Cell growth restoration and high level protein expression by the promoter of hexose transporter, HXT7, from Saccharomyces cerevisiae

Abstract Production of chemicals and biofuels through microbial fermentation is an economical and sustainable alternative for traditional chemical synthesis. Here we present the construction of a Saccharomyces cerevisiae platform strain for high-level production of very-long-chain fatty acid (VLCFA)-derived chemicals. Through rewiring the native fatty acid elongation system and implementing a heterologous Mycobacteria FAS I system, we establish an increased biosynthesis of VLCFAs in S. cerevisiae. VLCFAs can be selectively modified towards the fatty alcohol docosanol (C22H46O) by expressing a specific fatty acid reductase. Expression of this enzyme is shown to impair cell growth due to consumption of VLCFA-CoAs. We therefore implement a dynamic control strategy for separating cell growth from docosanol production. We successfully establish high-level and selective docosanol production of 83.5 mg l−1 in yeast. This approach will provide a universal strategy towards the production of similar high value chemicals in a more scalable, stable and sustainable manner.

Download Full-text

Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

Biochemical Journal ◽

10.1042/bj3390299 ◽

1999 ◽

Vol 339 (2) ◽

pp. 299-307 ◽

Cited By ~ 33

Author(s):

Arthur L. KRUCKEBERG ◽

Ling YE ◽

Jan A. BERDEN ◽

Karel van DAM

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Green Fluorescent Protein ◽

Glucose Transport ◽

Cellular Localization ◽

Fluorescent Protein ◽

Hexose Transporter ◽

High Concentrations ◽

Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

Download Full-text

Tempo and Mode of Ty Element Evolution in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/151.4.1341 ◽

1999 ◽

Vol 151 (4) ◽

pp. 1341-1351 ◽

Cited By ~ 2

Author(s):

I King Jordan ◽

John F McDonald

Keyword(s):

Saccharomyces Cerevisiae ◽

Negative Selection ◽

Sequence Variation ◽

Size Variation ◽

Sequence Comparisons ◽

Ty Elements ◽

Ty Element ◽

Amino Acid Sequence Variation ◽

Element Evolution ◽

High Level

Abstract The Saccharomyces cerevisiae genome contains five families of long terminal repeat (LTR) retrotransposons, Ty1–Ty5. The sequencing of the S. cerevisiae genome provides an unprecedented opportunity to examine the patterns of molecular variation existing among the entire genomic complement of Ty retrotransposons. We report the results of an analysis of the nucleotide and amino acid sequence variation within and between the five Ty element families of the S. cerevisiae genome. Our results indicate that individual Ty element families tend to be highly homogenous in both sequence and size variation. Comparisons of within-element 5′ and 3′ LTR sequences indicate that the vast majority of Ty elements have recently transposed. Furthermore, intrafamily Ty sequence comparisons reveal the action of negative selection on Ty element coding sequences. These results taken together suggest that there is a high level of genomic turnover of S. cerevisiae Ty elements, which is presumably in response to selective pressure to escape host-mediated repression and elimination mechanisms.

Download Full-text

Molecular Basis of Fructose Utilization by the Wine Yeast Saccharomyces cerevisiae: a Mutated HXT3 Allele Enhances Fructose Fermentation

Applied and Environmental Microbiology ◽

10.1128/aem.02269-06 ◽

2007 ◽

Vol 73 (8) ◽

pp. 2432-2439 ◽

Cited By ~ 67

Author(s):

Carole Guillaume ◽

Pierre Delobel ◽

Jean-Marie Sablayrolles ◽

Bruno Blondin

Keyword(s):

Saccharomyces Cerevisiae ◽

Molecular Basis ◽

Alcoholic Fermentation ◽

Wine Yeast ◽

Glycolytic Flux ◽

Hexose Transporter ◽

Wine Yeasts ◽

Yeast Saccharomyces Cerevisiae ◽

High Fructose ◽

Fructose Fermentation

ABSTRACT Fructose utilization by wine yeasts is critically important for the maintenance of a high fermentation rate at the end of alcoholic fermentation. A Saccharomyces cerevisiae wine yeast able to ferment grape must sugars to dryness was found to have a high fructose utilization capacity. We investigated the molecular basis of this enhanced fructose utilization capacity by studying the properties of several hexose transporter (HXT) genes. We found that this wine yeast harbored a mutated HXT3 allele. A functional analysis of this mutated allele was performed by examining expression in an hxt1-7Δ strain. Expression of the mutated allele alone was found to be sufficient for producing an increase in fructose utilization during fermentation similar to that observed in the commercial wine yeast. This work provides the first demonstration that the pattern of fructose utilization during wine fermentation can be altered by expression of a mutated hexose transporter in a wine yeast. We also found that the glycolytic flux could be increased by overexpression of the mutant transporter gene, with no effect on fructose utilization. Our data demonstrate that the Hxt3 hexose transporter plays a key role in determining the glucose/fructose utilization ratio during fermentation.

