Enhanced β-carotene production by promoting the multivesicular body (MVB) pathway in Yarrowia lipolytica CURRENT

Background: β-carotene is a precursor of vitamin A and has great commercial value as an additive in foods and feeds. Many pathways not directly related to the β-carotene synthesis affect β-carotene production since the interactions among metabolic fluxes of cells confer a complex regulatory network. Engineered Y. lipolytica strain has excellent potential for β-carotene production as oleaginous yeast. Optimizing indirectly metabolic pathways in Y. lipolytica may offer a new strategy for making the β-carotene production achieve a commercially viable yield. Results: In this study, we found that the proper promotion of the multivesicular body (MVB) sorting pathway elevated the production of β-carotene by 1.58 fold when overexpressing one copy of the Did2 gene in Y. lipolytica . Through the measurement of ATP, NADPH, the mRNA, and protein level of key genes in the β-carotene synthesis pathway, the reason for β-carotene elevated was deuced that the protein level of the key enzymes (tHMG and CarA) was increased. When overexpressing two copies of the Did2 gene, the transcription level of the key genes was all elevated. However, the protein level of key enzymes in the β-carotene synthesis pathway was reduced compared with the overexpressing one copy of the Did2 gene, which resulted in reduced β-carotene content. Conclusion: This study suggests that the MVB sorting pathway is not responsible for sorting protein but has a crucial regulating effect on protein abundance in cells. sorting other high-value Moreover, manipulation of indirectly related metabolic pathways also is a critical in research. transcriptional YL-C32ts protein YL-C32bs YL-C31bs These results indicate that the protein level of key enzymes (tHMG-strepII, carB-strepII) in the β-carotene synthesis pathway was lower in the YL-C32 strain with overexpressing two copies of the Did2 gene.

that could increase β-carotene synthesis. Many genes from other pathways have significant and unexpected effects on β-carotene synthesis. The genes Cab1, Nsg1, Erg13, and Erg27 could promote β-carotene biosynthesis in S. cerevisiae as these genes involved in lipid biosynthesis [12]. The VOA1 improves the production of β-carotene in S. cerevisiae because VOA1 may result in a low pH of cell membranes [13]. Impressively, the gene Did2 related to protein metabolism increases β-carotene yield by 2.1 times in S. cerevisiae.
The amount of protein in cells is vital for the synthesis of β-carotene. The HMGR is a key enzyme of the β-carotene pathway. By substituting the entire sequence with a catalytic domain, the stability of protein HMGR was improved [14]. The production of β-carotene was significantly enhanced after overexpressing tHMG [15]. Overexpressing the Did2 gene has been shown to improve the production of β-carotene by promoting the transcription of β-carotene pathway genes (Hmg1, Erg12, Erg20, Erg8, Bts1, crtYB, and crtI). For example, the gene Did2, which is related to the protein trafficking, plays a role in the multivesicular body (MVB) sorting pathway. The MVB sorting pathway has two core components: ESCRTIII and Vps4. The Did2 recruits Vps4 to dissociate ESCRTIII from endosomes for the next cycle and increases the efficiency of the Vsp4 in the MVB sorting pathway [16]. However, the protein level in cells was not measured after overexpression of the Did2 gene, and the reason for the improved β-carotene synthesis by MVB sorting way was not given.
In the present study, we integrated both one copy and two copies of the Did2 gene into the Y. lipolytica chromosome to explore the effect of overexpressing the Did2 gene on the β-carotene synthesis and the mechanism of how the Did2 gene affects β-carotene synthesis. We discovered that overexpressing the Did2 gene prompted the MVB sorting pathway, subsequently improved the protein level of key enzymes in β-carotene synthesis, which led to the improvement of β-carotene production. We have provided evidence that Y. lipolytica cells coordinate transcriptional regulation and protein degradation to control protein abundance. Our study provides an excellent start to explore the potential of the yeast used for β-carotene synthesis by further manipulating other metabolic pathways in yeast cells. In addition, as the protein mediates most biological processes, our research also provides new knowledge for better utilization of the MVB sorting pathway to improve other high-valued biosynthetic products.

Strains and media
All strains used in this study are listed in Table S1. E. coli DH5α was used for routine cloning procedures, growing in Luria-Bertani (LB) medium at 37 °C, 220 rpm with 100 µg/mL of ampicillin when necessary. The Y. Lipolytica was cultivated in YPD medium or SD-Leu − medium. Y. Lipolytica was cultivated in YPD medium and incubated at 30 ℃, 150 rpm in 250 mL Erlenmeyer flasks containing 50% fermentation medium, and 2% inoculum. The Y. Lipolytica was precultured in test tubes containing 3 ml SD-Leu − medium at 30 °C, 200 rpm for 48 h, and then the cells were inoculated into a fresh medium with 2% inoculum.

