Hybrid promoter engineering strategies in Yarrowia lipolytica: isoamyl alcohol production as a test study

Hybrid promoter engineering strategies in Yarrowia lipolytica: isoamyl alcohol production as a test study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Hybrid promoter engineering strategies in Yarrowia lipolytica : isoamyl alcohol production as a test study Yu Zhao, Shiqi Liu, Zhihui Lu, Baixiang Zhao, Shuhui Wang, Cuiying Zhang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-369447/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background In biological cells, promoters drive gene expression by binding to RNA polymerase specifically. They determine the starting position, timing and level of gene expression. Therefore, rational fine-tuning of promoters to regulate the expression levels of target genes for metabolic engineering applications to optimize biosynthetic pathways has recently become an active area of research. Results In this study, we systematically detected and characterized the common promoter elements in the unconventional yeast Yarrowia lipolytica , and constructed an artificial hybrid promoter library that covers a wide range of promoter strength. We also report for the first time that upstream activation sequences (UAS) of Saccharomyces cerevisiae promoters can be functionally transferred to Y. lipolytica . Subsequently, using the production of a versatile platform chemical isoamyl alcohol as a test study, the hybrid promoter library was applied to optimize the biosynthesis pathway expression in Y. lipolytica . Under the control of P UAS1B8−LEUm , the strongest promoter we constructed, overexpression of a key pathway gene led to 7.7-fold increase in the titer of isoamyl alcohol. Interestingly, a much weaker promoter P UAS1B4−EXPm increase the isoamyl alcohol titer by 30.3-fold. These results suggest that our hybrid promoter library can be a powerful toolkit for identifying optimum promoters for expressing metabolic pathways in Y. lipolytica . Conclusion We envision that this promoter engineering strategy and the rationally engineered promoters constructed in this study could also be extended to other non-model fungi for strain improvement. Biotechnology and Bioengineering Y. lipolytica metabolic engineering hybrid promoter isoamyl alcohol synthetic promoter Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Promoters are one of the most important components of synthetic biology, and well-controlled promoters are very critical for regulating gene expression in eukaryotes. The activity of a promoter is co-regulated by various elements. In yeast, the common promoter elements usually include upstream activation sequences (UAS), TATA box and core promoter [ 1 , 2 ]. By rational modification of these elements, the activity of promoters can be fine-tuned. At the beginning of transcription, regulatory signals are transmitted from the UAS to the core promoter, then transcription factors and RNA polymerase Ⅱ assembly combined with the transcription preinitiation complex (PIC) [ 3 ]. The core promoter significantly contributes to the regulation of gene expression and is also the key factor determining the promoter strength. Although the core promoters were initially thought to be invariant, researchers have found that they exhibit great structural and functional diversity [ 4 , 5 ]. TATA box, the recognition site of the transcription factor TATA binding protein (TBP), is one of the first kind of functional elements identified to regulate the promoter strength of the core promoter and typically located 40–120 bp upstream of the transcription start site [ 3 , 6 ]. Mutations in the TATA box usually alter the promoter strength [ 1 , 7 – 10 ]. The UAS, which is usually located at the 5’ of the promoter [ 3 , 11 ], is also known to affect the strength of the promoter by varying its copy number. By analysing the function of the endogenous alkaline extracellular protease 2 (XPR2) gene promoter P XPR2 of Yarrowia lipolytica , Madzak et al. identified UAS1B as the most significant functional element that activates the promoter P XPR2 [ 12 ]. Subsequently, evaluation of a hybrid promoter library consisting a minimal P LEU fragment and different copy numbers of the UAS1B indicates that enhancement in promoter strength is correlated to increased copy number of UAS1B [ 12 ]. When present in a promoter, different types of UAS can cooperate to control transcription. For example, by combining different UAS elements (UASTEF and UAS1B) in Y. lipolytica , the expression level of a constructed promoter was 7-fold higher than the wild-type promoter [ 2 ]. Taken together, by exploring the synergy between various promoter elements and understanding the working mechanism of the promoter, promoters with stronger activity and wider expression range can be constructed. In this study, in order to explore the mechanism of synergy between various elements in Y. lipolytica , the promoter elements were characterized and rearranged. Consequently, a library of hybrid promoters that enables stable expression and covers a wide range of promoter strength was constructed. Subsequently, we employed the hybrid promoters for promoter engineering of the pathway genes in isoamyl alcohol biosynthesis. Isoamyl alcohol, an important platform chemical, is widely applied in the production of biofuels, fragrances, medicines and fine chemicals [ 13 ] and has been produced in recent years by metabolic engineering of diverse microbial cells, such as Escherichia coli , Corynebacterium glutamicum and Aspergillus oryzae [ 14 – 16 ]. While there is a report on improving the production of isoamyl alcohol in Y. lipolytica by metabolic engineering, a native promoter was employed [ 17 ]. Therefore, we used this pathway as a testbed and demonstrated the efficacy of our promoter library for optimizing metabolic pathways by significantly improving the isoamyl alcohol titer (Fig. 1 ). The outcome of this work shows that promoter engineering is an effective strategy for facilitating metabolic engineering efforts to biosynthesize valuable chemicals and our hybrid promoter library is a powerful toolkit for future metabolic engineering work in Y. lipolytica . Results And Discussion 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 P UAS1B4−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. 3 a). The results indicate that fluorescence was not detected in the control strain Po1g KU70 Δ 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. 3 a). 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 P LEU , export protein (EXP) promoter P EXP and translation elongation factor-1α (TEF1) promoter P TEF . Based on the results of our experiments (Fig. 3 b), the relative fluorescence intensities of the corresponding strains from high to low are P TEF > P EXP > P LEU , whereby the strength of P TEF is about an order stronger than both P EXP and P LEU . 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’ [ 3 ]. 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 [ 21 ]. 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. 3 c). 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. 3 c). 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 [ 1 ]. Therefore, a series of TATA boxes (Table 2 ) were selected to study their specific performance in promoters in Y. lipolytica . P UAS1B4+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. 3 d). 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 [ 26 ], UASCLB [ 27 ] and UASCIT [ 28 ]. 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 P XPR2 and one P LEU core promoter have been combined to construct the strong constitutive promoter P UAS1B4+LEUm [ 18 ]. We increased the copy number of UAS and verified that the copy number of UAS is proportional to the hybrid promoter strength (Fig. 3 e), which corroborates with published data [ 18 ]. In addition, while it has been shown that synthetic terminators can be efficiently transferred in S. cerevisiae and Y. lipolytica [ 29 ], 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 P UAS1B4+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. 3 f). 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 KU70 Δ and driven by the constitutive promoter P UAS1B4+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 KU70 Δ (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 KU70 Δ (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 + P UAS1B4+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 KU70 Δ 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 [ 31 ]. In addition, we found that although the activity of P EXP was low, several strains containing P EXP elements (P EXP , P UAS1B4−EXPm , P UAS1B4+TATAEXP−LEUm ) had higher titers of isoamyl alcohol, suggesting that the elements of the P EXP 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 P EXP 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. Conclusions Promoters are one of the most important components of synthetic biology for determining protein expression. Compared to prokaryotes, the regulatory mechanism of the promoter structure in eukaryotes is extremely complex [ 1 , 32 ]. Increasing the promoter strength is a common method to improve gene transcription and protein expression level. However, recent studies have shown that not all strong promoters can achieve the highest protein expression and activity [ 31 ]. We explored the structure and functional characteristics of the promoters of Y. lipolytica , and subsequently constructed a series of constitutive promoters which are stable and efficient. Firstly, different variants of green fluorescent protein were screened in Y. lipolytica to identify a reporter gene that can be stably expressed. Among different transformants, the codon optimized hrGFP ( hrGFPO ) not only expressed at a high level but also expressed stably in Y. lipolytica . Therefore, the hrGFPO gene was used for subsequent promoter characterization experiments. Three native promoters of Y. lipolytica , P LEU , P TEF and P EXP , were characterized and the results showed that the strengths of these promoters are evidently different. These promoters were dissected into three parts, namely UAS, TATA box and Core promoter, and these elements were combinatorially arranged with other studied promoter elements to construct a constitutive promoter library that contains 21 stable hybrid promoters. It is the first time that the T content upstream of the TSS has been shown to positively correlate with the hybrid promoter strength in Y. lipolytica . It is worth noting that some core promoter elements, such as POX2m and LEUm, did not conform to the trend. Therefore, the relationship between the T content upstream of the TSS and the promoter strength in Y. lipolytica needs to be further studied. Next, the effects of different UAS elements from S. cerevisiae and Y. lipolytica on promoter strength were investigated and we discovered for the first time that UAS elements can be transferred between yeast species. These findings lay the groundwork for the development of hybrid promoters which can be efficiently transferred across diverse yeast species. To demonstrate application of our hybrid promoter library, the isoamyl alcohol production pathway was constructed to serve as a testbed by co-expression of multiple genes from S. cerevisiae and Y. lipolytica . ScAOR10 , the key gene of the isoamyl alcohol pathway, was selected as the test gene for expression under