RESUMEN
3-Hydroxypropionic acid (3-HP) production from renewable feedstocks is of great interest in efforts to develop greener processes for obtaining this chemical platform. Here we report an engineered E. coli strain for 3-HP production through the ß-alanine pathway. To obtain a new strain capable of producing 3-HP, the pathway was established by overexpressing the enzymes pyruvate aminotransferase, 3-hydroxyacid dehydrogenase, and L-aspartate-1-decarboxylase. Further increase of the 3-HP titer was achieved using evolutionary optimizations of a genome-scale metabolic model of E. coli containing the adopted pathway. From these optimizations, three non-intuitive targets for in vivo assessment were identified: L-alanine aminotransferase and alanine racemase overexpression, and L-valine transaminase knock-out. The implementation of these targets in the production strain resulted in a 40% increase in 3-HP titer. The strain was further engineered to overexpress phosphoenolpyruvate carboxylase, reaching 0.79 ± 0.02 g/L of 3-HP when grown using glucose. Surprisingly, this strain produced 63% more of the desired product when grown using a mixture of glucose and xylose (1:1, C-mol), and gene expression analysis showed that the cellular adjustment to consume xylose had a positive impact on 3-HP accumulation. Fed-batch culture with xylose feeding led to a final titer of 29.1 g/L. These results reinforce the value of computational methods in strain engineering, enabling the design of more efficient strategies to be assessed. Moreover, higher production of 3-HP under a sugar mixture condition points towards the development of bioprocesses based on renewable resources, such as hemicellulose hydrolysates.
Asunto(s)
Escherichia coli , Ingeniería Metabólica , Escherichia coli/genética , Escherichia coli/metabolismo , Ingeniería Metabólica/métodos , Ácido Láctico , Xilosa/metabolismo , Glucosa/metabolismoRESUMEN
We studied the expression of Bacillus amyloliquefaciens transglutaminase cloned in Escherichia coli BL21(DE3)pLysS harboring the plasmid pBAD/3C/bTGase, a bicistronic expression system, in bioreactor cultivation. Batch and fed-batch controlled as DO-stat strategies were employed for the production of the recombinant enzyme. In 30 h-batch cultivations using Terrific broth (TB), 6 g/L of biomass and 3.12 U/mgprotein of transglutaminase activity were obtained. DO-stat fed-batch cultivations under the control of oxygen concentration (DO-stat) using TB as medium but fed with glucose allowed the increment in biomass formation (17.5 g/L) and enzyme activity (6.43 U/mgprotein). DO-stat fed-batch using mineral medium (M9) and fed with glucose under the same conditions produced even higher enzymatic activity (9.14 U/mgprotein). The pH effect was investigated, and the best enzymatic activity could be observed at pH 8. In all cultivations, the bicistronic system remained stable, with 100% of plasmid-bearing cells. These results show that E. coli bearing bicistronic plasmid constructs to express recombinant TGase could be cultivated in bioreactors under DO-stat fed-batch using mineral medium and it is a promising strategy in future optimizations to produce this important enzyme.
Asunto(s)
Escherichia coli/enzimología , Transglutaminasas/biosíntesis , Bacillus amyloliquefaciens/enzimología , Bacillus amyloliquefaciens/genética , Reactores Biológicos , Medios de Cultivo , Escherichia coli/genética , Glucosa , Plásmidos/genética , Transglutaminasas/genéticaRESUMEN
This work investigated the application of a thermophilic (55⯰C) anaerobic reactor with immobilized biomass, mechanically stirred and operated in sequential batch and fed batch (AnSBBR) for environmental compliance and methane production by co-digesting cheese whey (W) and sugarcane vinasse (V). The assays were performed in four steps. In the first step the composition of 75%W:25%V (on a COD basis) was determined to be the most adequate for the anaerobic process. In the second step the applied volumetric organic load (AVOL) was increased and in the third step the feed strategy was modified achieving best results at AVOL of 25 gCOD.m-3.d-1, in which the removed organic matter efficiency was 72%, the molar productivity was 278 molCH4.m-3.d-1 and methane yield was 15.3 mmolCH4.gCOD-1. In the fourth step the temperature was modified to 50⯰C and 45⯰C, achieving worse results. From the kinetic model adjusted to experimental data it was identified that the acetoclastic route was predominant in methane generation. The estimated energy recovered by co-digesting cheese whey and sugarcane vinasse using industrial information was 2.2â¯×â¯104â¯MW h per month, equivalent (in Brazil) to the electricity consumption of about 135â¯×â¯103 inhabitants or monthly savings of US$ 1,653,000 replacing the diesel oil consumed in the industry.
Asunto(s)
Saccharum , Suero Lácteo , Anaerobiosis , Reactores Biológicos , Brasil , Metano , TemperaturaRESUMEN
One of the most important events in fed-batch fermentations is the definition of the moment to start the feeding. This paper presents a methodology for a rational selection of the architecture of an artificial intelligence (AI)system, based on a neural network committee (NNC),which identifies the end of the batch phase. The AI systemwas successfully used during high cell density cultivations of recombinant Escherichia coli. The AI algorithm wasvalidated for different systems, expressing three antigens to be used in human and animal vaccines: fragments of surface proteins of Streptococcus pneumoniae (PspA), clades 1 and 3, and of Erysipelothrix rhusiopathiae (SpaA). Standard feed-forward neural networks (NNs), with a single hidden layer, were the basis for the NNC. The NN architecture with best performance had the following inputs: stirrer speed, inlet air, and oxygen flow rates, carbon dioxide evolution rate, and CO2 molar fraction in the exhaust gas.