An artificial biocatalytic cascade for efficient synthesis of norepinephrine by combination of engineered L-threonine transaldolase with multi-enzyme expression fine-tuning.

Norepinephrine, a kind of ß-adrenergic receptor agonist, is commonly used for treating shocks and hypotension caused by a variety of symptoms. The development of a straightforward, efficient and environmentally friendly biocatalytic route for manufacturing norepinephrine remains a challenge. Here, we designed and realized an artificial biocatalytic cascade to access norepinephrine starting from 3, 4-dihydroxybenzaldehyde and L-threonine mediated by a tailored-made L-threonine transaldolase PsLTTA-Mu1 and a newly screened tyrosine decarboxylase ErTDC. To overcome the imbalance of multi-enzymes in a single cell, engineering of PsLTTA for improved activity and fine-tuning expression mode of multi-enzymes in single E.coli cells were combined, leading to a robust whole cell biocatalyst ES07 that could produce 100 mM norepinephrine with 99% conversion, delivering a highest time-space yield (3.38 g/L/h) ever reported. To summarized, the current study proposed an effective biocatalytic approach for the synthesis of norepinephrine from low-cost substrates, paving the way for industrial applications of enzymatic norepinephrine production.

A chemoenzymatic process for preparation of highly purified dehydroepiandrosterone in high space-time yield.

Dehydroepiandrosterone (DHEA) is an important neurosteroid hormone to keep human hormonal balance and reproductive health. However, DHEA was always produced with impurities either by chemical or biological method and required high-cost purification before the medical use. To address this issue, a novel chemoenzymatic process was proposed and implemented to produce DHEA. An acetoxylated derivate of 4-androstene-3,17-dione (4-AD) was generated by chemical reaction and converted into DHEA by an enzyme cascade reaction combining a hydrolysis reaction with a reduction reaction. The hydrolysis reaction was catalyzed by a commercial esterase Z03 while the reduction reaction was catalyzed by E. coli cells co-expressing a 3ß-hydroxysteroid dehydrogenase SfSDR and a glucose dehydrogenase BtGDH. After the condition optimization, DHEA was synthesized at a 100 mL scale under 100 mM of substrate loading and purified as white powder with the highest space-time yield (4.80 g/L/h) and purity (99 %) in the biosynthesis of DHEA. The successful attempt in this study provides a new approach for green synthesis of highly purified DHEA in the pharmaceutical industry.

Histone methylation in pancreatic cancer and its clinical implications.

Pancreatic cancer (PC) is an aggressive human cancer. Appropriate methods for the diagnosis and treatment of PC have not been found at the genetic level, thus making epigenetics a promising research path in studies of PC. Histone methylation is one of the most complicated types of epigenetic modifications and has proved crucial in the development of PC. Histone methylation is a reversible process regulated by readers, writers, and erasers. Some writers and erasers can be recognized as potential biomarkers and candidate therapeutic targets in PC because of their unusual expression in PC cells compared with normal pancreatic cells. Based on the impact that writers have on the development of PC, some inhibitors of writers have been developed. However, few inhibitors of erasers have been developed and put to clinical use. Meanwhile, there is not enough research on the reader domains. Therefore, the study of erasers and readers is still a promising area. This review focuses on the regulatory mechanism of histone methylation, and the diagnosis and chemotherapy of PC based on it. The future of epigenetic modification in PC research is also discussed.

Efficient biosynthesis of (2S, 3R)-4-methylsulfonylphenylserine by artificial self-assembly of enzyme complex combined with an intensified acetaldehyde elimination system.

(2S, 3R)-4-methylsulfonylphenylserine [(2S, 3R)-MPS], a key chiral precursor for antibiotics florfenicol and thiamphenicol, could be asymmetrically synthesized by l-threonine transaldolase (LTTA) coupled with an acetaldehyde elimination system. The low efficiency of acetaldehyde elimination system blocked further accumulation of (2S, 3R)-MPS. To address this issue, strengthening acetaldehyde elimination system and enzyme self-assembly strategy were combined to accelerate biosynthesis of (2S, 3R)-MPS. The new multi-enzyme cascade with intensified acetaldehyde elimination system BL21 (PsLTTAD2/ScADH/BtGDH) could produce (2S, 3R)-MPS with a titer of 157.6 mM, 1.7-folds than that produced by the original system BL21 (PsLTTAD2/ApADH/CbFDH). Moreover, self-assembly of PsLTTAD2 and ScADH by respective fusion of SpyTag and SpyCatcher were carried out to develop a self-assembled multi-enzyme cascade BL21 (ST-PsLTTAD2/SC-ScADH/BtGDH). As a result, the yield of (2S, 3R)-MPS was up to 248.1 mM with 95% de. As far as we knew, that represented the highest yield of (2S, 3R)-MPS by enzymatic synthesis, and therefore was a promising and green route for industrial production of this valuable compound.