various hybrid promoters from our library to optimize the enzyme’s expression and activity for enhance isoamyl alcohol production. Consequently, the titer of the isoamyl alcohol increased from 0.37 mg/L to 11.57 mg/L, which was 30.3-fold higher than the control strain Po1g KU70 Δ. To date, isoamyl alcohol has been successfully produced by metabolic engineering in several studies [ 17 , 33 , 34 ]. Although the titer of isoamyl alcohol from Y. lipolytica is lower compared with other studies, it is the first time that the promoter engineering has been applied for the biosynthesis of isoamyl alcohol to provide an advanced solution for the biosynthesis of biofuels and alcohols. Regulation of expression by promoters involves various factors, such as temperature, pH and substrate [ 1 , 35 ]. In the future, we will further study the mechanisms of promoters to construct hybrid promoters with stronger activity and wider expression range for optimum expression of biosynthesis pathway genes to achieve high-level production of value-added chemicals. Materials And Methods Strains and media Escherichia coli strain DH5α was used for all cloning and plasmid propagation, and DH5α was grown at 37°C in Luria Bertani (LB), and supplemented with ampicillin to final concentration of 100 µg/mL for plasmid propagation. Y. lipolytica strain Po1g KU70 Δ, a leucine auxotroph devoid of any secreted protease activity, was used as the base strain in this study. Y. lipolytica Po1g KU70 Δ containing plasmid was routinely cultivated at 28°C and 225 rpm with YPD media consisting of 20 g/L glucose, 20 g/L peptone, and 10 g/L yeast extract. In this study, PCR primers were synthesized by Genewiz (Jiangsu, China) and are listed in Table S1, plasmids are listed in Table S2 and strains used are listed in Table S3. Chemicals and enzymes All restriction enzymes were purchased from New England Biolabs (Beijing, China), 2×Phanta® max master mix, 2× Rapid Taq master mix, ClonExpress® II one step cloning kit, FastPure® Plasmid Mini Kit and FastPure® Gel DNA Extraction Mini Kit were purchased from Vazyme Biotech Co., Ltd. (Nanjing, China), peptone and yeast extract were purchased from Thermo Scientific Oxoid Microbiology Products (Basingstoke, England), isoamyl alcohol and n-dodecane were purchased from Aladdin® (Shanghai, China). Plasmid construction of promoter library The GFPuv gene was preserved in this laboratory, and cloned into pYLEX1 with primers GFPuv-F/GFPuv-R (Table S1) yield plasmid pYLGFPuv (Table S2). The hrGFP gene and hrGFPO gene were synthesized and cloned into pYLEX1 to yield plasmids pYLhrGFP and pYLhrGFPO (Table S2), respectively, by Genewiz (Jiangsu, China). The UASCIT S.c. 4, UASCLB S.c. 4, UASTEF S.c. 4, UASTEF Y.l. 4, UAS1B6 and UAS1B8 motifs were synthesized and cloned into plasmids pYLhrGFPO to replace UAS1B4 to yield plasmids pYLP UASCITSC4−LEUm +hrGFPO, pYLP UASCLBSC4−LEUm +hrGFPO, pYLP UASTEFSC4−LEUm +hrGFPO, pYLP UASTEFYL4−LEUm +hrGFPO, pYLP UAS1B6−LEUm +hrGFPO and pYLP UAS1B8−LEUm +hrGFPO (Table S2), respectively, by Genewiz (Jiangsu, China). Three endogenous promoters P LEU , P TEF and P EXP were cloned into vector pYLhrGFPO with primers PLEU-F/LEU-hrGFPO-R, PTEF-F/TEF-hrGFPO-R and PEXP-F/EXP-hrGFPO-R (Table S1) yield plasmids pYLP LEU +hrGFPO, pYLP TEF +hrGFPO and pYLP EXP +hrGFPO (Table S2), respectively. The Core promoters were amplified by primer pairs PAT1m-F/PAT1-hrGFPO-R, POX2m-F/POX2-hrGFPO-R, EXPm-F/EXP-hrGFPO-R, TEFm111-F/TEF-hrGFPO-R, TEF136-F/TEF-hrGFPO-R and TEFm175-F/TEF-hrGFPO-R (Table S1), and then replace the core promoter LEU in P UAS1B4−LEU . These promoters were ligated to pYLhrGFPO in place of the P UAS1B4−LEU to yield plasmids pYLP UAS1B4−PAT1m +hrGFPO, pYLP UAS1B4−POX2m +hrGFPO, pYLP UAS1B4−EXP1m +hrGFPO, pYLP UAS1B4−TEF111 +hrGFPO, pYLP UAS1B4−TEF136 +hrGFPO and pYLP UAS1B4−TEF175 +hrGFPO (Table S2), respectively. The TATA box LEU in P UAS1B4−LEU was replaced by TATA box TEF, EXP, PAT1 and POX2 using primer pairs TATA TEF-F/LEU-hrGFPO-R, TATA EXP-F/LEU-hrGFPO-R, TATA PAT1-F/LEU-hrGFPO-R and TATA POX2-F/LEU-hrGFPO-R (Table S1). These hybrid promoters were ligated to pYLhrGFPO in place of the P UAS1B4−LEU to yield plasmids pYLP UAS1B4−TATATEF−LEU +hrGFPO, pYLP UAS1B4−TATAEXP−LEU +hrGFPO, pYLP UAS1B4−TATAPAT1−LEU +hrGFPO and pYLP UAS1B4−TATAPOX2−LEU +hrGFPO (Table S2), respectively. All plasmids, linearized by Not I or Spe I, were transformed into competent cells of Y. lipolytica strains using the lithium acetate method [36]. Plasmid construction of exogenous isoamyl alcohol pathway The transaminase gene ( BAT1 , GenBank ID: 856615), decarboxylase gene ( ARO10 , GenBank ID: 851987) and alcohol dehydrogenase gene ( ADH2 , GenBank ID: 855349) from S. cerevisiae S288C were codon-optimized and synthesized and cloned into pYLEX1 to yield plasmids pYLSCBAT1, pYLSCARO10 and pYLSCADH2 (Table S2), respectively, by Genewiz (Jiangsu, China). In Y. lipolytica , the homologous sequences that YlBAT1-1 and YlBAT1-2 of ScBAT1 were cloned into pYLEX1 with primers YLBAT1-1-F/YLBAT1-1-R and YLBAT1-2-F/YLBAT1-2-R (Table S1) to yield plasmids pYLYLBAT1-1 and pYLYLBAT1-2 (Table S2), respectively. The homologous sequences that YlARO10-1 and YlARO10-2 of ScARO10 were cloned into pYLEX1 with primers YLARO10-1-F/YLARO10-1-R and YLARO10-2-F/YLARO10-2-R (Table S1) to yield plasmids pYLYLARO10-1 and pYLYLARO10-2 (Table S2), respectively. The homologous sequences that YlADH2-1, YlADH2-2, YlADH2-3, YlADH2-4 and YlADH2-5 of ScADH2 were cloned into pYLEX1 with primers YLADH2-1-F/YLADH2-1-R, YLADH2-2-F/YLADH2-2-R, YLADH2-3-F/YLADH2-3-R, YLADH2-4-F/YLADH2-4-R and YLADH2-5-F/YLADH2-5-R (Table S1) to yield plasmids pYLYLADH2-1, pYLYLADH2-2, pYLYLADH2-3, pYLYLADH2-4 and pYLYLADH2-5 (Table S2), respectively. The expression cassettes of ScARO10 and ScADH2 were cloned into pYLSCBAT1 with primers BDH-ADH2-F/BDH-ADH2-R and BDH-ARO10-F/ BDH-ARO10-R (Table S1) to yield plasmid pYLBAA (Table S2). All plasmids, linearized by Not I or Spe I, were transformed into competent cells of Y. lipolytica strains using the lithium acetate method [36]. Expressing the isoamyl alcohol synthesis pathway using the promoter library Several promoters from the promoter library were used to express the ARO10 gene which is the key gene in the isoamyl alcohol pathway. The promoters P EXP and P UAS1B4+EXPm were amplified by primers BDH-ARO10-F/PEXP-ARO10-R (Table S1), and then ligated to ScARO10 in pYLBAA to yield plasmid pYLBA + P EXP +ARO10 and pYLBA + P UAS1B4−EXPm +ARO10 (Table S2), respectively. The promoters P UAS1B4−POX2m and P UAS1B4−TEF136 were amplified by primers BDH-ARO10-F/POX2-ARO10-R and BDH-ARO10-F/PTEF-ARO10-R (Table S1), and then ligated to ScARO10 in pYLBAA to yield plasmid pYLBA + P UAS1B4−POX2m +ARO10 and pYLBA + P UAS1B4−TEF136 +ARO10 (Table S2), respectively. The promoters P UASTEFLY4−LEUm , P UAS1B4−TATAEXP−LEU , P UAS1B4−TATATEF−LEU and P UAS1B8− LEUm were amplified by primers BDH-ARO10-F/PLEU-ARO10-R (Table S1), and then ligated to ScARO10 in pYLBAA to yield plasmid pYLBA + P UASTEFLY4−LEUm +ARO10, pYLBA + P UAS1B4−TATAEXP−LEU +ARO10, pYLBA + P UAS1B4−TATATEF−LEU +ARO10 and pYLBA + P UAS1B8−LEUm +ARO10 (Table S2), respectively. All plasmids, linearized by Not I or Spe I, were transformed into competent cells of Y. lipolytica strains using the lithium acetate method [37]. Yeast strain construction The competent cell scheme and transformation method are referred to Pang, et al. [38]. After selection, the following engineered Y. lipolytica strains were generated: Po1g P UAS1B4−LEUm +GFPuv, Po1g P UAS1B4−LEUm +hrGFP, Po1g P UAS1B4−LEUm +hrGFPO, Po1g P UAS1B6−LEUm +hrGFPO, Po1g P UAS1B8−LEUm +hrGFPO, Po1g P LEUm +hrGFPO, Po1g P LEU +hrGFPO, Po1g P EXP +hrGFPO, Po1g P TEF +hrGFPO, Po1g P UAS1B4−EXPm +hrGFPO, Po1g P UAS1B4−POX2m +hrGFPO, Po1g P UAS1B4−PAT1m +hrGFPO, Po1g P UAS1B4−TEF111 +hrGFPO, Po1g P UAS1B4−TEF136 +hrGFPO, Po1g P UAS1B4−TEF175 +hrGFPO, Po1g P UAS1B4−TATAPAT1−LEUm +hrGFPO, Po1g P UAS1B4−TATAPOX2−LEUm +hrGFPO, Po1g P UAS1B4−TATAEXP−LEUm +hrGFPO, Po1g P UAS1B4−TATATEF−LEUm +hrGFPO, Po1g P UASTEFSC4−LEUm +hrGFPO, Po1g P UASCLBSC4−LEUm +hrGFPO, Po1g P UASTEFYL4−LEUm +hrGFPO, Po1g ScBAT1, Po1g YlBAT1-1, Po1g YlBAT1-2, Po1g ScARO10, Po1g YlARO10-1, Po1g YlARO10-2, Po1g ScADH2, Po1g YlADH2-1, Po1g YlADH2-2, Po1g YlADH2-3, Po1g YlADH2-4, Po1g YlADH2-5, Po1g BAA, Po1g BA + P EXP +ARO10, Po1g BA + P UAS1B4−EXPm +ARO10, Po1g BA + P UAS1B4−POX2m +ARO10, Po1g BA + P UASTEFYL4−LEUm +ARO10, Po1g BA + P UAS1B4−TEF136 +ARO10, Po1g BA + P UAS1B4−LEUm +ARO10, Po1g BA + P UAS1B4−TATAEXP−LEU +ARO10, Po1g BA + P UAS1B4−TATATEF−LEU +ARO10, Po1g BA + P UAS1B8−LEUm +ARO10 (Table S3). Flow cytometry The green fluorescent protein GFPuv, hrGFP and hrGFPO were selected as reporter proteins. The colonies of transformants were selected from plates and grew in 5 mL of fresh YPD medium in tube for 24 h. After that, the seed culture solution was inoculated to 250 mL flasks which contain 40 mL YPD medium, starting from OD 600 0.1. Cultures were cultivated at rotary shaker at 225 rpm and 28°C. Before flow cytometry analysis, the cultures were centrifuged at 12000 rpm for 1 min, and washed in 0.1 M phosphate-buffered saline (PBS), then resuspended in PBS. There were 10,000 cell count that were analysed with the BD Accuri C6 flow cytometer (BD Biosciences) using 488-nm excitation wavelength and FL1 channel for fluorescence detection. The CFlow software was used to analysed the data and compute mean fluorescence values. GC/MS analysis of isoamyl alcohol produced in the engineered Y. lipolytica strains The engineered Y. lipolytica transformants were selected from plate and prepared in 5 mL of fresh YPD medium in tube for 24 h. The seed culture solution was inoculated to 250 mL flasks containing 40 mL of YPD medium, starting from OD 600 0.1. The cultures were shaken at 225 rpm and 28°C for 3 days. In order to extract isoamyl alcohol from the cultures, 10% n -dodecane was added to the cultures, and the mixture was vortexed for 3 minutes, then centrifuged at 7500 rpm for 5 minutes. The organic phase of 1ul was detected by GC/MS using an Agilent 7890B GC with an 5977B MSD equipped with a HP-5MS column (60 m × 0.25 mm × 0.25 µm, Agilent, Santa Clara, CA, USA). GC oven temperature was initially held at 60°C for 2 min, and then ramped to 140°C at a rate of 5°C/min. It was then subsequently ramped at 10°C/min to 280°C and held for 5 min. The split ratio was 10:1. Helium was used as the carrier gas, with an inlet pressure of 13.8 psi. The injector was maintained at 280°C and the ion source temperature was set to 230°C. Final data analysis was achieved using MassHunter Workstation Software (Agilent, Santa Clara, CA, USA). Abbreviations GC/MS gas chromatography/mass spectrometry; OD 600 optical density at 600 nm; LB medium 0.5% yeast extract, 1% tryptone and 1% NaCl; YPD medium 1% yeast extract, 2% peptone and 2% glucose; YNB plate 2% glucose, 0.67% yeast nitrogen base without amino acids and 2% agar; PCR polymerase chain reaction. YlBAT1-1 YALI0_D01265g YlBAT1-2 YALI0_F19910g YlARO10-1 YALI0_D06930g YlARO10-2 YALI0_E07325g YlADH2-1 YALI0_A16379g YlADH2-2 YALI0_D25630g YlADH2-3 YALI0_E17787g YlADH2-4 YALI0_A15147g YlADH2-5 YALI0_E07766g Declarations Ethics approval and consent to participate This manuscript does not contain any studies with human participants or animals performed by any of the authors. Consent for publication All authors give consent to publish