scholarly journals A synthetic promoter library for constitutive gene expression in Lactobacillus plantarum

Microbiology ◽  
2006 ◽  
Vol 152 (4) ◽  
pp. 1011-1019 ◽  
Author(s):  
Ida Rud ◽  
Peter Ruhdal Jensen ◽  
Kristine Naterstad ◽  
Lars Axelsson
Keyword(s):  
Gene Expression ◽  
Lactobacillus Plantarum ◽  
Protein Production ◽  
Consensus Sequence ◽  
Fine Tuning ◽  
Stable Gene ◽  
Synthetic Promoter ◽  
Synthetic Promoters ◽  
Promoter Library ◽  
Wide Range

A synthetic promoter library (SPL) for Lactobacillus plantarum has been developed, which generalizes the approach for obtaining synthetic promoters. The consensus sequence, derived from rRNA promoters extracted from the L. plantarum WCFS1 genome, was kept constant, and the non-consensus sequences were randomized. Construction of the SPL was performed in a vector (pSIP409) previously developed for high-level, inducible gene expression in L. plantarum and Lactobacillus sakei. A wide range of promoter strengths was obtained with the approach, covering 3–4 logs of expression levels in small increments of activity. The SPL was evaluated for the ability to drive β-glucuronidase (GusA) and aminopeptidase N (PepN) expression. Protein production from the synthetic promoters was constitutive, and the most potent promoters gave high protein production with levels comparable to those of native rRNA promoters, and production of PepN protein corresponding to approximately 10–15 % of the total cellular protein. High correlation was obtained between the activities of promoters when tested in L. sakei and L. plantarum, which indicates the potential of the SPL for other Lactobacillus species. The SPL enables fine-tuning of stable gene expression for various applications in L. plantarum.

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

2021 ◽  
Author(s):  
Yu Zhao ◽  
Shiqi Liu ◽  
Zhihui Lu ◽  
Baixiang Zhao ◽  
Shuhui Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Yarrowia Lipolytica ◽  
Isoamyl Alcohol ◽  
Fine Tuning ◽  
Promoter Strength ◽  
Promoter Engineering ◽  
Test Study ◽  
Promoter Library ◽  
Wide Range ◽  
Hybrid Promoter

Abstract Background: In biological cells, promoters drive gene expression by specific binding of RNA polymerase. 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 optimizing biosynthetic pathways in metabolic engineering 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. By expressing the key pathway gene, ScARO10, with the promoter library, 1.1-30.3 folds increase in the isoamyl alcohol titer over that of the control strain Y. lipolytica Po1g KU70∆ was achieved. Interestingly, the highest titer increase was attained with a weak promoter PUAS1B4-EXPm to express ScARO10. 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.

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

2021 ◽  
Author(s):  
Yu Zhao ◽  
Shiqi Liu ◽  
Zhihui Lu ◽  
Baixiang Zhao ◽  
Shuhui Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Yarrowia Lipolytica ◽  
Isoamyl Alcohol ◽  
Fine Tuning ◽  
Promoter Strength ◽  
Promoter Engineering ◽  
Test Study ◽  
Promoter Library ◽  
Wide Range ◽  
Hybrid Promoter

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 PUAS1B8−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 PUAS1B4−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.

scholarly journals The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters

1998 ◽  
Vol 64 (1) ◽  
pp. 82-87 ◽  
Author(s):  
Peter Ruhdal Jensen ◽  
Karin Hammer
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Dna Sequencing ◽  
Lactococcus Lactis ◽  
Entire Range ◽  
Fine Tuning ◽  
Activity Increase ◽  
Consensus Sequences ◽  
Synthetic Promoters ◽  
Cell Factories

ABSTRACT We constructed a library of synthetic promoters forLactococcus lactis in which the known consensus sequences were kept constant while the sequences of the separating spacers were randomized. The library consists of 38 promoters which differ in strength from 0.3 up to more than 2,000 relative units, the latter among the strongest promoters known for this organism. The ranking of the promoter activities was somewhat different when assayed inEscherichia coli, but the promoters are efficient for modulating gene expression in this bacterium as well. DNA sequencing revealed that the weaker promoters (which had activities below 5 relative units) all had changes either in the consensus sequences or in the length of the spacer between the −35 and −10 sequences. The promoters in which those features were conserved had activities from 5 to 2,050 U, which shows that by randomizing the spacers, at least a 400-fold change in activity can be obtained. Interestingly, the entire range of promoter activities is covered in small steps of activity increase, which makes these promoters very suitable for quantitative physiological studies and for fine-tuning of gene expression in industrial bioreactors and cell factories.

