► A novel activity toward levulinic acid was generated by engineering of 3HBDH. ► Levulinic acid was converted to 4-hydroxyvaleric acid by the engineered 3HBDH. ► Engineering strategy was proposed ...and validated by molecular docking simulation. ► Approximately 60% conversion of levulinic acid was achieved by the engineered enzyme.
Enzymatic reduction of levulinic acid (LA) was performed for the synthesis of 4-hydroxyvaleric acid (4HV) – a monomer of bio-polyester and a precursor of bio-fuels – using 3-hydroxybutyrate dehydrogenase (3HBDH) from Alcaligenes faecalis. Due to the catalytic inactivity of the wild-type enzyme toward LA, engineering of the substrate specificity of the enzyme was performed. A rational design approach with molecular docking simulation was applied, and a double mutant, His144Leu/Trp187Phe, which has catalytic activity (kcat/Km=578.0min−1M−1) toward LA was generated. Approximately 57% conversion of LA to 4HV was achieved with this double mutant in 24h, while no conversion was achieved with the wild-type enzyme.
A series of new fungicides that can inhibit the succinate dehydrogenase (SDH) was classified and named as SDH inhibitors by the Fungicide Resistance Action Committee in 2009. To develop more ...potential SDH inhibitors, we designed and synthesized a novel series of
-(substituted pyridine-4-yl)-1-(substituted phenyl)-5-trifluoromethyl-1
-pyrazole-4-carboxamide derivatives,
-
, namely,
-
,
-
, and
-
. The bioassay results demonstrated that some title compounds exhibited excellent antifungal activity against four tested phytopathogenic fungi (
,
,
, and
). The EC
values were 1.8 μg/mL for
against
, 1.5 and 3.6 μg/mL for
against
and
, respectively, and 6.8 μg/mL for
against
. The SDH enzymatic activity testing revealed that the IC
values of
,
,
, and penthiopyrad were 12.5, 135.3, 6.9, and 223.9 μg/mL, respectively. The molecular docking results of this series of title compounds with SDH model demonstrated that the compounds could completely locate inside of the pocket, the body fragment formed H bonds, and the phenyl ring showed a π-π interaction with Arg59, suggesting that these novel 5-trifluoromethyl-pyrazole-4-carboxamide derivatives might target SDH. These results could provide a benchmark for understanding the antifungal activity against the phytopathogenic fungus
and prompt us to discover more potent SDH inhibitors.
Polyhydroxybutyrates (PHB) are biodegradable polymers that are produced by various microbes, including
Ralstonia, Pseudomonas
, and
Bacillus
species. In this study, a
Vibrio proteolyticus
strain, ...which produces a high level of polyhydroxyalkanoate (PHA), was isolated from the Korean marine environment. To determine optimal growth and production conditions, environments with different salinity, carbon sources, and nitrogen sources were evaluated. We found that the use of a medium containing 2% (w/v) fructose, 0.3% (w/v) yeast extract, and 5% (w/v) sodium chloride (NaCl) in M9 minimal medium resulted in high PHA content (54.7%) and biomass (4.94 g/L) over 48 h. Addition of propionate resulted in the production of poly(3-hydroxybutyrate-
co
-3-hydroxyvalerate) (P(HB-co-HV)) copolymer as propionate acts as a precursor for the HV unit. In these conditions, the bacteria produced poly(3-hydroxybutyrate-
co
-3-hydroxyvalerate) containing a 15.8% 3HV fraction with 0.3% propionate added as the substrate. To examine the possibility of using unsterilized media with high NaCl content for PHB production,
V. proteolyticus
was cultured in sterilized and unsterilized conditions. Our results indicated a higher growth, leading to a dominant population in unsterilized conditions and higher PHB production. This study showed the conditions for halophilic PHA producers to be later implemented at a larger scale.
•Levulinic acid was converted to 4-hydroxyvaleric acid by the engineered AKR7A5.•Structure-based rational design approach was applied for the engineering of AKR7A5.•7.0-fold increased activity toward ...levulinic acid was achieved.•Approximately 60% conversion of levulinic acid was achieved by the engineered enzyme.