the research in Biotechnology for Biofuels. Availability of data and material All relevant data generated or analysed during this study were included in this published article. Competing interests The authors declare that they have no competing interests. Funding The Natural Science Foundation of Tianjin, China (17JCYBJC40800), the Research Foundation of Tianjin Municipal Education Commission, China (2017ZD03), the Innovative Research Team of Tianjin Municipal Education Commission, China (TD13-5013), Tianjin Municipal Science and Technology Project (18PTSYJC00140, 19PTSYJC00060), Startup Fund for “Haihe Young Scholars” of Tianjin University of Science and Technology, the Thousand Young Talents Program of Tianjin, China. Authors' contributions AQY, JLF, DGX and CYZ conceived and designed the study. YZ, SQL, ZHL, BXZ and SHW performed plasmid and strain construction, and fermentation experiments. AQY, JLF, DGX, CYZ revised the manuscript. All authors read and approved the final manuscript. Acknowledgements This work was supported by the Natural Science Foundation of Tianjin, China (17JCYBJC40800), the Research Foundation of Tianjin Municipal Education Commission, China (2017ZD03), the Innovative Research Team of Tianjin Municipal Education Commission, China (TD13-5013), Tianjin Municipal Science and Technology Project (18PTSYJC00140, 19PTSYJC00060), Startup Fund for ‘Haihe Young Scholars’ of Tianjin University of Science and Technology, the Thousand Young Talents Program of Tianjin, China. Authors' information a State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, No.29 the 13th Street TEDA, Tianjin 300457, PR China b Synthetic Biology Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119228, Singapore c NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), National University of Singapore, Singapore 117456, Singapore d Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore References Shabbir Hussain M, Gambill L, Smith S, Blenner MA. Engineering promoter architecture in oleaginous yeast Yarrowia lipolytica . ACS Synth Biol. 2016;5(3):213–23. Blazeck J, Reed B, Garg R, Gerstner R, Pan A, Agarwala V, Alper HS. 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Mol Cell Biol. 1990;10(9):4795–806. Damude HG, Gillies PJ, Macool DJ, Picataggio SK, Pollak DWM, Ragghianti JJ, Xue Z. High eicosapentaenoic acid producing strains of Yarrowia lipolytica . US. 2013;US8518674:B2. tables Table 1 List of promoters used in this study Promoters UAS type TATA box Core promoter Strength Reference LEU + [ 39 ] EXP + + [ 40 ] TEF + + + [ 40 ] LEUm LEU LEU + [ 12 ] UASTEFSC4-LEUm UASTEFSC4 LEU LEU + + This study UASCLBSC4-LEUm UASCLBSC4 LEU LEU + + This study UASCITSC4-LEUm UASCITSC4 LEU LEU + + This study UAS1B4-EXPm UAS1B4 EXP EXP + + This study UAS1B4-POX2m UAS1B4 POX2 POX2 + + + This study UAS1B4-TATAPAT1-LEU UAS1B4 PAT1 LEU + + + This study UAS1B4-PAT1m UAS1B4 PAT1 PAT1 + + + This study UASTEFYL4-LEUm UASTEFYL4 LEU LEU + + + + This study UAS1B4-TEF175 UAS1B4 TEF TEF175 + + + + This study UAS1B4-TEF136 UAS1B4 TEF TEF136 + + + + This study UAS1B4-TEF111 UAS1B4 TEF TEF111 + + + + This study UAS1B4-LEUm UAS1B4 LEU LEU + + + + [ 23 ] UAS1B4-TATAPOX2-LEU UAS1B4 POX2 LEU + + + + This study UAS1B4-TATAEXP-LEU UAS1B4 EXP LEU + + + + This study UAS1B6-LEUm UAS1B6 LEU LEU + + + + + This study UAS1B4-TATATEF-LEU UAS1B4 TEF LEU + + + + + This study UAS1B8-LEUm UAS1B8 LEU LEU + + + + + [ 18 ] Table 2 TATA box tested in this study TATA box Sequence LEU TATATATA TEF TATAAAA EXP ATTATATATAA PAT1 TATATACC POX2 GTATACTTATATA Supplementary Files AdditionalfileforBforB.docx Additional file 1: Table S1. Primers used in PCR. Table S2. Plasmids used in this study. Table S3. Strains used in this study. Figure S1. Map of the plasmid pYLEX1. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-369447","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":19340869,"identity":"4dea7dc0-bead-4abb-b35d-582a6db05a18","order_by":0,"name":"Yu Zhao","email":"","orcid":"","institution":"Tianjin University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Zhao","suffix":""},{"id":19340870,"identity":"48e269a0-e861-4794-a7aa-da8bce4313ef","order_by":1,"name":"Shiqi Liu","email":"","orcid":"","institution":"Tianjin University of Science and 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19:07:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-369447/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-369447/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":7624903,"identity":"ecc27a89-b8c3-4e5b-a6a1-726813087c22","added_by":"auto","created_at":"2021-04-02 23:56:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":822595,"visible":true,"origin":"","legend":"The strategy of promoter engineering using isoamyl alcohol production as the test study\nAn artificial hybrid promoter library that covers a wide range of promoter strength was constructed, and applied to optimize the isoamyl alcohol synthesis pathway in Y. lipolytica.\n","description":"","filename":"OnlineFig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-369447/v1/4167b79a9236becc43f7120c.png"},{"id":7624902,"identity":"7c131323-05fd-4f48-9d3c-1e919d63661a","added_by":"auto","created_at":"2021-04-02 23:56:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":406516,"visible":true,"origin":"","legend":"The screening of suitable reporter gene and the characterization of promoter library strength\na. The fluorescence image of Y.lipolytica Po1g KU70Δ with the integrative plasmid pYLEX1.\nb. The fluorescence image of Y.lipolytica Po1g KU70Δ with the integrative plasmid pYLGFPuv.\nc. The fluorescence image of Y.lipolytica Po1g KU70Δ with the integrative plasmid pYLhrGFP.\nd. The fluorescence image of Y.lipolytica Po1g KU70Δ with the integrative plasmid pYLhrGFPO.\n","description":"","filename":"OnlineFig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-369447/v1/ce49b0ff10cf22788c358594.png"},{"id":7624901,"identity":"6018719a-ebe7-48ee-b00b-b4e20ab586a9","added_by":"auto","created_at":"2021-04-02 23:56:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":31791,"visible":true,"origin":"","legend":"The fluorescence strength of the promoters\na. Characterization of different green fluorescent proteins by promoter PUAS1B4-LEUm.\nb. Characterization of the native promoters PLEU, PEXP and PTEF.\nc. Characterization of different core promoters, and the relationship between T content upstream of TSS and relative mean fluorescence. Bars represent relative mean fluorescence and lines represent percentage of T content upstream of TSS.\nd. Characterization of different TATA boxes.\ne. The relationship between the copy number of UAS and relative mean fluorescence.\nf. Characterization of different UASs from S. cerevisiae and Y. lipolytica. \n","description":"","filename":"OnlineFig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-369447/v1/e4797f123dda3aed5fcbd25a.png"},{"id":7624905,"identity":"33dacaa4-2faf-4bb5-9674-67010954fe16","added_by":"auto","created_at":"2021-04-02 23:56:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":116496,"visible":true,"origin":"","legend":"The strategy of engineering hybrid promoter architecture\nDifferent promoter elements (UAS, TATA box and core promoter) were tested and ligated to the upstream of the reporter gene hrGFPO to characterize promoter strength in this study.\n","description":"","filename":"OnlineFig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-369447/v1/53b27ff76e96f57119001dda.png"},{"id":7624657,"identity":"55deafd6-d7b5-433f-a33e-2fd554f1e8fc","added_by":"auto","created_at":"2021-04-02 23:53:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":20210,"visible":true,"origin":"","legend":"The fluorescence of the promoter library constructed in this study\nThe hrGFPO was used as reporter gene for the promoter library constructed, and the fluorescence were detected by the BD Accuri C6 flow cytometer (BD Biosciences) using 488-nm excitation wavelength and FL1 channel.\n","description":"","filename":"OnlineFig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-369447/v1/57aa4a485d7ddc1452278c79.png"},{"id":7624661,"identity":"5e21a9f1-fea7-4e11-9919-8b58d6cdf72d","added_by":"auto","created_at":"2021-04-02 23:53:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":21079,"visible":true,"origin":"","legend":"The production of isoamyl alcohol in engineered Y. lipolytica\nThe cultures were grown in 40 mL YPD medium with an initial OD600 of 0.1 and 10% of n-dodecane in a 250 mL shake flask at 225 rpm and 28 °C for 3 days. The organic phase was analysed by GC/MS. ScARO10 is the key gene in the heterologous isoamyl alcohol production pathway. The isoamyl alcohol titer of Po1g ScARO10, which expressed ScARO10 under PUAS1B4-LEUm, was 1.36 mg/L, which was 2.7-fold higher than the control strain Po1g KU70Δ. The strain Po1g BAA, which co-expressed ScBAT1, ScARO10 and ScADH2 under PUAS1B4-LEUm, achieved an isoamyl alcohol titer of 1.8 mg/L, which was 3.9-fold that of control strain Po1g KU70Δ.\n","description":"","filename":"OnlineFig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-369447/v1/f881930e11cf592e81838213.png"},{"id":7624658,"identity":"d6c0bf2b-4d5a-4ab2-bd37-0d66ae10017a","added_by":"auto","created_at":"2021-04-02 23:53:42","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":14783,"visible":true,"origin":"","legend":"Comparison of partial promoter strength with isoamyl alcohol titer\nSeveral promoters were selected to replace PUAS1B4-LEUm for overexpressing ScARO10 in Po1g BAA. The strain with PUAS1B4-EXPm achieved the highest isoamyl alcohol titer of 11.57 mg/L, which was approximately 30.3-fold higher than that of Po1g KU70Δ and 5.4-fold that of Po1g BAA.\n","description":"","filename":"OnlineFig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-369447/v1/6c53be50e1be4495dd13bdda.png"},{"id":15672670,"identity":"87a3710c-4a4a-44c3-9738-94cd3b4e1c46","added_by":"auto","created_at":"2021-11-18 14:13:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1872720,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-369447/v1/cc237e72-6bea-4c59-8ea5-ac31437562e1.pdf"},{"id":7625326,"identity":"f09eff81-df90-4ec3-a125-53afd3eb73f8","added_by":"auto","created_at":"2021-04-02 23:59:42","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":67832,"visible":true,"origin":"","legend":"Additional file 1: Table S1. Primers used in PCR. Table S2. Plasmids used in this study. Table S3. Strains used in this study. Figure S1. Map of the plasmid pYLEX1.","description":"","filename":"AdditionalfileforBforB.docx","url":"https://assets-eu.researchsquare.com/files/rs-369447/v1/70b8072a8b1f802c656eb721.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eHybrid promoter engineering strategies in \u003cem\u003eYarrowia lipolytica\u003c/em\u003e:\u0026nbsp;isoamyl alcohol\u0026nbsp;production as a test study\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003ePromoters are one of the most important components of synthetic biology, and well-controlled promoters are very critical for regulating gene expression in eukaryotes. The activity of a promoter is co-regulated by various elements. In yeast, the common promoter elements usually include upstream activation sequences (UAS), TATA box and core promoter [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. By rational modification of these elements, the activity of promoters can be fine-tuned.