Multi-enzyme cascade for improving ß-hydroxy-α-amino acids production by engineering L-threonine transaldolase and combining acetaldehyde elimination system.

L-threonine transaldolase(PsLTTA) could asymmetric synthesize ß-hydroxy-α-amino acids (HAAs) with excellentstereoselectivity, while the poor yield limited its further application. Here we provided a combinatorial strategy to improve HAAs production, by directed evolution of PsLTTA towards enhanced activity and introducing an acetaldehyde elimination system to avoid acetaldehyde over-accumulation. A novel high throughput screening (HTS) method for evaluating PsLTTA activity was developed andapplied for directed evolution of PsLTTA. Subsequently, we co-expressedalcohol dehydrogenase andformate dehydrogenase to construct an acetaldehyde elimination system toremove acetaldehyde inhibition.Moreover, the above positive strategies were integrated. As a result,the (2S,3R)-p-methylsulfonyl phenylserine yield reached 154.0 mM andwith 94.6% devalue, the highest productivity and stereoselectivity of (2S,3R)-HAAs reported by enzymatic synthesis so far. Taken together, our studies provided an efficient and green route for chiral synthesis of (2S,3R)-HAAs, which might contribute to the industrialization production of these useful building blocks.

Mutagenesis on the surface of a ß-agarase from Vibrio sp. ZC-1 increased its thermo-stability.

The recombinant rAgaZC-1 was a family GH50 ß-agarase from Vibrio sp. ZC-1 (CICC 24670). In this paper, the mutant D622G (i.e., mutate the aspartic acid at position 622 to glycine) had better thermo-stability than rAgaZC-1, showing 1.5℃ higher T5010 (the temperature at which the half-time is 10 min) and 4-folds of half-time at 41℃, while they had almost same optimum temperature (38.5℃), optimum pH (pH6.0) and catalytic efficiency. Thermal deactivation kinetical analysis showed that D622G had higher activation energy for deactivation, enthalpy and Gibbs free energy than rAgaZC-1, indicating that more energy is required by D622G for deactivation. Substrate can protect agarase against thermal inactivation, especially D622G. Hence the yield of agarose hydrolysis catalyzed by D622G was higher than that by rAgaZC-1. The models of D622G and rAgaZC-1 predicted by homology modeling were compared to find that it is the improved distribution of surface electrostatic potential, great symmetric positive potential and more hydrophobic interactions of D622G that enhance the thermo-stability.

Preparation of bioactive neoagaroligosaccharides through hydrolysis of Gracilaria lemaneiformis agar: A comparative study.

Hydrolysis of Gracilaria lemaneiformis agar by ß-agarase was compared with HCl hydrolysis. The results showed that optimum catalysis conditions for the ß-agarase were pH 7.0 at 45°C. Mass spectroscopy, thin-layer chromatography and GPC results showed that the polymerization degrees of the hydrolysis products by the ß-agarase were mainly four, six and eight (more specific than the hydrolysate by HCl). The enzymatic degradation products of agar were distinctly different from those of HCl hydrolysis in the ratios among galactose and 3,6-anhydro-galactose and sulfate group contents. The NMR spectrometry proved that the products of ß-agarase were neoagaroligosaccharides, which was not found in the agarolytic products by HCl. The neoagarotetraose inhibited tyrosinase activity competitively with the KI value of 16.0mg/ml. Hydroxyl radical-scavenging ability of neoagaroligosaccharides was much greater than that of agar HCl hydrolysate. This work suggests that neoagaroligosaccharide products produced by our ß-agarase could be more effective in function than products from acid hydrolysis.

Inactivation kinetics and conformation change of Hypocrea orientalis ß-glucosidase with guanidine hydrochloride.

