In the animal feed industries, there is a global need for adding certain nutritional ingredients to augment deficits usually associated with plant-based materials. As a result, the industrial practices require direct addition of ingredients such as amino acids and vitamins. One of the key ingredients in this context is lysine. Alternately, the same goal can be achieved indirectly through in situ co-culturing of microorgan-isms. The focus of this thesis was genetic improvement of bacterial and /or fungal mutants, which could over-produce lysine. The accumulation of free lysine during microbial growth serves this end based on de-regulation of the lysine biosynthetic pathway. Microorganisms used in this thesis were nine species of lactobacilli and <i>Aspergillus ficuum</i>. Having in mind the highly complex nutritional requirements of lacto-bacilli, the assessment of possible lysine auxotrophy was performed. No lysine auxotrophs were found and the choice of <i>Lactobacillus plantarum</i> as the working species among nine others was based on its higher growth rate in minimal medium. Selection of mutants that overproduced lysine was carried out in the minimal medium supplemented with the following lysine analogs: S-aminoethyl-L-cysteine (AEC), DL-aspartic acid-Ò-hydroxamate (DL-ASP), Ò -fluoropyruvic-acid (FPA), L-lysine hydroxamate (LHX) and diaminopimelic acid (DAP). In L. plantarum, LHX was shown to be the most potent inhibitor; although, the bacterium demonstrated high resistance to all the analogs tested. The inhibition by LHX was obtained only after significant alteration of the minimal medium M3. Furthermore, the mutant # 34, resistant to 2 mM of LHX, secreted only 4.52 £gM of lysine in M3. To address the question of low lysine yield obtained by L. plantarum, thorough study of the regulation of aspartokinase (AK) was performed. It was found that AK exists as four isozymes, threonine sensitive, methionine sensitive and two lysine sensitive isozymes. Activity differed with respect to the growth stage of L. plantarum. Beside lysine, threonine and methionine have influenced the repression of AK isozymes, which suggested that effective lysine over-production could be obtained only if AK is simultaneously resistant to threonine and methionine analogs. In the case of <i>A. ficuum</i>, mutant #5-10 secreted 29.25 £gM of lysine in the minimal medium, which was approximately 30 % higher than that of the wild type. DL-ASP was found as the most potent inhibitor only after the conidia were soaked for 8 h in 0.03 % Tween 80. Ammonium phosphate as a nitrogen source enhanced lysine secretion in <i>A. ficuum</i> compared to five other nitrogen sources tested.

In the animal feed industries, there is a global need for adding certain nutritional ingredients to augment deficits usually associated with plant-based materials. As a result, the industrial practices require direct addition of ingredients such as amino acids and vitamins. One of the key ingredients in this context is lysine. Alternately, the same goal can be achieved indirectly through in situ co-culturing of microorgan-isms. The focus of this thesis was genetic improvement of bacterial and /or fungal mutants, which could over-produce lysine. The accumulation of free lysine during microbial growth serves this end based on de-regulation of the lysine biosynthetic pathway. Microorganisms used in this thesis were nine species of lactobacilli and <i>Aspergillus ficuum</i>. Having in mind the highly complex nutritional requirements of lacto-bacilli, the assessment of possible lysine auxotrophy was performed. No lysine auxotrophs were found and the choice of <i>Lactobacillus plantarum</i> as the working species among nine others was based on its higher growth rate in minimal medium. Selection of mutants that overproduced lysine was carried out in the minimal medium supplemented with the following lysine analogs: S-aminoethyl-L-cysteine (AEC), DL-aspartic acid-Ò-hydroxamate (DL-ASP), Ò -fluoropyruvic-acid (FPA), L-lysine hydroxamate (LHX) and diaminopimelic acid (DAP). In L. plantarum, LHX was shown to be the most potent inhibitor; although, the bacterium demonstrated high resistance to all the analogs tested. The inhibition by LHX was obtained only after significant alteration of the minimal medium M3. Furthermore, the mutant # 34, resistant to 2 mM of LHX, secreted only 4.52 £gM of lysine in M3. To address the question of low lysine yield obtained by L. plantarum, thorough study of the regulation of aspartokinase (AK) was performed. It was found that AK exists as four isozymes, threonine sensitive, methionine sensitive and two lysine sensitive isozymes. Activity differed with respect to the growth stage of L. plantarum. Beside lysine, threonine and methionine have influenced the repression of AK isozymes, which suggested that effective lysine over-production could be obtained only if AK is simultaneously resistant to threonine and methionine analogs. In the case of <i>A. ficuum</i>, mutant #5-10 secreted 29.25 £gM of lysine in the minimal medium, which was approximately 30 % higher than that of the wild type. DL-ASP was found as the most potent inhibitor only after the conidia were soaked for 8 h in 0.03 % Tween 80. Ammonium phosphate as a nitrogen source enhanced lysine secretion in <i>A. ficuum</i> compared to five other nitrogen sources tested.

