| Abstract [eng] |
Nowadays the lactic acid is one of the most commercially utilized chemical compounds as it finds application in the food, pharmaceutical, cosmeceutical, and chemistry industries. The lactic acid is a building block for one of the widely manufactured polymers, a polylactic acid (PLA) that can be used for developing various packaging materials, which are currently made from petrochemicals and are harmful to the environment. In order to improve today’s materials and to encourage industries to become greener, biobased strategies for the production of lactic acid and structurally similar compounds are being developed. One of the lactic acid-related compounds is glycolic acid that could be a potential platform chemical to replace the PLA with a polyglycolic acid (PGA) or both, lactic and glycolic acid, can be combined into one polymer to develop environment-friendly materials for various packaging solutions. Since microorganisms do not naturally produce glycolic acid under typical growth conditions, its biotechnological production requires metabolic engineering. To facilitate the improvement of genetically engineered microorganisms or to examine their cultivation in industrial conditions, high-throughput analytical methods and techniques are required. Transcription factor-based biosensors are one of such possible tools, which could help to investigate microorganisms in a laboratory inefficient way. They can be used in various applications including, for example, adaptive evolution or dynamic pathway control, to customize microorganism strains to industrial needs, and to perform real-time monitoring of chemical production in the high-throughput mode. There has been no transcription factor-based biosensor responding to the glycolic acid developed so far. Therefore, the aim of this research project was to characterize and use a glycolic acid-inducable gene expression system for developing a whole-cell biosensor. To this end, several plasmids with inducible system variants were constructed and investigated. To better understand and identify genetic elements and genes involved in the glycolic acid catabolism, bioinformatics analyses were applied. Using information obtained by bioinformatics searches and through the literature review, Glc operon was identified in E. coli MG1655 and P. putida KT2440 strains. Corresponding intergenic regions containing promoters and transcription regulator binding sequences, as well as genes encoding transcription regulators were determined and used for engineering the whole-cell biosensors. Subsequently, three different transcription factor-based biosensors were developed and evaluated. Whole-cell biosensor-based on pSS003 construct containing only intergenic regions of glcC/glcD revealed the highest fold induction amongst all three biosensors. Whole-cell biosensor-based on pSS004 construct showed the strongest relative normalized fluorescence response to the glycolic acid. Whereas, the addition of synthetic P13 promoter to increase the transcription regulator expression did not improve characteristics of whole-cell biosensor containing pSS004A construct. All three whole-cell biosensors were specific to glycolic acid and showed a minor response to D- lactic and 3-hydroxy propionic acids. However, they should be further improved to meat research and industrial application needs. |
