Screening of the fluorescent reporter gene in Y. lipolytica
Efficient engineering of microbial cell factories relies on optimizing the genetic construct of metabolic pathways to direct the carbon flux toward the desired product of interest. The key to achieving this goal is to eliminate metabolic bottlenecks and tune the expression of target gene precisely. To this end, we aim to construct a hybrid promoter library. For characterization of the promoters, it is critical to develop a stable, reliable and sensitive reporting system to monitor gene expression. A series of studies have evaluated the effectiveness of some reporters in the unconventional yeast Y. lipolytica for examining the strength of promoters, such as green fluorescent protein (GFP) and β-galactosidase [18, 19]. GFP is the most commonly used reporter gene to characterize promoter strength because of its convenience in detection. However, there are many variants of GFP and their applicability to Y. lipolytica needs to be assessed. Therefore, in this study, we systematically characterized the expression of different GFPs to investigate which gene may function as an ideal reporter gene in the yeast strain Y. lipolytica Po1g KU70∆ that was used as a host system for this work. Firstly, expression of different fluorescent reporter genes GFPuv (differs from the wild-type GFP by the amino acid replacements Val163Ala, Met153Thr, and Phe99Ser), hrGFP (humanized Renilla reinformis GFP) and hrGFPO (codon optimized hrGFP for Y. lipolytica) [1, 18, 20] was driven by the hybrid promoter PUAS1B4−LEUm, which is the promoter on the commercial integrative vector pYLEX1 for Y. lipolytica. Expression of the GFPs were detected by fluorescence microscopy and flow cytometry (Fig. 2, Fig. 3a). The results indicate that fluorescence was not detected in the control strain Po1g KU70Δ while the Y. lipolytica strains carrying integrated GFPuv or hrGFP gene fluoresced at different intensities. However, the fluorescence produced in the strain carrying the GFPuv gene was relatively weak, indicating that this is not a good reporter gene for Y. lipolytica. In contrast, the detected fluorescence intensity of strains that harbored the hrGFPO gene was more stable than strains that possessed the hrGFP gene (Fig. 3a). Henceforth, hrGFPO was selected as the reporter gene for promoter characterization in this work.
Characterization of native promoters as a basis for the construction of hybrid promoters for Y. lipolytica
The strengths of different native promoters are known to vary greatly in microbes. To form a basis for our hybrid promoter library, we sought to use the hrGFPO reporter gene to evaluate the promoter strengths of several commonly used native Y. lipolytica promoters: β-isopropylmalate dehydrogenase (LEU2) promoter PLEU, export protein (EXP) promoter PEXP and translation elongation factor-1α (TEF1) promoter PTEF. Based on the results of our experiments (Fig. 3b), the relative fluorescence intensities of the corresponding strains from high to low are PTEF > PEXP > PLEU, whereby the strength of PTEF is about an order stronger than both PEXP and PLEU. Subsequently, these promoters were dissected into the various promoter elements, i.e. UAS, TATA box and core promoter, and based on the structures of these native promoters, other known promoter elements were added to build hybrid promoters. In most previous studies on the construction on hybrid promoters, the focus was mainly on the utilization of UAS and there were few studies on varying the other promoter elements. Thus, in this study, we explored the mixing of promoter constituent elements and investigated the influence of the various combinations on the promoter strengths of the resulting hybrid promoters in Y. lipolytica (Table 1, Fig. 4).
Characterization of features in core promoters that influence promoter strength
The core promoter, first identified in the mammalian gene regulatory region, plays a very important role in the regulatory initiation of genes and is defined as ‘the smallest DNA element for transcription’ . In yeast systems, a large number of studies have shown that the regulation mechanism of the core promoter has a very complex impact on the activity and strength of the promoter, and thus modulate gene expression. For example, in S. cerevisiae, the T content in the core promoter upstream of the transcription start site (TSS) has a great influence on the promoter activity. When the gene expression was high, the T content upstream of the TSS was abundant, and the A content downstream of TSS was rich . Thus, we hypothesize that a similar trend exists in Y. lipolytica. Therefore, a series of endogenous core promoters of different lengths and contain TATA box, namely LEU, TEF, EXP, POX2 and PAT1, were selected to calculate the content of T upstream of the TSS and verify the functions of the core promoters in Y. lipolytica. To confirm the function of core promoters, the UAS1B elements which advance gene transcription were linked to the upstream of the core promoter to express the hrGFPO reporter gene for characterizing the promoter strengths by fluorescence. The results indicated that the hybrid promoters we constructed in general followed the trend that the promoter strength increases with the T content upstream of the TSS, with two exceptions, namely LEUm and POX2m (Fig. 3c). We also analysed the length of the TEF core promoter, and found that the shorter the core promoter is, the stronger the hybrid promoter (Fig. 3c). These data suggest that expression level of genes can be regulated largely by both the types and length of core promoters. While there appears to be a relationship between T content and promoter strength in Y. lipolytica, further studies are required to elucidate the specific relationship between base content and promoter strength.
