Estrategias up-stream para a otimização da produção de ramnolipídios a partir de fontes alternativas de carbono: um enfoque na linhagem
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Data
2021-12-03
Autores
Salazar-Bryam, Ana Maria [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Os ramnolipídeos são biosurfactantes que podem ser produzidos por microorganismos utilizando substratos alternativos, porém a sua produção ainda precisa ser otimizada pois os custos de produção são altos. O objetivo deste projeto foi abordar estratégias up-stream para melhorar a produção de ramnolipídios ao usar fontes de carbono hidrofílicas presentes em resíduos da agroindústria. Foi realizado um processo de otimização do meio de cultivo por meio de planejamento experimental, para encontrar as melhores condições a partir de um meio de cultivo existente, para produzir ramnolipídios a partir de glicerol utilizando a linhagem P. aeruginosa LBI 2A1, foi otimizada a produção e em escala de biorreator e foram obtidos 5,99 g/L de ramnolipídio, um rendimento Yp/s = 0,126, ao utilizar uma proporção C/N de 18. A partir do meio otimizado, foi proposto um processo de evolução adaptativa visando uma melhora no consumo de glicerol para aumentar indiretamente a produção de ramnolipídio; em comparação com a cepa Parental, P. aeruginosa LBI 2A1- T16A, após 65 gerações, aumentou a sua capacidade de produção de ramnolipídio 1,7 vezes (10,21 g/L) utilizando 5% de glicerol (condição inicial do processo de evolução). O experimento de evolução adaptativa permitiu melhorar o consumo de glicerol, direcionando maior fluxo de carbono à síntese da porção hidrofílica (dTPD-L-ramnose), e o crescimento da linhagem estudada, aumentando de forma indireta a produção de ramnolipídios. Como ultima estratégia foi proposta P. fluorescens DSM 50090 pSynPro_8o como um possível candidato para produção heteróloga de ramnolipídios utilizando glicerol e açucares presentes em hidrolisados de biomassa utilizada na agroindústria, respectivamente. Usando glicerol como fonte de carbono foi encontrado que o micro-organismo é um bom candidato para utilizar este substrato na produção de ramnolipídios, foram obtidos 1,65 g/L de ramnolipídio utilizando nitrato de sódio como fonte de nitrogênio e 10 g/L de glicerol. Finalmente, arabinose, glicose, manose e xilose foram avaliadas como possíveis fontes de carbono e P. fluorescens DSM 50090pSynPro_8o produziu 1,37 g/L utilizando glicose como fonte de carbono, e o micro- organismo foi capaz de consumir tanto hexoses quanto pentoses. Ao utilizar um meio simulando as proporções dos açucares de cultivo energético P. fluorescens DSM 50090pSynPro_8o produziu 0,65 g/L de ramnolipídios. Foi possível estabelecer a aptidão da Pseudomonas fluorescens para expressão heteróloga de ramnolipídios utilizando fontes de carbono não convencionais que favorecem o desenvolvimento da bioeconomia.
Rhamnolipids are biosurfactants that can be produced by microorganisms using alternative substrates, however their production still needs to be optimized because production costs are high. The objective of this project was to address up-stream strategies to improve rhamnolipid production by using hydrophilic carbon sources present in agroindustry waste. A medium optimization process was performed using experimental planning to find the best conditions from an existing culture medium to produce rhamnolipids from glycerol using the P. aeruginosa strain LBI 2A1, production was optimized and at bioreactor scale and 5.99 g/L of rhamnolipid, a yield Yp/s = 0.126, was obtained when using a C/N ratio of 18. From the optimized medium, an adaptive evolution process was proposed aiming at an improvement in glycerol consumption to indirectly increase rhamnolipid production; compared to the Parental strain, P. aeruginosa LBI 2A1-T16A, after 65 generations, increased its rhamnolipid production capacity 1.7-fold (10.21 g/L) using 5% glycerol (initial condition of the evolution process). The adaptive evolution experiment allowed to improve glycerol consumption, directing more carbon flux to the synthesis of the hydrophilic portion (dTPD-L-ramnose), and the growth of the strain studied, indirectly increasing the production of rhamnolipids. As a last strategy, P. fluorescens DSM 50090 pSynPro_8o was proposed as a possible candidate for heterologous production of rhamnolipids using glycerol and sugars present in hydrolysates of biomass used in agroindustry, respectively. Using glycerol as a carbon source it was found that the microorganism is a good candidate for using this substrate for rhamnolipid production, 1.65 g/L of rhamnolipid was obtained using sodium nitrate as nitrogen source and 10 g/L of glycerol. Finally, arabinose, glucose, mannose and xylose were evaluated as possible carbon sources and P. fluorescens DSM 50090pSynPro_8o produced 1.37 g/L using glucose as a carbon source, and the microorganism was able to consume both hexoses and pentoses. When using a medium simulating the proportions of the energy culture sugars P. fluorescens DSM 50090pSynPro_8o produced 0.65 g/L of rhamnolipids. It was possible to establish the suitability of Pseudomonas fluorescens for heterologous expression of rhamnolipids using unconventional carbon sources that favor the development of the bioeconomy.
Rhamnolipids are biosurfactants that can be produced by microorganisms using alternative substrates, however their production still needs to be optimized because production costs are high. The objective of this project was to address up-stream strategies to improve rhamnolipid production by using hydrophilic carbon sources present in agroindustry waste. A medium optimization process was performed using experimental planning to find the best conditions from an existing culture medium to produce rhamnolipids from glycerol using the P. aeruginosa strain LBI 2A1, production was optimized and at bioreactor scale and 5.99 g/L of rhamnolipid, a yield Yp/s = 0.126, was obtained when using a C/N ratio of 18. From the optimized medium, an adaptive evolution process was proposed aiming at an improvement in glycerol consumption to indirectly increase rhamnolipid production; compared to the Parental strain, P. aeruginosa LBI 2A1-T16A, after 65 generations, increased its rhamnolipid production capacity 1.7-fold (10.21 g/L) using 5% glycerol (initial condition of the evolution process). The adaptive evolution experiment allowed to improve glycerol consumption, directing more carbon flux to the synthesis of the hydrophilic portion (dTPD-L-ramnose), and the growth of the strain studied, indirectly increasing the production of rhamnolipids. As a last strategy, P. fluorescens DSM 50090 pSynPro_8o was proposed as a possible candidate for heterologous production of rhamnolipids using glycerol and sugars present in hydrolysates of biomass used in agroindustry, respectively. Using glycerol as a carbon source it was found that the microorganism is a good candidate for using this substrate for rhamnolipid production, 1.65 g/L of rhamnolipid was obtained using sodium nitrate as nitrogen source and 10 g/L of glycerol. Finally, arabinose, glucose, mannose and xylose were evaluated as possible carbon sources and P. fluorescens DSM 50090pSynPro_8o produced 1.37 g/L using glucose as a carbon source, and the microorganism was able to consume both hexoses and pentoses. When using a medium simulating the proportions of the energy culture sugars P. fluorescens DSM 50090pSynPro_8o produced 0.65 g/L of rhamnolipids. It was possible to establish the suitability of Pseudomonas fluorescens for heterologous expression of rhamnolipids using unconventional carbon sources that favor the development of the bioeconomy.
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Palavras-chave
Biossurfactantes, Planejamento experimental, Evolução adaptativa, Análise de fluxo metabólico, Produção heteróloga de ramnolipídios, Biosurfactants, Design of experiments, Adaptive evolution, Flux metabolic analysis, Heterologous production of rhamnolipids