Expression of Yl-SPT15 mutant libraries in assembled cassettes
According to the amino acid sequence of Spt15 in S. cerevisiae, we searched the conserved domains in Y. lipolytica by BLAST in NCBI database and got a Spt15-like protein named as Yl-Spt15 coded by YALIOB23056g on chromosome B (Fig. 1a and Additional file 1: Figure S1). This Yl-Spt15 owned the same conserved regions of “repeat element 1,” “helix 2,” “repeat element 2,” and “helix 2'” with Spt15 (Additional file 1: Figure S1, Additional file 2). Since the mutants of Spt15 in S. cerevisiae were proved effective to promote phenotypic evolution in many ways, we decided to construct the mutants of its potential counterpart Yl-Spt15 in Y. lipolytica to test its effects on product synthesis such as lipophilic products [10, 11, 34]. The manipulated Yl-SPT15 mutant libraries (see “Methods” for detailed construction process) were inserted in designed five expression cassettes and assembled meanwhile integrated in yeast chromosomal GUT2 site by in vivo homologous recombination (HR) (Fig. 1b, Additional file 1: Figure S2a, b and “Methods”). The correct rate of three-cassette assembly and integration at GUT2 site was about 7.3% and could be further improved to 18.0% in the strain with ku70 knockout (Additional file 1: Figure S3). Although the efficiency was not so high, this site primarily could be employed for integration of multiple Yl-SPT15 mutants. This kind of combinatorial mutagenesis of Yl-SPT15 permitted increased frequencies of both wild-type copies and mutant-type copies, offering expanded tuning range to identify dominant mutations that cause new functions in the presence of other unaltered genes.
To test whether the design of combinatorial Yl-SPT15 mutation was effective to tune metabolism of lipophilic products, we constructed a heterologous hybrid pathway to synthesize beta-carotene, a typical lipophilic product. Beta-carotene was used here for its lipophilic trait and visible color convenient for available yeast colony selection [35]. The heterologous carotene would introduce a change in the metabolism and composition of cell’s lipophilic properties, and through selection of drastically changed carotene production in yeast colonies, we obtained a chance to learn how cells tuned both global gene transcription and intrinsic lipophilicity. The carotene pathway containing four modules of H0-EXP1p-crtE-XPR2t-H1, H1-TEFp-crtB-LIP2t-H2, H2-GPDp-crtI-OCTt-H3, H3-GPATp-crtY-PEX16t-H4 and a left module “rDNAL-Ura3-H0” and a right module “H4-rDNAR” were assembled and integrated in chromosomal rDNA site (Additional file 1: Figure S2c, the strain was named as Yl_ini). Both “rDNAL” and “rDNAR” were 700 bp referenced to Gao’s design [17]. We got a similar phenomenon that the integration in rDNA was actually not exactly single copy. Quantitative PCR (Q-PCR) indicated that Yl_ini slightly underwent unexpected additional integrations of partial cassettes containing TEFp, GPDp, and GPATp (Additional file 1: Figure S4, Additional file 3: Table S1, “Methods”). However, we considered that these already obtained integrations were stable based on the nature of rDNA site, and the initial carotene production was also stable as detected. Based on this Yl_ini strain, we assembled sequential five cassettes of mutant libraries in yeast cells and successfully selected obvious color-changed yeast colonies. A most “enhanced” colony and a most “weakened” colony was selected by eye judgement for further analysis. It was proved that only cassettes of wild-type Yl-SPT15 did not affect colony’s color and carotene production (Additional file 1: Figure S5a).
Effects of combinatorial mutagenesis of Yl-SPT15 on beta-carotene production
The control strain containing cassettes of wild-type Yl-SPT15 was named as Yl_5_1, the visible “enhanced” strain was named as Yl_5_2, and the visible “weakened” strain was named as Yl_5_3 as follows (Fig. 2a). Firstly, the genotypes of selected strains were tested. According to the results of normal PCR and sequencing with special primers (“Methods” and Additional file 3: Table S2), the left integration site was exactly located upstream of GUT2’s open read frame (ORF), but the right integration site was not completely explicit. The designed homologous arms (H8) were 70% in accordance with the sequenced PCR products, but GUT2R could not be verified in alignment. This unexpected integration might lead to partial gene knockdown instead of knockout. The results of Q-PCR proved this speculation, as the expression of GUT2 was only partially suppressed to nearly half of previous level (Additional file 1: Figure S6 and Additional file 3: Table S1, “Methods”). By contrast, the expression of Yl-SPT15 was almost at the same level across all the tested strains including Yl_ini, Yl_5_1, Yl_5_2, and Yl_5_3, meaning the cells kept the Yl-SPT15’s transcription at a conservative level, although extra five cassettes were integrated.
