ADH1 Promoter Research Articles

Engineering the xylose metabolism of Saccharomyces cerevisiae for ethanol and single cell protein bioconversion

Xylose isomerase (XI) pathway has been widely employed to enable Saccharomyces cerevisiae to convert xylose and glucose into commercially feasible lignocellulosic ethanol products. Nevertheless, studies about the effect of different promoters to the expression of xylA are lacking. Therefore, five strains with ADH1, GAPDH, PDC1, PGK, TEF1 promoters were constructed. Among them, S. cerevisiae INVSc1/pHM368-PADH1-xylA generated with xylA driven by ADH1 promoter displayed the highest xylose utilization rate (approximately 19.98 %) using xylose as the only carbon source. With 4 g/L glucose and 10 g/L xylose as the carbon sources, the xylose utilization rate was 60.04 %. Moreover, the utilization rate increased to 64.04 % with fermentation temperature elevated from 28 °C to 30 °C and reached 83.09 % with peptone and yeast extract as the nitrogen sources. The ethanol titer reached 1.74 g/L with a yield of 0.38 g/g sugar under this condition. In Comparison with direct fermentation, the single cell protein (SCP) was 1.27-fold higher during aerobic fed-batch fermentation. Furthermore, INVSc1/pHM368-PADH1-xylA attains high ethanol productivities and yields by converting glucose and xylose from non-detoxified bagasse hydrolysates as carbon sources. The results extend our understanding of the xylose metabolism of S. cerevisiae and provide a platform for biomass conversion to ethanol and SCP, hence paving the way for the development of a more economical and sustainable approach to co-fermentation performance and capabilities for future engineering.

Engineering of the hypoxia-induced Pichia stipitis ADH2 promoter to construct a promoter library for Pichia pastoris.

Hypoxia-inducible promoters of a wide range of activities are desirable for fine-tuning gene expression in response to oxygen limitation, especially for the Crabtree negative yeast Pichia pastoris (Komagataella phaffii) with a high oxygen consumption rate in large-scale fermentations. Here we constructed a hypoxia-inducible promoter library for P. pastoris through error-prone PCR of Pichia stipitis ADH2 promoter (PsADH2). The library of 30 selected promoters showing 0.4- to 5.5-fold of the PsADH2 activity was obtained through high-throughput screening in microplates using the reporter yeast-enhanced green fluorescent protein. Two strong promoters, AM23 and AM30, were further characterized in shake flask cultures at high and low dissolved oxygen levels. They responded more sensitively to the low dissolved oxygen level, achieving a 4.6-, 7.9-fold and 3.6-, 7.7-fold higher fluorescence intensity and transcript level, respectively, than the wild-type PsADH2. Their hypoxia-inducible properties were confirmed with two additional reporters: β-galactosidase and Vitreoscilla hemoglobin, to demonstrate the broad applicability of the promoter library. During the typical fermentation process in shake flasks, the promoter AM30 showed strong expression with cell growth and decreased oxygen levels, without any additional chemical inducers or operations. Since the potent industrial host P. pastoris is recognized as an easy to scale-up system, it is reasonable to expect that the obtained hypoxia-inducible promoter library may have great potential to enable convenient regulation of gene expression under industrial fermentations which are usually run under oxygen limitation due to high cell density cultivations.

Optimization of the CRISPR/Cas9 system using adh1 promoter derivatives in fission yeast.

The CRSIPR/Cas9 system has been applied to fission yeast, but there remain some rooms for improvement. Here we report that the weaker versions of the adh1 + promoter, adh11 and adh41 promoters, for the potentially cytotoxic Cas9 achieved highly efficient mutagenesis and gene deletion at the ade6 + locus. Employing a drug-selectable marker instead of conventional auxotrophic markers, our new vector system is compatible with a variety of experimental settings including prototrophic/auxotrophic strains and complete/minimal media.

Hierarchical dynamic regulation of Saccharomyces cerevisiaefor enhanced lutein biosynthesis.

