scholarly journals Engineered reversal of function in glycolytic yeast promoters

2019 ◽  
Author(s):  
Arun S. Rajkumar ◽  
Emre Özdemir ◽  
Alicia V. Lis ◽  
Konstantin Schneider ◽  
Michael K. Jensen ◽  
...  
Keyword(s):  
Carbon Sources ◽  
Regulatory Elements ◽  
Regulatory Function ◽  
Cell Factory ◽  
Diauxic Shift ◽  
Cell Factories ◽  
Expression Of Genes ◽  
Alternate Promoters ◽  
Precise Expression ◽  
Heterologous Pathway

ABSTRACTPromoters are key components of cell factory design, allowing precise expression of genes in a heterologous pathway. Several commonly-used promoters in yeast cell factories belong to glycolytic genes, highly expressed in actively-growing yeast when glucose is used as a carbon source. However, their expression can be suboptimal when alternate carbon sources are used, or if there is a need to decouple growth from production. Hence, there is a need for alternate promoters for different carbon sources and production schemes. In this work, we demonstrate a reversal of regulatory function in two glycolytic yeast promoters by replacing glycolytic regulatory elements with ones induced by the diauxic shift. We observe a shift in induction from glucose-rich to glucose-poor medium without loss of regulatory activity, and strong ethanol induction. Applications of these promoters were validated for expression of the vanillin biosynthetic pathway, reaching production of vanillin comparable to pathway designs using strong constitutive promoters.

scholarly journals Organic Wastes as Feedstocks for Non-Conventional Yeast-Based Bioprocesses

2019 ◽  
Vol 7 (8) ◽  
pp. 229 ◽  
Author(s):  
Diem T. Hoang Do ◽  
Chrispian W. Theron ◽  
Patrick Fickers
Keyword(s):  
Carbon Sources ◽  
Value Added ◽  
Building Blocks ◽  
Organic Wastes ◽  
Cell Factory ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Alternative Feedstocks ◽  
Global Population ◽  
By Products

Non-conventional yeasts are efficient cell factories for the synthesis of value-added compounds such as recombinant proteins, intracellular metabolites, and/or metabolic by-products. Most bioprocess, however, are still designed to use pure, ideal sugars, especially glucose. In the quest for the development of more sustainable processes amid concerns over the future availability of resources for the ever-growing global population, the utilization of organic wastes or industrial by-products as feedstocks to support cell growth is a crucial approach. Indeed, vast amounts of industrial and commercial waste simultaneously represent an environmental burden and an important reservoir for recyclable or reusable material. These alternative feedstocks can provide microbial cell factories with the required metabolic building blocks and energy to synthesize value-added compounds, further representing a potential means of reduction of process costs as well. This review highlights recent strategies in this regard, encompassing knowledge on catabolic pathways and metabolic engineering solutions developed to endow cells with the required metabolic capabilities, and the connection of these to the synthesis of value-added compounds. This review focuses primarily, but not exclusively, on Yarrowia lipolytica as a yeast cell factory, owing to its broad range of naturally metabolizable carbon sources, together with its popularity as a non-conventional yeast.

scholarly journals Saccharomyces cerevisiae JEN1 Promoter Activity Is Inversely Related to Concentration of Repressing Sugar

2004 ◽  
Vol 70 (1) ◽  
pp. 8-17 ◽  
Author(s):  
Prima Chambers ◽  
Aminatu Issaka ◽  
Sean P. Palecek
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Concentration ◽  
Promoter Activity ◽  
Fluorescent Protein ◽  
Carbon Sources ◽  
Regulatory Elements ◽  
Dependent Manner ◽  
Diauxic Shift ◽  
Reporter System ◽  
Genomic Expression

ABSTRACT When carbon sources are changed, Saccharomyces cerevisiae transcriptional patterns drastically change. To identify genes whose transcription can be used to quantitatively measure sugar concentrations, we searched genomic expression databases for a set of genes that are highly induced during the diauxic shift, and we used the promoters from these genes to drive expression of green fluorescent protein (GFP). Certain sugars, including glucose, fructose, and mannose, repress the promoter of JEN1, which encodes a lactate-pyruvate transporter, in a dose-dependent manner. Nonrepressing carbon sources include galactose, raffinose, ethanol, lactate, and glycerol. JEN1 promoter activity is a linear function of glucose concentration when organisms are grown at a steady-state glucose concentration below 1 g/liter. JEN1 promoter repression is specific to carbon source; heat or cold shock, osmotic stress, DNA damage, and nitrogen starvation do not significantly affect promoter activity. Activation of the JEN1 promoter requires the Snf1 protein kinase, but multiple regulatory elements most likely combine to provide the linear relationship between JEN1 promoter activity and sugar concentration. Thus, a JEN1 promoter-reporter system appears to provide a good living cell biosensor for the concentration of certain sugars. The JEN1 promoter also permits quantitative regulation of cellular functions not normally controlled by sugar concentrations. For example, a strain expressing FLO1 under control of the JEN1 promoter flocculates at a low glucose concentration.