\u003c/p\u003e \u003cp\u003eAt the beginning of transcription, regulatory signals are transmitted from the UAS to the core promoter, then transcription factors and RNA polymerase Ⅱ assembly combined with the transcription preinitiation complex (PIC) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The core promoter significantly contributes to the regulation of gene expression and is also the key factor determining the promoter strength. Although the core promoters were initially thought to be invariant, researchers have found that they exhibit great structural and functional diversity [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. TATA box, the recognition site of the transcription factor TATA binding protein (TBP), is one of the first kind of functional elements identified to regulate the promoter strength of the core promoter and typically located 40\u0026ndash;120 bp upstream of the transcription start site [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Mutations in the TATA box usually alter the promoter strength [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The UAS, which is usually located at the 5\u0026rsquo; of the promoter [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], is also known to affect the strength of the promoter by varying its copy number. By analysing the function of the endogenous alkaline extracellular protease 2 (XPR2) gene promoter P\u003csub\u003e\u003cem\u003eXPR2\u003c/em\u003e\u003c/sub\u003e of \u003cem\u003eYarrowia lipolytica\u003c/em\u003e, Madzak et al. identified UAS1B as the most significant functional element that activates the promoter P\u003csub\u003e\u003cem\u003eXPR2\u003c/em\u003e\u003c/sub\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Subsequently, evaluation of a hybrid promoter library consisting a minimal P\u003csub\u003e\u003cem\u003eLEU\u003c/em\u003e\u003c/sub\u003e fragment and different copy numbers of the UAS1B indicates that enhancement in promoter strength is correlated to increased copy number of UAS1B [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. When present in a promoter, different types of UAS can cooperate to control transcription. For example, by combining different UAS elements (UASTEF and UAS1B) in \u003cem\u003eY. lipolytica\u003c/em\u003e, the expression level of a constructed promoter was 7-fold higher than the wild-type promoter [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Taken together, by exploring the synergy between various promoter elements and understanding the working mechanism of the promoter, promoters with stronger activity and wider expression range can be constructed.\u003c/p\u003e \u003cp\u003eIn this study, in order to explore the mechanism of synergy between various elements in \u003cem\u003eY. lipolytica\u003c/em\u003e, the promoter elements were characterized and rearranged. Consequently, a library of hybrid promoters that enables stable expression and covers a wide range of promoter strength was constructed. Subsequently, we employed the hybrid promoters for promoter engineering of the pathway genes in isoamyl alcohol biosynthesis. Isoamyl alcohol, an important platform chemical, is widely applied in the production of biofuels, fragrances, medicines and fine chemicals [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and has been produced in recent years by metabolic engineering of diverse microbial cells, such as \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eCorynebacterium glutamicum\u003c/em\u003e and \u003cem\u003eAspergillus oryzae\u003c/em\u003e [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. While there is a report on improving the production of isoamyl alcohol in \u003cem\u003eY. lipolytica\u003c/em\u003e by metabolic engineering, a native promoter was employed [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, we used this pathway as a testbed and demonstrated the efficacy of our promoter library for optimizing metabolic pathways by significantly improving the isoamyl alcohol titer (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The outcome of this work shows that promoter engineering is an effective strategy for facilitating metabolic engineering efforts to biosynthesize valuable chemicals and our hybrid promoter library is a powerful toolkit for future metabolic engineering work in \u003cem\u003eY. lipolytica\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e "},{"header":"Results And Discussion","content":"\u003ch2\u003eScreening of the fluorescent reporter gene in \u003cspan class=\"BoldItalic\"\u003eY. lipolytica\u003c/span\u003e\u003c/h2\u003e\n\u003cp\u003eEfficient 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 \u003cem\u003eY. lipolytica\u003c/em\u003e for examining the strength of promoters, such as green fluorescent protein (GFP) and \u0026beta;-galactosidase [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. 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 \u003cem\u003eY. lipolytica\u003c/em\u003e 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 \u003cem\u003eY. lipolytica\u003c/em\u003e Po1g \u003cem\u003eKU70\u003c/em\u003e∆ that was used as a host system for this work. Firstly, expression of different fluorescent reporter genes \u003cem\u003eGFPuv\u003c/em\u003e (differs from the wild-type GFP by the amino acid replacements Val163Ala, Met153Thr, and Phe99Ser), \u003cem\u003ehrGFP\u003c/em\u003e (humanized \u003cem\u003eRenilla reinformis\u003c/em\u003e GFP) and \u003cem\u003ehrGFPO\u003c/em\u003e (codon optimized \u003cem\u003ehrGFP\u003c/em\u003e for \u003cem\u003eY. lipolytica\u003c/em\u003e) [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e] was driven by the hybrid promoter P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e, which is the promoter on the commercial integrative vector pYLEX1 for \u003cem\u003eY. lipolytica\u003c/em\u003e. Expression of the GFPs were detected by fluorescence microscopy and flow cytometry (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). The results indicate that fluorescence was not detected in the control strain Po1g \u003cem\u003eKU70\u003c/em\u003e\u0026Delta; while the \u003cem\u003eY. lipolytica\u003c/em\u003e strains carrying integrated \u003cem\u003eGFPuv\u003c/em\u003e or \u003cem\u003ehrGFP\u003c/em\u003e 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 \u003cem\u003eY. lipolytica\u003c/em\u003e. In contrast, the detected fluorescence intensity of strains that harbored the \u003cem\u003ehrGFPO\u003c/em\u003e gene was more stable than strains that possessed the \u003cem\u003ehrGFP\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). Henceforth, \u003cem\u003ehrGFPO\u003c/em\u003e was selected as the reporter gene for promoter characterization in this work.\u003c/p\u003e\n\u003ch2\u003eCharacterization of native promoters as a basis for the construction of hybrid promoters for \u003cspan class=\"BoldItalic\"\u003eY. lipolytica\u003c/span\u003e\u003c/h2\u003e\n\u003cp\u003eThe 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 \u003cem\u003ehrGFPO\u003c/em\u003e reporter gene to evaluate the promoter strengths of several commonly used native \u003cem\u003eY. lipolytica\u003c/em\u003e promoters: \u0026beta;-isopropylmalate dehydrogenase (LEU2) promoter P\u003csub\u003e\u003cem\u003eLEU\u003c/em\u003e\u003c/sub\u003e, export protein (EXP) promoter P\u003csub\u003e\u003cem\u003eEXP\u003c/em\u003e\u003c/sub\u003e and translation elongation factor-1\u0026alpha; (TEF1) promoter P\u003csub\u003e\u003cem\u003eTEF\u003c/em\u003e\u003c/sub\u003e. Based on the results of our experiments (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb), the relative fluorescence intensities of the corresponding strains from high to low are P\u003csub\u003e\u003cem\u003eTEF\u003c/em\u003e\u003c/sub\u003e \u0026gt; P\u003csub\u003e\u003cem\u003eEXP\u003c/em\u003e\u003c/sub\u003e \u0026gt; P\u003csub\u003e\u003cem\u003eLEU\u003c/em\u003e\u003c/sub\u003e, whereby the strength of P\u003csub\u003e\u003cem\u003eTEF\u003c/em\u003e\u003c/sub\u003e is about an order stronger than both P\u003csub\u003e\u003cem\u003eEXP\u003c/em\u003e\u003c/sub\u003e and P\u003csub\u003e\u003cem\u003eLEU\u003c/em\u003e\u003c/sub\u003e. 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 \u003cem\u003eY. lipolytica\u003c/em\u003e (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eCharacterization of features in core promoters that influence promoter strength\u003c/h2\u003e\n\u003cp\u003eThe 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 \u0026lsquo;the smallest DNA element for transcription\u0026rsquo; [\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e]. 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 \u003cem\u003eS. cerevisiae\u003c/em\u003e, 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 [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. Thus, we hypothesize that a similar trend exists in \u003cem\u003eY. lipolytica\u003c/em\u003e. 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 \u003cem\u003eY. lipolytica\u003c/em\u003e. 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 \u003cem\u003ehrGFPO\u003c/em\u003e 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.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec). 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.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec). 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 \u003cem\u003eY. lipolytica\u003c/em\u003e, further studies are required to elucidate the specific relationship between base content and promoter strength.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eModulating the promoter strength by varying the TATA box\u003c/h2\u003e\n\u003cp\u003eFunctional 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 [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. 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 [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. 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 [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]. Therefore, a series of TATA boxes (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) were selected to study their specific performance in promoters in \u003cem\u003eY. lipolytica\u003c/em\u003e. P\u003csub\u003e\u003cem\u003eUAS1B4+LEU\u003c/em\u003e\u003c/sub\u003e, 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 \u003cem\u003ehrGFPO\u003c/em\u003e 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.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed). 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.\u003c/p\u003e\n\u003ch2\u003eConstruction of promoters with various UAS elements from \u003cspan class=\"BoldItalic\"\u003eY. lipolytic\u003c/span\u003e and \u003cspan class=\"BoldItalic\"\u003eS. cerevisiae\u003c/span\u003e\u003c/h2\u003e\n\u003cp\u003eThe 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 \u003cem\u003eS. cerevisiae\u003c/em\u003e, such as UASTEF [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e], UASCLB [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e] and UASCIT [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, only a few UASs were identified in \u003cem\u003eY. lipolytica\u003c/em\u003e, 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 [\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]. Four tandem UAS1B from P\u003csub\u003e\u003cem\u003eXPR2\u003c/em\u003e\u003c/sub\u003e and one P\u003csub\u003e\u003cem\u003eLEU\u003c/em\u003e\u003c/sub\u003e core promoter have been combined to construct the strong constitutive promoter P\u003csub\u003e\u003cem\u003eUAS1B4+LEUm\u003c/em\u003e\u003c/sub\u003e [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. We increased the copy number of UAS and verified that the copy number of UAS is proportional to the hybrid promoter strength (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ee), which corroborates with published data [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. In addition, while it has been shown that synthetic terminators can be efficiently transferred in \u003cem\u003eS. cerevisiae\u003c/em\u003e and \u003cem\u003eY. lipolytica\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e], there is no research on the transferability of promoter elements across diverse yeast species. Therefore, different UASs (UASCIT \u003cem\u003eS.c\u003c/em\u003e., UASCLB \u003cem\u003eS.c.