ABSTRACT
l-Methionine biosynthesis in Eschericha coli consists of multiple unit modules with various enzymes involved and the imbalance between different modules always restricted its productivity. In this study, the key enzymes participating in the pathway were investigated for their effect on l-methionine production and the pivotal enzyme homoserine O-succinyltransferase (MetA) was designed to be regulated. The surface amino acid residues of MetA were effectively modified through site-saturation mutagenesis and single mutants L63F, A28V, P298L and double mutant L63F/A28V were obtained with improved l-methionine productivity. The structure analysis revealed that the involved residues were on the surface loop regions, which was proposed to be conducive to the refolding of MetA and thus reduce the inhibition effect caused by l-methionine. After expression of the selected single mutant L63F in engineered E. coli ΔIJA-HFEBC strain with l-methionine efflux pump and mutated 3-phosphoglycerate dehydrogenase, the l-methionine production was significantly improved, with a final yield of 3528 mg/L. The results demonstrated the efficiency of MetA regulation for enhanced production of l-methionine and meanwhile provided important guidance for further engineering of MetA with increased l-methionine productivity.
Subject(s)
Escherichia coli/metabolism , Homoserine O-Succinyltransferase/genetics , Homoserine O-Succinyltransferase/metabolism , Methionine/biosynthesis , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial , Metabolic Engineering/methods , Metabolic Networks and Pathways/genetics , Mutagenesis, Site-Directed , Phosphoglycerate Dehydrogenase/genetics , Phosphoglycerate Dehydrogenase/metabolismABSTRACT
BACKGROUND: To investigate the risk factors and changes in serum inflammatory factors in puerperal infection, and propose clinical prevention measures. METHODS: A total of 240 subjects with suspected puerperal infection treated in our hospital from January 2017 to December 2017 were collected, among which puerperal infection was definitely diagnosed in 40 cases, and it was excluded in 40 cases. Levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and high-sensitivity C-reactive protein (hs-CRP) were compared between the two groups, and the change trends of IL-6 and hs-CRP were recorded. RESULTS: Levels of IL-6, hs-CRP, and TNF-α in puerperal infection group were higher than those in non-infection group (P < .05). Levels of IL-6 and hs-CRP at enrollment and 1-3 days after enrollment in infection group were higher than those in non-infection group (P < .05). The body mass index >25, placenta previa, placenta accreta, postpartum hemorrhage, premature rupture of membrane, gestational diabetes mellitus, and anemia during pregnancy were relevant and independent risk factors for puerperal infection. Puerperal infection occurred in uterine cavity, vagina, pelvic peritoneum, pelvic tissue, incision, urinary system, etc, and gram-negative (G+) bacteria were dominated in pathogens. CONCLUSION: The inflammatory response of patients with puerperal infection is significantly enhanced.
Subject(s)
Inflammation Mediators/blood , Puerperal Infection/blood , Puerperal Infection/prevention & control , Adult , C-Reactive Protein/metabolism , Female , Humans , Interleukin-6/blood , Logistic Models , Multivariate Analysis , Puerperal Infection/epidemiology , Puerperal Infection/microbiology , Risk Factors , Young AdultABSTRACT
OBJECTIVE: L-methionine is an important sulfur-containing amino acid essential for humans and animals. Its biosynthesis pathway is complex and highly regulated. This study aims to explore the bottleneck limiting the improvement of L-methionine productivity and apply efficient strategies to increase L-methionine production in engineered E. coli. RESULTS: The enzyme O-succinylhomoserine sulfhydrylase involved in thiolation of OSH to form homocysteine was overexpressed in the engineered strain E. coli W3110 IJAHFEBC/PAm, resulting in L-methionine production increased from 2.8 to 3.22 g/L in shake flask cultivation. By exogenous addition of L-glycine as the precursor of one carbon unit, the titer of L-methionine was increased to 3.68 g/L. The glycine cleavage system was further strengthened for the efficient one carbon unit supply and a L-methionine titer of 3.96 g/L was obtained, which was increased by 42% compared with that of the original strain. CONCLUSIONS: Insufficient supply of one carbon unit was found to be the issue limiting the improvement of L-methionine productivity and its up-regulation significantly promoted the L-methionine production in the engineered E. coli.
