Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-06-14
2001-02-20
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S254110, C435S254210
Reexamination Certificate
active
06190914
ABSTRACT:
This application is the U.S. National Phase entry of PCT/NL97/00688 filed Dec. 12, 1997 and has benefit of priority from that filing date.
The present invention relates to the field of biotechnology, in particular to the field of culturing micro-organisms, in particular yeast.
Culturing of micro-organisms is a relatively old technique which is well established and well understood by persons skilled in this art. It usually involves bringing a micro-organism of interest into a culture medium wherein it can survive, grow and divide. The culture medium usually comprises all the necessary nutrients for the micro-organism to be able to do this.
Micro-organisms are cultured for many different purposes. These include the production of biomass, the production of antibiotics, the production of useful proteins expressed by micro-organisms (be it naturally or through genetic engineering), the production of micro-organisms useful themselves (for instance in brewing or baking bread, leavening of dough, etc.) Because of its relatively long history and its many uses the techniques for culturing micro-organisms have been very well optimised, so that further gains in yield or growth rate of the micro-organism to be cultured are difficult to achieve. However, because of the cost of culturing micro-organisms and the large amounts needed, such improvements (however small percentage-wise) remain very desirable.
One of the problems of culturing micro-organisms is that they often show preference for certain carbon sources, which carbon sources do not result in the best yields and/or growth rates of the micro-organism. Often the availability of such a preferred carbon source will lead to repression of the metabolism of other available carbon sources (which other carbon sources often do result in higher yields). For instance,
S. cerevisiae
, as many other micro-organisms, shows marked preferences for certain sources of carbon, nitrogen and energy. One such preference concerns the use of glucose above other fermentable and non-fermentable carbon compounds (see 1,2). This behaviour causes diauxic growth of this yeast when cultured on mixtures of carbon sources that include glucose. Yeast cells growing on glucose display high growth rates, presumably related to the ease with which intermediates can be derived from the catabolism of this sugar. Glucose has radical consequences for the enzyme complement and metabolic patterns in the yeast cell (FIG.
1
). During growth on this sugar, enzymes required for metabolism of other carbon sources are either absent or strongly reduced in amount as a result of active degradation of mRNAs or proteins (catabolite inactivation), repressed synthesis of mRNAs (catabolite repression), or both. Such enzymes include permeases and key enzymes involved in the utilization of various sugars, enzymes of gluconeogenesis and the glyoxylate cycle. In addition, synthesis of components of the mitochondrial respiratory chain is repressed, resulting in a low respiratory capacity. Glucose-repressed cells, or cells pulsed briefly with excess glucose produce ethanol by decarboxylation of cytosolic pyruvate and subsequent action of alcohol dehydrogenase. This series of reactions, known as the Crabtree-effect, regenerates cytosolic NAD
+
required for glycolysis. Although the basis of the Crabtree response is largely unknown (3), the occurrence of these reactions during large scale production of
S. cerevisiae
is undesirable because it reduces cell yield.
Suppression of ethanol production by yeast cells growing on glucose is currently achieved by limitation of the supply rate of the sugar. This procedure is only partially successful in that incomplete mixing can trigger a short-term Crabtree response. Additionally it suffers from the drawback that cells are forced to grow below their maximum capacity.
These or similar problems occur in many micro-organisms, particularly in eukaryotes, more in particular in yeasts. The present invention provides a general mechanism by which such problems may be solved in that it provides a method for producing a micro-organism of which a metabolic pathway has been modulated, for instance a micro-organism of which the sugar/glucose metabolism has been shifted from aerobic fermentation towards oxidation, comprising providing said micro-organism with the capability of inhibition or circumvention of the repression of the oxidative metabolism of glucose induced by the availability for the micro-organism of said carbon source. Surprisingly we have found that by simply interfering at a single (well-chosen) spot in the complex regulatory mechanisms of metabolic routes in (in particular) eukaryotic micro-organisms, it is possible to redirect said metabolic routes from one mechanism (fermentation) to another (oxidation). Because of the complexity of the regulatory systems surrounding metabolism it is generally believed that interference at a single point would be unlikely to be of any significance (because of all kinds of positive and negative homeostatic mechanisms which would restore the original situation) or would be deleterious if not disastrous (if it were capable of disrupting the feedback-mechanisms). We have found that a simple modification, in particular at the level of transcription activation does lead to the desired switch in metabolic mechanism, without disrupting the metabolism of the micro-organism.
In particular, the invention solves this problem when the preferred carbon source of the micro-organism is glucose, which is the case for many micro-organisms, in particular yeasts. One of the most important yeasts in industry is (of course)
Saccharomyces cerevisiae
. For that reason we have chosen this micro-organism as a model for explaining our invention. Because of its importance it is of course also a highly preferred embodiment of the present invention. Other well known industrial yeast strains such as Hansenula, Kluyveromyces and other strains are of course also within the scope of the present invention.
It will be understood that the result of the preference for glucose leads the metabolism down the aerobic fermentative pathway in many cases, as will be explained below. It will also be clear that for yield in biomass and/or production of useful proteins, etc. the oxidative/gluconeogenesis pathway is to be preferred. This pathway is often part of the metabolism that is repressed when glucose is available and which is used when other carbon sources are available. Thus an important aspect of the present invention is to provide a method according whereby the repressed metabolism is restored to a significant extent by activation of the pathways for metabolism for the non-preferred carbon sources.
It is preferred that said activation is achieved by providing the micro-organism with at least one transcriptional activator for at least one gene encoding an enzyme in said pathways.
A very suitable and preferred way of achieving said activation is one whereby the transcriptional activator is provided by introduction into the micro-organism of a recombinant nucleic acid encoding said activator. Said recombinant nucleic acid is preferably an expression vector. Such a vector may be an autonomously replicating vector, but it is preferred to use vectors that integrate in the host genome. However, it may also be achieved by other means, such as mutation (site directed).
There are two ways of having the transcriptional activator expressed. In one embodiment of the invention the transcriptional activator is constitutively expressed by said micro-organism. In an alternative embodiment the transcriptional activator can be expressed by the micro-organism upon induction, for instance by the presence of glucose. The person skilled in the art will be able to determine which one is to be used for different circumstances and desired end results. In many instances it will be preferred that the vector used to introduce the activator is capable of integration into the genome of the host. In other embodiments a self-replicating vector may be used.
A very efficient way of
Blom Jolanda
Grivell Leslie Alan
Teixeira De Mattos Maarten Joost
Morrison & Foerster / LLP
Universiteit Van Amsterdam
Yucel Remy
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