Lutein, as a carotenoid with strong antioxidant capacity and an important component of macular pigment in the retina, has wide applications in pharmaceutical, food, feed, and cosmetics industries. Besides extraction from plant and algae, microbial fermentation using engineered cell factories to produce lutein has emerged as a promising route. However, intra-pathway competition between the lycopene cyclases and the conflict between cell growth and production are two major challenges. In our previous study, de novo synthesis of lutein had been achieved in Saccharomyces cerevisiae by dividing the pathway into two stages (δ-carotene formation and conversion) using temperature as the input signal to realize sequential cyclation of lycopene. However, lutein production was limited to microgram level, which is still too low to meet industrial demand. In this study, a dual-signal hierarchical dynamic regulation system was developed and applied to divide lutein biosynthesis into three stages in response to glucose concentration and culture temperature. By placing the genes involved in δ-carotene formation under the glucose-responsive ADH2 promoter and genes involved in the conversion of δ-carotene to lutein under temperature-responsive GAL promoters, the growth-production conflict and intra-pathway competition were simultaneously resolved. Meanwhile, the rate-limiting lycopene ε-cyclation and carotene hydroxylation reactions were improved by screening for lycopene ε-cyclase with higher activity and fine tuning of the P450 enzymes and their redox partners. Finally, a lutein titer of 19.92 mg/L (4.53 mg/g DCW) was obtained in shake-flask cultures using the engineered yeast strain YLutein-3S-6, which is the highest lutein titer ever reported in heterologous production systems.

MEK1/2 inhibitors induce class I alcohol dehydrogenase (ADH1) expression by regulating farnesoid X receptor in hepatic cell lines and C57BL/6J mouse.

Alcohol is mainly catabolized by class I alcohol dehydrogenase (ADH1) in liver. ADH deficiency can aggravate ethanol-induced tissue injury. Extracellular signal-regulated kinases 1/2 (ERK1/2) is involved in alcohol metabolism. However, the relationship between ERK1/2 and ADH1 remains unclear. To inhibit ERK1/2, HepG2 and BNL cells were treated with mitogen-activated protein kinases 1/2 (MEK1/2) inhibitors (U0126 and PD98059), and C57BL/6J mice were fed U0126. After treatment, the protein and mRNA expression of ADH1 were determined by Western blot and quantitative real time-PCR. The activity of ADH1 promoter was detected using luciferase assay. The results showed MEK1/2 inhibitors significantly increased ADH1 protein expression by inducing its transcription activity. Then we demonstrated a farnesoid X receptor (FXR) response element (FXRE) in ADH1 promoter by ChIP assay. To test whether FXR mediates the induction of MEK1/2 inhibitors on ADH1, HepG2 cells were transfected with FXR siRNA or ADH1 promoters with FXRE mutation. We found both FXR siRNA and FXRE mutation in ADH1 promoter abolished MEK1/2 inhibitors-induced ADH1 expression, indicating the activation of MEK1/2 inhibitors on ADH1 depends on FXR. Our findings revealed inhibition of ERK1/2 can significantly increase ADH1 expression, indicating MEK1/2 inhibitors may possess potential application in alcohol-related diseases.

Deletion analysis of Pichia pastoris alcohol dehydrogenase 2 (ADH2) promoter and development of synthetic promoters.

Pichia pastoris (Komagataella phaffii) is a non-conventional Crabtree-negative yeast with the capability of reaching very high cell densities in a fed-batch fermentation process. The alcohol dehydrogenase (ADH) genes of P. pastoris involved in ethanol metabolism were identified and were previously characterized. This work aimed to extend current knowledge of the regulation of the ADH2 promoter. To this end, we first determined the upstream activator (UAS) and repressor (URS) sequences of the promoter by deletion assays. Two upstream activator sites have been identified, positioned between -900 and -801bp,and -284 and -108bp upstream of the ADH2 transcription start site. The sequences positioned between -361 and -262bp had a negative effect on the promoter activity and designated a repressor sequence (URS). We then demonstrated that Mxr1 (methanol expression regulator 1) transcription factor activates the ADH2 promoter through the direct interaction with UAS regions in response to ethanol. Furthermore, five different synthetic promoters were constructed by adding or deleting the regulatory sites. These synthetic promoters were tested for extracellular xylanase production at shake flask level by inducing with ethanol. These promoter variants improved the xylanase production ranging between 165% and 200% of the native promoter. The synthetic promoter 5 (SNT5) that displayed the highest activity was further evaluated at the fermenter scale. The modification in the promoter features might have several implications for industrial processes where decoupling the cell growth and product formation is advantageous.

Engineering of cis-Element in Saccharomyces cerevisiae for Efficient Accumulation of Value-Added Compound Squalene via Downregulation of the Downstream Metabolic Flux.

