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Background: l-Leucine is a high-value amino acid with promising applications in the medicine and feed industries. However, the complex metabolic network and intracellular redox imbalance in fermentative microbes limit their efficient biosynthesis of l-leucine. Results: In this study, we applied rational metabolic engineering and a dynamic regulation strategy to construct a plasmid-free, non-auxotrophic Escherichia coli strain that overproduces l-leucine. First, the l-leucine biosynthesis pathway was strengthened through multi-step rational metabolic engineering. Then, a cooperative cofactor utilization strategy was designed to ensure redox balance for l-leucine production. Finally, to further improve the l-leucine yield, a toggle switch for dynamically controlling sucAB expression was applied to accurately regulate the tricarboxylic acid cycle and the carbon flux toward l-leucine biosynthesis. Strain LEU27 produced up to 55 g/L of l-leucine, with a yield of 0.23 g/g glucose. Conclusions: The combination of strategies can be applied to the development of microbial platforms that produce l-leucine and its derivatives. [ABSTRACT FROM AUTHOR]

Industrial amino acid production strains of Corynebacterium glutamicum are usually obtained by mutagenesis. However, the genetic and metabolic characteristics and the efficient synthesis mechanism of the selected mutants are unclear. The aims of this study were (1) to determine the gene transcriptional patterns and intracellular metabolite levels of an L-leucine-producing mutant C. glutamicum CP and an L-valine-producing mutant C. glutamicum XV referring to wild type, and (2) to understand the efficient synthesis mechanism of target product of these mutants. For this purpose, transcriptomic and metabolomics analyses were combined to investigate the association between intracellular patterns and product synthesis. The high intracellular level of glucose and the low intracellular level of metabolites in the central carbon metabolism meant the glucose metabolism rate of two mutants was lower than wild type. However, the increased intracellular pentose level and gene transcription in the pentose phosphate pathway (PPP) indicated that the PPP of mutants was more active. Furthermore, the mutants showed higher intracellular level of NADPH, which was mainly generated in PPP. In the specific pathway for the synthesis of L-leucine and L-valine, the transcriptional level of most genes was upregulated in the mutants. However, the transcription of transaminase C coding gene Cgl2844 was downregulated in CP but upregulated in XV. The upregulation of Cgl2844 might benefit to the synthesis of L-valine and cause the significant decrease of intracellular level of L-alanine and L-glutamate of XV. These characteristics of the mutants provided insight into changes that could be made to systematically optimize the metabolic pathways for the production of L-leucine and L-valine. [ABSTRACT FROM AUTHOR]

Background: Inadequate protein intake (PI), containing a sub-optimal source of essential amino acids (EAAs), and reduced appetite are contributing factors to age-related sarcopenia. The satiating effects of dietary protein per se may negatively affect energy intake (EI), thus there is a need to explore alternative strategies to facilitate PI without compromising appetite and subsequent EI.Methods: Older women completed two experiments (EXP1 and EXP2) where they consumed either a Bar (565 kJ), a Gel (477 kJ), both rich in EAAs (7.5 g, 40% L-leucine), or nothing (Control). In EXP1, participants (n = 10, 68 ± 5 years, mean ± SD) consumed Bar, Gel or Control with appetite sensations and appetite-related hormonal responses monitored for one hour, followed by consumption of an ad libitum breakfast (ALB). In EXP2, participants (n = 11, 69 ± 5 years) ingested Bar, Gel or Control alongside an ALB.Results: In EXP1, EI at ALB was not different (P = 0.674) between conditions (1179 ± 566, 1254 ± 511, 1206 ± 550 kJ for the Control, Bar, and Gel respectively). However, total EI was significantly higher in the Bar and Gel compared to the Control after accounting for the energy content of the supplements (P < 0.0005). Analysis revealed significantly higher appetite Area under the Curve (AUC) (P < 0.007), a tendency for higher acylated ghrelin AUC (P = 0.087), and significantly lower pancreatic polypeptide AUC (P = 0.02) in the Control compared with the Bar and Gel. In EXP2, EI at ALB was significantly higher (P = 0.028) in the Control (1282 ± 513 kJ) compared to the Bar (1026 ± 565 kJ) and Gel (1064 ± 495 kJ). However, total EI was significantly higher in the Bar and Gel after accounting for the energy content of the supplements (P < 0.007).Conclusions: Supplementation with either the Bar or Gel increased total energy intake whether consumed one hour before or during breakfast. This may represent an effective nutritional means for addressing protein and total energy deficiencies in older women.Trial Registration: Clinical trial register: retrospectively registered, ISRCTN12977929 on. [ABSTRACT FROM AUTHOR]

Background: Progressive decline in skeletal muscle mass and function are growing concerns in an aging population. Diet and physical activity are important for muscle maintenance but these requirements are not always met. This highlights the potential for nutritional supplementation. As a primary objective, we sought to assess the effect of a novel combination of L-Carnitine, creatine and leucine on muscle mass and performance in older subjects. Method: Forty-two healthy older adults aged 55-70 years were randomized to receive either a novel L-Carnitine (1500 mg), L-leucine (2000 mg), creatine (3000 mg), Vitamin D3 (10 µg) (L-Carnitine-combination) product (n =14),L- Carnitine (1500 mg) (n =14),oraplacebo(n = 14) for eight weeks. We evaluated body mass by DXA, upper and lower strength by dynamometry, and walking distance by a 6-min walk test at baseline and after eight weeks of intervention. These measures, reflecting muscle mass, functional strength and mobility have been combined to generate a primary composite score. Quality of life, blood safety markers, and muscle biopsies for protein biomarker analysis were also conducted at baseline and the end of the study. Results: The primary composite outcome improved by 63.5 percentage points in the L-Carnitine-combination group vs. placebo (P = 0.013). However, this composite score did not change significantly in the L-Carnitine group (P= 0.232), and decreased slightly in the placebo group (P= 0.534). Participants supplemented with the L-Carnitine-combination showed a 1.0 kg increase in total lean muscle mass (P = 0.013), leg lean muscle mass (0.35 kg, P= 0.005), and a 1.0 kg increase in lower leg strength (P = 0.029) at week 8. In addition, these increases were significant when compared to the placebo group (P = 0.034, P= 0.026, and P= 0.002, respectively). Total mTOR protein expression was increased in participants in the L-Carnitine-combination group at the end of the study compared to the baseline (P = 0.017). This increase was also significant when compared to the placebo (P= 0.039), suggesting that the increase in muscle mass and strength was due to new protein synthesis and mTOR pathway activation. Conclusions: The trial did reach its primary objective. L-Carnitine combined with creatine and L-leucine significantly improved the composite score which reflects muscle mass and strength, at the end of the study compared to placebo. The combination showed an increase in mTOR protein level, a driver for increased muscle mass which translated to an improvement in muscle strength. This new combination may provide a potential nutritional intervention to promote muscle growth and improved physical functioning in older adults. [ABSTRACT FROM AUTHOR]