The sequencing results showed that Yl_5_2 did receive more than one mutation in different cassettes, one (Glu208Ala) localized in the first module and three (Pro25Thr, Ala213Thr, Ala216Val) localized in the fifth module (Fig. 2b, Additional file 3: Table S3). The Yl_5_3 contained only one mutation (Phe194Leu) localized in the second module. All of the other modules at the expected locations in strain Yl_5_1, Yl_5_2, and Yl_5_3 were the cassettes of wild-type Yl-SPT15. As seen, four of all five mutations existed in the conserved transient region across “repeat element 2” (beta sheets) to “helix 2’” (alpha helix) [32, 36], indicating that this region was necessary to determine TF’s activity, similar within S. cerevisiae [6].
The production of beta-carotene was tested for these three strains (Fig. 2c). We chose defined media instead of rich media to remove interference caused by rich nutritional conditions. The carotene production in Yl_5_1 was 3.03 mg/L (0.87 mg/g DCW). Yl_5_2 got enhanced production as 12.34 mg/L (4.09 mg/g DCW, 4.7-fold of the Yl_5_1’s value), and Yl_5_3 got decreased production as 0.40 mg/L (0.11 mg/g DCW, 0.13-fold of the Yl_5_1’s value). The distinguished productions were directly reflected by the varied depth of strains’ yellow color (Fig. 2a). The growth rate was slightly different between strains (Additional file 1: Figure S5b). The Yl_5_2 with highest carotene production got lowest OD600, but the Yl_5_3 with lowest carotene production got highest OD600. An evaluation of the effects of each mutation on carotene production was also done. The exact single or combinatorial point mutants were introduced to natural Yl-SPT15 gene by PCR instead of directly cloning the sequencing-verified mutants from genome. Their individual expression cassette on plasmid pLD-EcYl was transformed into Yl_5_1. The carotene detection results suggested that the mutants in module 5 in Yl_5_2 and the mutant in module 2 in Yl_5_3 played significant role of impacting carotene production in each strain. It was also proved that the enhanced phenotype in Yl_5_2 was a result of combined efforts from different mutants (Additional file 1: Figure S7).
To test the influences of different levels of glucose, the strains were cultivated under 20, 30, and 40 g/L glucose keeping other conditions constant (Fig. 2d). Generally, the carotene yields increased as the glucose concentration increased, but there was slight impact on milligram per gram DCW values. When the glucose concentration was 40 g/L, we got the most enhanced production of carotene as 15.29 mg/L (4.29 mg/g DCW) in strain Yl_5_2, a limited 23.96% improvement. It was supposed that Y. lipolytica strains under SC-Ura-Leu fermentation could not present highest milligram per liter yield, and the SC other than YPD medium was used here for clearly detecting and analyzing cells’ internal change [37].
Exploration of key changed genes and pathways by transcriptome analysis
In a recent work, the transcriptome analysis was done for Y. lipolytica strains producing heterologous lycopene or not [38]. Our work focused on the changes in different strains producing varied levels of beta-carotene. To reveal the effects of combinatorial mutation of Yl-SPT15 on global transcription, the transcriptome analysis was done for the three strains of Yl_5_1 (as control), Yl_5_2, and Yl_5_3. Primarily, according to the Pearson Correlation analysis, the reasonableness of samples and reliability of the detection were confirmed (Additional file 1: Figure S8). The transcriptome data was analyzed under two conditions, a normal condition of p < 0.05, and a stringent condition of p < 0.001 and change-fold < 0.5 or > 2.0. According to the normal condition (p < 0.05) results, both Yl_5_2 and Yl_5_3 exhibited changed expressions of almost 3000 genes, nearly half of the total genes throughout genome (Additional file 1: Figure S9; Additional file 4). This ratio was coincident with the ratio of the genes under the control of Polymerase II transcriptional system in S. cerevisiae. This highly proved that Yl-SPT15 did own the function of global TF. When the condition was stringent (p < 0.001 and fold < 0.5 or > 2.0), Yl_5_2 and Yl_5_3 exhibited altered expression of hundreds of genes throughout genome compared with the control (Fig. 3). Only one-tenth of these transcription-modulated genes (39 out of total 416) in two strains were in accordance, quite differentiated with that of normal condition (1943 out of total 4687). Most of the genes (164 out of total 196) with differential expression in Yl_5_2 were up-regulated, while the majority in Yl_5_3 (201 out of total 259) were down-regulated, indicating a whole “enhanced” transcription in Yl_5_2 but a whole “weakened” transcription in Yl_5_3. The highest two up-regulated targets in Yl_5_2 were YALI0D17556g(RER2) that expressed a major enzyme participating in polyprenol synthesis in both endoplasmic reticulum (ER) and lipid droplets, and YALI0F19184g(RGT2) that was plasma membrane high glucose sensor for regulating glucose transport.
