L-Homoserine is a non-essential amino acid that is often used as an important platform compound and additive in industrial production. To improve the production efficiency, a previously constructed L-homoserine producing strain E. coli H0-0 was used as a chassis for further metabolic modification. Firstly, the ppc and pyccgP458S genes were overexpressed to optimize the Kreb's cycle. Subsequently, thrAC1034T and lysCcgC932T were overexpressed to improve the product synthesis, followed by inactivation of iclR gene to reduce the accumulation of by-products. The introduction of three sucrose metabolism genes, scrA, scrB and scrK, enabled E. coli to ferment sucrose. The titer of L-homoserine increased from 3.2 g/L to 11.1 g/L.
Escherichia coli/genetics* ; Homoserine ; Metabolic Engineering ; Serine

L-homoserine and its derivatives (O-succinyl-L-homoserine and O-acetyl-L-homoserine) are precursors for the biosynthesis of L-methionine, and various C4 compounds (isobutanol, γ-butyrolactone, 1, 4-butanediol, 2, 4-dihydroxybutyric acid) and L-phosphinothricin. Therefore, the fermentative production of L-homoserine and its derivatives became the research hotspot in recent years. However, the low fermentation yield and conversion rate, and the unclear regulation mechanism for the biosynthesis of L-homoserine and its derivatives, hamper the development of an efficient production process for L-homoserine and its derivatives. This review summarized the advances in the biosynthesis of L-homoserine and its derivatives by metabolic engineering of Escherichia coli from the aspects of substrate uptake, redirection of carbon flow at the key nodes, recycle of NADPH and export of target products. This review may facilitate subsequent metabolic engineering and biotechnological production of L-homoserine and its derivatives.
Escherichia coli/metabolism* ; Metabolic Engineering ; Homoserine/metabolism* ; Escherichia coli Proteins/metabolism* ; Fermentation

OBJECTIVETo construct luxS mutant aften luxS gene of Streptococcus mutans (S. mutans) was knocked out, and examine their ability of biofilm formation.

METHODSA recombinant plasmid containing the flanking fragment of luxS of S. mutans was transformed into S. mutans UA159, and selected by brain heart infusion (BHI) agar medium with kanamicin. The luxS mutant further confirmed via polymerase chain reaction (PCR) and the autoinducer-2 (AI-2) bioluminescence assay of Vibrio harveyi (V. harveyi), and ability of luxS mutant and S. mutans UA159 biofilm formation was examined in different phases, in BHI medium with 1% sucrose and 1% glycose by scanning electron microscopy (SEM).

RESULTSLuxS-deficient S. mutans strains were successfully constructed. Compared with S. mutans UA159, the luxS mutant maintained in BHI medium containing 1% sucrose displayed an apparent defect in biofilm formation, while they showed no significant deviation in BHI medium containing 1% glycose.

CONCLUSIONluxS gene in S.mutans can play a role in dental plaque biofilm formation, and the luxS gene is possible to regulate sucrose-dependent biofilm formation.

Bacterial Proteins ; Biofilms ; Carbon-Sulfur Lyases ; Culture Media ; Dental Plaque ; Homoserine ; analogs & derivatives ; Humans ; Lactones ; Streptococcus mutans

Cystathionine is well-known intermediate in the metabolism of methionine. It is cleaved to cysteine and homoserine by gamma-cystathionase. This enzyme utilize pyridoxal 5'-phosphate as coenzyme. gamma-cystathionase deficiency leads to persistent excretion of large amount of cystathionine in urine, as well as to accumulation of cystathionine in body tissues and fluids. It is inherited as an autosomal recessive trait and shows wide variety of clinical manifestations. No clinical abnormality seems to be specifically associated with gamma-cystathionase deficiency. The majority of patients responded to high dose administration of pyridoxine. We report the first case of cystathioninuric patient in Korea, 19 months of female with developmental delay. In brain MRI, there was generalized mild brain atrophy. There were several times of brief paroxysmal generalized polyspike and wave discharges in electroencephalography(EEG). In amino acid analysis of urine, there was elevated level of cystathionine. She was treated with high dose of pyridoxine. In follow up analysis of urinary amino acid, the cystathionine level was markedly decreased to normal range, and EEG was normalized. Her development shows improvement.
Atrophy ; Brain ; Cystathionine ; Cystathionine gamma-Lyase ; Cysteine ; Electroencephalography ; Female ; Follow-Up Studies ; Homoserine ; Humans ; Korea ; Magnetic Resonance Imaging ; Metabolism ; Methionine ; Pyridoxal ; Pyridoxine ; Reference Values

OBJECTIVETo detect the AI-2 quorum-sensing pathway and construct the luxS g-ene allelic exchange plasmid of Streptococcus mutans.

METHODSTo detect AI-2 pathway in Streptococcus mutans, the Vibrio harveyi BB170 was used as reporter strain. The PCR fragments of the upstream and downstream regions of luxS and the Erythromycin resistance gene were amplified with the primers respectively, and these fragments were ligated into pUC19 vector with double endonuclease reaction sequentially, the ligated DNAs were transformed into Escherichia coli DH5alpha, then the reconstructed plasmids were isolated and identified by restricted endonuclease digestions.

RESULTSStreptococcus mutans Ingbritt C could induce luminescence of BB170, suggesting the presence of AI-2 quorum sensing pathway in Streptococcus mutans, and such stimulatory activity was maximal at the mid-log growth phase. The recombinant plasmid pUCluxKO was digested by PstI-BamHI, and the digest product were 1000 bp and 5000 bp. When the pUCluxKO was digested by BamHI-KpnI, the digest product were 1500 bp and 4500 bp. While it was digested by KpnI-EcoRI, the digest product were 1000 bp and 5000 bp. All PCR product was in a single belt respectively.

CONCLUSIONSThe recombinant plasmid was cloned effectively and can be used in the construction of S.mutans luxS mutant.

Bacterial Proteins ; genetics ; Carbon-Sulfur Lyases ; genetics ; Gene Expression Regulation, Bacterial ; Genetic Vectors ; Homoserine ; genetics ; Plasmids ; Quorum Sensing ; genetics ; Streptococcus mutans ; genetics

Bacterial communities usually develop biofilms abound in nature niche. The development of biofilm is a highly dynamic and complex process coordinated by multiple mechanisms, of which two-component system and quorum sensing are two well-defined systems. Biofilm is involved in the virulence of many pathogens. Therefore, targeting the key factors involved in the biofilm formation represents a novel and promising avenue for developing better antibiotics.
Acyl-Butyrolactones ; metabolism ; Bacteria ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Biofilms ; growth & development ; Drug Delivery Systems ; Gene Expression Regulation, Bacterial ; Homoserine ; analogs & derivatives ; metabolism ; Lactones ; metabolism ; Quorum Sensing ; Signal Transduction