RESUMEN
1,4-Butanediol (1,4-BDO) is currently produced from succinate via six enzymatic reactions in an engineered Escherichia coli strain. Butyraldehyde dehydrogenase (Bld) and butanol dehydrogenase of Clostridium saccharoperbutylacetonicum were selected based on their activities of catalyzing the final two reactions in the 1,4-BDO pathway. To fit Bld into the non-natural 1,4-BDO pathway, we engineered it through random mutagenesis. Five Bld mutants were then isolated using a colorimetric Schiff's reagent-based method. Subsequent site-directed mutagenesis of Bld generated the two best Bld mutants, L273I and L273T, which produced 1,4-BDO titers fourfold greater than those of wild-type Bld. The enhanced 1,4-BDO titers obtained using L273I and L273T clearly correlated with their enhanced activities, which were caused by amino acid mutations at position 273 of Bld. The highest titer of 1,4-BDO (660 ± 40 mg/L) was obtained in a knock-out E. coli strain [ΔldhA ΔpflB ΔadhE ΔlpdA::K. lpd(E354K) Δmdh ΔarcA gltA(R164L)] coexpressing Bld273T+Bdh.
Asunto(s)
Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/metabolismo , Butileno Glicoles/metabolismo , Clostridium/enzimología , Escherichia coli/metabolismo , Ingeniería Metabólica/métodos , Redes y Vías Metabólicas/genética , Clostridium/genética , Escherichia coli/genética , Mutagénesis , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismoRESUMEN
Menaquinone-8 (MK-8, vitamin K) is composed of a non-polar side chain and a polar head group. Escherichia coli was chosen and metabolically engineered as a microbial platform for production of MK-8. MK-8 content in E. coli was significantly enhanced by modulating two precursor pools, which supply a non-polar side chain and a polar head group, and further increased by blocking formation of the competitor ubiquinone-8 (Q-8). Overexpression of E. coli IspA, DXR, or IDI increased MK-8 content up to twofold. A similar positive effect was also observed when E. coli MenA, MenB, MenC, MenD, MenE, MenF, or UbiE was overexpressed. The Q-8-deficient ubiCA mutant enhanced MK-8 content by 30% compared to wild-type E. coli. When MenA or MenD was overexpressed, MK-8 content was enhanced fivefold compared with wild-type E. coli.
Asunto(s)
Vías Biosintéticas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Vitamina K 2/análogos & derivados , Vitamina K/biosíntesis , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/genética , Expresión Génica , Ingeniería Genética , Vitamina K 2/metabolismoRESUMEN
The ent-kaurene is a dedicated precursor pool and is responsible for synthesizing natural sweeteners such as steviol glycosides. In this study, to produce ent-kaurene in Escherichia coli, we modularly constructed and expressed two ent-kaurene genes encoding ent-copalyl diphosphate synthase (CPPS) and ent-kaurene synthase (KS) from Stevia rebaudiana known as a typical plant producing steviol glycoside. The CPPS and KS from S. rebaudiana were functionally expressed in a heterologous host E. coli. Furthermore, in order to enhance ent-kaurene production in E. coli, six geranylgeranyl diphosphate synthases (GGPPS) from various microorganisms and eight strains of E. coli as host were compared by measuring ent-kaurene production. The highest ent-kaurene production of approximately 41.1mg/L was demonstrated in E. coli strain MG1655 co-expressing synthetic CPPS-KS module and GGPPS from Rhodobacter sphaeroides. The ent-kaurene production was further increased up to 179.6 mg/L by overexpression of the three key enzymes for isoprenoid precursor, 1-deoxyxylulose-5-phosphate synthase (DXS), farnesyl diphosphate synthase (IspA) and isopentenyl diphosphate isomerase (IDI) from E. coli. Finally, the highest titer of ent-kaurene (578 mg/L) with a specific yield of ent-kaurene of 143.5mg/g dry cell weight was obtained by culturing E. coli strain MG1655 co-expressing the ent-kaurene module, DXS, IDI and IspA in 1L bioreactor containing 20 g/L glycerol.
Asunto(s)
Diterpenos de Tipo Kaurano/genética , Diterpenos de Tipo Kaurano/metabolismo , Escherichia coli/genética , Ingeniería Metabólica/métodos , Stevia/genética , Escherichia coli/metabolismo , Fermentación , Redes y Vías Metabólicas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Osteoarthritis (OA) is the most common rheumatic pathology. One of the major objectives of OA research is the development of early diagnostic strategies such as those using proteomic technology. Synovial fluid (SF) in OA patients is a potential source of biomarkers for OA. The efficient and reliable preparation of SF proteomes is a critical step towards biomarker discovery. In this study, we have optimized a pretreatment method for two-dimensional gel electrophoresis (2DE) separation of the SF proteome, by enriching low-abundance proteins and simultaneously removing hyaluronic acid, albumin, and IgG. SF samples pretreated using this optimized method were then evaluated by 1DE and 2DE separation followed by immunodetection of cartilage oligomeric matrix protein (COMP), a known OA biomarker, and by the identification of 3 proteins (apolipoprotein, haptoglobin precursor, and fibrinogen D fragment) that are related to joint diseases.