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 A tunable anthranilate-inducible gene expression system for Pseudomonas species

2020 ◽  
Vol 105 (1) ◽  
pp. 247-258
Author(s):  
Lena Hoffmann ◽  
Michael-Frederick Sugue ◽  
Thomas Brüser
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
Ant System ◽  
Plant Host ◽  
Inducible Gene Expression ◽  
Gene Expression System ◽  
Expression Levels ◽  
Key Points ◽  
Wide Range ◽  
Inducible Gene

Abstract Pseudomonads are among the most common bacteria in soils, limnic ecosystems, and human, animal, or plant host environments, including intensively studied species such as Pseudomonas aeruginosa, P. putida, or P. fluorescens. Various gene expression systems are established for some species, but there is still a need for a simple system that is suitable for a wide range of pseudomonads and that can be used for physiological applications, i.e., with a tuning capacity at lower expression levels. Here, we report the establishment of the anthranilate-dependent PantA promoter for tunable gene expression in pseudomonads. During studies on P. fluorescens, we constructed an anthranilate-inducible AntR/PantA-based expression system, named pUCP20-ANT, and used GFP as reporter to analyze gene expression. This system was compared with the rhamnose-inducible RhaSR/PrhaB-based expression system in an otherwise identical vector background. While the rhamnose-inducible system did not respond to lower inducer concentrations and always reached high levels over time when induced, expression levels of the pUCP20-ANT system could be adjusted to a range of distinct lower or higher levels by variation of anthranilate concentrations in the medium. Importantly, the anthranilate-inducible expression system worked also in strains of P. aeruginosa and P. putida and therefore will be most likely useful for physiological and biotechnological purposes in a wide range of pseudomonads. Key points • We established an anthranilate-inducible gene expression system for pseudomonads. • This system permits tuning of gene expression in a wide range of pseudomonads. • It will be very useful for physiological and biotechnological applications.

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 Independent evolution of transcript abundance and gene regulatory dynamics

2020 ◽  
Author(s):  
Gat Krieger ◽  
Offir Lupo ◽  
Avraham A. Levy ◽  
Naama Barkai
Keyword(s):  
Gene Expression ◽  
Transcript Abundance ◽  
Species Variation ◽  
Promoter Strength ◽  
Limited Information ◽  
Regulate Gene Expression ◽  
Transcription Network ◽  
Expression Levels ◽  
Gene Positioning ◽  
Regulatory Dynamics

AbstractChanges in gene expression drive novel phenotypes, raising interest in how gene expression evolves. In contrast to the static genome, cells regulate gene expression to accommodate changing conditions. Previous comparative studies focused on specific conditions, describing inter-species variation in expression levels, but providing limited information about variations in gene regulation. To close this gap, we profiled gene expression of related yeast species in hundreds of conditions, and used co-expression analysis to distinguish variations in transcription regulation from variations in expression levels or environmental perception. The majority of genes whose expression varied between the species maintained a conserved transcriptional regulation. Profiling the interspecific hybrid provided insights into the basis of variations, showed that trans-varying alleles interact dominantly, and revealed complementation of cis-variations by variations in trans. Our data suggests that gene expression diverges primarily through changes in promoter strength that do not alter gene positioning within the transcription network.

scholarly journals iCodon: ideal codon design for customized gene expression

2021 ◽  
Author(s):  
Ariel Bazzini
Keyword(s):  
Gene Expression ◽  
Mrna Stability ◽  
Messenger Rna ◽  
Synonymous Codon ◽  
Biological Research ◽  
Dependent Manner ◽  
Coding Sequence ◽  
Expression Levels ◽  
Codon Composition ◽  
Wide Range

Abstract Messenger RNA (mRNA) stability substantially impacts steady-state gene expression levels in a cell. mRNA stability, in turn, is strongly affected by codon composition in a translation dependent manner across species, through a mechanism termed codon optimality. We have developed iCodon (www.iCodon.org), an algorithm for customizing mRNA expression through the introduction of synonymous codon substitutions into the coding sequence. iCodon is optimized for four vertebrate transcriptomes: mouse, human, frog, and fish. Users can predict the mRNA stability of any coding sequence based on its codon composition and subsequently generate more stable (optimized) or unstable (deoptimized) variants encoding for the same protein. Further, we show that codon optimality predictions correlate with expression levels using fluorescent reporters and endogenous genes in human cells and zebrafish embryos. Therefore, iCodon will benefit basic biological research, as well as a wide range of applications for biotechnology and biomedicine.

The development of the DCIS score: Scaling and normalization in the Marin General Hospital cohort.