scholarly journals Dual UTR-A novel 5′ untranslated region design for synthetic biology applications

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Simone Balzer Le ◽  
Ingerid Onsager ◽  
Jon Andreas Lorentzen ◽  
Rahmi Lale
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
De Novo ◽  
Expression Profiles ◽  
Regulation Of Gene Expression ◽  
Design Concept ◽  
Fine Tuning ◽  
The Novel ◽  
Wide Range ◽  
Application Potential

Abstract Bacterial 5′ untranslated regions of mRNA (UTR) involve in a complex regulation of gene expression; however, the exact sequence features contributing to gene regulation are not yet fully understood. In this study, we report the design of a novel 5′ UTR, dual UTR, utilizing the transcriptional and translational characteristics of 5′ UTRs in a single expression cassette. The dual UTR consists of two 5′ UTRs, each separately leading to either increase in transcription or translation of the reporter, that are separated by a spacer region, enabling de novo translation initiation. We rationally create dual UTRs with a wide range of expression profiles and demonstrate the functionality of the novel design concept in Escherichia coli and Pseudomonas putida using different promoter systems and coding sequences. Overall, we demonstrate the application potential of dual UTR design concept in various synthetic biology applications ranging from fine-tuning of gene expression to maximization of protein production.

scholarly journals Fine-tuning the expression of pathway gene in yeast using a regulatory library formed by fusing a synthetic minimal promoter with different Kozak variants

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Liping Xu ◽  
Pingping Liu ◽  
Zhubo Dai ◽  
Feiyu Fan ◽  
Xueli Zhang
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Fine Tuning ◽  
Minimal Promoter ◽  
Pathway Engineering ◽  
Gfp Expression ◽  
Chimeric Promoter ◽  
Promoter Library ◽  
Pathway Gene ◽  
Chimeric Promoters

Abstract Background Tailoring gene expression to balance metabolic fluxes is critical for the overproduction of metabolites in yeast hosts, and its implementation requires coordinated regulation at both transcriptional and translational levels. Although synthetic minimal yeast promoters have shown many advantages compared to natural promoters, their transcriptional strength is still limited, which restricts their applications in pathway engineering. Results In this work, we sought to expand the application scope of synthetic minimal yeast promoters by enhancing the corresponding translation levels using specific Kozak sequence variants. Firstly, we chose the reported UASF-E-C-Core1 minimal promoter as a library template and determined its Kozak motif (K0). Next, we randomly mutated the K0 to generate a chimeric promoter library, which was able to drive green fluorescent protein (GFP) expression with translational strengths spanning a 500-fold range. A total of 14 chimeric promoters showed at least two-fold differences in GFP expression strength compared to the K0 control. The best one named K528 even showed 8.5- and 3.3-fold increases in fluorescence intensity compared with UASF-E-C-Core1 and the strong native constitutive promoter PTDH3, respectively. Subsequently, we chose three representative strong chimeric promoters (K540, K536, and K528) from this library to regulate pathway gene expression. In conjunction with the tHMG1 gene for squalene production, the K528 variant produced the best squalene titer of 32.1 mg/L in shake flasks, which represents a more than 10-fold increase compared to the parental K0 control (3.1 mg/L). Conclusions All these results demonstrate that this chimeric promoter library developed in this study is an effective tool for pathway engineering in yeast.

scholarly journals CRISPR/Transposon gene integration (CRITGI) can manage gene expression in a retrotransposon-dependent manner

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Miki Hanasaki ◽  
Hiroshi Masumoto
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
Consensus Sequence ◽  
Marker Gene ◽  
Gene Activation ◽  
Fine Tuning ◽  
Selection Marker ◽  
Regulation System ◽  
Dependent Manner ◽  
Amino Acid Depletion