scholarly journals The Coordinated Upregulated Expression of Genes Involved in MEP, Chlorophyll, Carotenoid and Tocopherol Pathways, Mirrored the Corresponding Metabolite Contents in Rice Leaves during De-Etiolation

Plants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1456
Author(s):  
Xin Jin ◽  
Can Baysal ◽  
Margit Drapal ◽  
Yanmin Sheng ◽  
Xin Huang ◽  
...  
Keyword(s):  
Higher Plants ◽  
Expression Patterns ◽  
Regulatory Elements ◽  
Isoprenoid Biosynthesis ◽  
Promoter Regions ◽  
Expression Of Genes ◽  
Coordinated Expression ◽  
Rice Leaves ◽  
Mechanistic Basis ◽  
Phosphate Synthase

Light is an essential regulator of many developmental processes in higher plants. We investigated the effect of 4-hydroxy-3-methylbut-2-enyl diphosphate reductase 1/2 genes (OsHDR1/2) and isopentenyl diphosphate isomerase 1/2 genes (OsIPPI1/2) on the biosynthesis of chlorophylls, carotenoids, and phytosterols in 14-day-old etiolated rice (Oyza sativa L.) leaves during de-etiolation. However, little is known about the effect of isoprenoid biosynthesis genes on the corresponding metabolites during the de-etiolation of etiolated rice leaves. The results showed that the levels of α-tocopherol were significantly increased in de-etiolated rice leaves. Similar to 1-deoxy-D-xylulose-5-phosphate synthase 3 gene (OsDXS3), both OsDXS1 and OsDXS2 genes encode functional 1-deoxy-D-xylulose-5-phosphate synthase (DXS) activities. Their expression patterns and the synthesis of chlorophyll, carotenoid, and tocopherol metabolites suggested that OsDXS1 is responsible for the biosynthesis of plastidial isoprenoids in de-etiolated rice leaves. The expression analysis of isoprenoid biosynthesis genes revealed that the coordinated expression of the MEP (2-C-methyl-D-erythritol 4-phosphate) pathway, chlorophyll, carotenoid, and tocopherol pathway genes mirrored the changes in the levels of the corresponding metabolites during de-etiolation. The underpinning mechanistic basis of coordinated light-upregulated gene expression was elucidated during the de-etiolation process, specifically the role of light-responsive cis-regulatory motifs in the promoter region of these genes. In silico promoter analysis showed that the light-responsive cis-regulatory elements presented in all the promoter regions of each light-upregulated gene, providing an important link between observed phenotype during de-etiolation and the molecular machinery controlling expression of these genes.

scholarly journals Translation of ABCE1 Is Tightly Regulated by Upstream Open Reading Frames in Human Colorectal Cells

Biomedicines ◽  
2021 ◽  
Vol 9 (8) ◽  
pp. 911
Author(s):  
Joana Silva ◽  
Pedro Nina ◽  
Luísa Romão
Keyword(s):  
Translational Regulation ◽  
Regulatory Elements ◽  
Ribosome Profiling ◽  
Leader Sequence ◽  
Open Reading Frames ◽  
Regulatory Function ◽  
Coding Sequence ◽  
Abc Protein ◽  
Upstream Open Reading Frames ◽  
Reading Frames

ATP-binding cassette subfamily E member 1 (ABCE1) belongs to the ABC protein family of transporters; however, it does not behave as a drug transporter. Instead, ABCE1 actively participates in different stages of translation and is also associated with oncogenic functions. Ribosome profiling analysis in colorectal cancer cells has revealed a high ribosome occupancy in the human ABCE1 mRNA 5′-leader sequence, indicating the presence of translatable upstream open reading frames (uORFs). These cis-acting translational regulatory elements usually act as repressors of translation of the main coding sequence. In the present study, we dissect the regulatory function of the five AUG and five non-AUG uORFs identified in the human ABCE1 mRNA 5′-leader sequence. We show that the expression of the main coding sequence is tightly regulated by the ABCE1 AUG uORFs in colorectal cells. Our results are consistent with a model wherein uORF1 is efficiently translated, behaving as a barrier to downstream uORF translation. The few ribosomes that can bypass uORF1 (and/or uORF2) must probably initiate at the inhibitory uORF3 or uORF5 that efficiently repress translation of the main ORF. This inhibitory property is slightly overcome in conditions of endoplasmic reticulum stress. In addition, we observed that these potent translation-inhibitory AUG uORFs function equally in cancer and in non-tumorigenic colorectal cells, which is consistent with a lack of oncogenic function. In conclusion, we establish human ABCE1 as an additional example of uORF-mediated translational regulation and that this tight regulation contributes to control ABCE1 protein levels in different cell environments.