Transcriptional downregulation is widely used for metabolic flux control. Here, marO, a cis-element of Escherichia coli mar operator, was explored to engineer promoters of Saccharomyces cerevisiae for downregulation. First, the ADH1 promoter (PADH1) and its enhanced variant PUADH1 were engineered by insertion of marO into different sites, which resulted in decrease in both gfp5 transcription and GFP fluorescence intensity to various degrees. Then, marO was applied to engineer the native ERG1 and ERG11 promoters due to their importance for accumulation of value-added intermediates squalene and lanosterol. Elevated squalene content (4.9-fold) or lanosterol content (4.8-fold) and 91 or 28% decrease in ergosterol content resulted from the marO-engineered promoter PERG1(M5) or PERG11(M3), respectively, indicating the validity of the marO-engineered promoters in metabolic flux control. Furthermore, squalene production of 3.53 g/L from cane molasses, a cheap and bulk substrate, suggested the cost-effective and promising potential for squalene production.

Reciprocal relation between reporter gene transcription and translation efficiency in fission yeast

The fission yeast, Schizosaccharomyces pombe, is an excellent model for basic research but is not useful for commercial scale protein expression due to lack of strong expression vectors. Earlier, we showed that the lsd90 promoter elicited significantly greater GFP expression level than the adh1 and nmt1 promoters, albeit in different vector backbones. Here, we have systematically investigated the contribution of selectable markers, LEU2 and URA3m to GFP expression: while LEU2 elicited very low expression, the URA3m gene, with truncated promoter, elicited much greater GFP expression level with all promoters. Paradoxically, an inverse correlation was observed between the GFP transcription and translation efficiency. This system can be useful for understanding the factors governing recombinant gene expression and optimization of protein production.

Characterization of Viral Genome Encapsidated in Adeno-associated Recombinant Vectors Produced in Yeast Saccharomyces cerevisiae.

Adeno-associated virus (AAV) is a small, non-enveloped virus used as vector in gene therapy, mainly produced in human cells and in baculovirus systems. Intense studies on these platforms led to the production of vectors with titers between 103 and 105 viral genomes (vg) per cells. In spite of this, vector yields need to be improved to satisfy the high product demands of clinical trials and future commercialization. Our studies and those of other groups have explored the possibility to exploit the yeast Saccharomyces cerevisiae to produce rAAV. We previously demonstrated that yeast supports AAV genome replication and capsid assembly. The purpose of this study was to evaluate the quality of the encapsidated AAV DNA. Here, we report the construction of a yeast strain expressing Rep68/40 from an integrated copy of the Rep gene under the control of the yeast constitutive ADH promoter and Capsid proteins from the Cap gene under the control of an inducible GAL promoter. Our results indicate that a portion of AAV particles generated by this system contains encapsidated AAV DNA. However, the majority of encapsidated DNA consists of fragmented regions of the transgene cassette, with ITRs being the most represented sequences. Altogether, these data indicate that, in yeast, encapsidation occurs with low efficiency and that rAAVs resemble pseudo-vectors that are present in clinical-grade rAAV preparations.

SpEDIT: A fast and efficient CRISPR/Cas9 method for fission yeast.

The CRISPR/Cas9 system allows scarless, marker-free genome editing. Current CRISPR/Cas9 systems for the fission yeast Schizosaccharomyces pomberely on tedious and time-consuming cloning procedures to introduce a specific sgRNA target sequence into a Cas9-expressing plasmid. In addition, Cas9 endonuclease has been reported to be toxic to fission yeast when constitutively overexpressed from the strong adh1promoter. To overcome these problems we have developed an improved system, SpEDIT, thatuses a synthesised Cas9 sequencecodon-optimised for S. pombeexpressed from the medium strength adh15promoter.The SpEDITsystem exhibits a flexible modular design where the sgRNA is fused to the 3' end of the self-cleaving hepatitis delta virus (HDV) ribozyme, allowing expression of the sgRNA cassette to be driven by RNA polymerase III from a tRNA gene sequence.Lastly, the inclusion of sites for the BsaI type IIS restriction enzyme flanking a GFP placeholder enables one-step Golden Gate mediated replacement of GFP with synthesized sgRNAs for expression. The SpEDITsystem allowed a 100% mutagenesis efficiency to be achieved when generating targeted pointmutants in the ade6 + or ura4 +genes by transformation of cells from asynchronous cultures. SpEDITalso permitted insertion, tagging and deletion events to be obtained with minimal effort. Simultaneous editing of two independent non-homologous loci was also readily achieved. Importantly the SpEDITsystem displayed reduced toxicity compared to currently available S. pombeediting systems. Thus, SpEDITprovides an effective and user-friendly CRISPR/Cas9 procedure that significantly improves the genome editing toolbox for fission yeast.

