Engineering of Methionine Adenosyltransferase toward Mitigated Product Inhibition for Efficient Production of -Adenosylmethionine.

-Adenosylmethionine (SAM) is a crucial metabolic intermediate playing irreplaceable roles in organismal activities. However, the synthesis of SAM by methionine adenosyltransferase (MAT) is hindered by low conversion due to severe product inhibition. Herein structure-guided semirational engineering was conducted on MAT from (MAT) to mitigate the product inhibitory effect.

Engineering of Methionine Adenosyltransferase Reveals Key Roles of Electrostatic Interactions in Enhanced Catalytic Activity.

As an important dietary supplement, S-adenosylmethionine (SAM) is currently synthesized by methionine adenosyltransferase (MAT) using ATP and methionine as substrates. However, the activity of MAT is severely inhibited by product inhibition, which limits the industrial production of SAM. Here, MAT from Bacteroides fragilis (BfMAT), exhibiting relatively low product inhibition and moderate specific activity, was identified by gene mining.

[Commentary: polymer binding modules accelerate enzymatic degradation of poly(ethylene terephthalate)].

The large scale production and indiscriminate use of plastics led to serious environmental pollution. To reduce the negative effects of plastics waste on the environment, an approach of enzymatic degradation was put forward to catalyze plastics degradation. Protein engineering strategies have been applied to improve the plastics degrading enzyme properties such as activity and thermal stability.

Efficient production of hyaluronic acid by Streptococcus zooepidemicus using two-stage semi-continuous fermentation.

Hyaluronic acid is a kind of mucopolysaccharide that has wide applications in cosmetics, health food, and orthopedics. Using Streptococcus zooepidemicus ATCC 39920 as parent, a beneficial mutant SZ07 was obtained by UV mutagenesis, giving 1.42 g/L hyaluronic acid in shake flasks.

Self-Sufficient In Vitro Multi-Enzyme Cascade for Efficient Synthesis of Danshensu from l-DOPA.

Danshensu (DSS), a traditional Chinese medicine, is widely used for the treatment of cardiovascular and cancer diseases. Here, a one-pot multi-enzyme cascade pathway was designed for DSS synthesis from l-DOPA using tyrosine aminotransferase from (TyrB) and d-isomer-specific 2-hydroxyacid dehydrogenase from (D2-HDH). Glutamate dehydrogenase from (gluD) was also introduced for a self-sufficient system of α-ketoglutaric acid and NADH.

[Engineering the 182 site of cyclodextrin glucosyltransferase for glycosylated genistein synthesis].

Genistein and its monoglucoside derivatives play important roles in food and pharmaceuticals fields, whereas their applications are limited by the low water solubility. Glycosylation is regarded as one of the effective approaches to improve water solubility. In this paper, the glycosylation of sophoricoside (genistein monoglucoside) was investigated using a cyclodextrin glucosyltransferase from (CGTase).

Sustainable one-pot chemo-enzymatic synthesis of chiral furan amino acid from biomass via magnetic solid acid and threonine aldolase.

Sustainable synthesis of valuable noncanonical amino acids from renewable feedstocks is of great importance. Here, a feasible chemo-enzymatic procedure was developed for the synthesis of chiral β-(2-furyl)serine from biomass catalyzed by a solid acid catalyst and immobilized E. coli whole-cell harboring l-threonine aldolase.

[Engineering ω-transaminase by random mutagenesis and semi-rational design for the synthesis of (R)-(+)-1-(1-naphthyl)ethylamine].

(R)-(+)-1-(1-naphthyl)ethylamine is a key chiral intermediate for the synthesis of calcimimetic drug cinacalcet hydrochloride. ω-Transaminase has been considered to be potential for producing (R)-(+)-1-(1-naphthyl)ethylamine by asymmetric reduction of 1-acetonaphthone. Here, ω-transaminase from Arthrobacter sp.

High-Throughput Screening Method for Directed Evolution and Characterization of Aldol Activity of D-Threonine Aldolase.

