Effects of Exogenous Isosteviol on the Physiological Characteristics of Brassica napus Seedlings under Salt Stress.

In this paper, the effect of isosteviol on the physiological metabolism of Brassica napus seedlings under salt stress is explored. Brassica napus seeds (Qinyou 2) were used as materials, and the seeds were soaked in different concentrations of isosteviol under salt stress. The fresh weight, dry weight, osmotic substance, absorption and distribution of Na+, K+, Cl-, and the content of reactive oxygen species (ROS) were measured, and these results were combined with the changes shown by Fourier transform infrared spectroscopy (FTIR). The results showed that isosteviol at an appropriate concentration could effectively increase the biomass and soluble protein content of Brassica napus seedlings and reduce the contents of proline, glycine betaine, and ROS in the seedlings. Isosteviol reduces the oxidative damage to Brassica napus seedlings caused by salt stress by regulating the production of osmotic substances and ROS. In addition, after seed soaking in isosteviol, the Na+ content in the shoots of the Brassica napus seedlings was always lower than that in the roots, while the opposite was true for the K+ content. This indicated that under salt stress the Na+ absorbed by the Brassica napus seedlings was mainly accumulated in the roots and that less Na+ was transported to the shoots, while more of the K+ absorbed by the Brassica napus seedlings was retained in the leaves. It is speculated that this may be an important mechanism for Brassica napus seedlings to relieve Na+ toxicity. The spectroscopy analysis showed that, compared with the control group (T1), salt stress increased the absorbance values of carbohydrates, proteins, lipids, nucleic acids, etc., indicating structural damage to the plasma membrane and cell wall. The spectra of the isosteviol seed soaking treatment group were nearly the same as those of the control group (T1). The correlation analysis shows that under salt stress the Brassica napus seedling tissues could absorb large amounts of Na+ and Cl- to induce oxidative stress and inhibit the growth of the plants. After the seed soaking treatment, isosteviol could significantly reduce the absorption of Na+ by the seedling tissues, increase the K+ content, and reduce the salt stress damage to the plant seedlings. Therefore, under salt stress, seed soaking with isosteviol at an appropriate concentration (10-9~10-8 M) can increase the salt resistance of Brassica napus seedlings by regulating their physiological and metabolic functions.

Engineering Corynebacterium glutamicum for de novo production of 2-phenylethanol from lignocellulosic biomass hydrolysate.

BACKGROUND: 2-Phenylethanol is a specific aromatic alcohol with a rose-like smell, which has been widely used in the cosmetic and food industries. At present, 2-phenylethanol is mainly produced by chemical synthesis. The preference of consumers for "natural" products and the demand for environmental-friendly processes have promoted biotechnological processes for 2-phenylethanol production. Yet, high 2-phenylethanol cytotoxicity remains an issue during the bioproduction process. RESULTS: Corynebacterium glutamicum with inherent tolerance to aromatic compounds was modified for the production of 2-phenylethanol from glucose and xylose. The sensitivity of C. glutamicum to 2-phenylethanol toxicity revealed that this host was more tolerant than Escherichia coli. Introduction of a heterologous Ehrlich pathway into the evolved phenylalanine-producing C. glutamicum CALE1 achieved 2-phenylethanol production, while combined expression of the aro10. Encoding 2-ketoisovalerate decarboxylase originating from Saccharomyces cerevisiae and the yahK encoding alcohol dehydrogenase originating from E. coli was shown to be the most efficient. Furthermore, overexpression of key genes (aroGfbr, pheAfbr, aroA, ppsA and tkt) involved in the phenylpyruvate pathway increased 2-phenylethanol titer to 3.23 g/L with a yield of 0.05 g/g glucose. After introducing a xylose assimilation pathway from Xanthomonas campestris and a xylose transporter from E. coli, 3.55 g/L 2-phenylethanol was produced by the engineered strain CGPE15 with a yield of 0.06 g/g xylose, which was 10% higher than that with glucose. This engineered strain CGPE15 also accumulated 3.28 g/L 2-phenylethanol from stalk hydrolysate. CONCLUSIONS: In this study, we established and validated an efficient C. glutamicum strain for the de novo production of 2-phenylethanol from corn stalk hydrolysate. This work supplied a promising route for commodity 2-phenylethanol bioproduction from nonfood lignocellulosic feedstock.