\u003c/em\u003e, UASTEF \u003cem\u003eS.c.\u003c/em\u003e and UASTEF \u003cem\u003eY.l.\u003c/em\u003e) [\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e] from \u003cem\u003eS. cerevisiae\u003c/em\u003e and \u003cem\u003eY. lipolytica\u003c/em\u003e with the same copy number as P\u003csub\u003e\u003cem\u003eUAS1B4+LEUm\u003c/em\u003e\u003c/sub\u003e were used to replace UAS1B4 to explore the influence of UAS types and origin on hybrid promoter activity. By expressing the \u003cem\u003ehrGFPO\u003c/em\u003e 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\u0026thinsp;\u0026gt;\u0026thinsp;UASTEF \u003cem\u003eY.l.\u003c/em\u003e\u0026gt; UASCIT \u003cem\u003eS.c.\u003c/em\u003e\u0026gt; UASCLB \u003cem\u003eS.c.\u003c/em\u003e\u0026gt; UASTEF \u003cem\u003eS.c.\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ef). These results demonstrated for the first time that UAS from \u003cem\u003eS. cerevisiae\u003c/em\u003e are functional in \u003cem\u003eY. lipolytica\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eTaken 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.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e and Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. 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.\u003c/p\u003e\n\u003ch2\u003eConstruction of the isoamyl alcohol overexpression pathway in \u003cspan class=\"BoldItalic\"\u003eY. lipolytica\u003c/span\u003e\u003c/h2\u003e\n\u003cp\u003eAs an important platform chemical, isoamyl alcohol is a promising biofuel and biochemical with huge market demand. However, in \u003cem\u003eY. lipolytica\u003c/em\u003e, the titer of isoamyl alcohol natively is quite low at a mere 0.37 mg/L (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). 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.\u003c/p\u003e\n\u003cp\u003eIn yeast, isoamyl alcohol is generally produced through the Ehrlich pathway, which usually involves three reaction steps: transamination, decarboxylation and reduction. Twelve genes encoding transaminases (\u003cem\u003eScBAT1\u003c/em\u003e, \u003cem\u003eYlBAT1-1\u003c/em\u003e and \u003cem\u003eYlBAT1-2\u003c/em\u003e), decarboxylases (\u003cem\u003eScARO10\u003c/em\u003e, \u003cem\u003eYlARO10-1\u003c/em\u003e and \u003cem\u003eYlARO10-2\u003c/em\u003e) and alcohol dehydrogenases (\u003cem\u003eScADH2\u003c/em\u003e, \u003cem\u003eYlADH2-1\u003c/em\u003e, \u003cem\u003eYlADH2-2\u003c/em\u003e, \u003cem\u003eYlADH2-3\u003c/em\u003e, \u003cem\u003eYlADH2-4\u003c/em\u003e and \u003cem\u003eYlADH2-5\u003c/em\u003e) 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 \u003cem\u003eY. lipolytica\u003c/em\u003e Po1g \u003cem\u003eKU70\u003c/em\u003e\u0026Delta; and driven by the constitutive promoter P\u003csub\u003e\u003cem\u003eUAS1B4+LEUm\u003c/em\u003e\u003c/sub\u003e. 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 \u003cem\u003eKU70\u003c/em\u003e\u0026Delta; (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). 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 \u003cem\u003eScARO10\u003c/em\u003e-overexpressed strain, which reach 1.36 mg/L. The strains which overexpressed transaminase gene \u003cem\u003eScBAT1\u003c/em\u003e and dehydrogenase gene \u003cem\u003eScADH2\u003c/em\u003e also increased the harvest of isoamyl alcohol moderately. Therefore, to further improve the yield of isoamyl alcohol, the genes \u003cem\u003eScBAT1\u003c/em\u003e, \u003cem\u003eScARO10\u003c/em\u003e and \u003cem\u003eScADH2\u003c/em\u003e 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 \u003cem\u003eKU70\u003c/em\u003e\u0026Delta; (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). Thus, the strain Po1g BAA was selected for subsequent engineering by promoter replacement with our hybrid promoter library.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eApplication of the hybrid promoter library to improve the isoamyl alcohol biosynthesis pathway\u003c/h2\u003e\n\u003cp\u003eIn metabolic engineering, studies have shown that the yield of the target product can be increased by replacing promoters for pathway genes with stronger ones [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. 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 \u003cem\u003eScARO10\u003c/em\u003e in the isoamyl alcohol pathway. These constructed strains were cultured for 3 days, and the titer of the isoamyl alcohol was quantified (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). 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\u0026thinsp;+\u0026thinsp;P\u003csub\u003e\u003cem\u003eUAS1B4+EXPm\u003c/em\u003e\u003c/sub\u003e+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 \u003cem\u003eKU70\u003c/em\u003e\u0026Delta; and 5.4-fold that of Po1g BAA. This result is consistent with the opinion of Dulermo, \u003cem\u003eet al.\u003c/em\u003e that stronger promoters do not necessarily increase the expression level and/or function of a protein [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. In addition, we found that although the activity of P\u003csub\u003e\u003cem\u003eEXP\u003c/em\u003e\u003c/sub\u003e was low, several strains containing P\u003csub\u003e\u003cem\u003eEXP\u003c/em\u003e\u003c/sub\u003e elements (P\u003csub\u003e\u003cem\u003eEXP\u003c/em\u003e\u003c/sub\u003e, P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;EXPm\u003c/em\u003e\u003c/sub\u003e, P\u003csub\u003e\u003cem\u003eUAS1B4+TATAEXP\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e) had higher titers of isoamyl alcohol, suggesting that the elements of the P\u003csub\u003e\u003cem\u003eEXP\u003c/em\u003e\u003c/sub\u003e have greater beneficial effects to the expression of the \u003cem\u003eARO10\u003c/em\u003e gene, which encodes a key enzyme of the isoamyl alcohol pathway. More studies are needed to better understand the mechanism between the elements of P\u003csub\u003e\u003cem\u003eEXP\u003c/em\u003e\u003c/sub\u003e 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.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusions","content":" \u003cp\u003ePromoters are one of the most important components of synthetic biology for determining protein expression. Compared to prokaryotes, the regulatory mechanism of the promoter structure in eukaryotes is extremely complex [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Increasing the promoter strength is a common method to improve gene transcription and protein expression level. However, recent studies have shown that not all strong promoters can achieve the highest protein expression and activity [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. We explored the structure and functional characteristics of the promoters of \u003cem\u003eY. lipolytica\u003c/em\u003e, and subsequently constructed a series of constitutive promoters which are stable and efficient. Firstly, different variants of green fluorescent protein were screened in \u003cem\u003eY. lipolytica\u003c/em\u003e to identify a reporter gene that can be stably expressed. Among different transformants, the codon optimized \u003cem\u003ehrGFP\u003c/em\u003e (\u003cem\u003ehrGFPO\u003c/em\u003e) not only expressed at a high level but also expressed stably in \u003cem\u003eY. lipolytica\u003c/em\u003e. Therefore, the \u003cem\u003ehrGFPO\u003c/em\u003e gene was used for subsequent promoter characterization experiments.\u003c/p\u003e \u003cp\u003eThree native promoters of \u003cem\u003eY. lipolytica\u003c/em\u003e, P\u003csub\u003e\u003cem\u003eLEU\u003c/em\u003e\u003c/sub\u003e, P\u003csub\u003e\u003cem\u003eTEF\u003c/em\u003e\u003c/sub\u003e and P\u003csub\u003e\u003cem\u003eEXP\u003c/em\u003e\u003c/sub\u003e, were characterized and the results showed that the strengths of these promoters are evidently different. These promoters were dissected into three parts, namely UAS, TATA box and Core promoter, and these elements were combinatorially arranged with other studied promoter elements to construct a constitutive promoter library that contains 21 stable hybrid promoters. It is the first time that the T content upstream of the TSS has been shown to positively correlate with the hybrid promoter strength in \u003cem\u003eY. lipolytica\u003c/em\u003e. It is worth noting that some core promoter elements, such as POX2m and LEUm, did not conform to the trend. Therefore, the relationship between the T content upstream of the TSS and the promoter strength in \u003cem\u003eY. lipolytica\u003c/em\u003e needs to be further studied. Next, the effects of different UAS elements from \u003cem\u003eS. cerevisiae\u003c/em\u003e and \u003cem\u003eY. lipolytica\u003c/em\u003e on promoter strength were investigated and we discovered for the first time that UAS elements can be transferred between yeast species. These findings lay the groundwork for the development of hybrid promoters which can be efficiently transferred across diverse yeast species.\u003c/p\u003e \u003cp\u003eTo demonstrate application of our hybrid promoter library, the isoamyl alcohol production pathway was constructed to serve as a testbed by co-expression of multiple genes from \u003cem\u003eS. cerevisiae\u003c/em\u003e and \u003cem\u003eY. lipolytica\u003c/em\u003e. \u003cem\u003eScAOR10\u003c/em\u003e, the key gene of the isoamyl alcohol pathway, was selected as the test gene for expression under various hybrid promoters from our library to optimize the enzyme\u0026rsquo;s expression and activity for enhance isoamyl alcohol production. Consequently, the titer of the isoamyl alcohol increased from 0.37 mg/L to 11.57 mg/L, which was 30.3-fold higher than the control strain Po1g \u003cem\u003eKU70\u003c/em\u003eΔ. To date, isoamyl alcohol has been successfully produced by metabolic engineering in several studies [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Although the titer of isoamyl alcohol from \u003cem\u003eY. lipolytica\u003c/em\u003e is lower compared with other studies, it is the first time that the promoter engineering has been applied for the biosynthesis of isoamyl alcohol to provide an advanced solution for the biosynthesis of biofuels and alcohols. Regulation of expression by promoters involves various factors, such as temperature, pH and substrate [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In the future, we will further study the mechanisms of promoters to construct hybrid promoters with stronger activity and wider expression range for optimum expression of biosynthesis pathway genes to achieve high-level production of value-added chemicals.