Metabolic engineering of Pseudomonas putida for production of the natural sweetener 5-ketofructose from fructose or sucrose by periplasmic oxidation with a heterologous fructose dehydrogenase.

5-Ketofructose (5-KF) is a promising low-calorie natural sweetener with the potential to reduce health problems caused by excessive sugar consumption. It is formed by periplasmic oxidation of fructose by fructose dehydrogenase (Fdh) of Gluconobacter japonicus, a membrane-bound three-subunit enzyme containing FAD and three haemes c as prosthetic groups. This study aimed at establishing Pseudomonas putida KT2440 as a new cell factory for 5-KF production, as this host offers a number of advantages compared with the established host Gluconobacter oxydans. Genomic expression of the fdhSCL genes from G. japonicus enabled synthesis of functional Fdh in P. putida and successful oxidation of fructose to 5-KF. In a batch fermentation, 129 g l-1 5-KF were formed from 150 g l-1 fructose within 23 h, corresponding to a space-time yield of 5.6 g l-1 h-1 . Besides fructose, also sucrose could be used as substrate for 5-KF production by plasmid-based expression of the invertase gene inv1417 from G. japonicus. In a bioreactor cultivation with pulsed sucrose feeding, 144 g 5-KF were produced from 358 g sucrose within 48 h. These results demonstrate that P. putida is an attractive host for 5-KF production.

Novel plasmid-free Gluconobacter oxydans strains for production of the natural sweetener 5-ketofructose.

BACKGROUND: 5-Ketofructose (5-KF) has recently been identified as a promising non-nutritive natural sweetener. Gluconobacter oxydans strains have been developed that allow efficient production of 5-KF from fructose by plasmid-based expression of the fructose dehydrogenase genes fdhSCL of Gluconobacter japonicus. As plasmid-free strains are preferred for industrial production of food additives, we aimed at the construction of efficient 5-KF production strains with the fdhSCL genes chromosomally integrated. RESULTS: For plasmid-free 5-KF production, we selected four sites in the genome of G. oxydans IK003.1 and inserted the fdhSCL genes under control of the strong P264 promoter into each of these sites. All four recombinant strains expressed fdhSCL and oxidized fructose to 5-KF, but site-specific differences were observed suggesting that the genomic vicinity influenced gene expression. For further improvement, a second copy of the fdhSCL genes under control of P264 was inserted into the second-best insertion site to obtain strain IK003.1::fdhSCL2. The 5-KF production rate and the 5-KF yield obtained with this double-integration strain were considerably higher than for the single integration strains and approached the values of IK003.1 with plasmid-based fdhSCL expression. CONCLUSION: We identified four sites in the genome of G. oxydans suitable for expression of heterologous genes and constructed a strain with two genomic copies of the fdhSCL genes enabling efficient plasmid-free 5-KF production. This strain will serve as basis for further metabolic engineering strategies aiming at the use of alternative carbon sources for 5-KF production and for bioprocess optimization.

Re-evaluation of cyanophycin synthesis in Corynebacterium glutamicum and incorporation of glutamic acid and lysine into the polymer.

Corynebacterium glutamicum was only examined in the early 2000s as a possible microorganism for the production of the polyamide cyanophycin (multi-L-arginyl-poly-[L-aspartic acid], CGP). CGP is a potential precursor for the synthesis of polyaspartic acid and CGP-derived dipeptides which may be of use in peptide-based clinical diets, as dietary supplements, or in livestock feeds. In the past, C. glutamicum was disregarded for CGP production due to low CGP contents and difficulties in isolating the polymer. However, considering recent advances in CGP research, the capabilities of this organism were revisited. In this study, several cyanophycin synthetases (CphA) as well as expression vectors and cultivation conditions were evaluated. The ability of C. glutamicum to incorporate additional amino acids such as lysine and glutamic acid was also examined. The strains C. glutamicum pVWEx1::cphAΔ1 and C. glutamicum pVWEx1::cphABP1 accumulated up to 14% of their dry weight CGP, including soluble CGP containing more than 40 mol% of the alternative side-chain amino acid lysine. The soluble, lysine-rich form of the polymer was not detected in C. glutamicum in previous studies. Additionally, an incorporation of up to 6 mol% of glutamic acid into the backbone of CGP synthesized by C. glutamicum pVWEx1::cphADh was detected. The strain accumulated up to 17% of its dry weight in soluble CGP. Although glutamic acid had previously been found to replace arginine in the side chain, this is the first time that glutamic acid was found to substitute aspartic acid in the backbone.