2012 ◽  
Vol 30 (27_suppl) ◽  
pp. 190-190 ◽  
Author(s):  
Frederick L. Baehner ◽  
Steven M Butler ◽  
Carl N. Yoshizawa ◽  
Che Prasad ◽  
Diana B. Cherbavaz ◽  
...  
Keyword(s):  
Gene Expression ◽  
General Hospital ◽  
Risk Group ◽  
Recurrence Score ◽  
Low Risk ◽  
Tumor Characteristics ◽  
Clinicopathologic Characteristics ◽  
Expression Levels ◽  
Wide Range ◽  
Gene Expression Levels

190 Background: In selected low-risk patients with DCIS treated with wide local excision without radiation, the DCIS score was validated as a predictor of 10 year risk of an ipsilateral breast event (IBE - recurrence of in situ or invasive carcinoma) (p = 0.02) (Solin; SABCS 2011). As part of the development of the DCIS score, scaling from 0 to 100 and determination of risk group cutoff values was done using 100 patient DCIS samples from Marin General Hospital (MGH) selected to have a wide range of tumor characteristics. Methods: 100 patient specimens diagnosed with DCIS were provided by MGH. The Oncotype DX assay was performed, normalized expression levels for the 16 cancer related genes were determined, the DCIS Score and Recurrence Score (RS) were calculated. Distributions of the DCIS score, RS, and individual gene expression levels were described overall and by tumor characteristics. Treatment and pt outcome data were not available. Results: Samples for 96 pts had sufficient tumor and were evaluable; 47% had high nuclear grade, 52% comedo necrosis, 32% tumor size >10 mm, and 9% were ER-negative by IHC. After scaling and risk group cutoff determination, the DCIS score was low risk (0-38) for 49%, intermediate risk (39-54) for 27%, and high risk (≥55) for 24% of pts (Table). The DCIS score was widely distributed within subgroups defined by each of the clinical and pathology characteristics. Proliferation gene expression levels were low in DCIS, on average, relative to prior studies in invasive breast cancer. 92% had a proliferation gene group score <6.5, the threshold used when the RS is calculated; no threshold is used in calculating the DCIS score. Conclusions: Optimal scaling and risk cutoff determination for a wide range of all clinicopathologic characteristics provides for a wide distribution for the DCIS Score. [Table: see text]

scholarly journals Optimized gene expression from bacterial chromosome by high-throughput integration and screening

2021 ◽  
Vol 7 (7) ◽  
pp. eabe1767
Author(s):  
Tatyana E. Saleski ◽  
Meng Ting Chung ◽  
David N. Carruthers ◽  
Azzaya Khasbaatar ◽  
Katsuo Kurabayashi ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Throughput ◽  
High Throughput Screening ◽  
Efficient Production ◽  
Expression Levels ◽  
Pathway Gene ◽  
Pathway Expression ◽  
Wide Range ◽  
Cell To Cell Variability ◽  
Gene Expression Levels

Chromosomal integration of recombinant genes is desirable compared with expression from plasmids due to increased stability, reduced cell-to-cell variability, and elimination of the need for antibiotics for plasmid maintenance. Here, we present a new approach for tuning pathway gene expression levels via random integration and high-throughput screening. We demonstrate multiplexed gene integration and expression-level optimization for isobutanol production in Escherichia coli. The integrated strains could, with far lower expression levels than plasmid-based expression, produce high titers (10.0 ± 0.9 g/liter isobutanol in 48 hours) and yields (69% of the theoretical maximum). Close examination of pathway expression in the top-performing, as well as other isolates, reveals the complexity of cellular metabolism and regulation, underscoring the need for precise optimization while integrating pathway genes into the chromosome. We expect this method for pathway integration and optimization can be readily extended to a wide range of pathways and chassis to create robust and efficient production strains.

scholarly journals Local adaptation contributes to gene expression divergence in maize

2021 ◽  
Vol 11 (2) ◽  
Author(s):  
Jennifer Blanc ◽  
Karl A G Kremling ◽  
Edward Buckler ◽  
Emily B Josephs
Keyword(s):  
Gene Expression ◽  
Local Adaptation ◽  
Cold Response ◽  
Expression Levels ◽  
Maize Populations ◽  
Maize Genotypes ◽  
Wide Range ◽  
History Of ◽  
Insight Into ◽  
Gene Expression Levels

Abstract Gene expression links genotypes to phenotypes, so identifying genes whose expression is shaped by selection will be important for understanding the traits and processes underlying local adaptation. However, detecting local adaptation for gene expression will require distinguishing between divergence due to selection and divergence due to genetic drift. Here, we adapt a QST−FST framework to detect local adaptation for transcriptome-wide gene expression levels in a population of diverse maize genotypes. We compare the number and types of selected genes across a wide range of maize populations and tissues, as well as selection on cold-response genes, drought-response genes, and coexpression clusters. We identify a number of genes whose expression levels are consistent with local adaptation and show that genes involved in stress response show enrichment for selection. Due to its history of intense selective breeding and domestication, maize evolution has long been of interest to researchers, and our study provides insight into the genes and processes important for in local adaptation of maize.

