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Analysis and engineering of biomolecules and microorganisms: from genome-scale study of pathogens to programming of DNA and cells
Citation Link: https://doi.org/10.15480/882.1668
Publikationstyp
Doctoral Thesis
Date Issued
2018
Sprache
English
Author(s)
Advisor
Referee
Title Granting Institution
Technische Universität Hamburg-Harburg
Place of Title Granting Institution
Hamburg
Examination Date
2018-05-25
Institut
TORE-DOI
This thesis is consisted of three major but different parts with the general aims of systems level evaluation and engineering of biomolecules and biological systems. In the first part of this thesis, comparative genomic studies of mutans streptococci strains, which are involved in the development of dental caries, were performed for better understanding their pathogenicity at the level of systems biology. A mosaic-like structure of genome arrangement was revealed by genome alignment analysis. Genes related to pathogenicity were found to have high variations among the strains, whereas genes for oxidative stress resistance are well conserved, indicating the importance of this trait in the dental biofilm community. Genome-scale metabolic network analysis revealed significant differences in 42 pathways. A striking dissimilarity is the unique presence of two lactate oxidases in S. sobrinus DSM 20742, probably indicating an unusual capability of this strain in producing H2O2 and expanding its ecological niche. In addition, lactate oxidases may form a unique energy-producing pathway with other enzymes in S. sobrinus DSM 20742 that can remedy its deficiency in citrate utilization pathway. An "open" pan-genome was inferred by pan-genome analysis using 67 S. mutans genomes currently available including the strains sequenced in this study. An online regulation database for S. mutans, named StrepReg, was constructed by integrating a transcription factor-based gene regulatory network, which was derived from time-series transcriptome analysis, with STRING protein-protein interaction information and KEGG pathway information (http://biosystem.bt1.tu-harburg.de:1555/homes/).
Although systems biology is a powerful tool in understanding the system level behaviors of biological systems, the establishment of predictive, multiscale models in systems biology is still a challenge due to the complexity of biological systems. For the same reason, mathematical models often fail in applications under physiological conditions, such as for identification of targets in metabolic engineering for the development of highly production strains. In the second part of this thesis, a novel multiple input-output (I/O) system was therefore proposed and verified, which allows the identification of limiting bioreactions or key enzymes in metabolic pathways and even the optimization of biomolecules in vivo. The basic idea is to design a multiple I/O system which can introduce various genetic manipulations (perturbations) into the cells and record the specific intracellular signal changes correspondingly. This was achieved by engineering the interactions of phage with E. coli cells. Specifically, a multiple I/O system was implemented using M13 phage derivatives which can introduce various perturbations into E. coli cells after infection, such as up- or down-regulation of specific gene expressions. Using a rationally designed biological circuit, the intracellular signal changes after introduction of the perturbations by the phage infection were linked to the phage reproduction process. This means, signal changes caused by specific perturbations are linked to the specific populations of phages introducing the corresponding perturbations. In this way, the various signals are ‘recorded’ in forms of corresponding populations of phage derivatives. The usefulness of the multiple I/O system was demonstrated with three applications, i.e. identification of beneficial genetic manipulations, parallel evaluation of various designs of enzymes, and parallel screening of key enzymes for L-lysine biosynthesis in E. coli. Various gene operations related or not related to L-lysine biosynthesis in E. coli were used as inputs and the intracellular lysine concentration changes were used to trigger output signals. Correct predictions of beneficial genetic manipulations for enhanced lysine production in E. coli were achieved. New and effective variants of a key enzyme aspartate kinase III (AK-III), which is strictly inhibited by L-lysine, were obtained and evaluated in parallel. Importantly, the I/O system shows a ultra-sensitivity in capturing signal changes caused by the certain perturbations introduced. The approach developed in this work opens up new possibilities in systems metabolic engineering and synthetic biology of industrial microorganisms for practical applications.