Modulating the promoter strength by varying the TATA box
Functional elements of the core promoter including TATA box, initiator element (Inr), downstream promoter element (DPE), TFIIB recognition element (BRE) and motif ten element (MTE) have been identified [22, 23]. The sequence lengths of these functional elements are short, the specificities are low and the combinations in various promoters are different. All these functional elements, except the TATA box, are clearly nonconservative in yeast [24, 25]. The TATA box, which is the binding site of TATA binding protein (TBP), is the first element identified in the core promoter. Previous studies have shown that TATA box has a significant effect on promoter strength . Therefore, a series of TATA boxes (Table 2) were selected to study their specific performance in promoters in Y. lipolytica. PUAS1B4+LEU, which has the highest activity in the previous section, was selected as the control for engineering. Firstly, we selected several TATA boxes to replace TATA LEU by site-directed mutagenesis. The expression of hrGFPO under the promoter variants was evaluated by fluorescence, which showed that strains with different TATA boxes significantly affected the promoter strength. The fluorescence intensity of the strain with the hybrid promoter containing TATA TEF was more than twice that of the control strain with TATA LEU (Fig. 3d). Therefore, the result validates the important role of TATA box in influencing the strength of a promoter and provides a theoretical basis for future promoter engineering studies.
Construction of promoters with various UAS elements from Y. lipolytic and S. cerevisiae
The process of transcriptional regulation begins with the recognition of specific sequences by transcription factors (TFs), such as the recognition of UASs by transcriptional activators and upstream repression sequences (URSs) by repressors. Many studies have shown that UAS has a powerful influence on transcriptional regulation. Several UASs have been identified in S. cerevisiae, such as UASTEF , UASCLB  and UASCIT . However, only a few UASs were identified in Y. lipolytica, among which the UAS1B is the most well-studied. In previous studies, it has been shown that the copy number of UAS has significant impact on hybrid promoter strength as well [2, 12]. Four tandem UAS1B from PXPR2 and one PLEU core promoter have been combined to construct the strong constitutive promoter PUAS1B4+LEUm . We increased the copy number of UAS and verified that the copy number of UAS is proportional to the hybrid promoter strength (Fig. 3e), which corroborates with published data . In addition, while it has been shown that synthetic terminators can be efficiently transferred in S. cerevisiae and Y. lipolytica , there is no research on the transferability of promoter elements across diverse yeast species. Therefore, different UASs (UASCIT S.c., UASCLB S.c., UASTEF S.c. and UASTEF Y.l.) [2, 26–28] from S. cerevisiae and Y. lipolytica with the same copy number as PUAS1B4+LEUm were used to replace UAS1B4 to explore the influence of UAS types and origin on hybrid promoter activity. By expressing the hrGFPO gene under the hybrid promoters with different UASs, the activities of promoters were shown to be significantly affected by the variation in UAS. The relative fluorescence intensity from the GFP expressed from the promoters containing various UASs, from high to low, is UAS1B > UASTEF Y.l.> UASCIT S.c.> UASCLB S.c.> UASTEF S.c. (Fig. 3f). These results demonstrated for the first time that UAS from S. cerevisiae are functional in Y. lipolytica.
Taken together, we have constructed a library of hybrid promoters with different promoter strengths using various combination of UASs, TATA boxes and core promoters, as summarized in Fig. 5 and Table 1. To demonstrate the application of our hybrid promoter library, as a testbed, we aimed to optimize a biosynthesis pathway, i.e. isoamyl alcohol production, by promoter engineering using our hybrid promoters to regulate gene expression and improve production level of the target compound.