Abstract The fine-tuning of gene expression contributes to both basic science and applications. Here, we develop a novel gene expression technology termed CRITGI (CRISPR/Transposon gene integration). CRITGI uses CRISPR/Cas9 to integrate multiple copies of the plasmid pTy1 into Ty1 loci, budding yeast retrotransposons. The pTy1 plasmid harbors a Ty1 consensus sequence for integration, a gene of interest with its own promoter and a selection marker gene. Interestingly, the expression of the pTy1 gene in Ty1 loci could be induced in synthetic complete amino acid depletion medium, which could activate the selection marker gene on pTy1. The induction or repression of the gene on pTy1 depended on Ty1 transcription. Activation of the selection marker gene on pTy1 triggered Ty1 transcription, which led to induction of the gene on pTy1. The gene on pTy1 was not transcribed with Ty1 mRNA; the transcription required its own promoter. Furthermore, the trimethylation of histone H3 on lysine 4, a landmark of transcriptionally active chromatin, accumulated at the 5′ end of the gene on pTy1 following selection marker gene activation. Thus, CRITGI is a unique gene regulation system to induce the genes on pTy1 in amino acid depletion medium and utilizes Ty1 transcription to create a chromatin environment favorable for the transcription of the genes on pTy1.

scholarly journals Robust gene expression control in human cells with a novel universal TetR aptamer splicing module

2019 ◽  
Vol 47 (20) ◽  
pp. e132-e132 ◽  
Author(s):  
Adam A Mol ◽  
Florian Groher ◽  
Britta Schreiber ◽  
Ciaran Rühmkorff ◽  
Beatrix Suess
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
Dynamic Range ◽  
Intron Retention ◽  
Human Cells ◽  
Fine Tuning ◽  
Genomic Context ◽  
Mammalian Cell Lines ◽  
Gene Expression Control ◽  
Wide Range

Abstract Fine-tuning of gene expression is desirable for a wide range of applications in synthetic biology. In this context, RNA regulatory devices provide a powerful and highly functional tool. We developed a versatile, robust and reversible device to control gene expression by splicing regulation in human cells using an aptamer that is recognized by the Tet repressor TetR. Upon insertion in proximity to the 5′ splice site, intron retention can be controlled via the binding of TetR to the aptamer. Although we were able to demonstrate regulation for different introns, the genomic context had a major impact on regulation. In consequence, we advanced the aptamer to develop a splice device. Our novel device contains the aptamer integrated into a context of exonic and intronic sequences that create and maintain an environment allowing a reliable and robust splicing event. The exon-born, additional amino acids will then be cleaved off by a self-cleaving peptide. This design allows portability of the splicing device, which we confirmed by demonstrating its functionality in different gene contexts. Intriguingly, our splicing device shows a high dynamic range and low basal activity, i.e. desirable features that often prove a major challenge when implementing synthetic biology in mammalian cell lines.

scholarly journals Stringency of synthetic promoter sequences inClostridiumrevealed and circumvented by tuning promoter library mutation rates

2017 ◽  
Author(s):  
Paweł M. Mordaka ◽  
John T. Heap
Keyword(s):  
Anaerobic Bacteria ◽  
Gene Promoter ◽  
Mutation Rates ◽  
Synthetic Promoter ◽  
Clostridium Sporogenes ◽  
Fluorescent Reporter ◽  
Promoter Sequences ◽  
E Coli ◽  
Synthetic Promoters ◽  
Wide Range

ABSTRACTCollections of characterized promoters of different strengths are key resources for synthetic biology, but are not well established for many important organisms, including industrially-relevantClostridiumspp. When generating promoters, reporter constructs are used to measure expression, but classical fluorescent reporter proteins are oxygen-dependent and hence inactive in anaerobic bacteria likeClostridium. We directly compared oxygen-independent reporters of different types inClostridium acetobutylicumand found that glucuronidase (GusA) fromE. coliperformed best. Using GusA, a library of synthetic promoters was first generated by a typical approach entailing complete randomization of a constitutive thiolase gene promoter (Pthl) except for the consensus -35 and -10 elements. In each synthetic promoter, the chance of each degenerate position matching Pthlwas 25%. Surprisingly, none of the synthetic promoters from this library were functional inC. acetobutylicum, even though they functioned as expected inE. coli. Next, instead of complete randomization, we specified lower promoter mutation rates using oligonucleotide primers synthesized using custom mixtures of nucleotides. Using these primers, two promoter libraries were constructed in which the chance of each degenerate position matching Pthlwas 79% or 58%, instead of 25% as before. Synthetic promoters from these ‘stringent’ libraries functioned well inC. acetobutylicum, covering a wide range of strengths. The promoters functioned similarly in the distantly-related speciesClostridium sporogenes, and allowed predictable metabolic engineering ofC. acetobutylicumfor acetoin production. Besides generating the desired promoters and demonstrating their useful properties, this work indicates an unexpected ‘stringency’ of promoter sequences inClostridium, not reported previously.GRAPHICAL ABSTRACT

scholarly journals In Vivo Piggybac-Based Gene Delivery towards Murine Pancreatic Parenchyma Confers Sustained Expression of Gene of Interest