In the third part of this thesis, a novel self-error-detecting, three-base block encoding scheme (SED3B), which takes full advantage of the inherent redundancy feature of DNA synthesis for error correction, was proposed for reliable information encoding in DNA of living cells. In addition to the high error tolerance, SED3B encoded sequences were shown to be orthogonal to natural DNA sequences, indicating for the first time a low biological relevance of the encoded sequences. Features such as effective error tolerance and low biological relevance make SED3B an appealing solution for orthogonal information encoding in living cells with low or no affections to their biological functions, e.g. as a comment language in programming cells in vivo and for biological barcode encoding. Based on error-prone PCR experiments it was estimated that more than 12,000 years of continuous replication would be required to make the SED3B encoded information in E. coli cells become unrecoverable. To facilitate the usage of SED3B as a comment and barcode encoding system in synthetic biology, an online encoding-decoding system was implemented and released at http://biosystem.bt1.tu-harburg.de/sed3b. In principle, SED3B is also applicable for in vitro large data storage in synthesized DNA. Although further investigation is required, preliminary analysis shows that SED3B has a great potential for increasing the storage density to over several exabytes (EBs) per gram DNA which is theoretically much higher than that of methods reported in literature so far.
Although systems biology is a powerful tool in understanding the system level behaviors of biological systems, the establishment of predictive, multiscale models in systems biology is still a challenge due to the complexity of biological systems. For the same reason, mathematical models often fail in applications under physiological conditions, such as for identification of targets in metabolic engineering for the development of highly production strains. In the second part of this thesis, a novel multiple input-output (I/O) system was therefore proposed and verified, which allows the identification of limiting bioreactions or key enzymes in metabolic pathways and even the optimization of biomolecules in vivo. The basic idea is to design a multiple I/O system which can introduce various genetic manipulations (perturbations) into the cells and record the specific intracellular signal changes correspondingly. This was achieved by engineering the interactions of phage with E. coli cells. Specifically, a multiple I/O system was implemented using M13 phage derivatives which can introduce various perturbations into E. coli cells after infection, such as up- or down-regulation of specific gene expressions. Using a rationally designed biological circuit, the intracellular signal changes after introduction of the perturbations by the phage infection were linked to the phage reproduction process. This means, signal changes caused by specific perturbations are linked to the specific populations of phages introducing the corresponding perturbations. In this way, the various signals are ‘recorded’ in forms of corresponding populations of phage derivatives. The usefulness of the multiple I/O system was demonstrated with three applications, i.e. identification of beneficial genetic manipulations, parallel evaluation of various designs of enzymes, and parallel screening of key enzymes for L-lysine biosynthesis in E. coli. Various gene operations related or not related to L-lysine biosynthesis in E. coli were used as inputs and the intracellular lysine concentration changes were used to trigger output signals. Correct predictions of beneficial genetic manipulations for enhanced lysine production in E. coli were achieved. New and effective variants of a key enzyme aspartate kinase III (AK-III), which is strictly inhibited by L-lysine, were obtained and evaluated in parallel. Importantly, the I/O system shows a ultra-sensitivity in capturing signal changes caused by the certain perturbations introduced. The approach developed in this work opens up new possibilities in systems metabolic engineering and synthetic biology of industrial microorganisms for practical applications.
In the third part of this thesis, a novel self-error-detecting, three-base block encoding scheme (SED3B), which takes full advantage of the inherent redundancy feature of DNA synthesis for error correction, was proposed for reliable information encoding in DNA of living cells. In addition to the high error tolerance, SED3B encoded sequences were shown to be orthogonal to natural DNA sequences, indicating for the first time a low biological relevance of the encoded sequences. Features such as effective error tolerance and low biological relevance make SED3B an appealing solution for orthogonal information encoding in living cells with low or no affections to their biological functions, e.g. as a comment language in programming cells in vivo and for biological barcode encoding. Based on error-prone PCR experiments it was estimated that more than 12,000 years of continuous replication would be required to make the SED3B encoded information in E. coli cells become unrecoverable. To facilitate the usage of SED3B as a comment and barcode encoding system in synthetic biology, an online encoding-decoding system was implemented and released at http://biosystem.bt1.tu-harburg.de/sed3b. In principle, SED3B is also applicable for in vitro large data storage in synthesized DNA. Although further investigation is required, preliminary analysis shows that SED3B has a great potential for increasing the storage density to over several exabytes (EBs) per gram DNA which is theoretically much higher than that of methods reported in literature so far.
Subjects
biotechnology
systems biology
synthetic biology
DDC Class
570: Biowissenschaften, Biologie
610: Medizin
More Funding Information
BMBF "Medical systems biology - MedSys" programme
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