Production of dihydroxyacetone from glycerol by engineered Escherichia coli cells co-expressing gldA and nox genes

Glycerol can be converted into more valuable compound dihydroxyacetone by the nicotinamide adenine dinucleotide (NAD+)-dependent glycerol dehydrogenase. However, it is economically prohibitive to produce dihydroxyacetone using purified glycerol dehydrogenase at the expense of a stoichiometric amount of the cofactor NAD+. In this study, Escherichia coli was engineered for dihydroxyacetone production by enhancing its glycerol dehydrogenase activity and introducing NADH oxidase activity. Under optimized conditions, dihydroxyacetone productivity reached 0.13 g/h/g wet cell mass by recombinant E. coli D4 (pET-24b-gldA+nox) cells co-expressing gldA gene from E. coli and nox gene from Enterococcus faecalis. It was interesting to note that exogenous NAD+ greatly improved dihydroxyacetone production for the whole-cell biotransformation process. These results should be useful for the development of advanced bioprocess in terms of glycerol utilization.Keywords: Dihydroxyacetone, Glycerol dehydrogenase, NAD+, whole-cell biotransformation, Escherichia coliAfrican Journal of Biotechnology Vol. 12(27), pp. 4387-439

Engineering NAD+ availability for Escherichia coli whole-cell biocatalysis: A case study for dihydroxyacetone production

Background: Whole-cell redox biocatalysis has been intensively explored for the production of valuable compounds because excellent selectivity is routinely achieved. Although the cellular cofactor level, redox state and the corresponding enzymatic activity are expected to have major effects on the performance of the biocatalysts, our ability remains limited to predict the outcome upon variation of those factors as well as the relationship among them. Results: In order to investigate the effects of cofactor availability on whole-cell redox biocatalysis, we devised recombinant Escherichia coli strains for the production of dihydroxyacetone (DHA) catalyzed by the NAD + -dependent glycerol dehydrogenase (GldA). In this model system, a water-forming NAD + oxidase (NOX) and a NAD + transporter (NTT4) were also co-expressed for cofactor regeneration and extracellular NAD + uptake, respectively. We found that cellular cofactor level, NAD + /NADH ratio and NOX activity were not only strain-dependent, but also growth condition-dependent, leading to significant differences in specific DHA titer among different whole-cell biocatalysts. The host E. coli DH5α had the highest DHA specific titer of 0.81\ua0g/g DCW with the highest NAD + /NADH ratio of 6.7 and NOX activity of 3900 U. The biocatalyst had a higher activity when induced with IPTG at 37\ub0C for 8\ua0h compared with those at 30\ub0C for 8\ua0h and 18\ua0h. When cells were transformed with the ntt4 gene, feeding NAD + during the cell culture stage increased cellular NAD(H) level by 1.44 fold and DHA specific titer by 1.58 fold to 2.13\ua0g/g DCW . Supplementing NAD + during the biotransformation stage was also beneficial to cellular NAD(H) level and DHA production, and the highest DHA productivity reached 0.76\ua0g/g DCW /h. Cellular NAD(H) level, NAD + /NADH ratio, and NOX and GldA activity dropped over time during the biotransformation process.Conclusions: High NAD + /NADH ratio driving by NOX was very important for DHA production. Once cofactor was efficiently cycled, high cellular NAD(H) level was also beneficial for whole-cell redox biocatalysis. Our results indicated that NAD + transporter could be applied to manipulate redox cofactor level for biocatalysis. Moreover, we suggested that genetically designed redox transformation should be carefully profiled for further optimizing whole-cell biocatalysis. \ua9 2013 Zhou et al.; licensee BioMed Central Ltd

Engineering yeast for high-level production of diterpenoid sclareol

The diterpenoid sclareol is an industrially important precursor for alternative sustainable supply of ambergris. However, its current production from plant extraction is neither economical nor environmental-friendly, since it requires laborious and cost-intensive purification procedures and plants cultivation is susceptible to environmental factors. Engineering cell factories for bio-manufacturing can enable sustainable production of natural products. However, stringent metabolic regulation poses challenges to rewire cellular metabolism for overproduction of compounds of interest. Here we used a modular approach to globally rewire the cellular metabolism for improving sclareol production to 11.4 g/L in budding yeast Saccharomyces cerevisiae, the highest reported diterpenoid titer in microbes. Metabolic flux analysis showed that modular balanced metabolism drove the metabolic flux toward the biosynthesis of targeted molecules, and transcriptomic analysis revealed that the expression of central metabolism genes was shaped for a new balanced metabolism, which laid a foundation in extensive metabolic engineering of other microbial species for sustainable bio-production

Amine-responsive bilayer films with improved illumination stability and electrochemical writing property for visual monitoring of meat spoilage

