Improvement of cadaverine production in whole cell system with baker's yeast for cofactor regeneration.

Cadaverine, 1,5-diaminopentane, is one of the most promising chemicals for biobased-polyamide production and it has been successfully produced up to molar concentration. Pyridoxal 5'-phosphate (PLP) is a critical cofactor for inducible lysine decarboxylase (CadA) and is required up to micromolar concentration level. Previously the regeneration of PLP in cadaverine bioconversion has been studied and salvage pathway pyridoxal kinase (PdxY) was successfully introduced; however, this system also required a continuous supply of adenosine 5'-triphosphate (ATP) for PLP regeneration from pyridoxal (PL) which add in cost. Herein, to improve the process further a method of ATP regeneration was established by applying baker's yeast with jhAY strain harboring CadA and PdxY, and demonstrated that providing a moderate amount of adenosine 5'-triphosphate (ATP) with the simple addition of baker's yeast could increase cadaverine production dramatically. After optimization of reaction conditions, such as PL, adenosine 5'-diphosphate, MgCl2, and phosphate buffer, we able to achieve high production (1740 mM, 87% yield) from 2 M L-lysine. Moreover, this approach could give averaged 80.4% of cadaverine yield after three times reactions with baker's yeast and jhAY strain. It is expected that baker's yeast could be applied to other reactions requiring an ATP regeneration system.

Production of L-Theanine Using Escherichia coli Whole-Cell Overexpressing γ-Glutamylmethylamide Synthetase with Bakers Yeast.

L-Theanine, found in green tea leaves has been shown to positively affect immunity and relaxation in humans. There have been many attempts to produce L-theanine through enzymatic synthesis to overcome the limitations of traditional methods. Among the many genes coding for enzymes in the L-theanine biosynthesis, glutamylmethylamide synthetase (GMAS) exhibits the greatest possibility of producing large amounts of production. Thus, GMAS from Methylovorus mays No. 9 was overexpressed in several strains including vectors with different copy numbers. BW25113(DE3) cells containing the pET24ma::gmas was selected for strains. The optimal temperature, pH, and metal ion concentration were 50°C, 7, and 5 mM MnCl2, respectively. Additionally, ATP was found to be an important factor for producing high concentration of L-theanine so several strains were tested during the reaction for ATP regeneration. Bakers yeast was found to decrease the demand for ATP most effectively. Addition of potassium phosphate source was demonstrated by producing 4-fold higher L-theanine. To enhance the conversion yield, GMAS was additionally overexpressed in the system. A maximum of 198 mM L-theanine was produced with 16.5 mmol/l/h productivity. The whole-cell reaction involving GMAS has greatest potential for scale-up production of L-theanine.

Simultaneous monitoring of the bioconversion from lysine to glutaric acid by ethyl chloroformate derivatization and gas chromatography-mass spectrometry.

Glutaric acid is a precursor of a plasticizer that can be used for the production of polyester amides, ester plasticizer, corrosion inhibitor, and others. Glutaric acid can be produced either via bioconversion or chemical synthesis, and some metabolites and intermediates are produced during the reaction. To ensure reaction efficiency, the substrates, intermediates, and products, especially in the bioconversion system, should be closely monitored. Until now, high performance liquid chromatography (HPLC) has generally been used to analyze the glutaric acid-related metabolites, although it demands separate time-consuming derivatization and non-derivatization analyses. To substitute for this unreasonable analytical method, we applied herein a gas chromatography - mass spectrometry (GC-MS) method with ethyl chloroformate (ECF) derivatization to simultaneously monitor the major metabolites. We determined the suitability of GC-MS analysis using defined concentrations of six metabolites (l-lysine, cadaverine, 5-aminovaleric acid, 2-oxoglutaric acid, glutamate, and glutaric acid) and their mass chromatograms, regression equations, regression coefficient values (R2), dynamic ranges (mM), and retention times (RT). This method successfully monitored the production process in complex fermentation broth.

Conversion of waste cooking oil into biodiesel using heterogenous catalyst derived from cork biochar.

In this study, a heterogeneous catalyst prepared by pyrolysis of waste cork (Quercus suber) was used for the transesterification of waste cooking oil (WCO). Physicochemical properties of the synthesized biochar catalyst were studied using BET, SEM, FTIR, and XRD. The experiment results demonstrate that heterogeneous catalyst synthesized at 600 °C showed maximum fatty acids methyl esters (FAMEs) conversion (98%) at alcohol:oil (25:1), catalyst loading (1.5% w/v) and temperature 65 °C. Biodiesel produced from WCO (Canola oil) mainly composed of FAMEs in following order C18:1 > C18:2 > C16:0 > C18:0 > C20:0. Properties of produced biodiesel were analysed as cetane number (CN) 50.56, higher heating value (HHV) 39.5, kinematic viscosity (ʋ) 3.9, and density (ρ) 0.87.

Enhanced production of cadaverine by the addition of hexadecyltrimethylammonium bromide to whole cell system with regeneration of pyridoxal-5'-phosphate and ATP.

Cadaverine, also known as 1,5-pentanediamine, is an important platform chemical with a wide range of applications and can be produced either by fermentation or bioconversion. Bioconversion of cadaverine from l-lysine is the preferred method because of its many benefits, including rapid reaction time and an easy downstream process. In our previous study, we replaced pyridoxal-5-phosphate (PLP) with pyridoxal kinase (PdxY) along with pyridoxal (PL) because it could achieve 80% conversion with 0.4 M of l-lysine in 6 h. However, conversion was sharply decreased in the presence of high concentrations of l-lysine (i.e., 1 M), resulting in less than 40% conversion after several hours. In this study, we introduced an ATP regeneration system using polyphosphate kinase (ppk) into systems containing cadaverine decarboxylase (CadA) and PdxY for a sufficient supply of PLP, which resulted in enhanced cadaverine production. In addition, to improve transport efficiency, the use of surfactants was tested. We found that membrane permeabilization via hexadecyltrimethylammonium bromide (CTAB) increased the yield of cadaverine in the presence of high concentrations of l-lysine. By combining these two strategies, the ppk system and addition of CTAB, we enhanced cadaverine production up to 100% with 1 M of l-lysine over the course of 6 h.