Ethanol fed-batch bioreactor operation to enhance therapeutic protein production in Pichia pastoris under hybrid-architectured ADH2 promoter

ABSTRACT We presented ethanol fed-batch bioreactor operations (FBBOs) designed for enhanced recombinant human growth hormone (rhGH) production in Pichia pastoris constructed with novel hybrid-architectured ADH2 promoter, PADH2-Cat8-L2. The parameters in the fed-batch fermentation of ethanol were investigated, and the boundaries of the production domain in terms of the design parameters were determined. Two FBBO design methods were used, and a platform aiming to adjust cellular metabolism for the generation of constant specific growth rate (μ) and substrate concentration (CEtOH) was established. The highest overall productivity (rP,ov) was generated with model-based continuous feed-stream (CFS) design with μpreD = 0.035 h-1 up to tmax ≤ 9 h as rP,ov = 3.0 mg L-1 h-1 and CrhGH = 34 mg L-1. The ethanol-stat FBBO at CEtOH,set = 0.5 g L-1 allowed increased product titer with high productivity and the highest CrhGH =90.1 mg L-1 at tmax = 24 h. The effect of CEtOH on specific rates for cell reactions was represented by the Haldane model in which the cell generation and rhGH production were simulated with low mean relative errors of 6.5 and 16%, respectively. The results indicate the need for an upgraded CFS design model with kinetic expressions involving substrate inhibition. We conclude that a two-step hybrid fed-batch design strategy to be started (i) with the initial-period of the model-based FBBO, in turn, proceeded (ii) with the controlled feeding with ethanol-stat FBBO enables a gradual approach to the hypothetical performance of FBBOs.

Understanding the Cell‐Cycle Dependence of Mus81‐Mms4 Recruitment and its Reliance on Slx4

DNA damage repair pathways are a key element in maintaining genomic stability and preventing cancer and other diseases, particularly given the prevalence of DNA damaging agents. One form of DNA damage is double‐strand breaks (DSBs), the repair of which utilizes numerous repair proteins regardless of which subpathway is implemented. One such repair protein in S. cerevisiae is Mus81, which forms a complex with Mms4, that functions as a structure‐specific endonuclease that cleaves flapped DNA structures present in intertwined chromosomes that have undergone DSB repair in late S and G2 phases.Here, we seek to understand the cell cycle dependence of Mus81‐ recruitment to DSBs and determine if this recruitment is Slx4‐dependent. This exploration requires complete cell cycle synchronization with □‐factor followed by release back into cell cycle following □‐factor removal. As they progress through the cell cycle, cultures are monitored by fluorescence microscopy and flow cytometry to properly correlate fluorescence data with discrete stages of the cell cycle. The yeast strain under study contains a Mus81 protein tagged with yEmRFP which enables its tracking during the fluorescence microscopy portion of the experiment. Preliminary microscopy experiments on a Mus81‐yEmRFP labeled strain have demonstrated that Mus81 is present in low abundance, making it difficult to track the RFP signal throughout the cell cycle. This creates the need for increased expression of Mus81 if it is to be investigated via fluorescence microscopy. Therefore, a plasmid has been constructed that incorporates an ADH1 promoter in front of the Mus81‐yEmRFP gene to enable overexpression. The use of this modified strain will allow for experimental correlation of Mus81‐Mms4 activity with a specific stage of the cell cycle.Support or Funding InformationNational Institutes of Health (NIH) (Grant SC1GM127204)NIH BUILD Program (NIH Grant 5TL4GM118977)

Modulating transcription through development of semi-synthetic yeast core promoters.

Altering gene expression regulation by promoter engineering is a very effective way to fine-tune heterologous pathways in eukaryotic hosts. Typically, pathway building approaches in yeast still use a limited set of long, native promoters. With the today’s introduction of longer and more complex pathways, an expansion of this synthetic biology toolbox is necessary. In this study we elucidated the core promoter structure of the well-characterized yeast TEF1 promoter and determined the minimal length needed for sufficient protein expression. Furthermore, this minimal core promoter sequence was used for the creation of a promoter library covering different expression strengths. This resulted in a group of short, 69 bp promoters with an 8.0-fold expression range. One exemplar had a two and four times higher expression compared to the native CYC1 and ADH1 promoter, respectively. Additionally, as it was described that the protein expression range could be broadened by upstream activating sequences (UASs), we integrated earlier described single and multiple short, synthetic UASs in front of the strongest yeast core promoter. This approach resulted to further variation in protein expression and an overall promoter library spanning a 20-fold activity range and covering a length from 69 bp to maximally 129 bp. Furthermore, the robustness of this library was assessed on three alternative carbon sources besides glucose. As such, the suitability of short yeast core promoters for metabolic engineering applications on different media, either in an individual context or combined with UAS elements, was demonstrated.