The KEGG pathway enrichment was done for each pair of Yl_5_2 relative to Yl_5_1 and Yl_5_3 relative to Yl_5_1 (p < 0.05). The “enhanced” strain Yl_5_2 obtained some prominent enrichment of up-regulated genes in special pathways such as synthesis and degradation of ketone bodies, alpha-linolenic acid metabolism (an actual non-existing pathway in Y. lipolytica but related to three disperse real reactions later explained), mismatch repair, and homologous recombination (rich factors (RF) > 0.5) (Additional file 1: Figure S10 and Additional file 3: Table S4). The “weakened” strain Yl_5_3 gained some prominent enrichment of down-regulated genes in special pathways such as 10 amino acid metabolisms (valine, leucine, isoleucine, tryptophan, phenylalanine, tyrosine, lysine, histidine, cysteine, and methionine) and fatty acid degradation (RF > 0.6) (Additional file 1: Figure S11). The two strains shared an essential commonly changed pathway, RNA polymerase. The related genes in Y_5_2 were obviously down-regulated (RF = 0.62), while that in Yl_5_3 were notably up-regulated (RF = 0.55) (Additional file 1: Figures S12, S13). As mentioned above, another work concluded that the strain highly producing lipid bodies also highly produced carotene. However, in our work, a somewhat paradoxical phenomenon was observed. The genes associated with fatty acid biosynthesis in Yl_5_2 were obviously down-regulated (RF = 0.80), including the genes of YALI0C11407g(ACC1), YALI0B15059g(FAS1), YALI0B19382g(FAS2), and YALI0D17864g(FAA) (Additional file 1: Figure S13, Additional file 3: Table S5). By contrast, the fatty acid degradation was down-regulated in Yl_5_3 (RF = 0.78), including the genes of YALI0D17864g(FAA), YALI0D24750(ACX), YALI0F10857g(ACX), YALI0B10406g(ECH), YALI0E18568g(POT1), YALI0E11099g(ERG10), YALI0B08536g(ERG10), and YALI0F23749g(ACDH) (Additional file 1: Figure S11, Additional file 3: Table S6). There were two basic possibilities. First, the total carbon distribution to fatty acid synthesis and mevalonic acid (MVA) synthesis was constant and the increment of carotene caused down-regulated synthesis of fatty acids and vice versa. Second, the quantitative changes of carotene and fatty acids might be in accordance. The contradictory transcription regulation might indicate negative feedback regulation which commonly existed in fatty acid metabolism. The latter possibility was verified by later detection of the amounts of cellular fatty acids.
Modulation of the amounts of total fatty acids, free fatty acids, and lipid bodies
As transcriptome analysis revealed that fatty acid metabolism was regulated in both “enhanced” strain Yl_5_2 and “weakened” strain Yl_5_3, it was necessary to detect the amounts of fatty acids and lipids in these strains. The concentration of total fatty acids in Yl_5_2 was enhanced to 174.76 mg/L (58.03 mg/g DCW), 1.14-fold (1.32-fold) of the production contained in Yl_5_1 as 153.34 mg/L (44.11 mg/g DCW) (Fig. 4a, Additional file 1: Figure S14). As for free fatty acids, the concentration in Yl_5_2 was 29.73 mg/L (10.14 mg/g DCW), 1.15-fold (1.19-fold) of that in Yl_5_1 as 25.75 mg/L (8.55 mg/g DCW) (Fig. 4b, Additional file 1: Figure S15). By contrast, the concentration of total fatty acids in Yl_5_3 was reduced to 131.02 mg/L (36.73 mg/g DCW), 0.85-fold (0.83-fold) of that in Yl_5_1 (Fig. 4a, Additional file 1: Figure S14), and the free fatty acids was 23.54 mg/L (6.88 mg/g DCW) in Yl_5_3, 0.91-fold (0.80-fold) of that in Yl_5_1 (Fig. 4b, Additional file 1: Figure S15). The composition of FFA and total fatty acids was consistent among different strains (Additional file 1: Figures S14, S15). The increased production of both total and free fatty acids in Yl_5_2 might explain why its fatty acid synthesis pathway was down-regulated because this pathway owned typical mode of drastic feedback regulation by end products. On the other hand, the decreased production of fatty acids in Yl_5_3 explained why the fatty acid degradation pathway was down-regulated as cells had to down-regulate fatty acid degradation to protect its inner lipophilic environment. The microscopic observation showed that Yl_5_2 got more droplets of lipids but not a larger one compared with Yl_5_1 and Yl_5_3, as the increased lipophilic compartments were beneficial to carotene preservation and was created by whole cell’s complex tuning forces (Fig. 4c and Additional file 1: Figure S12).
