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 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.

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 Gene Expression Space Shapes the Bioprocess Trade-Offs among Titer, Yield and Productivity

2021 ◽  
Vol 11 (13) ◽  
pp. 5859
Author(s):  
Fernando N. Santos-Navarro ◽  
Yadira Boada ◽  
Alejandro Vignoni ◽  
Jesús Picó
Keyword(s):  
Gene Expression ◽  
Gene Transcription ◽  
Performance Metrics ◽  
Cell Mass ◽  
Gene Copy ◽  
Substrate Uptake ◽  
Promoter Strength ◽  
Expression Levels ◽  
Trade Offs ◽  
Wide Range

Optimal gene expression is central for the development of both bacterial expression systems for heterologous protein production, and microbial cell factories for industrial metabolite production. Our goal is to fulfill industry-level overproduction demands optimally, as measured by the following key performance metrics: titer, productivity rate, and yield (TRY). Here we use a multiscale model incorporating the dynamics of (i) the cell population in the bioreactor, (ii) the substrate uptake and (iii) the interaction between the cell host and expression of the protein of interest. Our model predicts cell growth rate and cell mass distribution between enzymes of interest and host enzymes as a function of substrate uptake and the following main lab-accessible gene expression-related characteristics: promoter strength, gene copy number and ribosome binding site strength. We evaluated the differential roles of gene transcription and translation in shaping TRY trade-offs for a wide range of expression levels and the sensitivity of the TRY space to variations in substrate availability. Our results show that, at low expression levels, gene transcription mainly defined TRY, and gene translation had a limited effect; whereas, at high expression levels, TRY depended on the product of both, in agreement with experiments in the literature.

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 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 Tuning Gene Expression in Yarrowia lipolytica by a Hybrid Promoter Approach

2011 ◽  
Vol 77 (22) ◽  
pp. 7905-7914 ◽  
Author(s):  
John Blazeck ◽  
Leqian Liu ◽  
Heidi Redden ◽  
Hal Alper
Keyword(s):  
Gene Expression ◽  
Yarrowia Lipolytica ◽  
Core Promoter ◽  
Pathway Engineering ◽  
Mrna Levels ◽  
Enhancer Element ◽  
Content Type ◽  
Synthetic Promoters ◽  
Native Promoter ◽  
Hybrid Promoter

ABSTRACTThe development of strong and tunable promoter elements is necessary to enable metabolic and pathway engineering applications for any host organism. Here, we have expanded and generalized a hybrid promoter approach to produce libraries of high-expressing, tunable promoters in the nonconventional yeastYarrowia lipolytica. These synthetic promoters are comprised of two modular components: the enhancer element and the core promoter element. By exploiting this basic promoter architecture, we have overcome native expression limitations and provided a strategy for both increasing the native promoter capacity and producing libraries for tunable gene expression in a cellular system with ill-defined genetic tools. In doing so, this work has created the strongest promoters ever reported forY. lipolytica. Furthermore, we have characterized these promoters at the single-cell level through the use of a developed fluorescence-based assay as well as at the transcriptional and whole-cell levels. The resulting promoter libraries exhibited a range of more than 400-fold in terms of mRNA levels, and the strongest promoters in this set had 8-fold-higher fluorescence levels than those of typically used endogenous promoters. These results suggest that promoters inY. lipolyticaare enhancer limited and that this limitation can be partially or fully alleviated through the addition of tandem copies of upstream activation sequences (UASs). Finally, this work illustrates that tandem copies of UAS regions can serve as synthetic transcriptional amplifiers that may be generically used to increase the expression levels of promoters.

scholarly journals Mapping the complex transcriptional landscape of the phytopathogenic bacterium Dickeya dadantii

2020 ◽  
Author(s):  
Raphaël Forquet ◽  
Xuejiao Jiang ◽  
William Nasser ◽  
Florence Hommais ◽  
Sylvie Reverchon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Environmental Changes ◽  
Computational Method ◽  
Fine Tuning ◽  
Transcription Start ◽  
Dickeya Dadantii ◽  
Phytopathogenic Bacterium ◽  
Plant Infection ◽  
Wide Range ◽  
Variable Gene

AbstractDickeya dadantii is a phytopathogenic bacterium that causes soft rot in a wide range of plant hosts worldwide and a model organism for studying gene regulation during the pathogenic process. The present study provides a comprehensive and annotated transcriptomic map of D. dadantii obtained by a computational method combining three independent transcriptomic datasets covering a wide range of conditions which closely reproduce the variations of transcription occurring in the course of plant infection: (1) paired-end RNA-seq data for a precise reconstruction of the RNA landscape, (2) DNA microarray data reflecting gene response to sudden environmental shocks mimicking conditions encountered by bacteria in the plant, (3) dRNA-seq data for a specific high-resolution mapping of transcription start sites. We define transcription units throughout the genome, and map the associated transcription start and termination sites with a quantitative magnitude analysis. Our results show that transcription units sometimes coincide with predicted operons but are generally longer, most of them exhibiting internal promoters and terminators that generate alternative transcripts of variable gene composition. We characterize the occurrence of transcriptional read-through at terminators, which might play a basal regulation role and explain the extent of transcription beyond the scale of operons. We finally highlight the presence of noncontiguous operons and excludons in D. dadantii genome, novel genomic arrangements that might contribute to the basal coordination of transcription. The highlighted transcriptional organization may allow D. dadantii finely adjusting its gene expression program for a rapid adaptation to fast changing environment relevant to plant infection.ImportanceThis is the first transcriptomic map of a phytopathogen, characterized under physiological conditions encountered by the bacteria during the infection process. It might therefore significantly contribute to further progress in the field of phytopathogenicity. Our findings also provide insights into basal rules of coordination of transcription that might be valid for other bacteria, and may raise interest in the field of microbiology in general. In particular, we demonstrate that gene expression is coordinated at the scale of transcription units rather than operons, which are larger functional genomic units capable of generating transcripts with variable gene composition for a fine-tuning of gene expression in response to environmental changes. In line with recent studies, our findings indicate that the canonical operon model is insufficient to explain the complexity of bacterial transcriptomes.

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