2019 ◽  
Vol 20 (13) ◽  
pp. 3116 ◽  
Author(s):  
Masahiro Sato ◽  
Emi Inada ◽  
Issei Saitoh ◽  
Shingo Nakamura ◽  
Satoshi Watanabe
Keyword(s):  
Gene Expression ◽  
Gene Delivery ◽  
Fluorescent Protein ◽  
Consensus Sequence ◽  
Constitutive Expression ◽  
Endocrine System ◽  
Stable Gene ◽  
Pancreatic Cells ◽  
Glandular Organ

The pancreas is a glandular organ that functions in the digestive system and endocrine system of vertebrates. The most common disorders involving the pancreas are diabetes, pancreatitis, and pancreatic cancer. In vivo gene delivery targeting the pancreas is important for preventing or curing such diseases and for exploring the biological function of genes involved in the pathogenesis of these diseases. Our previous experiments demonstrated that adult murine pancreatic cells can be efficiently transfected by exogenous plasmid DNA following intraparenchymal injection and subsequent in vivo electroporation using tweezer-type electrodes. Unfortunately, the induced gene expression was transient. Transposon-based gene delivery, such as that facilitated by piggyBac (PB), is known to confer stable integration of a gene of interest (GOI) into host chromosomes, resulting in sustained expression of the GOI. In this study, we investigated the use of the PB transposon system to achieve stable gene expression when transferred into murine pancreatic cells using the above-mentioned technique. Expression of the GOI (coding for fluorescent protein) continued for at least 1.5 months post-gene delivery. Splinkerette-PCR-based analysis revealed the presence of the consensus sequence TTAA at the junctional portion between host chromosomes and the transgenes; however, this was not observed in all samples. This plasmid-based PB transposon system enables constitutive expression of the GOI in pancreas for potential therapeutic and biological applications.

scholarly journals GAPPromoter Library for Fine-Tuning of Gene Expression in Pichia pastoris

2011 ◽  
Vol 77 (11) ◽  
pp. 3600-3608 ◽  
Author(s):  
Xiulin Qin ◽  
Jiangchao Qian ◽  
Gaofeng Yao ◽  
Yingping Zhuang ◽  
Siliang Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pichia Pastoris ◽  
Fine Tuning ◽  
Promoter Strength ◽  
Control Of Gene Expression ◽  
Content Type ◽  
Expression Levels ◽  
Precise Control ◽  
Promoter Library

ABSTRACTA library of engineered promoters of various strengths is a useful genetic tool that enables the fine-tuning and precise control of gene expression across a continuum of broad expression levels. The methylotrophic yeastPichia pastorisis a well-established expression host with a large academic and industrial user base. To facilitate manipulation of gene expression spanning a wide dynamic range inP. pastoris, we created a functional promoter library through mutagenesis of the constitutiveGAPpromoter. Using yeast-enhanced green fluorescent protein (yEGFP) as the reporter, 33 mutants were chosen to form the functional promoter library. The 33 mutants spanned an activity range between ∼0.6% and 19.6-fold of the wild-type promoter activity with an almost linear fluorescence intensity distribution. After an extensive characterization of the library, the broader applicability of the results obtained with the yEGFP reporter was confirmed using two additional reporters (β-galactosidase and methionine adenosyltransferase [MAT]) at the transcription and enzyme activity levels. Furthermore, the utility of the promoter library was tested by investigating the influence of heterologous MAT gene expression levels on cell growth andS-adenosylmethionine (SAM) production. The extensive characterization of the promoter strength enabled identification of the optimal MAT activity (around 1.05 U/mg of protein) to obtain maximal volumetric SAM production. The promoter library permits precise control of gene expression and quantitative assessment that correlates gene expression level with physiologic parameters. Thus, it is a useful toolbox for both basic and applied research inP. pastoris.

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