Download Full-text

Effect of HXT1 and HXT7 hexose transporter overexpression on wild-type and lactic acid producing Saccharomyces cerevisiae cells

Microbial Cell Factories ◽

10.1186/1475-2859-9-15 ◽

2010 ◽

Vol 9 (1) ◽

pp. 15 ◽

Cited By ~ 17

Author(s):

Giorgia Rossi ◽

Michael Sauer ◽

Danilo Porro ◽

Paola Branduardi

Keyword(s):

Saccharomyces Cerevisiae ◽

Lactic Acid ◽

Hexose Transporter ◽

Wild Type

Download Full-text

MiR-137 promotes cell growth and inhibits extracellular matrix protein expression in H2O2-induced human trabecular meshwork cells by targeting Src

Neuroscience Letters ◽

10.1016/j.neulet.2021.135902 ◽

2021 ◽

Vol 755 ◽

pp. 135902

Author(s):

Liang Wang ◽

Ying Tian ◽

Yan Cao ◽

Qiang Ma ◽

Shuai Zhao

Keyword(s):

Extracellular Matrix ◽

Cell Growth ◽

Protein Expression ◽

Trabecular Meshwork ◽

Matrix Protein ◽

Extracellular Matrix Protein ◽

Trabecular Meshwork Cells

Download Full-text

The rye Mutants Identify a Role for Ssn/Srb Proteins of the RNA Polymerase II Holoenzyme During Stationary Phase Entry in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/157.1.17 ◽

2001 ◽

Vol 157 (1) ◽

pp. 17-26 ◽

Cited By ~ 3

Author(s):

Ya-Wen Chang ◽

Susie C Howard ◽

Yelena V Budovskaya ◽

Jasper Rine ◽

Paul K Herman

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Growth ◽

Rna Polymerase ◽

Stationary Phase ◽

Yeast Cell ◽

Rna Polymerase Ii ◽

Transcriptional Response ◽

Nutrient Deprivation ◽

Ras Signaling ◽

Rna Polymerase Ii Holoenzyme

Abstract Saccharomyces cerevisiae cells enter into a distinct resting state, known as stationary phase, in response to specific types of nutrient deprivation. We have identified a collection of mutants that exhibited a defective transcriptional response to nutrient limitation and failed to enter into a normal stationary phase. These rye mutants were isolated on the basis of defects in the regulation of YGP1 expression. In wild-type cells, YGP1 levels increased during the growth arrest caused by nutrient deprivation or inactivation of the Ras signaling pathway. In contrast, the levels of YGP1 and related genes were significantly elevated in the rye mutants during log phase growth. The rye defects were not specific to this YGP1 response as these mutants also exhibited multiple defects in stationary phase properties, including an inability to survive periods of prolonged starvation. These data indicated that the RYE genes might encode important regulators of yeast cell growth. Interestingly, three of the RYE genes encoded the Ssn/Srb proteins, Srb9p, Srb10p, and Srb11p, which are associated with the RNA polymerase II holoenzyme. Thus, the RNA polymerase II holoenzyme may be a target of the signaling pathways responsible for coordinating yeast cell growth with nutrient availability.

Download Full-text

The Ras/PKA Signaling Pathway May Control RNA Polymerase II Elongation via the Spt4p/Spt5p Complex in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/165.3.1059 ◽

2003 ◽

Vol 165 (3) ◽

pp. 1059-1070

Author(s):

Susie C Howard ◽

Arelis Hester ◽

Paul K Herman

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Growth ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Signaling Pathway ◽

Elongation Factor ◽

Ras Signaling ◽

Elongation Step ◽

Rna Polymerase Ii Transcription

Abstract The Ras signaling pathway in Saccharomyces cerevisiae controls cell growth via the cAMP-dependent protein kinase, PKA. Recent work has indicated that these effects on growth are due, in part, to the regulation of activities associated with the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. However, the precise target of these Ras effects has remained unknown. This study suggests that Ras/PKA activity regulates the elongation step of the RNA polymerase II transcription process. Several lines of evidence indicate that Spt5p in the Spt4p/Spt5p elongation factor is the likely target of this control. First, the growth of spt4 and spt5 mutants was found to be very sensitive to changes in Ras/PKA signaling activity. Second, mutants with elevated levels of Ras activity shared a number of specific phenotypes with spt5 mutants and vice versa. Finally, Spt5p was efficiently phosphorylated by PKA in vitro. Altogether, the data suggest that the Ras/PKA pathway might be directly targeting a component of the elongating polymerase complex and that this regulation is important for the normal control of yeast cell growth. These data point out the interesting possibility that signal transduction pathways might directly influence the elongation step of RNA polymerase II transcription.

Download Full-text