A rapid and reliable method for the determination of aldol condensation activity of threonine aldolases (TAs) toward aldehydes and glycine was developed. This 2,4-dinitrophenylhydrazine (DNPH) method has high sensitivity and low background disturbance and can be spectrophotometrically measured for high-throughput screening and characterization of TAs. For 4-methylsulfonyl benzaldehyde (MSB), the maximum absorbance peak was observed at around 485 nm.

Enhancing n-Butanol Tolerance of Escherichia coli by Overexpressing of Stress-Responsive Molecular Chaperones.

Microbial tolerance to organic solvents is critical for efficient production of biofuels. In this study, n-butanol tolerance of Escherichia coli JM109 was improved by overexpressing of genes encoding stress-responsive small RNA-regulator, RNA chaperone, and molecular chaperone. Gene rpoS, coding for sigma S subunit of RNA polymerase, was the most efficient in improving n-butanol tolerance of E.

Engineering of Cyclodextrin Glycosyltransferase Reveals pH-Regulated Mechanism of Enhanced Long-Chain Glycosylated Sophoricoside Specificity.

Sophoricoside glycosylated derivatives, especially long-chain glycosylated sophoricosides (LCGS), have greatly improved water solubility compared with sophoricoside. Here, cyclodextrin glycosyltransferase from (CGTase) was employed for sophoricoside glycosylation. Saturation mutagenesis of alanine 156, alanine 166, glycine 173, and leucine 174 was performed due to their nonconservative properties among α-, β-, and γ-CGTases with different product specificities.

Enhancing soluble expression of sucrose phosphorylase in Escherichia coli by molecular chaperones.

Sucrose phosphorylase (SPase, EC 2.4.1.

CRISPR-Cpf1-Assisted Engineering of Corynebacterium glutamicum SNK118 for Enhanced L-Ornithine Production by NADP-Dependent Glyceraldehyde-3-Phosphate Dehydrogenase and NADH-Dependent Glutamate Dehydrogenase.

Here, Corynebacterium glutamicum SNK118 was metabolically engineered for L-ornithine production through CRISPR-Cpf1-based genome manipulation and plasmid-based heterologous overexpression. Genes argF, argR, and ncgl2228 were deleted to block the degradation of L-ornithine, eliminate the global transcriptional repression, and alleviate the competitive branch pathway, respectively. Overexpression of CsgapC (NADP-dependent glyceraldehyde 3-phosphate dehydrogenases gene from Clostridium saccharobutylicum DSM 13864) and BsrocG (NADH-dependent glutamate dehydrogenase gene from Bacillus subtilis HB-1) resulted markedly increased ornithine biosynthesis.

Composite coal fly ash solid acid catalyst in synergy with chloride for biphasic preparation of furfural from corn stover hydrolysate.

A solid acid catalyst SO/SnO-AlO-CFA was synthesized based on industrial waste coal fly ash (CFA) as carrier and applied in the conversion of oxalic acid pretreated corn stover hydrolysate to produce furfural. Physical properties of the solid acid catalyst were characterized by SEM, FTIR, XRD, BET, EDAX, and NH-TPD. Highly wrinkled structure of SO/SnO-AlO-CFA could provide more specific surface area for the covalent linkage between SiO and SnO.

Enhancing butanol tolerance of reveals hydrophobic interaction of multi-tasking chaperone SecB.

Background: has been proved to be one promising platform chassis for the production of various natural products, such as biofuels. Product toxicity is one of the main bottlenecks for achieving maximum production of biofuels. Host strain engineering is an effective approach to alleviate solvent toxicity issue in fermentation.

Improving Soluble Expression of Tyrosine Decarboxylase from Lactobacillus brevis for Tyramine Synthesis with High Total Turnover Number.

The soluble expression of tyrosine decarboxylase (TDC) in heterologous host is often challenging. Here, acidic condition was found to be favorable for improving the soluble expression of TDC from Lactobacillus brevis in Escherichia coli, while addition of carbohydrates (such as glucose, arabinose, and fructose) was vital for decreasing the insoluble fraction. By simple pH control and addition of glucose, the specific activity of TDC in crude extract was enhanced to 46.

