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Producción de etanol a partir de pentosas con levaduras que contienen una fosfofructoquinasa modificada

Resumen

Tipo:
Oferta Tecnológica
Referencia:
TOSI20160330002
Publicado:
08/04/2016
Caducidad:
08/04/2017
Resumen:
Un instituto de investigación esloveno ha desarrollado un gen que codifica la 6-fosfofructo-1-quinasa, la enzima reguladora clave en la glucólisis. La glucólisis es una de las vías metabólicas centrales en cualquier organismo, responsable de generar energía a partir de azúcares. La enzima modificada permite utilizar azúcar de pentosa fermentativa y aumentar la producción de etanol con la levadura Saccharomyces cerevisiae. El instituto busca socios del sector de producción de bioetanol interesados en continuar con el desarrollo y establecer acuerdos de investigación, cooperación técnica y licencia.

Details

Tittle:
Ethanol production from pentoses by yeasts possessing a modified phosphofructokinase
Summary:
A Slovenian research institute has developed a gene encoding a modified 6-phosphofructo-1-kinase, the key regulatory enzyme in glycolysis. The modified enzyme enables fermentative pentose sugar utilization and enhanced production of ethanol by the yeast Saccharomyces cerevisiae. The researchers are looking for partners in the field of bioethanol production interested in further development of the technology through research or technical cooperation agreements, and/or license agreements.
Description:
Glycolysis is one of the central metabolic pathways in all organisms, responsible for generating energy from sugars. In yeast cells, pentose sugars such as xylose and xylitol are first catabolised to products entering the so-called pentose phosphate pathway. After the transformation, fructose-6-phosphate (F6P) is formed and enters glycolysis. The enzyme phosphofructokinase (PFK) has an important regulatory function as it catalyzes the transformation of F6P to the next intermediate, fructose-1,6-bisphosphate (F-1,6-BP), using adenosine triphosphate (ATP) in the reaction. PFK is stimulated by a further intermediate, fructose-2,6-bisphosphate (F-2,6-BP), whereas citrate and ATP act as strong inhibitors.

Experiments have shown that the native PFK has a low activity and low maximal velocity when pentose sugars are used as substrates for the yeast. The technology solves this problem by introducing gene fragments encoding modified PFK enzymes, all of which show higher activity compared to the native enzyme. The activator, F-2,6-BP, causes a marked increase in the maximal velocity of the modified PFK whereas no such effect could be recorded with the native enzyme. This is particularly relevant in situations where levels of F6P are low, in which case the F6P cannot be metabolized with the native PFK, but it is successfully metabolized by the modified PFK. Consequently, the yeast cells were able to anaerobically grow on pentose sugars such as xylose and xylitol.

Enhanced flux through the glycolysis pathway leads to increased biosynthesis rates, which in turn leads to higher biomass yields, and increased production of homologous and heterologous proteins, primary metabolites (such as ethanol, acetate, lactate, organic acids, amino acids, polyols), and/or secondary metabolites (such as antibiotics, ergot alkaloids, statins, vitamins, immunomodulators, cytostatics, insecticides or herbicides). Adding the modified PFK gene to already engineered yeast strains with altered genes for various enzymes might further improve fermentative bioethanol production form pentose carbohydrates by Saccharomyces cerevisiae. In addition to S. cerevisiae, increased activity of the modified PFK was initially shown in the fungus Aspergillus niger, and later in human cancer cells, indicating a wide array of applications of the use of PFK gene fragments, from enhanced production of secondary metabolites to investigating pathways for cancer treatment.

Since the technology aims to reach its full potential in an industrial-scale bioethanol production, industrial partners are sought to license in the technology. The technology is in the field of microbial biotechnology, therefore technical cooperation is sought in order to facilitate continuous development as well as routine production. License agreements and / or agreements for technical cooperation will enable the researchers to maintain their focus on the research behind the technology whereas up-scaling to industrial level will be carried out in the industrial partner´s setting. Research cooperation agreements will, conversely, aid the researchers in further development in the laboratory setting.

The researchers are among the leading scientists in their respective departments, and regularly publish in high-impact scientific journals. They are experts in biochemical processes, particularly molecular recognition, signal transduction, and primary metabolism. They were the first to show that the PFK enzyme can be posttranslationally modified.
Advantages and Innovations:
In the past, attempts were carried out to increase metabolic flow in microorganisms through glycolysis via manipulation of the PFK enzyme but were unsuccessful. The invention solves this problem through modification of the gene encoding the enzyme (rather than the enzyme itself). PFK is stimulated by fructose-2,6-bisphosphate (F-2,6-BP), whereas citrate and ATP act as strong inhibitors. While the native PFK needs to be activated in order to produce citrate, and is feedback-inhibited by citrate, the modified PFK does not require activation and is not inhibited by citrate or ATP. This allows for enhanced metabolism rates. Active truncated genes encoding short PFK fragments retain the positive regulatory properties of the native protein and are activated in the presence of specific activators, but are resistant to feedback inhibition.

The main advantages of the invention are:
- enhancing the rate of cell biomass synthesis
- enhancing the excretion of extracellular enzymes
- increasing the productivity of primary and secondary metabolites
Consequently, biotechnological processing time is reduced, as are the production costs.
Stage of Development:
Available for demonstration
IPs:
Patent(s) applied for but not yet granted,Patents granted
CommeR Statunts Regarding IPR Status:
Protection has been granted in Brazil, Slovenia and United States, while few national patent applications originating from European granted patent are still pending.

Partner sought

Type and Role of Partner Sought:
The researchers are looking for industry partners in the field of bioethanol production, interested in licensing in the technology, with the focus on, and capacity for, industrial-scale production.

Partners from universities / research institutions, interested in further development of the technology through research or technical cooperation agreements, are also sought, the focus being on research rather than mass production.

Client

Type and Size of Client:
R&D Institution
Already Engaged in Trans-National Cooperation:
No
Languages Spoken:
English

Keywords

Technology Keywords:
06003002 Expresión genética, investigación proteómica
06006011 Fermentación
06002004 Ingeniería de proteínas
06002005 Ingeniería genética
06002008 Microbiología