scholarly journals Oleaginous Yeasts as Cell Factories for the Sustainable Production of Microbial Lipids by the Valorization of Agri-Food Wastes

Fermentation ◽  
2021 ◽  
Vol 7 (2) ◽  
pp. 50
Author(s):  
Antonio Caporusso ◽  
Angela Capece ◽  
Isabella De De Bari
Keyword(s):  
Value Chain ◽  
Carbon Sources ◽  
Research Progress ◽  
Food Wastes ◽  
Microbial Lipids ◽  
Oleaginous Yeasts ◽  
Cell Factories ◽  
Side Stream ◽  
Versatile Tool ◽  
Single Cell Oils

The agri-food industry annually produces huge amounts of crops residues and wastes, the suitable management of these products is important to increase the sustainability of agro-industrial production by optimizing the entire value chain. This is also in line with the driving principles of the circular economy, according to which residues can become feedstocks for novel processes. Oleaginous yeasts represent a versatile tool to produce biobased chemicals and intermediates. They are flexible microbial factories able to grow on different side-stream carbon sources such as those deriving from agri-food wastes, and this characteristic makes them excellent candidates for integrated biorefinery processes through the production of microbial lipids, known as single cell oils (SCOs), for different applications. This review aims to present an extensive overview of research progress on the production and use of oleaginous yeasts and present discussions on the current bottlenecks and perspectives of their exploitation in different sectors, such as foods, biofuels and fine chemicals.

scholarly journals Bacterial Biopolymer: Its Role in Pathogenesis to Effective Biomaterials

Polymers ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1242
Author(s):  
Sreejita Ghosh ◽  
Dibyajit Lahiri ◽  
Moupriya Nag ◽  
Ankita Dey ◽  
Tanmay Sarkar ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Carbon Sources ◽  
Chemical Properties ◽  
Interdisciplinary Studies ◽  
Extracellular Dna ◽  
Antimicrobial Drugs ◽  
Cell Factories ◽  
Food Components ◽  
Innovative Biomaterials ◽  
And Chemical Properties

Bacteria are considered as the major cell factories, which can effectively convert nitrogen and carbon sources to a wide variety of extracellular and intracellular biopolymers like polyamides, polysaccharides, polyphosphates, polyesters, proteinaceous compounds, and extracellular DNA. Bacterial biopolymers find applications in pathogenicity, and their diverse materialistic and chemical properties make them suitable to be used in medicinal industries. When these biopolymer compounds are obtained from pathogenic bacteria, they serve as important virulence factors, but when they are produced by non-pathogenic bacteria, they act as food components or biomaterials. There have been interdisciplinary studies going on to focus on the molecular mechanism of synthesis of bacterial biopolymers and identification of new targets for antimicrobial drugs, utilizing synthetic biology for designing and production of innovative biomaterials. This review sheds light on the mechanism of synthesis of bacterial biopolymers and its necessary modifications to be used as cell based micro-factories for the production of tailor-made biomaterials for high-end applications and their role in pathogenesis.

scholarly journals Nanocellulose-based mechanically stable immobilization matrix for enhanced ethylene production: A framework for photosynthetic solid-state cell factories

2021 ◽  
Author(s):  
Ville Rissanen ◽  
Sindhujaa Vajravel ◽  
Sergey Kosourov ◽  
Suvi Arola ◽  
Eero Kontturi ◽  
...  
Keyword(s):  
Solid State ◽  
Ethylene Production ◽  
Cell Immobilization ◽  
Cell Factory ◽  
Cell Factories ◽  
Solid State Cell ◽  
Immobilization Matrix

Cell immobilization is a promising approach to create efficient photosynthetic cell factories for sustainable chemicals production. Here, we demonstrate a novel photosynthetic solid-state cell factory design for sustainable biocatalytic ethylene...

The positive regulatory function of the 5'-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences

1988 ◽  
Vol 8 (9) ◽  
pp. 3827-3836
Author(s):  
N P Williams ◽  
P P Mueller ◽  
A G Hinnebusch
Keyword(s):  
Translational Control ◽  
Normal Growth ◽  
Regulatory Elements ◽  
Growth Conditions ◽  
Open Reading Frames ◽  
Regulatory Function ◽  
Yeast Saccharomyces Cerevisiae ◽  
Reading Frame ◽  
Yeast Genes ◽  
Reading Frames

