ABSTRACT
BACKGROUND: ε-Poly-L-lysine (ε-PL) is a natural and safe food preservative that is mainly produced by filamentous and aerobic bacteria Streptomyces albulus. During ε-PL biosynthesis, a large amount of ATP is used for the polymerization of L-lysine. A shortage of intracellular ATP is one of the major factors limiting the increase in ε-PL production. In previous studies, researchers have mainly tried to increase the oxygen supply to enhance intracellular ATP levels to improve ε-PL production, which can be achieved through the use of two-stage dissolved oxygen control, oxygen carriers, heterologous expression of hemoglobin, and supplementation with auxiliary energy substrates. However, the enhancement of the intracellular ATP supply by constructing an ATP regeneration system has not yet been considered. RESULTS: In this study, a polyphosphate kinase (PPK)-mediated ATP regeneration system was developed and introduced into S. albulus to successfully improve ε-PL production. First, polyP:AMP phosphotransferase (PAP) from Acinetobacter johnsonii was selected for catalyzing the conversion of AMP into ADP through an in vivo test. Moreover, three PPKs from different microbes were compared by in vitro and in vivo studies with respect to catalytic activity and polyphosphate (polyP) preference, and PPK2Bcg from Corynebacterium glutamicum was used for catalyzing the conversion of ADP into ATP. As a result, a recombinant strain PL05 carrying coexpressed pap and ppk2Bcg for catalyzing the conversion of AMP into ATP was constructed. ε-PL production of 2.34 g/L was achieved in shake-flask fermentation, which was an increase of 21.24% compared with S. albulus WG608; intracellular ATP was also increased by 71.56%. In addition, we attempted to develop a dynamic ATP regulation route, but the result was not as expected. Finally, the conditions of polyP6 addition were optimized in batch and fed-batch fermentations, and the maximum ε-PL production of strain PL05 in a 5-L fermenter was 59.25 g/L by fed-batch fermentation, which is the highest ε-PL production reported in genetically engineered strains. CONCLUSIONS: In this study, we proposed and developed a PPK-mediated ATP regeneration system in S. albulus for the first time and significantly enhanced ε-PL production. The study provides an efficient approach to improve the production of not only ε-PL but also other ATP-driven metabolites.
Subject(s)
Adenosine Triphosphate , Polylysine , Fermentation , RegenerationABSTRACT
PURPOSE: Controversy remains as to whether enteral supplementation of ω-3 fatty acids (FA) could improve outcomes in patients with acute respiratory distress syndrome (ARDS). Thus, we did a meta-analysis and aimed to investigate the benefit and harm of enteral ω-3 FA supplementation in adult patients with ARDS. METHODS: Databases including PubMed, Embase, the Cochrane Register of Controlled Trials, and Google Scholar were searched to find relevant articles. Randomized controlled trials (RCTs) comparing enteral ω-3 FA supplementation with a control or placebo intervention in adult patients with ARDS were included. The primary outcome was all-cause 28-day mortality. We used the Cochrane Collaboration methodology. RESULTS: Seven RCTs with 955 adult patients qualified for inclusion, and all the selected trials were considered as at high risk of bias. The use of enteral ω-3 FA did not significantly reduce all-cause 28-day mortality [relative risk (RR), 0.90; 95 % confidence intervals (CI), 0.68-1.18; p = 0.44; I (2) = 31 %; random effects]. Trial sequential analysis indicated lack of firm evidence for a 20 % RR reduction in all-cause 28-day mortality. PaO2/FiO2 ratio was significantly increased in the ω-3 FA group on day 4 [weighted mean difference (WMD), 45.14; 95 % CI, 16.77-73.51; p = 0.002; I (2) = 86 %; random effects] and day 7 (WMD, 33.10; 95 % CI, 1.67-64.52; p = 0.04; I (2) = 88 %; random effects). Meta-analysis using a random effects model showed no significant differences in ventilator-free days (VFD) (WMD, 2.47 days; 95 % CI, -2.85 to 7.79; p = 0.36; I (2) = 91 %) or intensive care unit-free days (ICU) (WMD, 2.31 days; 95 % CI, -2.34 to 6.97; p = 0.33; I (2) = 89 %) between the two groups. CONCLUSIONS: Among patients with ARDS, enteral supplementation of ω-3 FA seemed ineffective regarding all-cause 28-day mortality, VFD, and ICU-free days. Routine use of enteral ω-3 FA cannot be recommended based on the available evidence.