Construction of the isoamyl alcohol overexpression pathway in Y. lipolytica
As an important platform chemical, isoamyl alcohol is a promising biofuel and biochemical with huge market demand. However, in Y. lipolytica, the titer of isoamyl alcohol natively is quite low at a mere 0.37 mg/L (Fig. 6). Thus, the production titer of isoamyl alcohol has much room for improvement and the biosynthesis pathway serves as a good testbed for optimization by promoter engineering using our hybrid promoter library.
In yeast, isoamyl alcohol is generally produced through the Ehrlich pathway, which usually involves three reaction steps: transamination, decarboxylation and reduction. Twelve genes encoding transaminases (ScBAT1, YlBAT1-1 and YlBAT1-2), decarboxylases (ScARO10, YlARO10-1 and YlARO10-2) and alcohol dehydrogenases (ScADH2, YlADH2-1, YlADH2-2, YlADH2-3, YlADH2-4 and YlADH2-5) were selected and individually overexpressed to determine the key genes of isoamyl alcohol biosynthesis in the Ehrlich pathway. For this purpose, twelve strains overexpressing native and heterologous genes in the Ehrlich pathway were constructed. All genes were individually integrated into the genome of Y. lipolytica Po1g KU70Δ and driven by the constitutive promoter PUAS1B4+LEUm. After 3 days of cultivation, individual overexpression of the pathway genes enhanced the isoamyl alcohol titer in the engineered strains compared to that of the control strain Po1g KU70Δ (Fig. 6). The results showed that among the three evaluated classes of enzymes in the Ehrlich pathway, the strains overexpressing decarboxylase genes resulted in the most significant increase in isoamyl alcohol production. Among them, the highest isoamyl alcohol production was obtained by the ScARO10-overexpressed strain, which reach 1.36 mg/L. The strains which overexpressed transaminase gene ScBAT1 and dehydrogenase gene ScADH2 also increased the harvest of isoamyl alcohol moderately. Therefore, to further improve the yield of isoamyl alcohol, the genes ScBAT1, ScARO10 and ScADH2 were chosen to construct strain Po1g BAA. After 3 days of cultivation, the titer of isoamyl alcohol reached 1.8 mg/L, which was 3.9-fold higher than that of the control strain Po1g KU70Δ (Fig. 6). Thus, the strain Po1g BAA was selected for subsequent engineering by promoter replacement with our hybrid promoter library.
Application of the hybrid promoter library to improve the isoamyl alcohol biosynthesis pathway
In metabolic engineering, studies have shown that the yield of the target product can be increased by replacing promoters for pathway genes with stronger ones [19, 30]. Therefore, to demonstrate the application of our promoter library for optimizing metabolic pathways, we employed some of our hybrid promoters in the heterologous isoamyl alcohol pathway of Po1g BAA. We chose from the promoter library nine promoters that cover a range of strengths to express the key gene ScARO10 in the isoamyl alcohol pathway. These constructed strains were cultured for 3 days, and the titer of the isoamyl alcohol was quantified (Fig. 7). It can be seen from the results that the isoamyl alcohol titer does not correlate to the strength of the promoter used. For example, strain Po1g BA + PUAS1B4+EXPm+ARO10 with a low-activity promoter had the highest isoamyl alcohol titer of 11.57 mg/L, which was about 30.3-fold higher than that of Po1g KU70Δ and 5.4-fold that of Po1g BAA. This result is consistent with the opinion of Dulermo, et al. that stronger promoters do not necessarily increase the expression level and/or function of a protein . In addition, we found that although the activity of PEXP was low, several strains containing PEXP elements (PEXP, PUAS1B4−EXPm, PUAS1B4+TATAEXP−LEUm) had higher titers of isoamyl alcohol, suggesting that the elements of the PEXP have greater beneficial effects to the expression of the ARO10 gene, which encodes a key enzyme of the isoamyl alcohol pathway. More studies are needed to better understand the mechanism between the elements of PEXP and gene expression which resulted in the improved production titer. Nevertheless, we demonstrated successful application of our hybrid promoter for identification of suitable promoters to improve metabolic pathways.