Construction and fermentation control of reductive TCA pathway for malic acid production in Saccharomyces cerevisiae / 生物工程学报

Malic acid is widely used in food, and chemical industries. Through overexpressing pyruvate carboxylase and malate dehydrogenase in pdc1-deficient Saccharomyces cerevisiae, malic acid was successfully produced through the reductive TCA pathway. No malic acid was detected in wild type Saccharomyces cerevisiae, however, 45 mmol/L malic acid was produced in engineered strain, and the concentration of byproduct ethanol also reduced by 18%. The production of malic acid enhanced 6% by increasing the concentration of Ca2+. In addition, the final concentration reached 52.5 mmol/L malic acid by addition of biotin. The increasing is almost 16% higher than that of the original strain.

Effect of co-expression of nicotinic acid phosphoribosyl transferase and pyruvate carboxylase on succinic acid production in Escherichia coli BA002 / 生物工程学报

Escherichia coli BA002, in which the ldhA and pflB genes are deleted, cannot utilize glucose anaerobically due to the inability to regenerate NAD+. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase (NAPRTase) encoded by the pncB gene, a rate-limiting enzyme of NAD(H) synthesis pathway, resulted in a significant increase in cell mass and succinate production under anaerobic conditions. However, a high concentration of pyruvate was accumulated. Thus, co-expression of NAPRTase and the heterologous pyruvate carboxylase (PYC) of Lactococcus lactis subsp. cremoris NZ9000 in recombinant E. coli BA016 was investigated. Results in 3 L fermentor showed that OD600 is 4.64 and BA016 consumed 35.00 g/L glucose and produced 25.09 g/L succinate after 112 h under anaerobic conditions. Overexpression of pncB and pyc in BA016, the accumulation of pyruvic acid was further decreased, and the formation of succinic acid was further increased.

Expression of heterogenous pyruvate carboxylase in Escherichia coli with lactose as inducer and its effect on succinate production / 生物工程学报

Escherichia coli strain DC1515, deficient in glucose phosphotransferase (ptsG), lactate dehydrogenase (ldhA) and pyruvate:formate lyase (pflA), is a promising candidate for the fermentative production of succinate. To further improve the succinate producing capability of DC1515, we constructed plasmid pTrchisA-pyc with heterogenous pyruvate carboxylase (pyc) from Bacillus subtilis 168 under the Trc promoter and introduced it into DC1515. We used lactose as a substitute of IPTG to induce pyc. We optimized the culture conditions such as the lactose addition time, the lactose concentration and the culture temperature after induction for succinate production. We also explored the effect of lactose supplement during the fermentation. The results showed that pyc can be expressed under lactose induction in the fermentative medium with 15 g/L glucose due to the deficient of ptsG in DC1515. Under optimized conditions, the final succinate concentration reached to 15.17 g/L, which was 1.78-fold higher than that of control strain. If complementing lactose twice to the concentration of 1 g/L during the fermentation, the final succinate concentration could further reach to 17.54 g/L. This work might provide valuable information for gene expression in E. coli strains using lactose as inducer for succinate production in a glucose-medium. Due to the reduced cost, E. coli is becoming a more promising strain for succinate production through fermentation.

Papel da geracao de oxaloacetato no exercicio fisico moderado em ratos: consequencias da suplementacao de aspartato, asparagina e carnitina / The role of the production of oxaloacetate in the moderate exercise in rats: consequence of the supplementation of aspartate, asparagine and carnitine

A importancia na geracao de oxaloacetato foi investigada atraves da determinacao da atividade da piruvato carboxilase nos musculos estriados e da suplementacao de seus precursores (aspartato e aspargina) na dieta de ratos. A atividade da piruvato carboxilase eleva-se durante o exercicio fisico e, portanto, deve fornecer mais oxaloacetato para a etapa inicial do ciclo de Krebs. A suplementacao cronica (5 semanas) de aspartato e aspargina promove aumento da resistencia ao esforco em ratos treinados em natacao durante 1 hora diaria por 5 semanas. Este efeito foi acompanhado de elevacao no numero e tamanho das mitocondrias e alteracao no metabolismo de glicose dos musculos esqueleticos (elevacao do conteudo de glicogenio e de sua sintese e diminuicao da glicolise). Esses resultados sugerem que a geracao de oxaloacetato desempenha papel fundamental na manutencao do esforco prolongado. A suplementacao de aspartato e aspargina na dieta melhora a performance nessas condicoes, porem causa lesoes na ultraestrutura muscular (mitocondrias, linha "Z" e microfiblilas)