\u003c/p\u003e "},{"header":"Materials And Methods","content":"\u003cdiv\u003e\n\u003ch2\u003eStrains and media\u003c/h2\u003e\n\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e strain DH5\u0026alpha; was used for all cloning and plasmid propagation, and DH5\u0026alpha; was grown at 37\u0026deg;C in Luria Bertani (LB), and supplemented with ampicillin to final concentration of 100 \u0026micro;g/mL for plasmid propagation. \u003cem\u003eY. lipolytica\u003c/em\u003e strain Po1g \u003cem\u003eKU70\u003c/em\u003e\u0026Delta;, a leucine auxotroph devoid of any secreted protease activity, was used as the base strain in this study. \u003cem\u003eY. lipolytica\u003c/em\u003e Po1g \u003cem\u003eKU70\u003c/em\u003e\u0026Delta; containing plasmid was routinely cultivated at 28\u0026deg;C and 225 rpm with YPD media consisting of 20 g/L glucose, 20 g/L peptone, and 10 g/L yeast extract. In this study, PCR primers were synthesized by Genewiz (Jiangsu, China) and are listed in Table S1, plasmids are listed in Table S2 and strains used are listed in Table S3.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n\u003ch2\u003eChemicals and enzymes\u003c/h2\u003e\n\u003cp\u003eAll restriction enzymes were purchased from New England Biolabs (Beijing, China), 2\u0026times;Phanta\u0026reg; max master mix, 2\u0026times; Rapid Taq master mix, ClonExpress\u0026reg; II one step cloning kit, FastPure\u0026reg; Plasmid Mini Kit and FastPure\u0026reg; Gel DNA Extraction Mini Kit were purchased from Vazyme Biotech Co., Ltd. (Nanjing, China), peptone and yeast extract were purchased from Thermo Scientific Oxoid Microbiology Products (Basingstoke, England), isoamyl alcohol and n-dodecane were purchased from Aladdin\u0026reg; (Shanghai, China).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n\u003ch2\u003ePlasmid construction of promoter library\u003c/h2\u003e\n\u003cp\u003eThe \u003cem\u003eGFPuv\u003c/em\u003e gene was preserved in this laboratory, and cloned into pYLEX1 with primers GFPuv-F/GFPuv-R (Table S1) yield plasmid pYLGFPuv (Table S2). The \u003cem\u003ehrGFP\u003c/em\u003e gene and \u003cem\u003ehrGFPO\u003c/em\u003e gene were synthesized and cloned into pYLEX1 to yield plasmids pYLhrGFP and pYLhrGFPO (Table S2), respectively, by Genewiz (Jiangsu, China). The UASCIT \u003cem\u003eS.c.\u003c/em\u003e4, UASCLB \u003cem\u003eS.c.\u003c/em\u003e4, UASTEF \u003cem\u003eS.c.\u003c/em\u003e4, UASTEF \u003cem\u003eY.l.\u003c/em\u003e4, UAS1B6 and UAS1B8 motifs were synthesized and cloned into plasmids pYLhrGFPO to replace UAS1B4 to yield plasmids pYLP\u003csub\u003e\u003cem\u003eUASCITSC4\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e+hrGFPO, pYLP\u003csub\u003e\u003cem\u003eUASCLBSC4\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e+hrGFPO, pYLP\u003csub\u003e\u003cem\u003eUASTEFSC4\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e+hrGFPO, pYLP\u003csub\u003e\u003cem\u003eUASTEFYL4\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e+hrGFPO, pYLP\u003csub\u003e\u003cem\u003eUAS1B6\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e+hrGFPO and pYLP\u003csub\u003e\u003cem\u003eUAS1B8\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e+hrGFPO (Table S2), respectively, by Genewiz (Jiangsu, China). Three endogenous promoters P\u003csub\u003e\u003cem\u003eLEU\u003c/em\u003e\u003c/sub\u003e, P\u003csub\u003e\u003cem\u003eTEF\u003c/em\u003e\u003c/sub\u003e and P\u003csub\u003e\u003cem\u003eEXP\u003c/em\u003e\u003c/sub\u003e were cloned into vector pYLhrGFPO with primers PLEU-F/LEU-hrGFPO-R, PTEF-F/TEF-hrGFPO-R and PEXP-F/EXP-hrGFPO-R (Table S1) yield plasmids pYLP\u003csub\u003e\u003cem\u003eLEU\u003c/em\u003e\u003c/sub\u003e+hrGFPO, pYLP\u003csub\u003e\u003cem\u003eTEF\u003c/em\u003e\u003c/sub\u003e+hrGFPO and pYLP\u003csub\u003e\u003cem\u003eEXP\u003c/em\u003e\u003c/sub\u003e+hrGFPO (Table S2), respectively. The Core promoters were amplified by primer pairs PAT1m-F/PAT1-hrGFPO-R, POX2m-F/POX2-hrGFPO-R, EXPm-F/EXP-hrGFPO-R, TEFm111-F/TEF-hrGFPO-R, TEF136-F/TEF-hrGFPO-R and TEFm175-F/TEF-hrGFPO-R (Table S1), and then replace the core promoter LEU in P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;LEU\u003c/em\u003e\u003c/sub\u003e. These promoters were ligated to pYLhrGFPO in place of the P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;LEU\u003c/em\u003e\u003c/sub\u003e to yield plasmids pYLP\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;PAT1m\u003c/em\u003e\u003c/sub\u003e+hrGFPO, pYLP\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;POX2m\u003c/em\u003e\u003c/sub\u003e+hrGFPO, pYLP\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;EXP1m\u003c/em\u003e\u003c/sub\u003e+hrGFPO, pYLP\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;TEF111\u003c/em\u003e\u003c/sub\u003e+hrGFPO, pYLP\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;TEF136\u003c/em\u003e\u003c/sub\u003e+hrGFPO and pYLP\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;TEF175\u003c/em\u003e\u003c/sub\u003e+hrGFPO (Table S2), respectively. The TATA box LEU in P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;LEU\u003c/em\u003e\u003c/sub\u003e was replaced by TATA box TEF, EXP, PAT1 and POX2 using primer pairs TATA TEF-F/LEU-hrGFPO-R, TATA EXP-F/LEU-hrGFPO-R, TATA PAT1-F/LEU-hrGFPO-R and TATA POX2-F/LEU-hrGFPO-R (Table S1). These hybrid promoters were ligated to pYLhrGFPO in place of the P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;LEU\u003c/em\u003e\u003c/sub\u003e to yield plasmids pYLP\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;TATATEF\u0026minus;LEU\u003c/em\u003e\u003c/sub\u003e+hrGFPO, pYLP\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;TATAEXP\u0026minus;LEU\u003c/em\u003e\u003c/sub\u003e+hrGFPO, pYLP\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;TATAPAT1\u0026minus;LEU\u003c/em\u003e\u003c/sub\u003e+hrGFPO and pYLP\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;TATAPOX2\u0026minus;LEU\u003c/em\u003e\u003c/sub\u003e+hrGFPO (Table S2), respectively.\u003c/p\u003e\n\u003cp\u003eAll plasmids, linearized by Not I or Spe I, were transformed into competent cells of \u003cem\u003eY. lipolytica\u003c/em\u003e strains using the lithium acetate method [36].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n\u003ch2\u003ePlasmid construction of exogenous isoamyl alcohol pathway\u003c/h2\u003e\n\u003cp\u003eThe transaminase gene (\u003cem\u003eBAT1\u003c/em\u003e, GenBank ID: 856615), decarboxylase gene (\u003cem\u003eARO10\u003c/em\u003e, GenBank ID: 851987) and alcohol dehydrogenase gene (\u003cem\u003eADH2\u003c/em\u003e, GenBank ID: 855349) from \u003cem\u003eS. cerevisiae\u003c/em\u003e S288C were codon-optimized and synthesized and cloned into pYLEX1 to yield plasmids pYLSCBAT1, pYLSCARO10 and pYLSCADH2 (Table S2), respectively, by Genewiz (Jiangsu, China). In \u003cem\u003eY. lipolytica\u003c/em\u003e, the homologous sequences that \u003cem\u003eYlBAT1-1\u003c/em\u003e and \u003cem\u003eYlBAT1-2\u003c/em\u003e of \u003cem\u003eScBAT1\u003c/em\u003e were cloned into pYLEX1 with primers YLBAT1-1-F/YLBAT1-1-R and YLBAT1-2-F/YLBAT1-2-R (Table S1) to yield plasmids pYLYLBAT1-1 and pYLYLBAT1-2 (Table S2), respectively. The homologous sequences that \u003cem\u003eYlARO10-1\u003c/em\u003e and \u003cem\u003eYlARO10-2\u003c/em\u003e of \u003cem\u003eScARO10\u003c/em\u003e were cloned into pYLEX1 with primers YLARO10-1-F/YLARO10-1-R and YLARO10-2-F/YLARO10-2-R (Table S1) to yield plasmids pYLYLARO10-1 and pYLYLARO10-2 (Table S2), respectively. The homologous sequences that \u003cem\u003eYlADH2-1, YlADH2-2, YlADH2-3, YlADH2-4\u003c/em\u003e and \u003cem\u003eYlADH2-5\u003c/em\u003e of \u003cem\u003eScADH2\u003c/em\u003e were cloned into pYLEX1 with primers YLADH2-1-F/YLADH2-1-R, YLADH2-2-F/YLADH2-2-R, YLADH2-3-F/YLADH2-3-R, YLADH2-4-F/YLADH2-4-R and YLADH2-5-F/YLADH2-5-R (Table S1) to yield plasmids pYLYLADH2-1, pYLYLADH2-2, pYLYLADH2-3, pYLYLADH2-4 and pYLYLADH2-5 (Table S2), respectively.\u003c/p\u003e\n\u003cp\u003eThe expression cassettes of \u003cem\u003eScARO10\u003c/em\u003e and \u003cem\u003eScADH2\u003c/em\u003e were cloned into pYLSCBAT1 with primers BDH-ADH2-F/BDH-ADH2-R and BDH-ARO10-F/ BDH-ARO10-R (Table S1) to yield plasmid pYLBAA (Table S2). All plasmids, linearized by Not I or Spe I, were transformed into competent cells of \u003cem\u003eY. lipolytica\u003c/em\u003e strains using the lithium acetate method [36].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n\u003ch2\u003eExpressing the isoamyl alcohol synthesis pathway using the promoter library\u003c/h2\u003e\n\u003cp\u003eSeveral promoters from the promoter library were used to express the \u003cem\u003eARO10\u003c/em\u003e gene which is the key gene in the isoamyl alcohol pathway. The promoters P\u003csub\u003e\u003cem\u003eEXP\u003c/em\u003e\u003c/sub\u003e and P\u003csub\u003e\u003cem\u003eUAS1B4+EXPm\u003c/em\u003e\u003c/sub\u003e were amplified by primers BDH-ARO10-F/PEXP-ARO10-R (Table S1), and then ligated to \u003cem\u003eScARO10\u003c/em\u003e in pYLBAA to yield plasmid pYLBA\u0026thinsp;+\u0026thinsp;P\u003csub\u003e\u003cem\u003eEXP\u003c/em\u003e\u003c/sub\u003e+ARO10 and pYLBA\u0026thinsp;+\u0026thinsp;P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;EXPm\u003c/em\u003e\u003c/sub\u003e+ARO10 (Table S2), respectively. The promoters P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;POX2m\u003c/em\u003e\u003c/sub\u003e and P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;TEF136\u003c/em\u003e\u003c/sub\u003e were amplified by primers BDH-ARO10-F/POX2-ARO10-R and BDH-ARO10-F/PTEF-ARO10-R (Table S1), and then ligated to \u003cem\u003eScARO10\u003c/em\u003e in pYLBAA to yield plasmid pYLBA\u0026thinsp;+\u0026thinsp;P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;POX2m\u003c/em\u003e\u003c/sub\u003e+ARO10 and pYLBA\u0026thinsp;+\u0026thinsp;P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;TEF136\u003c/em\u003e\u003c/sub\u003e+ARO10 (Table S2), respectively. The promoters P\u003csub\u003e\u003cem\u003eUASTEFLY4\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e, P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;TATAEXP\u0026minus;LEU\u003c/em\u003e\u003c/sub\u003e, P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;TATATEF\u0026minus;LEU\u003c/em\u003e\u003c/sub\u003e and P\u003csub\u003e\u003cem\u003eUAS1B8\u0026minus; LEUm\u003c/em\u003e\u003c/sub\u003e were amplified by primers BDH-ARO10-F/PLEU-ARO10-R (Table S1), and then ligated to \u003cem\u003eScARO10\u003c/em\u003e in pYLBAA to yield plasmid pYLBA\u0026thinsp;+\u0026thinsp;P\u003csub\u003e\u003cem\u003eUASTEFLY4\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e+ARO10, pYLBA\u0026thinsp;+\u0026thinsp;P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;TATAEXP\u0026minus;LEU\u003c/em\u003e\u003c/sub\u003e+ARO10, pYLBA\u0026thinsp;+\u0026thinsp;P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;TATATEF\u0026minus;LEU\u003c/em\u003e\u003c/sub\u003e+ARO10 and pYLBA\u0026thinsp;+\u0026thinsp;P\u003csub\u003e\u003cem\u003eUAS1B8\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e +ARO10 (Table S2), respectively.\u003c/p\u003e\n\u003cp\u003eAll plasmids, linearized by Not I or Spe I, were transformed into competent cells of \u003cem\u003eY. lipolytica\u003c/em\u003e strains using the lithium acetate method [37].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n\u003ch2\u003eYeast strain construction\u003c/h2\u003e\n\u003cp\u003eThe competent cell scheme and transformation method are referred to Pang, \u003cem\u003eet al.