Amine-responsive bilayer films were developed by using agar (AG), anthocyanins (AN), gellan gum (GG) and TiO2 nanoparticles for visual monitoring of meat spoilage. The AG-AN layer worked as the sensing layer to volatile amines, while GG-TiO2 layer served as the light barrier layer and simultaneously the conducting layer to improve the illumination stability and electrochemical writing ability of the AG-AN layer, respectively. The Scanning electron microscopy (SEM) images and X-ray diffraction (XRD) spectra indicated the successful fabrication of bilayer films. Illumination experiments showed that the incorporation of TiO2 in the GG-TiO2 layer significantly improved the illumination stability of AN in the AG-AN layer. Meanwhile, electrochemical writing process could be easily conducted on the AG-AN layer in the presence of GG-TiO2 layer, indicating the feasibility of ink-free printing on bilayer biopolymer films. The AG-AN/GG-2%TiO2 film presented a limit of detection of 0.018 mM to trimethylamine (TMA), a typical basic gas generated during meat spoilage. Based on its good illumination stability and sensing ability to basic gases, the AG-AN/GG-2%TiO2 film exhibited rose red-to-green color changes along with the spoilage of pork and silver carp, indicating its great potential for monitoring meat spoilage in intelligent food packaging

Morphology of Poly(Ethylene Oxide)b-Poly(epsilon-Caprolactone) Spherulites Formed Under Compressed CO2

Melt crystallization of PEO-b-PCL thin films was conducted under compressed CO2 and the morphology of the spherulites obtained at various pressures was investigated by polarizing optical microscopy (POM) and atomic force microscopy (AFM). At 3 MPa CO2 classical ring-banded spherulites with periodical twisting lamellae formed. However, in the spherulites with concentric ring-banded structures formed at 5 MPa CO2, the rings were formed due to periodical variations in thickness along the radial direction

Synthesis of novel pyridinium N-chloramine precursors and its antimicrobial application on cotton fabrics

To control pathogenic microbial contamination on polymeric material surface, it is pivotal to develop materials with efficacious antimicrobial activity. Two pyridinium N-chloramine precursors containing a siloxane handle were synthesized, characterized, and grafted onto cotton fabrics. The attenuated total reflectance spectra and scanning electron microscope photo analysis indicated that the cotton fabric surface was successfully modified. The resultant chlorinated fabric samples were challenged against bacteria Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923. Results showed that: (1) the surface modified cotton fabrics displayed satisfactory biocidal efficacy; (2) the precursor structure played a major role on surface grafting and antibacterial activity. This work provides two promising pyridinium N-chloramine precursors which hold potential application for preparing antibacterial textile materials. (C) 2017 Wiley Periodicals, Inc

Research and Application of a Quantitative Prediction Method for Sandstone Thickness in a Zone with Dense Well Pattern Development Based on an Objective Function

The seismic amplitude along a layer in a section can reveal lateral reservoir changes and is one of the important means of reservoir prediction, but it is often difficult to establish the quantitative relationship between the seismic amplitude and sandstone thickness in wells in blocks with denser development. The use of a lower-amplitude slice for precisely quantitatively predicting the sandstone thickness, based on the goal of obtaining data on layers in the development zone with a dense well pattern, is accurate and has a valuable advantage. The method of formation slice optimization based on an objective function is studied to improve the ability of reservoir characterization by seismic attributes. This method has been applied to the reservoir prediction of the G I2 sedimentary unit in the ZQX block of the SRT oilfield and has achieved good results

Expanding the product portfolio of fungal type I fatty acid synthases

Fungal type I fatty acid synthases (FASs) are mega-enzymes with two separated, identical compartments, in which the acyl carrier protein (ACP) domains shuttle substrates to catalytically active sites embedded in the chamber wall. We devised synthetic FASs by integrating heterologous enzymes into the reaction chambers and demonstrated their capability to convert acyl-ACP or acyl-CoA from canonical fatty acid biosynthesis to short/ medium-chain fatty acids and methyl ketones

Synthesis of pyridinium N-chloramines for antibacterial applications

To develop more efficacious antibacterial agents, a new type of cationic N-chloramines that contain a pyridinium moiety and a N-chloramine moiety covalently linked via an alkyl chain were prepared and characterized. Preliminary assays indicated that 1) the compound with a propylidene linker exhibited higher antibacterial activity than the quaternary ammonium counterpart; 2) the chain length of the alkyl linker had major effects on their biocidal properties. Our results may inspire exploration of more pyridinium N-chloramines for antibacterial applications. (C) 2016 Elsevier Ltd. All rights reserved

Size Dependence of Gold Nanorods for Efficient and Rapid Photothermal Therapy

In recent years, gold nanomaterials have become a hot topic in photothermal tumor therapy due to their unique surface plasmon resonance characteristics. The effectiveness of photothermal therapy is highly dependent on the shape and size of gold nanoparticles. In this work, we investigate the photothermal therapeutic effects of four different sizes of gold nanorods (GNRs). The results show that the uptake of short GNRs with aspect ratios 3.3–3.5 by cells is higher than that of GNRs with aspect ratios 4–5.5. Using a laser with single pulse energy as low as 28 pJ laser for 20 s can induce the death of liver cancer cells co-cultured with short GNRs. Long GNRs required twice the energy to achieve the same therapeutic effect. The dual-temperature model is used to simulate the photothermal response of intracellular clusters irradiated by a laser. It is found that small GNRs are easier to compact because of their morphological characteristics, and the electromagnetic coupling between GNRs is better, which increases the internal field enhancement, resulting in higher local temperature. Compared with a single GNR, GNR clusters are less dependent on polarization and wavelength, which is more conducive to the flexible selection of excitation laser sources