Among the transcriptional regulated genes, ACC1, FAS1, and FAS2 played essential roles in the biosynthesis of fatty acids. The carboxylation of acetyl-CoA by ACC1 was considered as rate-limiting step, and this gene was usually overexpressed for lipid accumulation in S. cerevisiae and Y. lipolytica [21, 24, 25, 39, 40]. Besides, the coordinate expression of FAS1 and FAS2 was necessary for heteromultimeric fatty acid synthase complex [41]. In Y. lipolytica, acyl-CoA was a key intermediate closely correlated with both free fatty acids and lipids (triacylglycerol, TAG), and its changing tendency was probably same as FFA and lipids (Fig. 4, Additional file 1: Figures S14, S15). As increased acyl-CoA plays the key role of negative feedback regulation of ACC1, it was understandable that the transcriptions of YALI0C11407g(ACC1), YALI0B15059g(FAS1), and YALI0B19382g(FAS2) were down-regulated in Yl_5_2 at the cell’s late growth stage. In addition, YALI0E11099g(ERG10) and YALI0F10857g(ACX) might play key roles as these two genes were regulated by obviously reverse trends in Yl_5_2 and Yl_5_3 (Figs. 4c, 5a, b). The two enzymes, acetyl-CoA C-acetyltransferase and acyl-CoA oxidase, also covered the crossroad locations for regulating distribution of acetyl-CoA flux in Y. lipolytica.
Enhancement of carotene production by overexpressing newly revealed intrinsic genes
As previously mentioned, in this work we revealed new up-regulated pathways in Yl_5_2 that might be correlated with the phenotype of higher carotene production. To determine these obviously up-regulated pathways in Yl_5_2 contributed to the improved beta-carotene production, we chose seven typical up-regulated genes from the two pathways and transformed their individual expression cassette on plasmids into Yl_5_2 (Fig. 5a, b, Additional file 1: Figure S2c). The results showed that all the seven transformed strains with recombinant plasmids (from Yl_5_2_1 to Yl_5_2_7) gained higher beta-carotene production than the initial strain Yl_5_2 with blank plasmid (Yl_5_2_0). These genes did increase the flux of acetyl-CoA towards terpene synthesis. The increment was up to about 50–60%, although all the values were slightly reduced perhaps owing to the influences of cell’s growth on the medium with hygromycin B (Fig. 5c). The collaboration of four enzymes expressed individually from YALI0E11099g(EGR10), YALI0F30481g(ERG13), YALI0B22550g(HMGL), and YALI0F26587g(OXCT) generated a special recycling of acetyl-CoA in Y. lipolytica and its flux was obviously up-regulated in Yl_5_2 for enhancing the pull of acetyl-CoA towards HMG-CoA and offering more precursors to MVA pathway (Additional file 3: Table S4). The single-gene overexpression proved that the flux of this cycling could be further enhanced for improving carotene synthesis and therein the YALI0E11099g(EGR10) might be the bottleneck. Another up-regulated pathway alpha-linolenic acid metabolism was actually a mistake of KEGG enrichment analysis as it did not exist in Y. lipolytica. The actual functions of the coupling of YALI0F10857g(ACX) and YALI0E18568(POT1) shall be reacting repeated beta-oxidation of acyl-CoA to produce final acetyl-CoA (Fig. 5b, Additional file 3: Table S4). Another gene YALI0F10010g(TGL4) actually charged degradation of multiple compounds to recycle free fatty acids (Fig. 5b, Additional file 3: Table S4). Single-gene especially YALI0E18568(POT1) overexpression showed that it was beneficial to further enhance this pathway to gain more carotene products.