scholarly journals Alcohol Acetyltransferase Eat1 Is Located in Yeast Mitochondria

2018 ◽  
Vol 84 (19) ◽  
Author(s):  
Aleksander J. Kruis ◽  
Astrid E. Mars ◽  
Servé W. M. Kengen ◽  
Jan Willem Borst ◽  
John van der Oost ◽  
...  
Keyword(s):  
Ethyl Acetate ◽  
Metabolic Pathways ◽  
Rational Design ◽  
Kluyveromyces Lactis ◽  
Efficient Production ◽  
Yeast Mitochondria ◽  
Acetate Production ◽  
Content Type ◽  
Alcohol Acetyltransferase ◽  
Yeast Strains

ABSTRACT Eat1 is a recently discovered alcohol acetyltransferase responsible for bulk ethyl acetate production in yeasts such as Wickerhamomyces anomalus and Kluyveromyces lactis. These yeasts have the potential to become efficient bio-based ethyl acetate producers. However, some fundamental features of Eat1 are still not understood, which hampers the rational engineering of efficient production strains. The cellular location of Eat1 in yeast is one of these features. To reveal its location, Eat1 was fused with yeast-enhanced green fluorescent protein (yEGFP) to allow intracellular tracking. Despite the current assumption that bulk ethyl acetate production occurs in the yeast cytosol, most of Eat1 localized to the mitochondria of Kluyveromyces lactis CBS 2359 Δku80. We then compared five bulk ethyl acetate-producing yeasts in iron-limited chemostats with glucose as the carbon source. All yeasts produced ethyl acetate under these conditions. This strongly suggests that the mechanism and location of bulk ethyl acetate synthesis are similar in these yeast strains. Furthermore, an in silico analysis showed that Eat1 proteins from various yeasts were mostly predicted as mitochondrial. Altogether, it is concluded that Eat1-catalyzed ethyl acetate production occurs in yeast mitochondria. This study has added new insights into bulk ethyl acetate synthesis in yeast, which is relevant for developing efficient production strains. IMPORTANCE Ethyl acetate is a common bulk chemical that is currently produced from petrochemical sources. Several Eat1-containing yeast strains naturally produce large amounts of ethyl acetate and are potential cell factories for the production of bio-based ethyl acetate. Rational design of the underlying metabolic pathways may result in improved production strains, but it requires fundamental knowledge on the function of Eat1. A key feature is the location of Eat1 in the yeast cell. The precursors for ethyl acetate synthesis can be produced in multiple cellular compartments through different metabolic pathways. The location of Eat1 determines the relevance of each pathway, which will provide future targets for the metabolic engineering of bulk ethyl acetate production in yeast.

scholarly journals Homolactic Acid Fermentation by the Genetically Engineered Thermophilic Homoacetogen Moorella thermoacetica ATCC 39073

2017 ◽  
Vol 83 (8) ◽  
Author(s):  
Yuki Iwasaki ◽  
Akihisa Kita ◽  
Koichiro Yoshida ◽  
Takahisa Tajima ◽  
Shinichi Yano ◽  
...  
Keyword(s):  
Wild Type Strain ◽  
Genetically Engineered ◽  
Efficient Production ◽  
Acetate Production ◽  
Acid Fermentation ◽  
Content Type ◽  
Moorella Thermoacetica ◽  
Double Deletion ◽  
Dehydrogenase Gene ◽  
Acetate Formation

ABSTRACT For the efficient production of target metabolites from carbohydrates, syngas, or H2-CO2 by genetically engineered Moorella thermoacetica, the control of acetate production (a main metabolite of M. thermoacetica) is desired. Although propanediol utilization protein (PduL) was predicted to be a phosphotransacetylase (PTA) involved in acetate production in M. thermoacetica, this has not been confirmed. Our findings described herein directly demonstrate that two putative PduL proteins, encoded by Moth_0864 (pduL1) and Moth_1181 (pduL2), are involved in acetate formation as PTAs. To disrupt these genes, we replaced each gene with a lactate dehydrogenase gene from Thermoanaerobacter pseudethanolicus ATCC 33223 (T-ldh). The acetate production from fructose as the sole carbon source by the pduL1 deletion mutant was not deficient, whereas the disruption of pduL2 significantly decreased the acetate yield to approximately one-third that of the wild-type strain. The double-deletion (both pduL genes) mutant did not produce acetate but produced only lactate as the end product from fructose. These results suggest that both pduL genes are associated with acetate formation via acetyl-coenzyme A (acetyl-CoA) and that their disruption enables a shift in the homoacetic pathway to the genetically synthesized homolactic pathway via pyruvate. IMPORTANCE This is the first report, to our knowledge, on the experimental identification of PTA genes in M. thermoacetica and the shift of the native homoacetic pathway to the genetically synthesized homolactic pathway by their disruption on a sugar platform.