\u003c/em\u003e[38]. After selection, the following engineered \u003cem\u003eY. lipolytica\u003c/em\u003e strains were generated: Po1g P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e+GFPuv, Po1g P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e+hrGFP, Po1g P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e+hrGFPO, Po1g P\u003csub\u003e\u003cem\u003eUAS1B6\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e+hrGFPO, Po1g P\u003csub\u003e\u003cem\u003eUAS1B8\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e+hrGFPO, Po1g P\u003csub\u003e\u003cem\u003eLEUm\u003c/em\u003e\u003c/sub\u003e+hrGFPO, Po1g P\u003csub\u003e\u003cem\u003eLEU\u003c/em\u003e\u003c/sub\u003e+hrGFPO, Po1g P\u003csub\u003e\u003cem\u003eEXP\u003c/em\u003e\u003c/sub\u003e+hrGFPO, Po1g P\u003csub\u003e\u003cem\u003eTEF\u003c/em\u003e\u003c/sub\u003e+hrGFPO, Po1g P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;EXPm\u003c/em\u003e\u003c/sub\u003e+hrGFPO, Po1g P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;POX2m\u003c/em\u003e\u003c/sub\u003e+hrGFPO, Po1g P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;PAT1m\u003c/em\u003e\u003c/sub\u003e+hrGFPO, Po1g P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;TEF111\u003c/em\u003e\u003c/sub\u003e+hrGFPO, Po1g P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;TEF136\u003c/em\u003e\u003c/sub\u003e+hrGFPO, Po1g P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;TEF175\u003c/em\u003e\u003c/sub\u003e+hrGFPO, Po1g P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;TATAPAT1\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e+hrGFPO, Po1g P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;TATAPOX2\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e+hrGFPO, Po1g P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;TATAEXP\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e+hrGFPO, Po1g P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;TATATEF\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e+hrGFPO, Po1g P\u003csub\u003e\u003cem\u003eUASTEFSC4\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e+hrGFPO, Po1g P\u003csub\u003e\u003cem\u003eUASCLBSC4\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e+hrGFPO, Po1g P\u003csub\u003e\u003cem\u003eUASTEFYL4\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e+hrGFPO, Po1g ScBAT1, Po1g YlBAT1-1, Po1g YlBAT1-2, Po1g ScARO10, Po1g YlARO10-1, Po1g YlARO10-2, Po1g ScADH2, Po1g YlADH2-1, Po1g YlADH2-2, Po1g YlADH2-3, Po1g YlADH2-4, Po1g YlADH2-5, Po1g BAA, Po1g BA\u0026thinsp;+\u0026thinsp;P\u003csub\u003e\u003cem\u003eEXP\u003c/em\u003e\u003c/sub\u003e+ARO10, Po1g BA\u0026thinsp;+\u0026thinsp;P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;EXPm\u003c/em\u003e\u003c/sub\u003e+ARO10, Po1g BA\u0026thinsp;+\u0026thinsp;P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;POX2m\u003c/em\u003e\u003c/sub\u003e+ARO10, Po1g BA\u0026thinsp;+\u0026thinsp;P\u003csub\u003e\u003cem\u003eUASTEFYL4\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e+ARO10, Po1g BA\u0026thinsp;+\u0026thinsp;P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;TEF136\u003c/em\u003e\u003c/sub\u003e+ARO10, Po1g BA\u0026thinsp;+\u0026thinsp;P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e+ARO10, Po1g BA\u0026thinsp;+\u0026thinsp;P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;TATAEXP\u0026minus;LEU\u003c/em\u003e\u003c/sub\u003e+ARO10, Po1g BA\u0026thinsp;+\u0026thinsp;P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;TATATEF\u0026minus;LEU\u003c/em\u003e\u003c/sub\u003e+ARO10, Po1g BA\u0026thinsp;+\u0026thinsp;P\u003csub\u003e\u003cem\u003eUAS1B8\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e+ARO10 (Table S3).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n\u003ch2\u003eFlow cytometry\u003c/h2\u003e\n\u003cp\u003eThe green fluorescent protein GFPuv, hrGFP and hrGFPO were selected as reporter proteins. The colonies of transformants were selected from plates and grew in 5 mL of fresh YPD medium in tube for 24 h. After that, the seed culture solution was inoculated to 250 mL flasks which contain 40 mL YPD medium, starting from OD\u003csub\u003e600\u003c/sub\u003e 0.1. Cultures were cultivated at rotary shaker at 225 rpm and 28\u0026deg;C. Before flow cytometry analysis, the cultures were centrifuged at 12000 rpm for 1 min, and washed in 0.1 M phosphate-buffered saline (PBS), then resuspended in PBS. There were 10,000 cell count that were analysed with the BD Accuri C6 flow cytometer (BD Biosciences) using 488-nm excitation wavelength and FL1 channel for fluorescence detection. The CFlow software was used to analysed the data and compute mean fluorescence values.\u003c/p\u003e\n\u003ch2\u003eGC/MS analysis of isoamyl alcohol produced in the engineered Y. lipolytica strains\u003c/h2\u003e\n\u003cp\u003eThe engineered \u003cem\u003eY. lipolytica\u003c/em\u003e transformants were selected from plate and prepared in 5 mL of fresh YPD medium in tube for 24 h. The seed culture solution was inoculated to 250 mL flasks containing 40 mL of YPD medium, starting from OD\u003csub\u003e600\u003c/sub\u003e 0.1. The cultures were shaken at 225 rpm and 28\u0026deg;C for 3 days. In order to extract isoamyl alcohol from the cultures, 10% \u003cem\u003en\u003c/em\u003e-dodecane was added to the cultures, and the mixture was vortexed for 3 minutes, then centrifuged at 7500 rpm for 5 minutes. The organic phase of 1ul was detected by GC/MS using an Agilent 7890B GC with an 5977B MSD equipped with a HP-5MS column (60 m \u0026times; 0.25 mm \u0026times; 0.25 \u0026micro;m, Agilent, Santa Clara, CA, USA). GC oven temperature was initially held at 60\u0026deg;C for 2 min, and then ramped to 140\u0026deg;C at a rate of 5\u0026deg;C/min. It was then subsequently ramped at 10\u0026deg;C/min to 280\u0026deg;C and held for 5 min. The split ratio was 10:1. Helium was used as the carrier gas, with an inlet pressure of 13.8 psi. The injector was maintained at 280\u0026deg;C and the ion source temperature was set to 230\u0026deg;C. Final data analysis was achieved using MassHunter Workstation Software (Agilent, Santa Clara, CA, USA).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Abbreviations","content":" \u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGC/MS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egas chromatography/mass spectrometry;\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOD\u003csub\u003e600\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eoptical density at 600 nm;\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLB medium\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e0.5% yeast extract, 1% tryptone and 1% NaCl;\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eYPD medium\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e1% yeast extract, 2% peptone and 2% glucose;\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eYNB plate\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2% glucose, 0.67% yeast nitrogen base without amino acids and 2% agar;\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epolymerase chain reaction.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eYlBAT1-1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eYALI0_D01265g\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eYlBAT1-2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eYALI0_F19910g\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eYlARO10-1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eYALI0_D06930g\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eYlARO10-2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eYALI0_E07325g\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eYlADH2-1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eYALI0_A16379g\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eYlADH2-2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eYALI0_D25630g\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eYlADH2-3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eYALI0_E17787g\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eYlADH2-4\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eYALI0_A15147g\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eYlADH2-5\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eYALI0_E07766g\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis manuscript does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors give consent to publish the research in Biotechnology for Biofuels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll relevant data generated or analysed during this study were included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Natural Science Foundation of Tianjin, China (17JCYBJC40800), the Research Foundation of Tianjin Municipal Education Commission, China (2017ZD03), the Innovative Research Team of Tianjin Municipal Education Commission, China (TD13-5013), Tianjin Municipal Science and Technology Project (18PTSYJC00140, 19PTSYJC00060), Startup Fund for \u0026ldquo;Haihe Young Scholars\u0026rdquo; of Tianjin University of Science and Technology, the Thousand Young Talents Program of Tianjin, China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAQY, JLF, DGX and CYZ conceived and designed the study. YZ, SQL, ZHL, BXZ and SHW performed plasmid and strain construction, and fermentation experiments. AQY, JLF, DGX, CYZ revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Natural Science Foundation of Tianjin, China (17JCYBJC40800), the Research Foundation of Tianjin Municipal Education Commission, China (2017ZD03), the Innovative Research Team of Tianjin Municipal Education Commission, China (TD13-5013), Tianjin Municipal Science and Technology Project (18PTSYJC00140, 19PTSYJC00060), Startup Fund for \u0026lsquo;Haihe Young Scholars\u0026rsquo; of Tianjin University of Science and Technology, the Thousand Young Talents Program of Tianjin, China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003eState Key Laboratory of Food Nutrition and Safety, Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, No.29 the 13th Street TEDA, Tianjin 300457, PR China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u003c/sup\u003eSynthetic Biology Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119228, Singapore\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ec\u003c/sup\u003eNUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), National University of Singapore, Singapore 117456, Singapore\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ed\u003c/sup\u003eDepartment of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eShabbir Hussain M, Gambill L, Smith S, Blenner MA. 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J Vis Exp. 2016;20(115):54371.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, Zhu K, Miao L, Rong L, Zhao Y, Li S, Ma L, Li J, Zhang C, Xiao D, Foo JL, Yu A. Simultaneous improvement of limonene production and tolerance in \u003cem\u003eYarrowia lipolytica\u003c/em\u003e through tolerance engineering and evolutionary engineering. ACS Synth Biol. 2021. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1021/acssynbio.1c00052\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePang Y, Zhao Y, Li S, Zhao Y, Li J, Hu Z, Zhang C, Xiao D, Yu A. Engineering the oleaginous yeast \u003cem\u003eYarrowia lipolytica\u003c/em\u003e to produce limonene from waste cooking oil. Biotechnol Biofuels. 2019;12:241.