Desenvolvimento de uma plataforma microbiana produtora de ácido lático utilizando estratégias de engenharia metabólica

Abstract

Lactic acid (LA) is an organic acid of great industrial interest, but its current production involves environmental impacts and economic bottlenecks that affect its applications, especially the production of polylactic acid (PLA). To overcome this, it is possible to use robust microorganisms tolerant to organic acids, such as the yeast Saccharomyces cerevisiae. In this context, this work aims to build a microbial strain capable of converting glucose (C6) and xylose (C5), sugars derived from agro-industrial residues, into L-lactic acid. Initially, tolerance tests were carried out with five yeast strains in a YPD medium with increasing concentrations of LA (20 to 80 g/L) at pH 3 and in lignocellulosic hydrolyzate of sugarcane bagasse solidified at pH 3 and 5. Then, using synthetic biology procedures, plasmids containing LA production enzyme expression cassettes (L-lactate dehydrogenase, LDH) from four different organisms were constructed: Bos taurus, Lactobacillus helveticus, Lactobacillus plantarum, and Lacticaseibacillus casei. In addition, vectors were also built to use the CRISPR-Cas9 system for the partial deletion of the alcoholic fermentation pathway and genomic integration of the chosen LDH. Then, the partial deletion of the ethanol pathway was performed and the different LDHs in the developed strain were tested by fermentation assays. Four yeast strains, S. cerevisiae PE-2, S. cerevisiae ATCC38555, Zygosaccharomyces bailii NCYC 1427, and Brettanomyces bruxellensis WLP 4639 were considered highly tolerant to lactic acid and only one, Z. bailli, showed good growth in lignocellulosic hydrolyzate, revealing its tolerance to the inhibitors present in this medium. As the microbial chassi chosen in this work is a xylosefermenting strain derived from PE-2 (BR-X5), this configures it as an ideal strain for this study. Obtaining the BR-X5Δpdc1 strain revealed that the deletion of the PDC1 gene, the main pyruvate decarboxylase expressed in yeast, was not sufficient for the carbon redirection of this pathway. The expression of the LDH vectors in this strain allowed the selection of L-LDH from B. taurus as the best enzyme for the production of LA. Finally, the assembly of genomic integration plasmids allows the development of strains with different copies of this bovine enzyme and the deletion of other genes of the ethanol pathway, such as ADH1. It was then possible to build a proof-of-principle strain producing 1.7 g/L of lactic acid, with a yield of 1.8% from 90 g/L of sugar (glucose and xylose) and productivity of 0.043 g/L. L.h. From this strain, it is possible to develop a microorganism that enables the efficient production of LA from a renewable resource and a more sustainable and efficient process.