Construção de linhagens recombinantes de Xanthomonas sp. para a produção de Goma Xantana

• Autor(a) principal: Oliveira, Davi Benedito
• Data de Publicação: 2021
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Repositório Institucional da UFSCAR
• Texto Completo: https://repositorio.ufscar.br/handle/ufscar/14400
• topic: Goma xantana; Xanthan gum; Xanthomonas; Vetores de expressão; Engenharia metabólica; Expression vectors; Metabolic engineering; CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR

Xanthan gum is a polysaccharide produced by bacteria from the genus Xanthomonas. This biopolymer has many industrial applications such as in food and cosmetics production, oil extraction process, among others. There is no national production of this compound, which leads us to import high volumes of xanthan gum annually. Currently, the development of production processes for this biopolymer has focused on the optimization of growth media and operational parameters, as well as on downstream steps to recover the product from the fermentation broth. However, a key aspect of this process is the development of optimized strains. In this context, a deterministic model in Python developed by Kundlascht (2017) identified the reactions catalyzed by UDP-Glucose pyrophosphorylase (UDPG-PP) and UDP-Glucose dehydrogenase (UDPG-deH) as being the bottleneck of the xanthan gum biosynthesis pathway. The model predicted the system behavior with the enzymes’ super-expression, indicating a significant rise in the gum monomers synthesis. From these results, the present work sought the hypothesis validation performing the cloning and the transformation of the genes galU (UDPG-PP) e Udg (UDPG-deH) in Xanthomonas campestris pv. campestris (XCC) using a broad host range plasmid as the backbone. Three plasmids were then produced containing each individual gene or both genes together, which were respectively named pLACR1_deH, pLACR1_PP and pLACR1_OP. To evaluate gum production by the new transformed strains with the constructed plasmids, cultures were performed in shake flasks with two different inducer concentrations. The results showed up to 21.7% more productivity (Pr) of xanthan gum for the best recombinant strain compared to the parental strain. This result validates the initial hypothesis about the pathway bottlenecks identified by in silico analysis.