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXuan JW, Fournier P, Declerck N, Chasles M, Gaillardin C. Overlapping reading frames at the LYS5 locus in the yeast \u003cem\u003eYarrowia lipolytica\u003c/em\u003e. Mol Cell Biol. 1990;10(9):4795\u0026ndash;806.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDamude HG, Gillies PJ, Macool DJ, Picataggio SK, Pollak DWM, Ragghianti JJ, Xue Z. High eicosapentaenoic acid producing strains of \u003cem\u003eYarrowia lipolytica\u003c/em\u003e. US. 2013;US8518674:B2.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"tables","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eList of promoters used in this study\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePromoters\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eUAS type\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTATA box\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCore promoter\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eStrength\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eReference\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLEU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eEXP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+ +\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTEF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+ + +\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLEUm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLEU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLEU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUASTEFSC4-LEUm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUASTEFSC4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLEU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLEU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+ +\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eThis study\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUASCLBSC4-LEUm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUASCLBSC4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLEU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLEU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+ +\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eThis study\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUASCITSC4-LEUm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUASCITSC4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLEU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLEU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+ +\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eThis study\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUAS1B4-EXPm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUAS1B4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eEXP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eEXP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+ +\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eThis study\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUAS1B4-POX2m\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUAS1B4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePOX2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePOX2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+ + +\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eThis study\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUAS1B4-TATAPAT1-LEU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUAS1B4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePAT1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLEU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+ + +\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eThis study\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUAS1B4-PAT1m\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUAS1B4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePAT1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePAT1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+ + +\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eThis study\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUASTEFYL4-LEUm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUASTEFYL4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLEU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLEU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+ + + +\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eThis study\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUAS1B4-TEF175\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUAS1B4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTEF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTEF175\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+ + + +\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eThis study\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUAS1B4-TEF136\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUAS1B4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTEF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTEF136\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+ + + +\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eThis study\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUAS1B4-TEF111\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUAS1B4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTEF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTEF111\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+ + + +\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eThis study\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUAS1B4-LEUm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUAS1B4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLEU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLEU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+ + + +\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUAS1B4-TATAPOX2-LEU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUAS1B4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePOX2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLEU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+ + + +\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eThis study\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUAS1B4-TATAEXP-LEU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUAS1B4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eEXP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLEU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+ + + +\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eThis study\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUAS1B6-LEUm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUAS1B6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLEU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLEU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+ + + + +\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eThis study\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUAS1B4-TATATEF-LEU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUAS1B4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTEF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLEU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+ + + + +\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eThis study\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUAS1B8-LEUm\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUAS1B8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLEU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLEU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+ + + + +\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eTATA box tested in this study\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTATA box\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSequence\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLEU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTATATATA\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTEF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTATAAAA\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eEXP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eATTATATATAA\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePAT1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTATATACC\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePOX2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGTATACTTATATA\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u0026nbsp;\u003c/div\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Y. lipolytica, metabolic engineering, hybrid promoter, isoamyl alcohol, synthetic promoter","lastPublishedDoi":"10.21203/rs.3.rs-369447/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-369447/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eIn biological cells, promoters drive gene expression by binding to RNA polymerase specifically. They determine the starting position, timing and level of gene expression. Therefore, rational fine-tuning of promoters to regulate the expression levels of target genes for metabolic engineering applications to optimize biosynthetic pathways has recently become an active area of research.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this study, we systematically detected and characterized the common promoter elements in the unconventional yeast \u003cem\u003eYarrowia lipolytica\u003c/em\u003e, and constructed an artificial hybrid promoter library that covers a wide range of promoter strength. We also report for the first time that upstream activation sequences (UAS) of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e promoters can be functionally transferred to \u003cem\u003eY. lipolytica\u003c/em\u003e. Subsequently, using the production of a versatile platform chemical isoamyl alcohol as a test study, the hybrid promoter library was applied to optimize the biosynthesis pathway expression in \u003cem\u003eY. lipolytica\u003c/em\u003e. Under the control of P\u003csub\u003e\u003cem\u003eUAS1B8\u0026minus;LEUm\u003c/em\u003e\u003c/sub\u003e, the strongest promoter we constructed, overexpression of a key pathway gene led to 7.7-fold increase in the titer of isoamyl alcohol. Interestingly, a much weaker promoter P\u003csub\u003e\u003cem\u003eUAS1B4\u0026minus;EXPm\u003c/em\u003e\u003c/sub\u003e increase the isoamyl alcohol titer by 30.3-fold. These results suggest that our hybrid promoter library can be a powerful toolkit for identifying optimum promoters for expressing metabolic pathways in \u003cem\u003eY. lipolytica\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eWe envision that this promoter engineering strategy and the rationally engineered promoters constructed in this study could also be extended to other non-model fungi for strain improvement.\u003c/p\u003e","manuscriptTitle":"Hybrid promoter engineering strategies in Yarrowia lipolytica:\u0026nbsp;isoamyl alcohol\u0026nbsp;production as a test study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-04-02 23:53:40","doi":"10.21203/rs.3.rs-369447/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ad3d6a5d-6dfd-4742-8ab3-de59b5285450","owner":[],"postedDate":"April 2nd, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":3372090,"name":"Biotechnology and Bioengineering"}],"tags":[],"updatedAt":"2021-05-05T12:49:44+00:00","versionOfRecord":[],"versionCreatedAt":"2021-04-02 23:53:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-369447","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-369447","identity":"rs-369447","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}