Construction Of Recombinant Plasmids And Strains
All the plasmids and primers used in this study are listed in Table 1 and Table S1, respectively. For gene integration, the plasmid pJN44-Did2 was constructed that the gene Did2 (YALI0C10098g) was amplified with primers Did2-F/R from the Y.lipolytica genome and inserted at smaI/HindIII site of plasmid pJN44. Then the plasmid pURA-∆Gut2::Did2 and pURA-∆Gut2::2Did2 were formed that the expression cassette 'pTEF-Did2-CYC1t' was amplified with primers G-Did2-F/R from pJN44-Did2 and inserted into plasmid pURA-∆Gut2 at the site of speI. For western blot, a fusion StrepII tag is needed to add to the 3' end of tHMG, carRA, and carB gene. The tHMG, carRA, and carB gene were amplified with primers w-tHMG-tag-F/R, w-carB-tag-F/R, and w-carB-tag-F/R and inserted into plasmid pJN44 at the site of smaI to form plasmid pJN44-tHMG-StrepII, pJN44-carRA-StrepII, and pJN44-carB-StrepII. Table 1 The content of β-carotene and the relative mRNA levels of the genes in the MVB sorting pathway (Did2, Vps4) and the β-carotene synthesis pathway (Thmg, Ggs1, CarRA, CarB) in engineered β-carotene strains YL-C31 and YL-C32. FPP, one precursor of the β-carotene synthesis pathway, was measured following a previously published method with some modifications [31]. Alkaline phosphatase and Pyrophosphatase cleave the phosphoric acid moieties of FPP, convert FPP to farnesol.

Strains
Using GC-MS detected the content of farnesol to reflect the amount of FPP. The cell pellets of 100 ml medium were harvested and suspended in a 2 ml buffer (1 M diethanolamine, 0.5 mM MgCl2, pH 9.8). After lysing cells with sonication for 30 minutes, the sample was centrifuged at 12000*g for 10 min. The supernatant was added to Pyrophosphatase (10 U) at 25 ℃ for 1 h, followed by the addition of Alkaline phosphatase (10 U) at 30 ℃ for 1 h.
Finally, the farnesol was extracted by N-hexane for GC-MS detection. The β-carotene and lycopene were determined using a previously published method [9].
Determination Of Coenzyme Factor (atp, Nadph) The ATP was determined using the ATP Assay Kit (Beyotime; Shanghai, China) with some modifications. The process that firefly luciferase catalyzes the production of fluorescence needs the participation of ATP. Using the fluorescence intensity reflects the amount of ATP. The content of NADPH was detected using NADP+ /NADPH Assay Kit (Beyotime; Shanghai. China), based on WST-8 color reaction with some modifications.

Statistical analysis
All experiments were repeated three times. Data from each treatment are presented as means ± standard deviation. Statistical analyses were conducted using SPSS 18.0 (SPSS Inc; Chicago, IL, USA). Data in Figs. 2, 3, 4, 5, 6, and 7 were analyzed using one-way ANOVA, followed by Duncan's multiple range tests to determine the significant difference. P < 0.05 was considered statistically significant. Origin software 8.0 (Origin Lab; USA) was used for graphs construction.