Streptomyces clavuligerus is a gram-positive filamentous bacterium well known for its ability to produce an array of &amp / #61538 / -lactam compounds (secondary metabolites) including cephamycin C, clavulanic acid and other structurally related clavams. Of these, cephamycin C is a second generation cephalosporin antibiotic having great medical significance. Biosynthesis of the &amp / #946 / -lactam nucleus begins with the non-ribosomal condensation of L-&amp / #945 / -aminoadipic acid (&amp / #945 / -AAA), L-cysteine and L-valine to form the tripeptide &amp / #945 / -aminoadipiyl-cysteinyl-valine (ACV). In Streptomyces clavuligerus, &amp / #945 / -aminoadipic acid (&amp / #945 / -AAA) is a catabolic product of L-lysine produced from the lysine branch of the aspartate pathway and its biosynthesis represents a key secondary metabolic regulatory step in carbon flow to &amp / #946 / -lactam synthesis through this core pathway. The ask (aspartokinase)-asd (aspartate semialdehyde dehydrogenase) gene cluster which encodes for the first key enzymes of aspartate pathway has already been cloned from S. clavuligerus, characterized and heterologously expressed for the first time in our laboratory. Amplification of ask-asd cluster or ask gene alone in a multi-copy Streptomyces plasmid vector and determination of the effects of multiple copies on cephamycin C biosynthesis were the goals of the present study. For this purpose, three different strategies were employed. Of these, two strategies involving the use of vector pIJ702 did not work because of the instability of resulting recombinant plasmids. In the third and last strategy, we used another multicopy Streptomyces vector, pIJ486, which we showed in this study to be very stable for the same purpose. Meanwhile, an efficient protoplast transformation protocol was developed in our laboratory. Ask gene was cloned into this vector and S. clavuligerus protoplasts were efficiently transformed with the recombinant plasmid (pTB486) using the newly-developed protocol. After stable recombinants were obtained, the effects of the multiple copies of ask gene on cephamycin C biosynthesis were determined. There was a profound reduction in the rate and extent of growth of Ask overproducers, as experienced by testing two independent ask-multicopy recombinants. Although one such recombinant strain (designated S. clavuligerus TB 3585) had a 5.5 fold increased level of Ask activity as compared to the parental strain, it displayed only a 1.1 fold increase in specific production of cephamycin C.

No description available.

Aspartokinase is the first enzyme of the aspartate family amino acids biosynthetic pathway. Cephamycin C is a &amp / #946 / -lactam antibiotic produced as a secondary metabolite via the enzymatic reactions in the lysine branch of this pathway in Streptomyces clavuligerus. The aspartokinase activity of S. clavuligerus is under concerted feedback inhibition by two of the end product amino acids, lysine plus threonine. It is also known that carbon flow through the lysine branch of the aspartate pathway is rate limiting step in the formation of cephamycin C. Therefore, genetic alterations in the regulatory regions of the aspartokinase enzyme are expected to lead to an increased cephamycin C production. The aim of this study was to obtain S. clavuligerus mutants that possess aspartokinase enzyme insensitive to feedback inhibition by lysine and threonine, identification of the mutation(s) accounting for the resistance being the ultimate goal. For this aim, chemical mutagenesis was employed to increase random mutation rate and a population of lysine anti-metabolite resistant S. clavuligerus mutants that can grow in the presence of S-(2-aminoethyl)-L-cysteine was obtained. The mutants were screened for their aspartokinase insensitivity via enzyme assays and one mutant exhibiting the highest level of deregulation was assessed for its cephamycin C production. The results revealed a 2-fold increase in specific production of the antibiotic. As a member of &amp / #946 / -lactam class antibiotics, cephamycin C has an importance in medicine. Therefore, the mutant strain obtained might be a candidate for industrial production of the compound.