The relationship between unfolding and inactivation of Hypocrea orientalis ß-glucosidase has been investigated for the first time. The secretion of ß-glucosidase from H. orientalis is induced by raw cassava residues. The enzyme was 75 kD without glycosylation. Guanidine hydrochloride (GuHCl) could reversibly inactivate the enzyme with an estimated IC50 value of 0.4 M. The inactivation kinetics model by GuHCl has been established and the microscopic inactivation rate constants are determined. The values of forward inactivation rate constants of free enzyme are found to be larger than that of substrate-enzyme complex suggesting the enzyme could be protected by substrate during denaturation. Conformational change of the enzyme during denaturation is observed as the intrinsic fluorescence emission peaks appeared red-shift (334-354 nm) with intensity decreased following increase of GuHCl concentrations. Inactivation extent is found to be greater than conformation change of the whole enzyme, indicating that the active site of H. orientalis ß-glucosidase might be a more flexible region than the whole enzyme.

Heat inactivation kinetics of Hypocrea orientalis ß-glucosidase with enhanced thermal stability by glucose.

Thermal inactivation kinetics of Hypocrea orientalis ß-glucosidase and effect of glucose on thermostability of the enzyme have been determined in this paper. Kinetic studies showed that the thermal inactivation was irreversible and first-order reaction. The microscopic rate constants for inactivation of free enzyme and substrate-enzyme complex were both determined, which suggested that substrates can protect ß-glucosidase against thermal deactivation effectively. On the other hand, glucose was found to protect ß-glucosidase from heat inactivation to remain almost whole activity below 70°C at 20mM concentration, whereas the apparent inactivation rate of BG decreased to be 0.3×10(-3)s(-1) in the presence of 5mM glucose, smaller than that of sugar-free enzyme (1.91×10(-3)s(-1)). The intrinsic fluorescence spectra results showed that glucose also had stabilizing effect on the conformation of BG against thermal denaturation. Docking simulation depicted the interaction mode between glucose and active residues of the enzyme to produce stabilizing effect.

Enzymatic saccharification of cassava residues and glucose inhibitory kinetics on ß-glucosidase from Hypocrea orientalis.

Cassava residues are byproducts of the starch industry containing abundant cellulose for bioproduction of green fuel. To obtain maximum sugar yields from cassava residues, the optimal conditions for hydrolyzing the residues were determined using cellulase prepared from a novel Hypocrea orientalis strain. The optimal pH value and optimal temperature for the cellulase hydrolysis were 5.0 and 50 °C, respectively. The concentration of NaOH was determined to be 1% for pretreatment of cassava residues to gain enough soluble sugars suitably. The yield of released sugars was 10 mg/mL in the optimal conditions after 24 h of reaction, which was similar to that of bagasse and wheat grass. Inhibition kinetics of H. orientalis ß-glucosidase (BG) by glucose was first studied using the progress-of-substrate-reaction method as described by Tsou (Tsou, C. L. Adv. Enzymol. Related Areas Mol. Biol. 1988, 61, 381-436), and the microscopic inhibition rate constants of glucose were determined. The results showed that glucose could inhibit BG reversibly and competitively. The rate constants of forward (k(+0)) and reverse (k(-0)) reaction were measured to be 4.88 × 10(-4) (mM·s)(-1) and 2.7 × 10(-4) s(-1), respectively. Meanwhile, the inhibition was more significant than that of L-glucose, D-mannose, D-galactose, D-aminoglucose, acetyl-D-glucose, and D-fructose. This work reveals how to increase sugar yields and reduce product inhibition during enzymatic saccharification of cellulose.

Cellulase hydrolysis of rice straw and inactivation of endoglucanase in urea solution.

In order to optimize the cellulase (from Aspergillus glaucus) hydrolysis of pretreated rice straw, the effects of varying enzyme concentration, temperature, and pH were studied. The best experimental conditions found to degrade the pretreated rice straws were 24 h of incubation at 55 °C and pH 5.0, with an enzyme concentration of 48 mg/L. Urea is one of the important nitrogen sources used in fungi culture, but it is also a denaturant. The model of denaturation of endoglucanase (EG) in urea solutions was established. The denaturation was a slow, reversible reaction. Determination of microscopic rate constants showed k(+0) > k'(+0), indicating that EG was protected by the substrate to a certain extent during denaturation. Comparison with the results from fluorescence emission spectroscopy revealed that the inactivation of EG occurred before the marked conformational changes could be detected.