Engineering enzyme substrate specificity is a promising approach that can expand the applicability of enzymes for the biocatalytic production of industrial chemicals and fuels. In this study, succinic semialdehyde reductase (AKR7A5) was engineered for the conversion of levulinic acid to 4-hydroxyvaleric acid. Levulinic acid is a derivative of cellulosic biomass, and 4-hydroxyvaleric acid is a potential precursor to bio-polymers and fuels. Therefore, the enzymatic conversion of levulinic acid to 4-hydroxyvaleric acid is of special significance in that this conversion could provide a meaningful basis for the bio-production of useful chemicals from cellulosic biomass. In engineering the substrate specificity of the AKR7A5, a rational design approach with the aid of enzyme-substrate interatomic contact analysis was applied. The Met13 residue was selected as a key mutation site, and substitutions of the residue with six hydrophobic amino acids were applied. As a result, four mutants with enhanced catalytic activity toward levulinic acid were obtained, and the most improved mutant, Met13Trp, exhibited a 7.0-fold increase in catalytic efficiency. Additionally, the structural effects of the positive mutations were investigated to analyze the structural basis for the enzyme substrate specificity with the target substrate.
The P-glycoprotein (P-gp) encoded by the MDR1 gene is a drug-exporting transporter located in the cellular membrane. P-gp induction is regarded as one of the main mechanisms underlying drug-induced ...resistance. Although there is great interest in the regulation of P-gp expression, little is known about its underlying regulatory mechanisms. In this study, we demonstrate that casein kinase 2 (CK2)-mediated phosphorylation of heat shock protein 90β (Hsp90β) and subsequent stabilization of PXR is a key mechanism in the regulation of MDR1 expression. Furthermore, we show that CK2 is directly activated by rifampin. Upon exposure to rifampin, CK2 catalyzes the phosphorylation of Hsp90β at the Ser-225/254 residues. Phosphorylated Hsp90β then interacts with PXR, causing a subsequent increase in its stability, leading to the induction of P-gp expression. In addition, inhibition of CK2 and Hsp90β enhances the down-regulation of PXR and P-gp expression. The results of this study may facilitate the development of new strategies to prevent multidrug resistance and provide a plausible mechanism for acquired drug resistance by CK2-mediated regulation of P-gp expression.
Background: Rifampin is a representative inducer of P-gp, and the only known mechanism is binding between rifampin and PXR.
Results: Rifampin directly activates CK2, which phosphorylates Hsp90β. Consequently, PXR increases, and P-gp expression is induced.
Conclusion: A mechanism for inducing P-gp expression by rifampin exposure is newly identified.
Significance: A strategy for overcoming P-gp-derived drug resistance is suggested.
Polyhydroxyalkanoate (PHA) is a family of biodegradable polymers, and incorporation of different monomers can alter its physical properties. To produce the copolymer poly(3-hydroxybutyrate
-co-
...3-hydroxyvalerate) (P(3HB-
co
-3HV)) containing a high level of 3-hydroxyvalerate (3HV) by altering acetyl-CoA pool levels, we overexpressed an acetyl-CoA acetyltransferase (
atoAD
) in an engineered
E. coli
strain, YH090, carrying PHA synthetic genes
bktB, phaB
, and
phaC
. It was found that, with introduction of
atoAD
and with propionate as a co-substrate, 3HV fraction in PHA was increased up to 7.3-fold higher than a strain without
atoAD
expressed
in trans
(67.9 mol%). By the analysis of CoA pool concentrations in vivo and in vitro using HPLC and LC-MS, overexpression of AtoAD was shown to decrease the amount of acetyl-CoA and increase the propionyl-CoA/acetyl-CoA ratio, ultimately resulting in an increased 3HV fraction in PHA. Finally, synthesis of P(3HB-
co
-3HV) containing 57.9 mol% of 3HV was achieved by fed-batch fermentation of YJ101 with propionate.
L-Theanine, found in green tea leaves has been shown to positively affect immunity and relaxation in humans. There have been many attempts to produce L-theanine through enzymatic synthesis to ...overcome the limitations of traditional methods. Among the many genes coding for enzymes in the L-theanine biosynthesis, glutamylmethylamide synthetase (GMAS) exhibits the greatest possibility of producing large amounts of production. Thus, GMAS from Methylovorus mays No. 9 was overexpressed in several strains including vectors with different copy numbers. BW25113(DE3) cells containing the pET24ma::gmas was selected for strains. The optimal temperature, pH, and metal ion concentration were 50℃, 7, and 5 mM MnCl 2 , respectively. Additionally, ATP was found to be an important factor for producing high concentration of L-theanine so several strains were tested during the reaction for ATP regeneration. Baker’s yeast was found to decrease the demand for ATP most effectively. Addition of potassium phosphate source was demonstrated by producing 4-fold higher L-theanine. To enhance the conversion yield, GMAS was additionally overexpressed in the system. A maximum of 198 mM L-theanine was produced with 16.5 mmol/l/h productivity. The whole-cell reaction involving GMAS has greatest potential for scale-up production of L-theanine.
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