Tuberculosis (TB) is an infectious and fatal disease that ranks as the second leading killer worldwide. It is caused by Mycobacterium tuberculosis (Mtb) which is an obligate intracellular parasite that colonizes the alveolar macrophages of the immune system. The major health concern associated with TB is its co-infection with HIV and the development of strains with multi-drug resistance. The elimination of TB has been hindered due to the lack of understanding of the survival strategies used by this pathogen. Thus, research towards discovering new effective antibacterial drugs is necessary and a group of Mtb kinase enzymes were targeted in this study because these enzymes are crucial for metabolism, pathogenesis and, hence, the survival of Mtb. Kinases are a group of structurally distinct and diverse proteins that catalyze the transfer of the phosphate group from high energy donor molecules such as ATP (or GTP) to a substrate. The phosphorylation of proteins modifies the activity of specific proteins which is subsequently used to control complex cellular processes within Mtb. The starting point of this research targeted eight specific Mtb kinases namely; Nucleoside diphosphokinase, Homoserine kinase, Acetate kinase, Glycerol kinase, Thiamine monophosphate kinase, Ribokinase, Aspartokinase and Shikimate kinase. The aim of this project was to subclone the gene sequences for these eight recombinant Mtb kinases and express them in Escherichia coli, to purify the proteins and determine their activity. In the effort to find new lead compounds, the final stage of this study focused on the basic screening of the TB kinases against an extract prepared from Pelargonium sidoides, a medicinal plant, to identify any inhibitory effects. Although this traditional medicinal plant has been broadly researched and extensively used to treat TB, there is still a lack of understanding of this plant’s scientific curative effect. Various molecular and biochemical methods were used to achieve the aims of this project. The putative gene sequence was obtained from the annotated genome of H37Rv, deposited at NCBI as NC_000962.2. The genes encoding the kinases were successfully PCR-amplified from genomic DNA, cloned into an expression vector in-frame with a C- or N-terminal 6-histidine-tag and expressed in E. coli BL21 (DE3). The purification of the protein was complex, but various different methods and techniques were explored to obtain sufficient amounts of protein. The functional characterization of the kinases involved an HPLC enzyme assay that showed that the recombinant kinases were active. These enzymes were then screened against the potential inhibitory compounds in P. sidoides using enzyme assays to generate dose-response curves. This allowed an effective comparison not only of the Mtb kinases’ activity under normal conditions but also the kinases’ activity in the presence of a potential inhibitor. Overall, the inhibition of the enzymes required the presence of higher concentrations of the P. sidoides extract. However, the SK enzyme results presented a significantly higher inhibition and the lowest IC50 value, in comparison to the other kinases, which makes this kinase an attractive potential drug target against TB. In summation, cloning and purification of SK was successful, resulting in a concentration of 2030 μg/ml of purified enzyme and its activity analysis demonstrated enzyme functionality. This activity was reduced to zero in the presence of 1 x 102 mg/ml dilution of P. sidoides plant extract. This research conducted has extended the quality of information available in this field of study. These interesting results, proposing and identifying SK as a suitable potential target can be a starting point to significantly contribute and progress in this field of research, with the eventual goal of developing a drug to combat this fatal disease. / Life Sciences / M. Sc. (Life Sciences)

Tuberculosis (TB) is an infectious and fatal disease that ranks as the second leading killer worldwide. It is caused by Mycobacterium tuberculosis (Mtb) which is an obligate intracellular parasite that colonizes the alveolar macrophages of the immune system. The major health concern associated with TB is its co-infection with HIV and the development of strains with multi-drug resistance. The elimination of TB has been hindered due to the lack of understanding of the survival strategies used by this pathogen. Thus, research towards discovering new effective antibacterial drugs is necessary and a group of Mtb kinase enzymes were targeted in this study because these enzymes are crucial for metabolism, pathogenesis and, hence, the survival of Mtb. Kinases are a group of structurally distinct and diverse proteins that catalyze the transfer of the phosphate group from high energy donor molecules such as ATP (or GTP) to a substrate. The phosphorylation of proteins modifies the activity of specific proteins which is subsequently used to control complex cellular processes within Mtb. The starting point of this research targeted eight specific Mtb kinases namely; Nucleoside diphosphokinase, Homoserine kinase, Acetate kinase, Glycerol kinase, Thiamine monophosphate kinase, Ribokinase, Aspartokinase and Shikimate kinase. The aim of this project was to subclone the gene sequences for these eight recombinant Mtb kinases and express them in Escherichia coli, to purify the proteins and determine their activity. In the effort to find new lead compounds, the final stage of this study focused on the basic screening of the TB kinases against an extract prepared from Pelargonium sidoides, a medicinal plant, to identify any inhibitory effects. Although this traditional medicinal plant has been broadly researched and extensively used to treat TB, there is still a lack of understanding of this plant’s scientific curative effect. Various molecular and biochemical methods were used to achieve the aims of this project. The putative gene sequence was obtained from the annotated genome of H37Rv, deposited at NCBI as NC_000962.2. The genes encoding the kinases were successfully PCR-amplified from genomic DNA, cloned into an expression vector in-frame with a C- or N-terminal 6-histidine-tag and expressed in E. coli BL21 (DE3). The purification of the protein was complex, but various different methods and techniques were explored to obtain sufficient amounts of protein. The functional characterization of the kinases involved an HPLC enzyme assay that showed that the recombinant kinases were active. These enzymes were then screened against the potential inhibitory compounds in P. sidoides using enzyme assays to generate dose-response curves. This allowed an effective comparison not only of the Mtb kinases’ activity under normal conditions but also the kinases’ activity in the presence of a potential inhibitor. Overall, the inhibition of the enzymes required the presence of higher concentrations of the P. sidoides extract. However, the SK enzyme results presented a significantly higher inhibition and the lowest IC50 value, in comparison to the other kinases, which makes this kinase an attractive potential drug target against TB. In summation, cloning and purification of SK was successful, resulting in a concentration of 2030 μg/ml of purified enzyme and its activity analysis demonstrated enzyme functionality. This activity was reduced to zero in the presence of 1 x 102 mg/ml dilution of P. sidoides plant extract. This research conducted has extended the quality of information available in this field of study. These interesting results, proposing and identifying SK as a suitable potential target can be a starting point to significantly contribute and progress in this field of research, with the eventual goal of developing a drug to combat this fatal disease. / Life Sciences / M. Sc. (Life Sciences)