PUB - Publikationen an der Universität Bielefeld

The identification of Corynebacterium glutamicum as a glutamate producer in the 1950’s was the start of its career as an amino acid producer. C. glutamicum has now been employed as cell factory for industrial amino acid production for over five decades and has a market size to reach $20 billion by 2020. As C. glutamicum was isolated for its natural ability to produce glutamate it makes it an excellent chassis for engineering it to produce its derivatives ornithine, proline, putrescine, citrulline, and arginine. These products are becoming increasingly important as they have a wide range of applications and the demand for these products is rising. The construction of a platform strain can reduce the time and resources required for strain development. Another approach to reduce the time and resources required for strain development is by developing new tools for metabolic engineering. These strategies were both explored to ease the process of strain construction. Proline, citrulline, and putrescine can be synthesized directly from ornithine, and three more reactions are required to synthesize arginine. Hence a strain engineered to produce ornithine can serve as a platform to produce the other four compounds. The first step was to establish proline and citrulline production by C. glutamicum as the rational engineering of C. glutamicum for production of these two amino acids had not previously been described. Proline is synthesized from glutamate via the proline biosynthetic pathway in most microorganisms, but with the help of the enzyme ornithine cyclodeaminase (Ocd) it can be synthesized from ornithine. Overexpression of putative ocd from C. glutamicum in an ornithine overproducing strain did not result in detectable proline production. Plasmid-based expression of ocd from Pseudomonas putida (ocdPp) on the other hand allowed accumulation of proline with a yield of 0.06 g proline / g glucose (g pro/g glc). Interestingly replacing the stop codon TGA of ocdPp with TAA resulted in a remarkable decrease in glutamate accumulation while the yield of proline increased to 0.25 g pro/g glc. The byproduct accumulation was further reduced by medium optimization, which also entailed a 25% higher proline yield. Lastly, the yield was boosted by overexpression of argBfbr encoding feedback-alleviated N-acetylglutamate kinase to 0.36 ± 0.01 g pro/g glc. A base strain suitable for the production of ornithine, proline, putrescine, citrulline, and arginine was constructed by deleting the repressor of the arginine pathway ArgR. Additionally the genes encoding enzymes of the arginine biosynthetic pathway were deleted, the deletion of argF allows ornithine to accumulate, and the deletion of argG allows citrulline to accumulate when argF is overexpressed. To add citrulline to the product spectrum of the platform strain we found it necessary to overexpress argBfbr along with argF. The strain accumulated citrulline with a yield of 0.38 ± 0.01 g citrulline / g glucose. The potential and versatility of the strain was demonstrated by producing citrulline from the alternative carbon sources starch, xylose, and glucosamine. After the product range of the base strain had been extended to include citrulline, metabolic engineering was performed to increase the ornithine yield of the strain. The initial assumption was that an increased ornithine yield could be translated into increased yields of the other four bioproducts. The ornithine yield was improved by 71% with a yield of 0.52 g ornithine / g glucose (g orn/g glc) compared to 0.31 g orn/g glc of the parent strain. This was achieved by feedback alleviation of N-acetylglutamate kinase, tuning of the promoter of gdh encoding glutamate dehydrogenase, lowering expression of pgi encoding phosphoglucoisomerase, along with the introduction of a second copy of the arginine biosynthetic operon argCJBfbrD into the chromosome. Strains capable of efficiently producing citrulline, proline, arginine or putrescine were derived from ornithine producing strains by plasmid-based overexpression of appropriate pathway modules with one to three genes. It was found that optimizing the base strain for ornithine production did not increase citrulline and arginine yields any further, indicating that the reaction converting ornithine into to citrulline is a bottleneck in citrulline and arginine production. Finally the popular CRISPR/dCas9 technology was adapted for the use of metabolic engineering in C. glutamicum. The system could be used to reversibly perturb gene expression. As proof of concept we targeted pgi in the lysine overproducing strain DM1729, where an almost complete repression of the transcription of the gene resulted in a 2-fold increase in the lysine titer. We also targeted the genes pck and pyk to increase glutamate production in wild-type C. glutamicum where we also observed nearly complete repression and increased glutamate titers. With this work it was shown how valuable the concept of using a platform strain for production of several industrially relevant bioproducts is. Moreover an efficient way to screen for new targets for metabolic engineering in C. glutamicum was demonstrated. From the initial cloning in Escherichia coli the C. glutamicum clones could be obtained in as little as four days by adapting the CRISPR/dCas9 system.