O efeito imediato da acupuntura e da eletroacupuntura no eletroencefalograma em indivíduos estressados / The immediate effect of acupuncture and electroacupuncture in the electroencephalogram in stressed individuals

O stress pode ser definido como uma reação complexa, necessária e benéfica ao organismo, porém quando permanece por um período prolongado cria-se um desequilíbrio no funcionamento global orgânico, enfraquecendo o sistema imunológico e deixando o indivíduo exposto a infecções e doenças oportunistas. Diversos tratamentos são propostos para o controle do stress dentre eles está à acupuntura, uma técnica milenar que consiste na inserção de agulhas em pontos específicos da pele. O eletroencefalograma (EEG) é um instrumento de registro gráfico das variações do potencial de membrana dos neurônios corticais, seu registro e sua interpretação é um processo rotineiro de diagnóstico neurológico e neste caso foi utilizado para verificar se existe alguma alteração no espectro de frequência do EEG imediatamente após a aplicação da acupuntura. Foram selecionados 38 sujeitos randomizados em grupo controle (n= 13), grupo acupuntura (n=13) e grupo eletroacupuntura (n=12). Antes das intervenções os sujeitos foram submetidos ao Inventário de Sintomas de Stress para adultos de Lipp (ISSL), a escala visual analógica (EVA) de stress, mensurados frequência cardíaca (FC) e pressão arterial (PA). As intervenções ocorreram durante vinte minutos e vinte minutos antes e depois das intervenções foram realizados os registros de EEG. Após o término do EEG os sujeitos novamente responderam ao EVA e foram mensurados FC e PA. Para análise dos dados foram utilizados ANOVA, correlação, BOOTSTRAP e teste t-student. Não foram encontradas diferenças significativas em relação ao espectro de frequência do EEG e a EVA antes e depois das intervenções, houve uma diminuição significante da FC após as intervenções. Não houve alteração da PA durante todo o processo. A acupuntura e a eletroacupuntura não apresentaram efeitos imediatos significativos após sua realização no espectro de frequência do EEG, embora tenha apresentado uma tendência a alterações. Não houve relação do espectro do EEG e os quesitos... (AU)

Stress can be defined as a complex, necessary and beneficial response by the body, but when it remains for a prolonged period creates an imbalance in the overall organ function, by weakening the immune system, leaving exposed the individual opportunistic infections and diseases. Several treatments are proposed for the control of stress among them is acupuncture, an ancient technique that involves inserting fine needles into specific points on the skin. The electroencephalogram (EEG) is a tool of graphic record of the variations of the membrane potential of cortical neurons registration and its interpretation is a routine process of neurological diagnosis and this case was used to verify if there is any change in the frequency spectrum of the EEG immediately after application of acupuncture. 38 subjects randomized into control group (n = 13), acupuncture group (n = 13) and electroacupuncture group (n = 12) were selected. Before the intervention the subjects were submitted to the Inventory of Stress Symptoms for adults Lipp (ISSL), a visual analog scale (VAS) of stress, measured heart rate (HR) and blood pressure (BP). The interventions occurred for twenty minutes and twenty minutes before and after interventions EEG records were performed. After the end of the EEG subjects again responded to the EVA and HR and BP were measured. For data analysis, ANOVA, correlation, BOOTSTRAP and student t test were used. No significant differences were found in relation to the frequency of the EEG spectrum and VAS before and after the interventions, there was a significant decrease in HR after the interventions. There was no change in BP during the entire process. Acupuncture and electroacupuncture showed no significant immediate effect after its completion in the EEG frequency spectrum, although it had a tendency to change. There was no relation of the EEG spectrum and the questions questionnaire Lipp and EVA