De novo Synthesis of 2-phenylethanol from Glucose by Metabolically Engineered Escherichia coli.

2-Phenylethanol (2- PE) is an aromatic alcohol with wide applications, but there is still no efficient microbial cell factory for 2-PE based on Escherichia coli. In this study, we constructed a metabolically engineered E. coli capable of de novo synthesis of 2-PE from glucose. Firstly, the heterologous styrene-derived and Ehrlich pathways were individually constructed in an L-Phe producer. The results showed that the Ehrlich pathway was better suited to the host than the styrene-derived pathway, resulting in a higher 2-PE titer of ∼0.76 ± 0.02 g/L after 72 h of shake flask fermentation. Furthermore, the phenylacetic acid synthase encoded by feaB was deleted to decrease the consumption of 2-phenylacetaldehyde, and the 2-PE titer increased to 1.75 ± 0.08 g/L. As phosphoenolpyruvate (PEP) is an important precursor for L-Phe synthesis, both the crr and pykF genes were knocked out, leading to ∼35% increase of the 2-PE titer, which reached 2.36 ± 0.06 g/L. Finally, a plasmid-free engineered strain was constructed based on the Ehrlich pathway by integrating multiple ARO10 cassettes (encoding phenylpyruvate decarboxylases) and overexpressing the yjgB gene. The engineered strain produced 2.28 ± 0.20 g/L of 2-PE with a yield of 0.076 g/g glucose and productivity of 0.048 g/L/h. To our best knowledge, this is the highest titer and productivity ever reported for the de novo synthesis of 2-PE in E. coli. In a 5-L fermenter, the 2-PE titer reached 2.15 g/L after 32 h of fermentation, suggesting that the strain has the potential to efficiently produce higher 2-PE titers following further fermentation optimization.

Increasing glycolysis by deletion of kcs1 and arg82 improved S-adenosyl-L-methionine production in Saccharomyces cerevisiae.

Reprogramming glycolysis for directing glycolytic metabolites to a specific metabolic pathway is expected to be useful for increasing microbial production of certain metabolites, such as amino acids, lipids or considerable secondary metabolites. In this report, a strategy of increasing glycolysis by altering the metabolism of inositol pyrophosphates (IPs) for improving the production of S-adenosyl-L-methionine (SAM) for diverse pharmaceutical applications in yeast is presented. The genes associated with the metabolism of IPs, arg82, ipk1 and kcs1, were deleted, respectively, in the yeast strain Saccharomyces cerevisiae CGMCC 2842. It was observed that the deletions of kcs1 and arg82 increased SAM by 83.3 % and 31.8 %, respectively, compared to that of the control. In addition to the improved transcription levels of various glycolytic genes and activities of the relative enzymes, the levels of glycolytic intermediates and ATP were also enhanced. To further confirm the feasibility, the kcs1 was deleted in the high SAM-producing strain Ymls1ΔGAPmK which was deleted malate synthase gene mls1 and co-expressed the Acetyl-CoA synthase gene acs2 and the SAM synthase gene metK1 from Leishmania infantum, to obtain the recombinant strain Ymls1Δkcs1ΔGAPmK. The level of SAM in Ymls1Δkcs1ΔGAPmK reached 2.89 g L-1 in a 250-mL flask and 8.86 g L-1 in a 10-L fermentation tank, increasing 30.2 % and 46.2 %, respectively, compared to those levels in Ymls1ΔGAPmK. The strategy of increasing glycolysis by deletion of kcs1 and arg82 improved SAM production in yeast.