Ethyl acetate production by the elusive alcohol acetyltransferase from yeast

2017 ◽  
Vol 41 ◽  
pp. 92-101 ◽  
Author(s):  
Aleksander J. Kruis ◽  
Mark Levisson ◽  
Astrid E. Mars ◽  
Max van der Ploeg ◽  
Fernando Garcés Daza ◽  
...  
Keyword(s):  
Ethyl Acetate ◽  
Acetate Production ◽  
Alcohol Acetyltransferase

Gradual enhancement of ethyl acetate production through promoter engineering in chinese liquor yeast strains

2018 ◽  
Vol 34 (2) ◽  
pp. 328-336 ◽  
Author(s):  
Jian Dong ◽  
Kun-Qiang Hong ◽  
Ai-Li Hao ◽  
Cui-Ying Zhang ◽  
Xiao-Meng Fu ◽  
...  
Keyword(s):  
Ethyl Acetate ◽  
Acetate Production ◽  
Chinese Liquor ◽  
Promoter Engineering ◽  
Yeast Strains

scholarly journals Improving Ethyl Acetate Production in Baijiu Manufacture by Wickerhamomyces anomalus and Saccharomyces cerevisiae Mixed Culture Fermentations

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Guangsen Fan ◽  
Chao Teng ◽  
Dai Xu ◽  
Zhilei Fu ◽  
Pengxiao Liu ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ethyl Acetate ◽  
Mixed Culture ◽  
Nutrient Competition ◽  
Acetate Production ◽  
Mixed Fermentation ◽  
Wickerhamomyces Anomalus ◽  
Flavor Perception ◽  
Positive Effect

Ethyl acetate content has strong influence on the style and quality of Baijiu. Therefore, this study investigated the effect of Saccharomyces cerevisiae Y3401 on the production of ethyl acetate by Wickerhamomyces anomalus Y3604. Analysis of cell growth showed that Y3401 influences Y3604 by nutrient competition and inhibition by metabolites, while the effect of Y3604 on Y3401 was mainly competition for nutrients. Mixed fermentation with two yeasts was found to produce more ethyl acetate than a single fermentation. The highest yield of ethyl acetate was 2.99 g/L when the inoculation ratio of Y3401:Y3604 was 1:2. Synergistic fermentation of both yeasts improved ethyl acetate production and increased the content of other flavor compounds in liquid and simulated solid-state fermentation for Baijiu. Saccharomyces cerevisiae had a positive effect on ethyl acetate production in mixed culture and provides opportunities to alter the aroma and flavor perception of Baijiu.

scholarly journals Site-Directed Mutagenesis of the Heterotrimeric Killer Toxin Zymocin Identifies Residues Required for Early Steps in Toxin Action

2014 ◽  
Vol 80 (20) ◽  
pp. 6549-6559 ◽  
Author(s):  
Sabrina Wemhoff ◽  
Roland Klassen ◽  
Friedhelm Meinhardt
Keyword(s):  
Kluyveromyces Lactis ◽  
Killer Toxin ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Target Cells ◽  
Directed Mutagenesis ◽  
Content Type ◽  
Protein Toxin ◽  
Assisted Delivery

ABSTRACTZymocin is aKluyveromyces lactisprotein toxin composed of αβγ subunits encoded by the cytoplasmic virus-like element k1 and functions by αβ-assisted delivery of the anticodon nuclease (ACNase) γ into target cells. The toxin binds to cells' chitin and exhibits chitinase activityin vitrothat might be important during γ import.Saccharomyces cerevisiaestrains carrying k1-derived hybrid elements deficient in either αβ (k1ORF2) or γ (k1ORF4) were generated. Loss of either gene abrogates toxicity, and unexpectedly, Orf2 secretion depends on Orf4 cosecretion. Functional zymocin assembly can be restored by nuclear expression of k1ORF2 or k1ORF4, providing an opportunity to conduct site-directed mutagenesis of holozymocin. Complementation required active site residues of α's chitinase domain and the sole cysteine residue of β (Cys250). Since βγ are reportedly disulfide linked, the requirement for the conserved γ C231 was probed. Toxicity of intracellularly expressed γ C231A indicated no major defect in ACNase activity, while complementation of k1ΔORF4 by γ C231A was lost, consistent with a role of β C250 and γ C231 in zymocin assembly. To test the capability of αβ to carry alternative cargos, the heterologous ACNase fromPichia acaciae(P. acaciaeOrf2 [PaOrf2]) was expressed, along with its immunity gene, in k1ΔORF4. While efficient secretion of PaOrf2 was detected, suppression of the k1ΔORF4-derived k1Orf2 secretion defect was not observed. Thus, the dependency of k1Orf2 on k1Orf4 cosecretion needs to be overcome prior to studying αβ's capability to deliver other cargo proteins into target cells.

scholarly journals Light-driven fine chemical production in yeast biohybrids

Science ◽  
2018 ◽  
Vol 362 (6416) ◽  
pp. 813-816 ◽  
Author(s):  
Junling Guo ◽  
Miguel Suástegui ◽  
Kelsey K. Sakimoto ◽  
Vanessa M. Moody ◽  
Gao Xiao ◽  
...  
Keyword(s):  
Energy Efficient ◽  
Rational Design ◽  
Light Harvesting ◽  
Shikimic Acid ◽  
Genetically Engineered ◽  
Efficient Production ◽  
Chemical Production ◽  
Photogenerated Electrons ◽  
Common Precursor ◽  
Hybrid Platform

Inorganic-biological hybrid systems have potential to be sustainable, efficient, and versatile chemical synthesis platforms by integrating the light-harvesting properties of semiconductors with the synthetic potential of biological cells. We have developed a modular bioinorganic hybrid platform that consists of highly efficient light-harvesting indium phosphide nanoparticles and genetically engineered Saccharomyces cerevisiae, a workhorse microorganism in biomanufacturing. The yeast harvests photogenerated electrons from the illuminated nanoparticles and uses them for the cytosolic regeneration of redox cofactors. This process enables the decoupling of biosynthesis and cofactor regeneration, facilitating a carbon- and energy-efficient production of the metabolite shikimic acid, a common precursor for several drugs and fine chemicals. Our work provides a platform for the rational design of biohybrids for efficient biomanufacturing processes with higher complexity and functionality.

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