol could be produced more efficiently in the 5-L bioreactor for large-scale production, which may be due to the high dissolved oxygen requirement of Y. lipolytica during the fermentation process. The limited dissolved oxygen in the shake flasks cannot meet the needs during the fermentation process.

Crude glycerol, a mixture of glycerol and other substances, was produced as a by-product along with biodiesel production. It is unclear if crude glycerol contains components that inhibit the growth and product production of engineered strains. To determine whether crude glycerol affects the erythritol production by the engineered strain Y-11, we fermented Y-11 and control strain under different carbon sources (Figure 6). The results revealed no significant difference in the growth of Y-11 and control strain in glucose, pure glycerol, and crude glycerol. In contrast, the erythritol titer, Y_ERY, and Q_ERY of Y-11 in crude glycerol were slightly increased compared to the glucose and pure glycerol. These results indicated that the waste substrate in crude glycerol had no negative effect on the cell growth and erythritol synthesis of strain Y-11. Furthermore, Y-11 was fermented with and without NaCl. The results revealed that the addition of NaCl effectively reduced the by-products mannitol and arabinitol by 46 and 30%, mainly because increased osmotic pressure affected the proportions of erythritol, mannitol, and arabinitol produced by Y. lipolytica. The same results were also obtained in previous studies (Mirończuk et al., 2017; Bilal et al., 2020).