Engineered E. coli for D-pantothenic acid production with an acetolactate isomeroreductase mutant.

D-Pantothenic acid, as a momentous vitamin, is extensively applied to feed, medicine, cosmetics and other fields. However, there are still limitations to produce D-pantothenic acid by microbial fermentation at present. In this paper, we constructed a recombinant strain for D-pantothenic acid production by blocking the organic acid pathway, boosting pyruvate biosynthesis, relieving feedback inhibition of acetolactate synthase, improving glucose intake capacity, and modifying essential genes in the metabolic pathway. In addition, a new acetolactate isomeroreductase mutant V412A origin from Escherichia coli (EcAHAIR) encoded by ilvC was obtained to explore its substrate promiscuity. Compared with the wild type, the variant EcAHAIR-V412A has reduced steric hindrance and enhanced intermolecular forces, resulting in a high affinity for 2-acetolactate. Eventually, the fermentation production of the final strain DPAN19/trc-ilvCV412A reached 4.65 g/L, increased by 192.5% compared with strain DPA8 in shake flask cultivation and produced 62.82 g/L D-pantothenic acid in a 5 L bioreactor. The metabolic engineering strategies and enzyme modification approaches described in this paper provide a particular perspective for the bio-manufacturing of D-pantothenic acid, branched-chain amino acids and its derivates.

Multiplex modification of Yarrowia lipolytica for enhanced erythritol biosynthesis from glycerol through modularized metabolic engineering.

Erythritol is a novelty 4-carbon sugar polyol and has great potential to be used as the precursor of some platform chemicals. The increasing cost of glucose poses researchers shifting insights to the cheaper biodiesel raw materials. Herein, we engineered a non-degradation, non-byproducts Yarrowia lipolytica for the erythritol production with high-titer from glycerol. Initially, the degradation and competition modules were blocked by URA3 counter-selection marker. Subsequently, a shortened biosynthetic pathway was explored to elevate its synthetic flux by multi-modules combination expression of functional genes. Furthermore, a screened glycerol transporter ScFPS1 was integrated into ERY6 genome to promote the glycerol uptake. The constructed strain ERY8 produced 176.66 g/L erythritol in the 5-L bioreactor with a yield and productivity of 0.631 g/g and 1.23 g/L/h, respectively, which achieved the highest fermentation production efficiency till date. This study proposed a novel multi-modules combination strategy for effectively engineering Y. lipolytica to produce erythritol using glycerol.

Recent advances in metabolic regulation and bioengineering of gibberellic acid biosynthesis in Fusarium fujikuroi.

The plant growth hormone gibberellic acid (GA3), as one of the representative secondary metabolites, is widely used in agriculture, horticulture and brewing industry. GA3 is detected in both plants and several fungi with the ability to stimulate plant growth. Currently, the main mode of industrial production of GA3 is depended on the microbial fermentation via long-period submerged fermentation using Fusarium fujikuroi as the only producing strain, qualified for its natural productivity. However, the demand of large-sale industrialization of GA3 was still restricted by the low productivity. The biosynthetic route of GA3 in F. fujikuroi is now well-defined. Furthermore, the multi-level regulation mechanisms involved in the whole network of GA3 production have also been gradually unveiled by the past two decades based on the identification and characterization of several global regulators and their mutual functions. Combined with the quick development of genetic manipulation techniques, the rational modification of producing strain F. fujikuroi development become practical for higher productivity achievement. Herein, we review the latest advances in the molecular regulation of GA3 biosynthesis in F. fujikuroi and conclude a comprehensive network involving nitrogen depression, global regulator, histone modification and G protein signaling pathway. Correspondingly, the bioengineering strategies covering conventional random mutation, genetic manipulating platform development, metabolic edition and fermentation optimization were also systematically proposed.

Module engineering coupled with omics strategies for enhancing D-pantothenate production in Escherichia coli.

Biosynthesis of D-pantothenate has been widely studied as D-pantothenate is one kind of important vitamins used in food and pharmaceuticals. However, the engineered strain for D-pantothenate production was focused solely on the main biosynthetic pathway, while other important factors such as one carbon unit were ignored. Here the systematic modular engineering on different factors coupled with omics analysis were studied in Escherichia coli for efficient D-pantothenate production. Through reinforcing the precursor pool, refactoring the one carbon unit generation pathway, optimization of reducing power and energy supply, the D-pantothenate titer reached 34.12 g/L with the yield at 0.28 g/g glucose under fed-batch fermentation in 5-L bioreactor. With a further comparative transcriptome and metabolomics studies, the addition of citrate was implemented and 45.35 g/L D-pantothenate was accumulated with a yield of 0.31 g/g glucose. The systematic modular engineering coupled with omics studies provide useful strategies for the industrial production of D-pantothenate.

Electrochemical preparation of surface molecularly imprinted poly(3-aminophenylboronic acid)/MWCNTs nanocomposite for sensitive sensing of epinephrine.