Results
Overexpression of the Did2 gene promoted β-carotene synthesis The optimization of metabolic pathways indirect related to β-carotene synthesis improves the production of β-carotene. Engineered Y. lipolytica strain is oleaginous yeast, has excellent potential for β-carotene production, and a high capacity to store β-carotene [17]. To further explore the potential of engineered Y. lipolytica strain for producing βcarotene, we studied the effects of the indirectly related metabolic pathways on the βcarotene synthesis pathway. Several genes outside the β-carotene synthesis pathway have been shown to affect the production of β-carotene in S. cerevisiae [ 12,18]. For example, the incorporation of the Did2 gene, a member of the MVB sorting pathway, led to the most significant improvement of β-carotene yield by engineered S. cerevisiae [ 13]. The YL-C1 strain is an engineered Y. lipolytica with basal β-carotene producing capacity. We integrated the Did2 gene into YL-C1 at the Gut2 site, resulting in stain YL-C31 to explore the effect of the Did2 gene on β-carotene synthesis in the engineered Y. lipolytica strain.
The strain YL-C2, which the Gut2 gene was knocked out, was used as second control since the integration of the Did2 gene interrupted the Gut2 gene. β-carotene content was analyzed using HPLC after 96 h of fermentation. The β-carotene content in YL-C1, YL-C2, and YL-C31 strains was 9.85, 8.87, and 15.63 mg/g DCW, respectively; and the β-carotene concentration was 49.86, 51.45, and 80.65 mg/L, respectively (Fig. 1a, 1b). Overexpression of the Did2 gene improved the mRNA level of the genes in the β-carotene synthesis pathway We measured the relative mRNA level of Thmg, Ggs1, CarRA, and CarB, key genes in the β-carotene synthesis pathway, to explore the reason that the overexpression of the Did2 gene improved the production of β-carotene in Y. lipolytica. The actin gene was used as an internal reference [10]. The mRNA of key genes in YL-C1 was set as 1. For Thmg, the mRNA in YL-C31 was increased (P < 0.05) by 17% compared to YL-C1, increased (P < 0.05) by 30% compared to YL-C2 (Fig. 3a). For Ggs1, the mRNA in YL-C31 was increased (P < 0.05) by 45% compared to YL-C1, increased (P < 0.05) by 35% compared to YL-C2 ( Fig. 3b). For CarRA, the mRNA in YL-C31 was increased (P < 0.05) by 78% compared to YL-C1, increased (P < 0.05) by 97% compared to YL-C2 (Fig. 3c). For CarB, the mRNA in YL-C31 was increased (P < 0.05) by 55% compared to YL-C1, increased (P < 0.05) by 91% compared to YL-C2 (Fig. 3d). So, the mRNA of Thmg, Ggs1, CarRA, and CarB genes were all higher (P < 0.05) in the YL-C31 strain with the overexpressed Did2 gene.
Overexpression of the Did2 gene improved the utilization of precursors in the β-carotene synthesis pathway The utilization of the precursors is directly linked to β-carotene synthesis. The sesquiterpenes, diterpenes, triterpenes, and tetraterpenes all compete with β-carotene for the precursor FPP [19]. The lycopene is the direct precursor of β-carotene. To ascertain the effect of overexpression of the Did2 gene on the utilization of precursors, we measured the concentration of FPP and lycopene. The FPP and lycopene levels in YL-C1 were considered as 1. For FPP, the utilization of FPP in YL-C31 was increased (P < 0.05) by 34% compared to YL-C1, increased (P < 0.05) by 37% compared to YL-C2 (Fig. 4a). For lycopene, the utilization in YL-C31 was increased (P < 0.05) by 8.1% compared to YL-C1, increased (P < 0.05) by 7.9% compared to YL-C2 (Fig. 4b). The utilization of both FPP and lycopene in YL-C31 was highest (P < 0.05) among strains YL-C1, YL-C2, and YL-C31.
Overexpression of the Did2 gene increased the protein level of the key enzymes in the βcarotene synthesis pathway We performed the Western blot assay to measure whether the amount of the key enzymes in the β-carotene synthesis pathway was affected by the overexpression of the Did2 gene.
carB-strepII protein has the highest amount when the pJN44-carB-s was expressed in YL-C31. These results demonstrate that the overexpression of the Did2 gene increased (P < 0.05) the level of key enzymes (tHMG-strepII, carB-strepII). For carRA-strepII, the exact bands cannot be identified by Western blotting (As shown in Fig. S1). The reason may be that the stability of carRA-strepII is weak, and the protein was degraded during the extraction.
Overexpression of the Did2 gene increased the transcription level of the Vps4 gene in the

MVB sorting pathway
The Did2 gene is a positive regulator of the MVB sorting pathway [20]. The Did2 protein, one subunit of the ESCRT protein complex, recruits Vps4 protein to bind ESCRT.
Meanwhile, the Vps4 protein is a core factor of the MVB sorting pathway [21]. To explore the effect of the overexpression of the Did2 gene on the MVB sorting pathway, we measured the mRNA levels of the Vps4 gene and the Did2 gene. The mRNA level of the Did2 gene increased (P < 0.05) by 43% compared to YL-C1, and increased (P < 0.05) by 68% compared to YL-C2 (Fig. 6a). The mRNA level of the Vps4 gene increased (P < 0.05) by 28% compared to YL-C1, and increased (P < 0.05) by 47% compared to YL-C2 (Fig. 6b).
These results demonstrate that the mRNA level of the Vps4 gene was increased (P < 0.05) by the overexpression of the Did2 gene.
Overexpression of two copies of the Did2 gene further stimulated the MVB sorting pathway but reduced the protein level of key enzymes in the β-carotene synthesis pathway To further explore the effect of the MVB sorting pathway on the protein level of key enzymes in the β-carotene synthesis pathway, the MVB sorting pathway was promoted by overexpressing two copies of the Did2 gene. Two copies of the Did2 gene were integrated into the engineered Y. lipolytica strain (YL-C1) genome at the Gut2, resulted in YL-C32.
The mRNA level of the Vps4 and Did2 genes in YL-C32 was further elevated (P < 0.05) by 50% and 33% compared to YL-C31 (Table 1), respectively. Meanwhile, compared to the overexpression of one copy of Did2 gene, the mRNA level of the Thmg, Ggs1, CarRA, and CarB, the key genes in β-carotene synthesis pathway, increased (P < 0.05) by 63%, 57%, 54%, and 85% (Table 1), respectively. However, the protein level of tHMG-strepII in YL-C32ts was reduced compared to YL-C31ts (Fig. 7). For carB-strepII, the protein level in YL-C32bs was also reduced compared to YL-C31bs (Fig. 7). These results indicate that the protein level of key enzymes (tHMG-strepII, carB-strepII) in the β-carotene synthesis pathway was lower in the YL-C32 strain with overexpressing two copies of the Did2 gene.

