Improving 3-hydroxypropionic acid production in E. coli by in silico prediction of new metabolic targets.

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.

Expression of Bacillus amyloliquefaciens transglutaminase in recombinant E. coli under the control of a bicistronic plasmid system in DO-stat fed-batch bioreactor cultivations.

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.

Biomethane production by thermophilic co-digestion of sugarcane vinasse and whey in an AnSBBR: Effects of composition, organic load, feed strategy and temperature.

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 2.2 × 104 MW h per month, equivalent (in Brazil) to the electricity consumption of about 135 × 103 inhabitants or monthly savings of US$ 1,653,000 replacing the diesel oil consumed in the industry.

Canthaxanthin biosynthesis by Dietzia natronolimnaea HS-1: effects of inoculation and aeration rate.

The interest in production of natural colorants by microbial fermentation has been currently increased. The effects of D-glucose concentration (3.18-36.82 g/L), inoculum size (12.5 × 10(9)-49.5 × 10(9) cfu cells/mL) and air-flow rate (1.95-12.05 L/L min) on the biomass, total carotenoid and canthaxanthin (CTX) accumulation of Dietzia natronolimnaea HS-1 in a batch bioreactor was scrutinized using a response surface methodology-central composite rotatable design (RSM-CCRD). Second-order polynomial models with high R (2) values ranging from 0.978 to 0.990 were developed for the studied responses using multiple linear regression analysis. The models showed the maximum cumulative amounts of biomass (7.85 g/L), total carotenoid (5.48 mg/L) and CTX (4.99 mg/L) could be achieved at 23.38 g/L of D-glucose, 31.2 × 10(9) cfu cells/mL of inoculation intensity and air-flow rate of 7.85 L/L min. The predicted values for optimum conditions were in good agreement with experimental data.

Canthaxanthin biosynthesis by Dietzia natronolimnaea HS-1: effects of inoculation and aeration rate

The interest in production of natural colorants by microbial fermentation has been currently increased. The effects of D-glucose concentration (3.18-36.82 g/L), inoculum size (12.5 x 10(9)-49.5 x 10(9) cfu cells/mL) and air-flow rate (1.95-12.05 L/L min) on the biomass, total carotenoid and canthaxanthin (CTX) accumulation of Dietzia natronolimnaea HS-1 in a batch bioreactor was scrutinized using a response surface methodology-central composite rotatable design (RSM-CCRD). Second-order polynomial models with high R² values ranging from 0.978 to 0.990 were developed for the studied responses using multiple linear regression analysis. The models showed the maximum cumulative amounts of biomass (7.85 g/L), total carotenoid (5.48 mg/L) and CTX (4.99 mg/L) could be achieved at 23.38 g/L of D-glucose, 31.2 x 10(9) cfu cells/mL of inoculation intensity and air-flow rate of 7.85 L/L min. The predicted values for optimum conditions were in good agreement with experimental data.

Canthaxanthin biosynthesis by Dietzia natronolimnaea HS-1: effects of inoculation and aeration rate

The interest in production of natural colorants by microbial fermentation has been currently increased. The effects of D-glucose concentration (3.18-36.82 g/L), inoculum size (12.5 x 10(9)-49.5 x 10(9) cfu cells/mL) and air-flow rate (1.95-12.05 L/L min) on the biomass, total carotenoid and canthaxanthin (CTX) accumulation of Dietzia natronolimnaea HS-1 in a batch bioreactor was scrutinized using a response surface methodology-central composite rotatable design (RSM-CCRD). Second-order polynomial models with high R² values ranging from 0.978 to 0.990 were developed for the studied responses using multiple linear regression analysis. The models showed the maximum cumulative amounts of biomass (7.85 g/L), total carotenoid (5.48 mg/L) and CTX (4.99 mg/L) could be achieved at 23.38 g/L of D-glucose, 31.2 x 10(9) cfu cells/mL of inoculation intensity and air-flow rate of 7.85 L/L min. The predicted values for optimum conditions were in good agreement with experimental data.

Biorreator à membrana em batelada sequencial aplicado ao tratamento de esgoto visando à remoção de nutrientes / Membrane sequencing batch reactor for the wastewater treatment aiming the nutrients removal

Neste trabalho, avaliou-se o desempenho de um biorreator à membrana em batelada sequencial para a remoção de nutrientes (nitrogênio e fósforo) de esgoto sanitário. O reator, construído em escala piloto, foi operado durante 241 dias com tempo total de ciclo de 4 horas, sendo 5 minutos para alimentação, 55 minutos para a fase anóxica e 180 minutos para as fases de aeração e filtração (simultaneamente). Ao longo do monitoramento, foram empregados dois fluxos de filtração: 5,55 e 11,1 L.m-2.h-1, que resultaram nas taxas de troca volumétrica de 5 e 10%, respectivamente. As eficiências médias de remoção de Demanda Química de Oxigênio total, nitrogênio amoniacal e nitrogênio total alcançadas foram de 99, 98 e 96%, respectivamente. Em relação à remoção de fósforo, observou-se inicialmente um baixo rendimento do reator, sendo verificado ao longo do tempo, no entanto, uma tendência de melhora na remoção desse nutriente, atingindo eficiência média de 74% entre os dias 158 e 241. A utilização do fluxo de filtração de 5,55 L.m-2.h-1 proporcionou uma operação estável ao biorreator à membrana em batelada sequencial no que se refere à pressão transmembrana, tendo sido atingido o valor limite de 0,7 bar apenas uma vez em 181 dias de operação, ao passo que, com fluxo de 11,1 L.m-2.h-1, esse limite foi atingido 3 vezes em 55 dias.

This study evaluated the performance of a membrane bioreactor sequencing batch, in pilot scale, to remove nutrients (nitrogen and phosphorus) from domestic wastewater. The reactor was operated for 241 days with a total cycle time of 4 hours, with 5 minutes for feeding, 55 minutes for the anoxic phase and 180 minutes for the aeration and filtration phases (simultaneously). Throughout the monitoring, two filtration flows were employed: 5.5 and 11.1 (critical flux) L.m-2.h-1, which resulted in the volume exchange rates of 5 and 10%, respectively. The removal efficiencies of total Chemical Oxygen Demand, ammonia nitrogen and total nitrogen were achieved by 99, 98 and 96%, respectively. Regarding phosphorus removal, a poor performance was observed in the beginning of the experiment; however, a tendency of improvement in the removal of this nutrient was further verified, reaching an efficiency average of 74% between the operational days 158 and 241. The flux filtration at 5.55 L.m-2.h-1 has provided a membrane bioreactor sequencing batch stable operation in relation to transmembrane pressure since it reached the limit value of 0.7 bar only once in 181 operational days, while for 11.1 L.m-2.h-1 it was observed 3 times in 55 days.

Robust artificial intelligence tool for automatic start-up of the supplementary medium feeding in recombinant E. colicultivations

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.