Study and Engineering of Gram- Negative Bacteria as Hosts for Production of Recombinant Proteins and Biopolymers
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Gram-negative bacteria are a large group of prokaryotic organisms that thrive in diverse environments and differ in terms of structure and metabolism. They are capable of producing a variety of natural compounds and biopolymers and many of them, especially Escherichia coli, are frequently used as microbial hosts for industrial bioprocesses due to their rapid growth, relative genetic simplicity, and ability to express a broad range of heterologous proteins. The development of Gramnegative microbial cell factories requires the integration of multi-omics approaches, expanding genetic and genome engineering toolboxes, and utilization of high-throughput genome sequencing. The positively regulated XylS/Pm expression system is a genetic tool commonly used for inducible fine-tuned gene expression in many different bacterial species (Paper I). In this PhD project, the system was used in a plasmid-based reporter system constructed to analyze cell physiology in terms of availability of translational resources and protein misfolding and aggregation (Paper II). As heterologous protein production is associated with adaptive readjustments of cellular metabolism and may trigger heat shock-like and stringent stress responses regulated by δ32 and ppGpp, respectively, the δ32-dependent synthetic tandem promoter Pibpfxs and the ppGpp-dependent ribosomal protein promoter PrpsJ were combined with the XylS/Pm in the reporter plasmid and used to control expression of three distinct fluorescent proteins. The activity of the PrpsJ in E. coli corresponded to the upregulation of chromosomal ribosome subunit gene rplC and was increased in response to sublethal doses of translation-inhibiting antibiotic. The responsiveness of the Pibpfxs was confirmed by detecting its upregulated activity likely caused by the formation of abnormal, misfolded proteins upon the incorporation of an amino acid analogue, azetidine. All three reporter units showed different activity under non-induced and induced cultivation conditions and functioned in three tested Gram-negative species. One of the premises of the presented thesis was to move beyond the transcriptional regulation of gene expression and demonstrate that the strategy based on the regulation of translational efficiency of endogenous proteins can be effectively used in the development of microbial cell factories. For this purpose, translation initiation rates of five native E. coli proteins involved in periplasmic protein folding or degradation were up- or downregulated by replacing corresponding ribosome binding sites with their synthetic variants designed by the RBS Calculator (Paper III). The effect of the targeted genome engineering on the production of translocated recombinant proteins was determined using two pharmaceutically relevant model proteins, scFv, and GM-CSF, both expressed under control of the XylS/Pm. Most of the created mutant strains showed improved periplasmic production of at least one of the model proteins. Besides engineering efforts, the development of Gram-negative microbial cell factories should involve the identification and characterization of new biosynthetic gene clusters and enzymes displaying novel activities. Therefore, in parallel to the work on the reporter system and improving periplasmic production of recombinant proteins, three mannuronan C-5-epimerase genes recently identified in the publicly available genome sequence of Azotobacter chroococcum were cloned and heterologously expressed in E. coli under control of the XylS/Pm (Paper IV). The purified enzymes were used to determine their potential epimerase and/or lyase activities, substrate specificities, and the structure and composition of their alginate products. One of the enzymes showed structural homology to Azotobacter vinelandii AlgE2 epimerase and was capable of generating long consecutive sequences of guluronic acid residues. Two other enzymes similar to A. vinelandii AlgE7 bifunctional protein displayed both epimerase and lyase activity. In summary, the presented PhD project delivers the broad-host-range genetic tool for highthroughput screening and on-line analysis of bacterial stress responses related to heterologous gene expression, a verification of the new approach for strain engineering for improved periplasmic production of recombinant protein based on translation regulation of endogenous proteins, and, finally, the set of newly characterized alginate-modifying enzymes with biotechnological potential.