Pichia pastoris Promoters.

The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is used as an expression system for recombinant protein production for a variety of applications. It grows rapidly on inexpensive media containing methanol, glucose, glycerol, or ethanol as a sole carbon source. P. pastoris makes many posttranslational modifications and produces recombinant proteins either intracellularly or extracellularly. Because of these properties, P. pastoris has become a highly preferred host organism for biotechnology, pharmaceutical industry, and researchers.Recombinant protein production is usually performed under the control of the promoter of the alcohol oxidase gene I (AOX1). The AOX1 promoter is induced by methanol and repressed by glucose and ethanol. The regulation mechanisms of the AOX1 promoter have been studied in recent years. Another promoter used in recombinant protein production is derived from glyceraldehyde 3-phosphate dehydrogenase (GAP). It is a constitutive promoter. Recent literature showed that newly identified promoters of P. pastoris are promising as well, in addition to pAOX1 and pGAP.In this chapter, the regulation mechanisms of inducible pAOX1 and constitutive pGAP promoters are discussed. In addition, here we present an overview about the novel ADH3 promoter and alternative promoters of P. pastoris.

A study on the use of strain-specific and homologous promoters for heterologous expression in industrial Saccharomyces cerevisiae strains

Polymorphism is well known in Saccharomyces cerevisiae strains used for different industrial applications, however little is known about its effects on promoter efficiency. In order to test this, five different promoters derived from an industrial and a laboratory (S288c) strain were used to drive the expression of eGFP reporter gene in both cells. The ADH1 promoter (PADH1) in particular, which showed more polymorphism among the promoters analyzed, also exhibited the highest differences in intracellular fluorescence production. This was further confirmed by Northern blot analysis. The same behavior was also observed when the gene coding for secreted α-amylase from Cryptococcus flavus was placed under the control of either PADH1. These results underline the importance of the careful choice of the source of the promoter to be used in industrial yeast strains for heterologous expression.

Efficient secretion of three fungal laccases from Saccharomyces cerevisiae and their potential for decolorization of textile industry effluent-A comparative study.

Laccases are enzymes with a broad range of biotechnological applications and have, for example, the ability to oxidize many xenobiotics including synthetic dyes. In order to obtain an efficient laccase for the decolorization of dyes which spoil wastewater from the textile industry, genes encoding three various laccase enzymes were expressed in Saccharomyces cerevisiae. The expression of laccases from ascomycete Myceliophthora thermophila (MtL), and two basidiomycetes Trametes versicolor (TvL) and Trametes trogii (TtL) was optimized via selection of plasmids, promoters, media composition, and cultivation conditions. For the first time, the activity of the three secreted laccases was directly compared with the use of various substrates, including different dyes and a wastewater sample. A strong constitutive ADH1 promoter, minimal growth medium, optimized combination of copper and organic nitrogen source, and low cultivation temperature were shown to significantly increase the yields and relative activities of secreted laccases. Heterologous expression of three fungal laccases was successfully achieved in S. cerevisiae being the highest for MtL and the lowest for TvL. MtL, and particularly TtL, showed the decolorization capacity. This is the first report which compared decolorization of synthetic dyes and wastewater by several recombinant laccases and suggested MtL and TtL to be applicable in the ecofriendly enzymatic treatment of colored industry effluent. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:69-80, 2018.

Strategies to optimize capsid protein expression and single-stranded DNA formation of adeno-associated virus in Saccharomyces cerevisiae.

Adeno-associated virus type 2 (AAV) is a nonpathogenic parvovirus that is a promising tool for gene therapy. We aimed to construct plasmids for optimal expression and assembly of capsid proteins and evaluate adenovirus (Ad) protein effect on AAV single-stranded DNA (ssDNA) formation in Saccharomyces cerevisiae. Yeast expression plasmids have been developed in which the transcription of AAV capsid proteins (VP1,2,3) is driven by the constitutive ADH1 promoter or galactose-inducible promoters. Optimal VP1,2,3 expression was obtained from GAL1/10 bidirectional promoter. Moreover, we demonstrated that AAP is expressed in yeast and virus-like particles (VLPs) assembled inside the cell. Finally, the expression of two Ad proteins, E4orf6 and E1b55k, had no effect on AAV ssDNA formation. This study confirms that yeast is able to form AAV VLPs; however, capsid assembly and ssDNA formation are less efficient in yeast than in human cells. Moreover, the expression of Ad proteins did not affect AAV ssDNA formation. New manufacturing strategies for AAV-based gene therapy vectors (rAAV) are needed to reduce costs and time of production. Our study explores the feasibility of yeast as alternative system for rAAV production.

