Expression and activity of the Hxt7 high‐affinity hexose transporter of <i>Saccharomyces cerevisiae</i>

Ye, Ling; Berden, J.A.; Dam, K. Van; Kruckeberg, Arthur L.

doi:10.1002/yea.771

Cited by 53 publications

(21 citation statements)

References 26 publications

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“…For that purpose, FAA1 was cloned on a CEN.ARS plasmid under the control of a copper-induced promoter ( CUP1 promoter) so that expression could be enabled by the addition of Cu 2+ to the culture medium at specific time points [22, 23]. In parallel, FAA1 was also put under control of a promoter induced by high glucose and repressed by low glucose concentrations ( HXT1 promoter) and a promoter repressed by high glucose and induced by low glucose conditions ( HXT7 promoter), respectively [24, 25]. As controls, an empty plasmid not expressing FAA1 (p413) and a plasmid with strong constitutive expression using the TEF1 promoter (pTEF1-FAA1) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…1b), followed by a tight repression during the ethanol phase, which leads to very low levels of Faa1 present in the cytosol through most of the culture. On the other hand, HXT7p is induced under low glucose conditions, while being repressed at high glucose levels [25]. This way, FAA1 is only expressed in the end of the glucose phase when glucose levels are very low so that FFAs are converted to acyl-CoA mostly during the ethanol phase.…”

Section: Resultsmentioning

confidence: 99%

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Dynamic regulation of fatty acid pools for improved production of fatty alcohols in Saccharomyces cerevisiae

et al. 2017

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BackgroundIn vivo production of fatty acid-derived chemicals in Saccharomyces cerevisiae requires strategies to increase the intracellular supply of either acyl-CoA or free fatty acids (FFAs), since their cytosolic concentrations are quite low in a natural state for this organism. Deletion of the fatty acyl-CoA synthetase genes FAA1 and FAA4 is an effective and straightforward way to disable re-activation of fatty acids and drastically increase FFA levels. However, this strategy causes FFA over-accumulation and consequential release to the extracellular medium, which results in a significant loss of precursors that compromises the process yield. In the present study, we aimed for dynamic expression of the fatty acyl-CoA synthetase gene FAA1 to regulate FFA and acyl-CoA pools in order to improve fatty alcohol production yields.ResultsWe analyzed the metabolite dynamics of a faa1Δ faa4Δ strain constitutively expressing a carboxylic acid reductase from Mycobacterium marinum (MmCAR) and an endogenous alcohol dehydrogenase (Adh5) for in vivo production of fatty alcohols from FFAs. We observed production of fatty acids and fatty alcohols with different rates leading to high levels of FFAs not being converted to the final product. To address the issue, we expressed the MmCAR + Adh5 pathway together with a fatty acyl-CoA reductase from Marinobacter aquaeolei to enable fatty alcohol production simultaneously from FFA and acyl-CoA, respectively. Then, we expressed FAA1 under the control of different promoters in order to balance FFA and acyl-CoA interconversion rates and to achieve optimal levels for conversion to fatty alcohols. Expressing FAA1 under control of the HXT1 promoter led to an increased accumulation of fatty alcohols per OD600 up to 41% while FFA levels were decreased by 63% compared with the control strain.ConclusionsFine-tuning and dynamic regulation of key metabolic steps can be used to improve cell factories when the rates of downstream reactions are limiting. This avoids loss of precursors to the extracellular medium or to competing reactions, hereby potentially improving the process yield. The study also provides knowledge of a key point of fatty acid regulation and homeostasis, which can be used for future design of cells factories for fatty acid-derived chemicals.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0663-3) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Dynamic regulation of fatty acid pools for improved production of fatty alcohols in Saccharomyces cerevisiae

et al. 2017

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“…the macronutrients glucose, nitrogen, phosphate and sulfate, and micronutrients like metal ions and vitamins. Limitation for glucose causes upregulation of Hxt6 and Hxt7 3, for nitrogen the general amino acid permease Gap1 4 and ammonium transporters Mep1 and Mep 2 5, for phosphate Pho84 6, for sulfate Sul1 and Sul2 7, for iron Ftr1 8 and for Zinc Zrt1 9. Even for non-essential nutrients like uracil, uracil permease is induced in media lacking uracil 10.…”

Section: Introductionmentioning

confidence: 99%

Identification of Ftr1 and Zrt1 as iron and zinc micronutrient transceptors for activation of the PKA pathway in Saccharomyces cerevisiae

Schothorst

Zeebroeck

Thevelein³

2017

Microb Cell

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Multiple types of nutrient transceptors, membrane proteins that combine a transporter and receptor function, have now been established in a variety of organisms. However, so far all established transceptors utilize one of the macronutrients, glucose, amino acids, ammonium, nitrate, phosphate or sulfate, as substrate. This is also true for the Saccharomyces cerevisiae transceptors mediating activation of the PKA pathway upon re-addition of a macronutrient to glucose-repressed cells starved for that nutrient, re-establishing a fermentable growth medium. We now show that the yeast high-affinity iron transporter Ftr1 and high-affinity zinc transporter Zrt1 function as transceptors for the micronutrients iron and zinc. We show that replenishment of iron to iron-starved cells or zinc to zinc-starved cells triggers within 1-2 minutes a rapid surge in trehalase activity, a well-established PKA target. The activation with iron is dependent on Ftr1 and with zinc on Zrt1, and we show that it is independent of intracellular iron and zinc levels. Similar to the transceptors for macronutrients, Ftr1 and Zrt1 are strongly induced upon iron and zinc starvation, respectively, and they are rapidly downregulated by substrate-induced endocytosis. Our results suggest that transceptor-mediated signaling to the PKA pathway may occur in all cases where glucose-repressed yeast cells have been starved first for an essential nutrient, causing arrest of growth and low activity of the PKA pathway, and subsequently replenished with the lacking nutrient to re-establish a fermentable growth medium. The broadness of the phenomenon also makes it likely that nutrient transceptors use a common mechanism for signaling to the PKA pathway.