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 Single Molecule-based FliFISH Validates Radial and Heterogeneous Gene Expression Patterns in Pancreatic Islet β Cells

2021 ◽  
Author(s):  
Fangjia Li ◽  
Dehong Hu ◽  
Cailin Dieter ◽  
Charles Ansong ◽  
Lori Sussel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pancreatic Islet ◽  
Spatial Organization ◽  
Single Cells ◽  
Expression Patterns ◽  
Gene Expression Patterns ◽  
Β Cells ◽  
Β Cell ◽  
Expression Levels ◽  
Wide Range

Single cell RNA sequencing (scRNA-Seq) technologies have greatly enhanced our understanding of islet cell transcriptomes and have revealed the existence of β cell heterogeneity. However, comparison of scRNA-Seq datasets from different groups have highlighted inconsistencies in gene expression patterns, primarily due to variable detection of lower abundance transcripts. Furthermore, such analyses are unable to uncover the spatial organization of heterogeneous gene expression. Here we used fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) to quantify transcripts in single cells in mouse pancreatic islet sections. We compared the expression patterns of <i>Insulin 2</i> (<i>Ins2)</i> with <i>Mafa</i> and <i>Ucn3</i> <i>–</i> two genes expressed in β cells as they mature, as well as <i>Rgs4 – </i>a factor with variably reported expression in the islet. This approach accurately quantified transcripts across a wide range of expression levels - from single copies to over hundred copies per cell in one islet. Importantly, fliFISH allowed evaluation of transcript heterogeneity in the spatial context of an intact islet. These studies confirm the existence of a high degree of heterogeneous gene expression levels within the islet and highlight relative and radial expression patterns that likely reflect distinct β cell maturation states along the radial axis of the islet.

100 GLOBAL H3K27me3 IS DISTINCT IN THE PORCINE EPIBLAST AND TROPHECTODERM AND IS POTENTIALLY CORRELATED TO X-INACTIVATION IN FEMALE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 155
Author(s):  
Y. Gao ◽  
V. Hall ◽  
P. Hyttel
Keyword(s):  
Gene Expression ◽  
Cell Fate ◽  
X Chromosome ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Male And Female ◽  
Epigenetic Marks ◽  
Expression Levels

During embryonic development in mammals, the first cell fate decision occurs at the morula stage, which leads to the establishment of the inner cell mass (ICM) and trophectoderm (TE). At this point of development, differential patterns of gene expression and epigenetic marks are observed within these two lineages. The ICM later differentiates to form the epiblast. Previous reports suggest that the distinct patterns of expression might be substantially regulated by epigenetic marks such as DNA methylation and post-transcriptional modifications. In this study, we examined the global H3K27me3 distribution, as well as the expression levels of H3K27me3 specific methyltransferases and XIST in the epiblast and TE of Day 10 in vivo porcine embryos. A total of 33 embryos were collected by non-surgical flushing from inseminated, and later, culled sows. The embryos were sexed by PCR (sequences of ZFX (X chromosome) and SRY (Y chromosome) genes were amplified by PCR using primers) because the H3K27me3 has been associated with X chromosome inactivation. For immunocytochemistry, a small piece of TE was removed before fixation and analysed for sexing by PCR. For comparative RT-PCR studies, embryos were mechanically separated, sexed, and then later pooled as male or female epiblast and TE (male = 6, female = 8). Global H3K27me3 was analysed by immunocytochemistry in 11 male and 8 female Day 10 embryos. Expression of methylases (EZH2, EED and SUZ12, three core components of PRC2), demethylases (JMJD3 and UTX) of H3K27me3, and XIST was performed on the pooled epiblasts and pooled TE. Expression levels were normalized to the reference gene, GAPDH, and was further normalized to Day 9 embryos. Our results show that high nuclear expression of H3K27me3 was observed in both male and female TE cells, with little to no observable expression in the epiblast. However, a single, small, punctate spot could be detected within the nuclei of the female epiblast and TE. XIST, a non-coding RNA associated with the initiation of X chromosome inactivation (XCI), was observed to be highly expressed in the female epiblast and TE, which suggests H3K27me3 punctate spots that presented in female epiblast or TE are potentially expressed on the inactive X chromosomes. We also detected higher expression of the H3K27me3 methylase (EZH2) and the methylase cofactors (EED, SUZ12) in both male and female TE. Of interest, EED expression was higher in the female epiblast and TE compared to the male epiblast and TE. This suggests that EED may play an important role in the initiation of XCI. The expression of H3K27me3 demethylases JMJD3 and UTX, were also higher in the TE compared to the epiblast, which indicates the trimethylation of H3K27 in the embryos is a dynamic process. We suggest that no, or extremely low, H3K27me3 in the porcine epiblast might be required for the cells to program gene expression towards different cell fates upon differentiation and the enrichment of H3K27me3 in the TE of Day 10 porcine embryos might reinforce the commitment towards the TE lineage.

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