Discussion
The optimization of the metabolic pathways indirect related to β-carotene synthesis makes the engineered Y. lipolytica strain beneficial for the expression β-carotene synthesis pathway. The engineered Y. lipolytica strain is oleaginous yeast, has excellent potential for β-carotene production since a lot of acetyl-CoA and a lipid body used to store βcarotene [9]. Previous strategies for improving the production of β-carotene focused mainly on enhancing the isoprenoid flux toward carotenoid production [22]. Many genes indirectly related to carotenoid synthesis have been shown to improve the production of carotenoid. The knockout of the gdhA gene, the enzyme responsible for converting alphaketoglutarate to glutamic acid, led to the increase of lycopene in S. cerevisiae [ 23,24].
Deletion of the genes Ser33 (related to amino acid synthesis), Prb1 (related to vacuolar protein degradation), and Rox1 (a transcription repress factor) improved the production of carotenoid [18]. Overexpression of the genes Tif 5 (a translation initiation factor), Voa1 (vacuolar H(C)-ATPase subunit 1), and Did2 (a subunit in the MVB sorting pathway) genes enhanced the production of β-carotene in S. cerevisiae [ 13]. Overexpression of the Did2 gene led to a 2.1-fold improvement of β-carotene production in S. cerevisiae [ 13]. In this study, we integrated the Did2 gene into the engineered Y. lipolytica strain and the production of β-carotene improved 1.58-fold. The reason for promotion could be deduced that the protein level of the key enzymes in the β-carotene synthesis pathway was increased by overexpression of Did2.
Proper promotion of the multivesicular body sorting pathway improves the protein level of key enzymes in β-carotene synthesis pathway. The cells control protein abundance by coordination of protein synthesis and degradation. When protein degradation was elevated, the cells regulate protein level by enhancing the mRNA expression to compensate for the effect of protein degradation [25]. The MVB sorting pathway plays a key role in protein degradation [26][27][28]. ESCRT, the key unit in the MVB sorting pathway, binds to endosomes to sort proteins for protein degradation or to transport to other organelles. The Did2 protein, one subunit of ESCRT protein complex, activates and recruits (tHMG-strepII and carB-strepII) was reduced. Analyzed the causes, we deduced that the transcription level of gene was not enough to compensate for the degradation of the corresponding protein though its mRNA level was elevated. Furthermore, the protein level of both tHMG-strepII and carB-strepII were all decreased, which resulted in the reduced the β-carotene content by 25%. Therefore, the overexpression of two copies Did2 gene was excessive for the promotion of the MVB sorting pathway. Furthermore, combined all results in this study, we can suggest the MVB sorting pathway is not responsible to protein degradation but has important regulating effects on protein abundance in cells.

Conclusion
In this study, we found the proper promotion of the MVB sorting pathway elevated the production of β-carotene in Y. lipolytica by overexpressing one copy of the Did2 gene. The reason for enhanced β-carotene production most likely is attributed to increased mRNA and protein levels of key genes, which resulted from the promotion of the MVB sorting pathway. These results suggest that engineering the MVB sorting pathway could potentially increase the production of other high-value products. Moreover, manipulating indirectly related metabolic pathways also is a critical strategy in metabolic engineering.  Table S1. Strains and plasmids used in this study Table S2. List of primers used in this study       The protein level of the key enzymes reduced after overexpressing two copies of the Did2 gene compared with the overexpression of one copy of the Did2 gene. Western blot result of the tHMG-strepII fusion protein in the strains YL-C31ts and YL-C32ts and the carB-strepII fusion protein in the strains YL-C31bs and YL-C32bs.