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“…When the glucose concentrations are lowered to around 0.1%, HXT2 and HXT4, encoding moderate-affinity glucose transporters (K m ϭ ϳ5 to 10 mM), are expressed (10). HXT6 and HXT7, encoding highly homologous glucose transporters with very high glucose affinity (K m ϭ ϳ1 mM), are expressed just before the depletion of glucose in the medium (11). Expression of HXT5, encoding a moderate-affinity transporter (K m ϭ ϳ10 mM), is not regulated by extracellular glucose concentrations but by various environmental conditions affecting growth rates (12,13).…”

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confidence: 99%

Improvement of Glucose Uptake Rate and Production of Target Chemicals by Overexpressing Hexose Transporters and Transcriptional Activator Gcr1 in Saccharomyces cerevisiae

Kim

Song

Hahn

2015

Appl Environ Microbiol

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Metabolic engineering to increase the glucose uptake rate might be beneficial to improve microbial production of various fuels and chemicals. In this study, we enhanced the glucose uptake rate in Saccharomyces cerevisiae by overexpressing hexose transporters (HXTs). Among the 5 tested HXTs (Hxt1, Hxt2, Hxt3, Hxt4, and Hxt7), overexpression of high-affinity transporter Hxt7 was the most effective in increasing the glucose uptake rate, followed by moderate-affinity transporters Hxt2 and Hxt4. Deletion of STD1 and MTH1, encoding corepressors of HXT genes, exerted differential effects on the glucose uptake rate, depending on the culture conditions. In addition, improved cell growth and glucose uptake rates could be achieved by overexpression of GCR1, which led to increased transcription levels of HXT1 and ribosomal protein genes. All genetic modifications enhancing the glucose uptake rate also increased the ethanol production rate in wild-type S. cerevisiae. Furthermore, the growth-promoting effect of GCR1 overexpression was successfully applied to lactic acid production in an engineered lactic acid-producing strain, resulting in a significant improvement of productivity and titers of lactic acid production under acidic fermentation conditions. Saccharomyces cerevisiae has been used as an important cell factory for production of fuels and chemicals (1, 2). Although yeast can utilize a wide range of carbon sources, glucose is the most widely preferred carbon source for yeast fermentation (3). Glucose uptake in S. cerevisiae is elaborately regulated to adapt cellular metabolism in response to ever-changing environmental conditions (3, 4). However, to achieve high titers and productivity of target chemicals using metabolically engineered yeast cells, it may be beneficial to inactivate, circumvent, or modify some of such natural regulatory mechanisms.The first regulatory step of glucose uptake is the facilitated diffusion of glucose through hexose transporters (HXTs) in the plasma membrane (4, 5). S. cerevisiae contains 18 HXT family members of hexose transporters, Hxt1 to Hxt17 and Gal2, among which Hxt1 to Hxt7 function as major glucose transporters (6). Expression of the HXTs, all of which exhibit different levels of glucose affinity, is differentially regulated depending on extracellular glucose concentrations (7-9). Hxt1 (K m ϭ ϳ100 mM) and Hxt3 (K m ϭ ϳ30 to 60 mM) are classified as low-affinity glucose transporters. HXT1 is highly induced in the presence of high (Ͼ1%) concentrations of glucose, whereas HXT3 is induced by both low and high levels of glucose. When the glucose concentrations are lowered to around 0.1%, HXT2 and HXT4, encoding moderate-affinity glucose transporters (K m ϭ ϳ5 to 10 mM), are expressed (10). HXT6 and HXT7, encoding highly homologous glucose transporters with very high glucose affinity (K m ϭ ϳ1 mM), are expressed just before the depletion of glucose in the medium (11). Expression of HXT5, encoding a moderate-affinity transporter (K m ϭ ϳ10 mM), is not regulated by extracellular glucose ...

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Expression and activity of the Hxt7 high‐affinity hexose transporter of Saccharomyces cerevisiae

Cited by 53 publications

References 26 publications

Dynamic regulation of fatty acid pools for improved production of fatty alcohols in Saccharomyces cerevisiae

Dynamic regulation of fatty acid pools for improved production of fatty alcohols in Saccharomyces cerevisiae

Identification of Ftr1 and Zrt1 as iron and zinc micronutrient transceptors for activation of the PKA pathway in Saccharomyces cerevisiae

Improvement of Glucose Uptake Rate and Production of Target Chemicals by Overexpressing Hexose Transporters and Transcriptional Activator Gcr1 in Saccharomyces cerevisiae

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