A nanocomposite with multi-walled carbon nanotubes (MWCNTs) coated with surface molecularly imprinted polymers (MIPs) poly(3-aminophenylboronic acid) (PAPBA) was successfully prepared via potentiodynamic electropolymerization and tested as an effective electrochemical material for epinephrine (EP) detection. The morphology and properties of the sensing material were characterized with scanning electron microscopy and electrochemical impedance spectroscopy. Compared with MWCNTs or non-imprinted polymers PAPBA modified MWCNTs electrodes, the PAPBA(MIPs)/MWCNTs modified electrode showed a lower charge transfer resistance and enhanced electrochemical performance for EP detection. The improved performance can be attributed to the large amount of specific imprinted cavities with boric acid group which can selectively adsorb EP molecule and the synergistic effect between MWCNTs and PAPBA(MIPs). The effects of pH, the molar ratio between monomer and template molecule, the cycle number of electropolymerization, and the accumulation time of the modified electrode on the sensing performance were investigated. It was found that under the optimal conditions, the PAPBA(MIPs)/MWCNTs sensor could effectively recognize EP from many possible interferents of higher concentration within a wide linear range of 0.2-800 µmol·L-1, with low detection limit of 35 nmol·L-1, high sensitivity and good discrimination. The detection of EP in human serum and real injection samples using the PAPBA(MIPs)/MWCNTs sensor also gave satisfactory results.

Electrochemical preparation of two nanostructured poly(sulfosalicylic acid) films with different morphologies and properties for selective sensing of dopamine: Comparative study.

Two nanostructured poly(sulfosalicylic acid) (PSA) films were synthesized from room temperature ionic liquid (RTIL) or aqueous solution on a glassy carbon electrode (GCE) via potentiodynamic electropolymerization. The morphology and properties of the PSA films were characterized with scanning electron microscopy (SEM), scanning probe microscopy (SPM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). It was found that solvent had a major influence on the morphology and electrochemical properties of the resultant PSA films. The PSA(Ι) film, which was prepared from RTIL, consists of granular particles with cracks, whereas the PSA(II) film prepared from aqueous solution consists of nano-triangles with a more compact surface. The blocking effect of the PSA(Ι) film for the [Fe(CN)6]3-/4- electrochemical probe is much stronger, and a remarkably enhanced voltammetric response of the [Ru(NH3)6]3+ electrochemical probe can be observed for the PSA(II) film. When it is used to detect dopamine in the presence of a high concentration of ascorbic acid, PSA(II)/GCE has three linear parts with better discrimination and a detection limit of 0.03µM. For PSA(Ι)/GCE, there are two linear parts with a detection limit of 0.05µM. However, the reproducibility and storage stability of PSA(Ι)/GCE are better than those of PSA(ΙI)/GCE.

Crystal structure of N-(7-di-bromo-methyl-5-methyl-1,8-naphthyridin-2-yl)benzamide-pyrrolidine-2,5-dione (1/1).

The title compound, C17H13Br2N3O·C4H5NO2, is a co-crystal of N-(7-di-bromo-methyl-5-methyl-1,8-naphthyridin-2-yl)benzamide and pyrrolidine-2,5-dione (succinimide). The benzamide mol-ecule exhibits pseudo-mirror symmetry, with an r.m.s. deviation of the non-H atoms of 0.09 Š(except for the two Br atoms). The angle between the least-squares planes of the two mol-ecules is 26.2 (2)°. In the crystal, the two mol-ecules are mutually linked by N-H⋯O and N-H⋯N hydrogen bonds. The packing is consolidated by C-H⋯(O,N) hydrogen bonds and π-π stacking inter-actions.

Crystal structure of 9-(di-bromo-meth-yl)-1,1-di-fluoro-3,7-dimethyl-1H-[1,3,5,2]oxadi-aza-borinino[3,4-a][1,8]naphthyridin-11-ium-1-uide.

The mol-ecule of the title 1,8-naphthyridine-BF2 derivative, C12H10BBr2F2N3O, is located on a mirror plane running parallel to the entire ring system and the attached methyl C atoms. Individual mol-ecules are stacked along the b-axis direction. The cohesion in the crystal structure is accomplished by C-H⋯F hydrogen bonds and additional off-set π-π inter-actions [centroid-to-centroid distance = 3.6392 (9) Å, slippage 0.472 Å], leading to the formation of a three-dimensional supra-molecular network.

A comparison between three-dimensional conformal radiotherapy combined with interventional treatment and interventional treatment alone for hepatocellular carcinoma with portal vein tumour thrombosis.

INTRODUCTION: We investigated the therapeutic effects of three-dimensional conformal radiotherapy combined with transcatheter arterial chemoembolisation (TACE) for hepatocellular carcinoma (HCC) with portal vein tumour thrombosis (PVTT). METHODS: Sixty-three HCC patients with PVTT were divided into two groups. Group A (30 patients) was treated with three-dimensional conformal radiotherapy followed by 2-3 series of TACE, while group B (33 patients) was only treated with TACE. RESULTS: The 1- and 2-year survival rates of group A were 62.40% and 20.81%, respectively, with a mean survival time of 13.0 months. The 1- and 2-year survival rates of group B were 56.49% and 18.83%, respectively, with a mean survival time of 9.0 months. There were significant differences between the two groups (log-rank chi-square value = 3.950, P = 0.047). CONCLUSION: Three-dimensional conformal radiotherapy combined with TACE can significantly improve clinical outcomes in patients with HCC and PVTT compared with TACE alone.