Lactic acid is the monomer unit of the bioplastic poly-lactic acid (PLA). One candidate organism for lactic acid production is Pichia pastoris, a yeast widely used for heterologous protein production. Nevertheless, this yeast has a poor fermentative capability that can be modulated by controlling oxygen levels. In a previous study, lactate dehydrogenase (LDH) activity was introduced into P. pastoris, enabling this yeast to produce lactic acid. The present study aimed to increase the flow of pyruvate towards the production of lactic acid in P. pastoris. To this end, a strain designated GLp was constructed by inserting the bovine lactic acid dehydrogenase gene (LDHb) concomitantly with the interruption of the gene encoding pyruvate decarboxylase (PDC). Aerobic fermentation, followed by micro-aerophilic culture two-phase fermentations, showed that the GLp strain achieved a lactic acid yield of 0.65 g/g. The distribution of fermentation products demonstrated that the acetate titer was reduced by 20% in the GLp strain with a concomitant increase in arabitol production: arabitol increased from 0.025 g/g to 0.174 g/g when compared to the GS115 strain. Taken together, the results show a significant potential for P. pastoris in producing lactic acid. Moreover, for the first time, physiological data regarding co-product formation have indicated the redox balance limitations of this yeast.

BackgroundCrude glycerol is the main byproduct of the biodiesel industry. Although it can have different applications, its purification is costly. Therefore, in this study a biotechnological route has been proposed for further utilization of crude glycerol in the fermentative production of lactic acid. This acid is largely utilized in food, pharmaceutical, textile, and chemical industries, making it the hydroxycarboxylic acid with the highest market potential worldwide. Currently, industrial production of lactic acid is done mainly using sugar as the substrate. Thus here, for the first time, Pichia pastoris has been engineered for heterologous l-lactic acid production using glycerol as a single carbon source. For that, the Bos taurus lactate dehydrogenase gene was introduced into P. pastoris. Moreover, a heterologous and a novel homologous lactate transporter have been evaluated for l-lactic acid production.ResultsBatch fermentation of the P. pastoris X-33 strain producing LDHb allowed for lactic acid production in this yeast. Although P. pastoris is known for its respiratory metabolism, batch fermentations were performed with different oxygenation levels, indicating that lower oxygen availability increased lactic acid production by 20 %, pushing the yeast towards a fermentative metabolism. Furthermore, a newly putative lactate transporter from P. pastoris named PAS has been identified by search similarity with the lactate transporter from Saccharomyces cerevisiae Jen1p. Both heterologous and homologous transporters, Jen1p and PAS, were evaluated in one strain already containing LDH activity. Fed-batch experiments of P. pastoris strains carrying the lactate transporter were performed with the batch phase at aerobic conditions followed by an aerobic oxygen-limited phase where production of lactic acid was favored. The results showed that the strain containing PAS presented the highest lactic acid titer, reaching a yield of approximately 0.7 g/g.ConclusionsWe showed that P. pastoris has a great potential as a fermentative organism for producing l-lactic acid using glycerol as the carbon source at limited oxygenation conditions (below 0.05 % DO in the bioreactor). The best strain had both the LDHb and the homologous lactate transporter encoding genes expressed, and reached a titer 1.5 times higher than the strain with the S. cerevisiae transporter. Finally, it was also shown that increased lactic acid production was concomitant to reduction of acetic acid formation by half.

Introduction Iontophoresis is a noninvasive technique used to increase transdermal penetration of substances through the skin layer (epidermis, dermis and hypodermis) in a controlled manner. Technological advance in recent decades have provided reduced cost of equipment needed for implementation, which allowed for the expansion of this technique. Objective The aim of this paper is to present the state of the art on iontophoresis, ranging from the atomic characteristics of the ion formation to the current applications of the technique. Methods Were researched papers from databases: IOP publishing, ScienceDirect, Pubmed, Springer, IEEE Xplore, Google Scholar and books with keywords iontophoresis, ions, topical applications between 1967 and 2010. Results Were selected (number of papers and database) 1 IOP Publishing, 1 from ScienceDirect, Central, 1 from Springer, 2 from PubMed, 11 from IEEE Xplore, 35 from Google Scholar, and 15 books, totaling 66 references and websites with nationally marketed electrotherapy products. Conclusion Iontophoresis is suitable for applications such as acetic acid (calcific tendinitis and myositis ossificans), calcium chloride and magnesium sulfate (control of musculoskeletal spasms), dexamethasone (inflammation), lidocaine (inflammation of soft tissues), zinc oxide (rheumatoid arthritis). It is also used in cosmetic applications with devices attached to the skin and for eye treatment aimed at specific tissues of the eye, providing a treatment option for various eye diseases, reducing the complications secondary to traditional methods of treatment. The advantages are the significant increase in the release and control of therapeutic agents, including drugs with high molecular weight. The disadvantages of iontophoresis are the complexity of the drug release system and prolonged exposure of the skin to an electrical current.

IntroductionThe National Committee for Health Technology Incorporation (CONITEC) evaluates health technologies to recommend their inclusion or exclusion within the Brazilian Public Health System (SUS), and uses the budget impact assessment to estimate costs to the system. The Ministry of Health (MS) guideline recommends treatment of phenylketonuria (PKU) with restricted phenylalanine diet and phenylalanine-free amino acid formula (PFAAf) supplementation. CONITEC evaluated the inclusion of sapropterin dihydrochloride for PKU in the SUS.MethodsThe population eligible for treatment was evaluated by the number of patients receiving PFAAf between 2014 and 2017 registered in the SUS. Patients were stratified by age/weight and a simple linear regression was performed to estimate the future population. The costs of treatment and testing the responsiveness of sapropterin dihydrochloride were estimated according to the recommended dosage guideline of the MS, leaflet and public purchasing prices. A univariate deterministic sensitivity analysis was performed to evaluate different prices, responsiveness test methods and variations in the reduction of formula use.ResultsThe incorporation of sapropterin dihydrochloride would generate an incremental budget impact in the SUS of around BRL 79 million (USD 21.7 million) in 2019 and BRL 300 million (USD 82.1 million) in five years (2019-2023). The univariate sensitivity analysis estimated that the incremental budget impact could be between BRL 66 and BRL 103 million (USD 18 and USD 28 million) in the first year and between BRL 251 and BRL 388 million (USD 69 and USD 106 million) in five years. Sensitivity analysis showed that the price of sapropterin dihydrochloride was the most sensitive variable in the model.ConclusionsThe incorporation of sapropterin dihydrochloride in the SUS represents a significant budgetary impact and covers a small number of patients. Sapropterin dihydrochloride was recommended by CONITEC for the treatment of women with PKU, with a positive drug responsiveness test, and who are in the preconception period or in the gestational period.

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