A stepwise control strategy for glutathione synthesis in Saccharomyces cerevisiae based on oxidative stress and energy metabolism.

A stepwise control strategy for enhancing glutathione (GSH) synthesis in yeast based on oxidative stress and energy metabolism was investigated. First, molasses and corn steep liquor were selected and fed as carbon source mixture at a flow rate of 1.5 g/L/h and 0.4 g/L/h, respectively, for increasing cell density in a 10 L fermenter. When the biomass reached 90 g/L, the KMnO4 sustained-release particles, composed of 1.5% KMnO4, 3% stearic acid, 2% polyethylene glycol and 3% agar powder, were prepared and added to the fermentation broth for maintaining the oxidative stress. The results showed that the maximum GSH accumulation of the group fed KMnO4 sustained-release particles was 39.0% higher than that of KMnO4-fed group. In addition to the improved average GSH productivity and average specific production rate, the activities of GSH peroxidase, γ-glutamylcysteine synthetase and GSH reductase, enzymes taking part in GSH metabolism, were also significantly enhanced by KMnO4 sustained-release particles feeding. Finally, 6 g/L sodium citrate fed as an energy adjuvant elevated the intracellular ATP level for further enhancing GSH production. Through the above stepwise strategy, the GSH accumulation reached 5.76 g/L, which was 2.84-fold higher than that of the control group. The stepwise control strategy based on oxidative stress and energy metabolism significantly improved GSH accumulation in yeast.

Reshaping the substrate binding region of (R)-selective ω-transaminase for asymmetric synthesis of (R)-3-amino-1-butanol.

(R)-Selective ω-transaminase (ω-TA) is a key enzyme for the asymmetric reductive amination of carbonyl compounds to produce chiral amines which are essential parts of many therapeutic compounds. However, its practical industrial applications are hindered by the low catalytic efficiency and poor thermostability of naturally occurring enzymes. In this work, we report the molecular modification of (R)-selective ω-TA from Aspergillus terreus (AtTA) to allow asymmetric reductive amination of 4-hydroxy-2-butanone, producing (R)-3-amino-1-butanol. Based on substrate docking analysis, 4 residues in the substrate tunnel and binding pocket of AtTA were selected as mutation hotspots. The screening procedure was facilitated by the construction of a "small-intelligent" library and the use of thin-layer chromatography for preliminary screening. The resulting mutant AtTA-M5 exhibited a 9.6-fold higher kcat/Km value and 9.4 °C higher [Formula: see text] than that of wild-type AtTA. Furthermore, the conversion of 20 and 50 g L-1 4-hydroxy-2-butanone by AtTA-M5 reached 90.8% and 79.1%, suggesting significant potential for production of (R)-3-amino-1-butanol. Under the same conditions, wild-type AtTA achieved less than 5% conversion. Moreover, the key mutation (S215P in AtTA) was validated in 7 other (R)-selective ω-TAs, indicating its general applicability in improving the catalytic efficiency of homologous (R)-selective ω-TAs.

Metabolic engineering of Escherichia coli and in silico comparing of carboxylation pathways for high succinate productivity under aerobic conditions.