Glandular trichome-specific expression of alcohol dehydrogenase 1 (ADH1) using a promoter-GUS fusion in Artemisia annua L.

Artemisinin, isolated from Artemisia annua L., is widely known as a functional anti-malaria drug. Due to the low content of artemisinin in A. annua plants, great efforts have been made to determine the artemisinin biosynthetic pathway by genetic engineering. ADH1, encoding an alcohol dehydrogenase, was cloned from the glandular secretory trichomes (GSTs) in A. annua. The gene expression analysis showed that ADH1 was predominately expressed in buds and young leaves, and the expression of ADH1 was the highest in the youngest leaves. To further investigate the expression pattern of ADH1 in A. annua, a 1070-bp promoter region of ADH1 was cloned. We found 14 putative cis-elements were presented in the ADH1 promoter sequence, indicating that ADH1 is complexly regulated. The ADH1 promoter sequence was fused to the β-glucuronidase reporter gene (GUS) and introduced into A. annua plants. GUS signals were only found in the glandular secretory trichomes of young tissues in transgenic A. annua plants. Besides, the treatment of A. annua seedlings with 100 μM methyl jasmonate (MeJA) and 100 μM abscisic acid (ABA), respectively, increased the ADH1 transcript levels. The dual luciferase (dual-LUC) assay demonstrated that the reported transcription factors, MYC2 and ERF1, activated the expression of ADH1 in vivo. Our study shows that ADH1 gene is exclusively expressed in the glandular secretory trichomes of young tissues of A. annua, it implies that the promoter of ADH1 gene could be used in engineering of A. annua for increasing artemisinin content.

Facile manipulation of protein localization in fission yeast through binding of GFP-binding protein to GFP.

GFP-binding protein (or GBP) has been recently developed in various systems and organisms as an efficient tool to purify GFP-fusion proteins. Due to the high affinity between GBP and GFP or GFP variants, this GBP-based approach is also ideally suited to alter the localization of functional proteins in live cells. In order to facilitate the wide use of the GBP-targeting approach in the fission yeast Schizosaccharomyces pombe, we developed a set of pFA6a-, pJK148- and pUC119-based vectors containing GBP- or GBP-mCherry-coding sequences and variants of inducible nmt1 or constitutive adh1 promoters that result in different levels of expression. The GBP or GBP-mCherry fragments can serve as cassettes for N- or C-terminal genomic tagging of genes of interest. We illustrated the application of these vectors in the construction of yeast strains with Dma1 or Cdc7 tagged with GBP-mCherry and efficient targeting of Dma1- or Cdc7-GBP-mCherry to the spindle pole body by Sid4-GFP. This series of vectors should help to facilitate the application of the GBP-targeting approach in manipulating protein localization and the analysis of gene function in fission yeast, at the level of single genes, as well as at a systematic scale.

English

Hybrid promoters are created by shuffling of DNA fragments while keeping intact regulatory regions crucial of promoter activity. Two fragments of alcohol dehydrogenase (Adh) promoter from Zea mays were selected to generate hybrid promoter. Sequence analysis of both alcohol dehydrogenase promoter fragments through bioinformatics tools identified several crucial cis regulatory elements and transcription factors binding sites. Both fragments were separately cloned in the TA vector (pTZ57R/T) and fused to get the complete hybrid promoter (Adh-H). Alcohol dehydrogenase hybrid promoter was further cloned in expression vector pGR1 through adaptor ligation. Transient β-glucuronidase (GUS) assay revealed that hybrid promoter exhibited high expression under anaerobic conditions in wheat tissues. From the study it is concluded that hybrid promoter (Adh-H) may be used to derive gene expression in monocots during anaerobic conditions. The present work also provides an important insight in the designing of hybrid monocot promoters to improve multiple traits in crops without facing intellectual property rights (IPRs) issues.   Key words: Hybrid promoter, histochemical β-glucuronidase (GUS) assay staining, cis regulatory elements, alcohol dehydrogenase, Zea mays.