Metabolic engineering of C. glutamicum for amino acid production improvement

Abstract

A central goal in metabolic engineering is the design of more productive biological systems by genetically modifying metabolic pathways. In this thesis we report such an optimization in the bacterial strain Corynebacterium glutamicum that is employed for the fermentative production of various amino acids such as lysine. The main goal of the research presented here was the application of metabolic and genetic engineering tools in order to investigate the role of the pyruvate node in cellular physiology. This was achieved by integrating the tools of bioinformatics, recombinant DNA technology, enzymology and classical bioengineering in the context of control and genetically engineered strains of C. glutamicum. First, the main anaplerotic pathway responsible for replenishing oxaloacetate, namely pyruvate carboxylase was targeted. After fruitless attempts to establish an in vitro enzymatic activity for this enzyme, our efforts were directed towards its gene identification. This was achieved by designing PCR primers corresponding to homologous regions among pyruvate carboxylases from other organisms. Utilizing these primers, a PCR fragment was isolated corresponding to part of the gene of the C. glutamicum pyruvate carboxylase. The sequence of the complete gene was finally obtained by screening a C. glutamicum cosmid library. In order to investigate the physiological effect that this enzyme has on lysine production, recombinant strains and deletion mutants were generated. The presence of the gene of pyruvate carboxylase in a multicopy plasmid is not sufficient to yield a significant overexpresssion of this enzyme in C. glutamicum. Contrary to our expectations, overexpression of pyruvate carboxylase has a negative effect on lysine production but improves significantly the growth properties of C. glutamicum. A metabolic model was developed according to which pyruvate carboxylase overexpression increases the carbon flux that enters the TCA cycle, thus the higher growth. However due to the presence of a rate-limiting step in the lysine biosynthesis pathway this increased carbon flux does not translate into higher lysine production. The role of aspartokinase, the first step in lysine biosynthesis, was explored as such a potential bottleneck. Its overexpression proves to increase the amount of lysine produced, however it leads to a lower growth and finally a lower productivity. Since pyruvate carboxylase and aspartokinase have opposite effects on cell physiology, the combination of the overexpression of these two enzymes was finally studied. By this simultaneous overexpression, we achieved to create a C. glutamicum recombinant strain with similar growth as that of the control but higher lysine production and productivity. In the context of exploring the physiological role of pyruvate carboxylase, a biotinylated enzyme, two other enzyme that utilize biotin were also investigated namely acetyl-CoA-carboxylase and biotin ligase. The first enzyme was purified to completion and its N-terminal as well internal amino acid sequences were obtained. A cosmid from the C. glutamicum cosmid library was identified that most likely contains the gene of the latter enzyme. In summary, in the present work we have achieved to prove unequivocally the presence of pyruvate carboxylase in C. glutamicum. We have also achieved to characterize the second biotinylated enzyme in this organism, namely acetyl-CoAcarboxylase. The physiological effect of both pyruvate carboxylase and aspartokinase was established and a metabolic model was developed based on these experimental results. This model finally led us to the construction of a new recombinant strain with improved lysine productivity. As such, this work stands as one of the few examples of a primary metabolite production improvement using metabolic engineering techniques.