Translational control of GCN4 expression in the yeast Saccharomyces cerevisiae is mediated by multiple AUG codons present in the leader of GCN4 mRNA, each of which initiates a short open reading frame of only two or three codons. Upstream AUG codons 3 and 4 are required to repress GCN4 expression in normal growth conditions; AUG codons 1 and 2 are needed to overcome this repression in amino acid starvation conditions. We show that the regulatory function of AUG codons 1 and 2 can be qualitatively mimicked by the AUG codons of two heterologous upstream open reading frames (URFs) containing the initiation regions of the yeast genes PGK and TRP1. These AUG codons inhibit GCN4 expression when present singly in the mRNA leader; however, they stimulate GCN4 expression in derepressing conditions when inserted upstream from AUG codons 3 and 4. This finding supports the idea that AUG codons 1 and 2 function in the control mechanism as translation initiation sites and further suggests that suppression of the inhibitory effects of AUG codons 3 and 4 is a general consequence of the translation of URF 1 and 2 sequences upstream. Several observations suggest that AUG codons 3 and 4 are efficient initiation sites; however, these sequences do not act as positive regulatory elements when placed upstream from URF 1. This result suggests that efficient translation is only one of the important properties of the 5' proximal URFs in GCN4 mRNA. We propose that a second property is the ability to permit reinitiation following termination of translation and that URF 1 is optimized for this regulatory function.

scholarly journals Computational Analysis of Dynamic Light Exposure of Unicellular Algal Cells in a Flat-Panel Photobioreactor to Support Light-Induced CO2 Bioprocess Development

2021 ◽  
Vol 12 ◽  
Author(s):  
Nicolò S. Vasile ◽  
Alessandro Cordara ◽  
Giulia Usai ◽  
Angela Re
Keyword(s):  
Light Transmission ◽  
Light Exposure ◽  
Model Organism ◽  
Cell Factory ◽  
Bioprocess Development ◽  
Cultivation Conditions ◽  
Flat Panel ◽  
Modeling Framework ◽  
Cell Factories ◽  
Cell Trajectories

Cyanobacterial cell factories trace a vibrant pathway to climate change neutrality and sustainable development owing to their ability to turn carbon dioxide-rich waste into a broad portfolio of renewable compounds, which are deemed valuable in green chemistry cross-sectorial applications. Cell factory design requires to define the optimal operational and cultivation conditions. The paramount parameter in biomass cultivation in photobioreactors is the light intensity since it impacts cellular physiology and productivity. Our modeling framework provides a basis for the predictive control of light-limited, light-saturated, and light-inhibited growth of the Synechocystis sp. PCC 6803 model organism in a flat-panel photobioreactor. The model here presented couples computational fluid dynamics, light transmission, kinetic modeling, and the reconstruction of single cell trajectories in differently irradiated areas of the photobioreactor to relate key physiological parameters to the multi-faceted processes occurring in the cultivation environment. Furthermore, our analysis highlights the need for properly constraining the model with decisive qualitative and quantitative data related to light calibration and light measurements both at the inlet and outlet of the photobioreactor in order to boost the accuracy and extrapolation capabilities of the model.

scholarly journals Enhancers mapping uncovers phenotypic heterogeneity and evolution in patients with luminal breast cancer

2017 ◽  
Author(s):  
Darren K. Patten ◽  
Giacomo Corleone ◽  
Balázs Győrffy ◽  
Edina Erdős ◽  
Alina Saiakhova ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Progression ◽  
Regulatory Elements ◽  
Phenotypic Heterogeneity ◽  
Endocrine Treatment ◽  
Clinical Samples ◽  
Luminal Breast Cancer ◽  
Breast Cancer Patients ◽  
Expression Of Genes

AbstractThe degree of intrinsic and interpatient phenotypic heterogeneity and its role in tumour evolution is poorly understood. Phenotypic divergence can be achieved via the inheritance of alternative transcriptional programs1,2. Cell-type specific transcription is maintained through the activation of epigenetically-defined regulatory regions including promoters and enhancers1,3,4. In this work, we annotated the epigenome of 47 primary and metastatic oestrogen-receptor (ERα)-positive breast cancer specimens from clinical samples, and developed strategies to deduce phenotypic heterogeneity from the regulatory landscape, identifying key regulatory elements commonly shared across patients. Highly shared regions contain a unique set of regulatory information including the motif for the transcription factor YY1. In vitro work shows that YY1 is essential for ERα transcriptional activity and defines the critical subset of functional ERα binding sites driving tumor growth in most luminal patients. YY1 also control the expression of genes that mediate resistance to endocrine treatment. Finally, we show that H3K27ac levels at active enhancer elements can be used as a surrogate of intra-tumor phenotypic heterogeneity, and to track expansion and contraction of phenotypic subpopulations throughout breast cancer progression. Tracking YY1 and SLC9A3R1 positive clones in primary and metastatic lesions, we show that endocrine therapies drive the expansion of phenotypic clones originally underrepresented at diagnosis. Collectively, our data show that epigenetic mechanisms significantly contribute to phenotypic heterogeneity and evolution in systemically treated breast cancer patients.

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