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Genes and Enzymes Involved in Pyruvate Metabolism in Coryneform Bacteria

#A-794.2


Investigation of Genes and Enzymes Involved in Pyruvate Metabolism in Coryneform Bacteria. Approaches to the Improvement of Amino Acid Strain Producers by Recombinant DNA Technology

Tech Area / Field

  • BIO-IND/Industrial Biotechnology/Biotechnology
  • BIO-CHM/Biochemistry/Biotechnology
  • BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
  • BIO-MIB/Microbiology/Biotechnology

Status
3 Approved without Funding

Registration date
22.03.2004

Leading Institute
Scientific and Production Center "Armbiotechnology" NAS RA, Armenia, Yerevan

Collaborators

  • Massachusetts Institute of Technology (MIT) / Department of Biology, USA, MA, Cambridge

Project summary

Goal of the Project: investigation of the pathways of enzyme biosynthesis and regulations of genes involved in pyruvate metabolism.

Coryneform bacteria are soil microorganisms. In natural conditions they have ability to produce certain quantity of amino acids from simple carbon and nitrogen sources, strictly limiting their reproduction. For construction of strains-producer of amino acids one needs a detailed investigation of pathways of enzyme biosynthesis and the ways of regulations of genes involved in biosynthesis of amino acids. Nowadays significant successes are achieved in the investigation of the molecular mechanisms of gene regulation. It is established that biosynthesis of amino acids is controlled by a complex system of regulation, with details that are different in various species. The common thing is that the regulation is fulfilled on the level of both genes and their products (enzymes of biosynthesis). Nowadays genes of biosynthesis of amino acids are isolated and investigated, vectors for cloning of genes are created and methods for transformation of corynebacteria are elaborated. Current achievements allow to use recombinant DNA technology for improvement of strain – producers. Therefore understanding of mechanisms of gene expression and their regulation as well as investigation of physiology and biochemistry of the strains constructed is very urgent today.

Amino acid super producers, which are widely used in biotechnology for industrial production of amino acids are obtained on the basis of coryneform bacteria by using genetical selection methods. However, amino acids overproduction depends not only on activity of enzymes, directly participating in the synthesis of amino acids, but also on metabolism of their precursors.

It is known that the majority of amino acids are synthesized either from pyruvate or from intermediate products of the Krebs cycle. Introduction of mutations in the cells, which increase pool of amino acids precursors, can significantly increase amino acid productivity of the strain. The four basic enzymes: phosphoenol pyruvate carboxylase (FEP-carboxylase), pyruvate carboxylase (P-carboxylase), pyruvate kinase (P-kinase) and pyruvate dehydrogenase (P-dehydrogenase) regulate pool of pyruvate and consequently quantity of the synthesized amino acids of pyruvate family. Change of the ratio of these enzymes by insertion of appropriate mutations in the genome can extensively change biosynthetical activity of the stains. Besides, the mutants with changed flux of pyruvate can be used for cloning of key genes of biosynthesis of pyruvate family amino acids.

We have obtained super active lysine and valine production strains. The stains-producers of lysine are implemented in lysine producing plants of CIS countries. During this process we have created a collection of strains with the changed ratio of enzymes involved in metabolism of pyruvate. The above mentioned changes were caused by the introduction of fluorpyruvate - FP sensitive mutation into the strain. According to our results sensitivity to FP influences on the ability of the stain to produce not only lysine, but also valine, alanine, glutamic acid and etc.

We have also constructed the shuttle vector, which is able to replicate in E. coli, as well as in different types of corynebacteria. Currently using this vector we have cloned genes of lysine biosynthesis and genes of pyruvate metabolism.

Objectives of this research are:

1. Investigation of FP sensitive mutants in Brevibacterium lactofermentum, classification of mutants according to their sensitivity towards different doses of FP and their biosynthetical activity.

2. Investigation of activity of PEP-carboxylase, P-dehydrogenase, P-kinase, P-carboxylase in mutants with different biosynthetical activity, identification of the mechanisms on dependence of mutants productivity from enzyme activities.

3. Construction of the high expressive vector specific for Corynebacteria.

4. Identification and investigation of mutant genes of pyruvate metabolism affected by the mutation of FP-sensitivity.


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