Metabolic engineering of Corynebacterium glutamicum for improved L-arginine synthesis by enhancing NADPH supply.

Corynebacterium glutamicum SNK 118 was metabolically engineered with improved L-arginine titer. Considering the crucial role of NADPH level in L-arginine production, pntAB (membrane-bound transhydrogenase) and ppnK (NAD kinase) were co-expressed to increase the intracellular NADPH pool. Expression of pntAB exhibited significant effects on NADPH supply and L-arginine synthesis.

Structural Insight into Enantioselective Inversion of an Alcohol Dehydrogenase Reveals a "Polar Gate" in Stereorecognition of Diaryl Ketones.

Diaryl ketones are important building blocks for synthesizing pharmaceuticals and are generally regarded as "difficult-to-reduce" ketones due to the large steric hindrance of their two bulky aromatic side chains. Alcohol dehydrogenase from Kluyveromyces polyspora ( KpADH) has been identified as a robust biocatalyst due to its high conversion of diaryl ketone substrate (4-chlorophenyl)(pyridine-2-yl)ketone (CPMK) with a moderate R-selectivity of 82% ee. To modulate the stereoselectivity of KpADH, a "polarity scanning" strategy was proposed, in which six key residues inside and at the entrance of the substrate binding pocket were identified.

Detoxification of furfural residues hydrolysate for butanol fermentation by Clostridium saccharobutylicum DSM 13864.

The toxicity of furfural residues (FRs) hydrolysate is a major obstacle in its application. This work focused on the detoxification of FRs hydrolysate and its application in butanol fermentation. Combination of activated carbon and resin 717 was appropriate for the detoxification of hydrolysate.

Identification of d-carbamoylase for biocatalytic cascade synthesis of d-tryptophan featuring high enantioselectivity.

In this study, an enantioselective d-carbamoylase (AcHyuC) was identified from Arthrobacter crystallopoietes with optimum pH of 8.5, much more compatible with hydantoinase process than other reported d-N-carbamoylases. AcHyuC has a substrate preference for aromatic carbamoyl-compounds.

High production of genistein diglucoside derivative using cyclodextrin glycosyltransferase from Paenibacillus macerans.

Genistein has been regarded as one important soy isoflavone with multiple health benefits, whereas its applications are limited by the low hydrophilicity. To improve the water solubility, codon optimized cyclodextrin glycosyltransferase from Paenibacillus macerans was employed for genistein transglycosylation in this study. At least four transglycosylation products were produced and identified by HPLC and LC-MS: genistein monoglucoside, diglucoside, triglucoside, and tetraglucoside derivatives.

Engineering coenzyme A-dependent pathway from Clostridium saccharobutylicum in Escherichia coli for butanol production.

Clostridium saccharobutylicum has been proved to be efficient in butanol fermentation from various feedstocks. Whereas, lack of genetic manipulation system has severely hindered the engineering of C. saccharobutylicum for more extensive applications.

Enhanced curdlan production with nitrogen feeding during polysaccharide synthesis by Rhizobium radiobacter.

Curdlan is a secondary metabolite synthesized by Agrobacterium sp. and some other bacteria. A newly isolated exopolysaccharide-producing strain was identified to be Rhizobium radiobacter CGMCC 12099.

Crystal structure of tyrosine decarboxylase and identification of key residues involved in conformational swing and substrate binding.

Tyrosine decarboxylase (TDC) is a pyridoxal 5-phosphate (PLP)-dependent enzyme and is mainly responsible for the synthesis of tyramine, an important biogenic amine. In this study, the crystal structures of the apo and holo forms of Lactobacillus brevis TDC (LbTDC) were determined. The LbTDC displays only 25% sequence identity with the only reported TDC structure.

DNA microarray of global transcription factor mutant reveals membrane-related proteins involved in n-butanol tolerance in Escherichia coli.

Background: Escherichia coli has been explored as a platform host strain for biofuels production such as butanol. However, the severe toxicity of butanol is considered to be one major limitation for butanol production from E. coli.