A novel aerobic succinate production system was strategically designed that allows Escherichia coli to produce and accumulate succinate with high specific productivity under aerobic conditions. Mutations in the tricarboxylic acid cycle (sdhA, iclR) and byproduct formation pathways (poxB, ackA-pta, mgsA) of E. coli were created to construct the glyoxylate cycle and oxidative branch of the TCA cycle for aerobic succinate production. Strain ZJG13 (ΔsdhA, ΔackA-pta, ΔpoxB, ΔmgsA, ΔiclR) exhibited normal growth behavior and accumulated succinate with an average specific productivity of 0.50mmolg CDW(-1)h(-1) during the fermentation. The glyoxylate shunt operon aceKBA was overexpressed by introducing plasmid pT9aceKAB to ZJG13; the resulting strain had minor effect on productivity improvement. To fully understand the effect of the carboxylation reactions on succinate production, three reactions catalyzed by pyruvate carboxylase (PYC), malic enzyme (MAEA) and phosphoenolpyruvate carboxylase (PPC) were analyzed by a Computational Approach for Strain Optimization aiming at high Productivity (CASOP). Based on the CASOP analysis, carboxylation reaction catalyzed by PYC was the most suitable one to obtain high productivity. When pyc was overexpressed in ZJG13, the specific succinate productivity further increased to 0.76mmolg CDW(-1)h(-1). Fed-batch culture of the strain ZJG13/pT184pyc led to a titer of 36.1g/L succinate, with a specific productivity of 2.75mmolg CDW(-1)h(-1) which stands for the highest value among currently reported aerobic bacterial succinate producers. These results indicate that the CASOP strategy is useful as a guiding tool for the rational strain design with high productivity.

Aerobic production of succinate from arabinose by metabolically engineered Corynebacterium glutamicum.

Arabinose is considered as an ideal feedstock for the microbial production of value-added chemicals due to its abundance in hemicellulosic wastes. In this study, the araBAD operon from Escherichia coli was introduced into succinate-producing Corynebacterium glutamicum, which enabled aerobic production of succinate using arabinose as sole carbon source. The engineered strain ZX1 (pXaraBAD, pEacsAgltA) produced 74.4 mM succinate with a yield of 0.58 mol (mol arabinose)(-1), which represented 69.9% of the theoretically maximal yield. Moreover, this strain produced 110.2 mM succinate using combined substrates of glucose and arabinose. To date, this is the highest succinate production under aerobic conditions in minimal medium.

Improved succinate production in Corynebacterium glutamicum by engineering glyoxylate pathway and succinate export system.

A dual route for anaerobic succinate production was engineered into Corynebacterium glutamicum. The glyoxylate pathway was reconstructed by overexpressing isocitrate lyase, malate synthase and citrate synthase. The engineered strain produced succinate with a yield of 1.34 mol (mol glucose)(-1). Further overexpression of succinate exporter, SucE, increased succinate yield to 1.43 mol (mol glucose)(-1). Metabolic flux analysis revealed that the glyoxylate pathway was further activated by engineering succinate export system. Using an anaerobic fed-batch fermentation process, the final strain produced 926 mM succinate (= 109 g l(-1)) with an overall volumetric productivity of 9.4 mM h(-1) and an average yield of 1.32 mol (mol glucose)(-1).

Engineering of acetate recycling and citrate synthase to improve aerobic succinate production in Corynebacterium glutamicum.

Corynebacterium glutamicum lacking the succinate dehydrogenase complex can produce succinate aerobically with acetate representing the major byproduct. Efforts to increase succinate production involved deletion of acetate formation pathways and overexpression of anaplerotic pathways, but acetate formation could not be completely eliminated. To address this issue, we constructed a pathway for recycling wasted carbon in succinate-producing C. glutamicum. The acetyl-CoA synthetase from Bacillus subtilis was heterologously introduced into C. glutamicum for the first time. The engineered strain ZX1 (pEacsA) did not secrete acetate and produced succinate with a yield of 0.50 mol (mol glucose)(-1). Moreover, in order to drive more carbon towards succinate biosynthesis, the native citrate synthase encoded by gltA was overexpressed, leading to strain ZX1 (pEacsAgltA), which showed a 22% increase in succinate yield and a 62% decrease in pyruvate yield compared to strain ZX1 (pEacsA). In fed-batch cultivations, strain ZX1 (pEacsAgltA) produced 241 mM succinate with an average volumetric productivity of 3.55 mM h(-1) and an average yield of 0.63 mol (mol glucose) (-1), making it a promising platform for the aerobic production of succinate at large scale.