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.

Improvement of polyhydroxybutyrate (PHB) plate-based screening method for PHB degrading bacteria using cell-grown amorphous PHB and recovered by sodium dodecyl sulfate (SDS).

Poly(3-hydroxybutyrate) (PHB) is a biobased and biodegradable plastic. Considering the environmental issues of petroleum-based plastics, PHB is promising as it can be degraded in a relatively short time by bacteria to water and carbon dioxide. Substantial efforts have been made to identify PHB-degrading bacteria. To identify PHB-degrading bacteria, solid-based growth or clear zone assays using PHB as the sole carbon source are the easiest methods; however, PHB is difficult to dissolve and distribute evenly, and bacteria grow slowly on PHB plates. Here, we suggest an improved PHB plate assay using cell-grown PHB produced by Halomonas sp. and recovered by sodium dodecyl sulfate (SDS). Preparation using SDS resulted in evenly distributed PHB plates that could be used for sensitive depolymerase activity screening in less time compared with solvent-melted pellet or cell-grown PHB. With this method, we identified 15 new strains. One strain, Cutibacterium sp. SOL05 (98.4% 16S rRNA similarity to Cutibacterium acne), showed high PHB depolymerase activity in solid and liquid conditions. PHB degradation was confirmed by clear zone size, liquid culture, scanning electron microscopy, and Fourier-transform infrared spectroscopy. The results indicate this method can be used to easily identify PHB-degrading bacteria from various sources to strengthen the benefits of bioplastics.

Revealing of sugar utilization systems in Halomonas sp. YLGW01 and application for poly(3-hydroxybutyrate) production with low-cost medium and easy recovery.

Poly(3-hydroxybutyrate) (PHB) is a common polyhydroxyalkanoate (PHA) with potential as an alternative for petroleum-based plastics. Previously, we reported a new strain, Halomonas sp. YLGW01, which hyperproduces PHB with 94% yield using fructose. In this study, we examined the PHB production machinery of Halomonas sp. YLGW01 in more detail by deep-genome sequencing, which revealed a 3,453,067-bp genome with 65.1% guanine-cytosine content and 3054 genes. We found two acetyl-CoA acetyltransferases (Acetoacetyl-CoA thiolase, PhaA), one acetoacetyl-CoA reductase (PhaB), two PHB synthases (PhaC1, PhaC2), PHB depolymerase (PhaZ), and Enoyl-CoA hydratase (PhaJ) in the genome, along with two fructose kinases and fructose transporter systems, including the phosphotransferase system (PTS) and ATP-binding transport genes. We then examined the PHB production by Halomonas sp. YLGW01 using high-fructose corn syrup (HFCS) containing fructose, glucose, and sucrose in sea water medium, resulting in 7.95 ± 0.11 g/L PHB (content, 67.39 ± 0.34%). PHB was recovered from Halomonas sp. YLGW01 using different detergents; the use of Tween 20 and SDS yielded micro-sized granules with high purity. Overall, these results reveal the distribution of PHB synthetic genes and the sugar utilization system in Halomonas sp. YLGW01 and suggest a possible method for PHB recovery.

Fructose based hyper production of poly-3-hydroxybutyrate from Halomonas sp. YLGW01 and impact of carbon sources on bacteria morphologies.

Polyhydroxyalkanoates (PHA), such as poly (3-hydroxybutyrate) (PHB), have emerged as potential alternatives to petroleum-based plastics and can be produced through the appropriate selection of marine bacteria that are already adapted to high salt and low temperature conditions without the requirement of antibiotic treatment. The present study, thus, aimed to screen and characterize thirteen PHA-producing microbial strains isolated from the Gwangalli beach in Busan, South Korea. Among them, Halomonas sp. YLGW01 produced the highest amount of PHB (94.6 ± 1.8% (w/w)) using fructose. Interestingly Halomonas sp. YLGW01 showed increase in cell size (8.39 ± 3.63 µm) with fructose as carbon source as compared to glucose (2.34 ± 0.44 µm). Fructose syrup was investigated as carbon source under unsterilized conditions and 95.26 ± 1.78% of PHB was produced. Overall, this strain showed the highest PHB contents in halotolerant bacteria.

Production of glutaric acid from 5-aminovaleric acid by robust whole-cell immobilized with polyvinyl alcohol and polyethylene glycol.

Glutaric acid is an attractive C5 dicarboxylic acid with wide applications in the biochemical industry. Glutaric acid can be produced by fermentation and bioconversion, and several of its biosynthesis pathways have been well characterized, especially the simple pathway involving glutaric acid from l-lysine using 5-aminovaleric acid. We previously reported the production of glutaric acid using 5-aminovaleric acid and α-ketoglutaric acid by a whole-cell reaction, resulting in a high conversion yield. In this study, we sought to enhance the stability and reusability of this whole-cell system for realizing the efficient production of glutaric acid under harsh reaction conditions. To this end, various matrices were screened to immobilize Escherichia coli whole-cell overexpressing 4-aminobutyrate aminotransferase (GabT), succinate semi-aldehyde dehydrogenase (GabD), and NAD(P)H oxidase (NOX). We ultimately selected a PVA-PEG gel (LentiKats®) for cell entrapment, and several factors of the reaction were optimized. The optimal temperature and pH were 35 °C and 8.5, respectively. Treatment with Tween 80 as a surfactant, as well as additional NOX, was found to be effective. Under the optimized conditions, an immobilized cell retained 55% of its initial activity even after the eighth cycle, achieving 995.2 mM accumulated glutaric acid, whereas free cell lost most of their activity after only two cycles. This optimized whole-cell system can be used in the large-scale production of glutaric acid.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) terpolymer production from volatile fatty acids using engineered Ralstonia eutropha.

One of the advantages of microbial synthesis of polyhydroxyalkanoates (PHAs) is the production of diverse polymers with different properties by the addition of different monomers, such as 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx). Considering the number of possible variables, terpolymers can have more variations than copolymers. In this study, we aimed to synthesize the terpolymer P(3HB-co-3HV-co-3HHx) from volatile fatty acids such as propionate and butyrate using the recombinant Ralstonia eutropha strain (Re2133/pCB81), containing deletions in the phaB1, phaB2, and phaB3 genes, and overexpression of synthetic PHA operon (phaC2, phaA, phaJ). This strain produced terpolymers depending on the ratio of two different carbon sources, namely, propionic and butyric acids; however, wild type R. eutropha could not produce the same type of polymer. The incorporation of 3-hydroxyvalerate and 3-hydroxyhexanoate monomers was confirmed by gas chromatography and H-nuclear magnetic resonance spectroscopy, and the parameters affecting the terpolymer composition were obtained based on regression. In addition, the thermal analysis showed that this terpolymer has properties different from those of the copolymer, obtained from the same composition of volatile acids. Depending on the ratio of two volatile acids, the composition of the terpolymer can be regulated resulting in different properties.

Bioconversion of barley straw lignin into biodiesel using Rhodococcus sp. YHY01.

Rhodococcus sp. YHY01 was studied to utilize various lignin derived aromatic compounds. It was able to utilize p-coumaric acid, cresol, and 2,6 dimethoxyphenol and resulted in biomass production i.e. 0.38 g dcw/L, 0.25 g dcw/L and 0.1 g dcw/L, and lipid accumulation i.e. 49%, 40%, 30%, respectively. The half maximal inhibitory concentration (IC50) value for p-coumaric acid (13.4 mM), cresol (7.9 mM), and 2,6 dimethoxyphenol (3.4 mM) was analyzed. Dimethyl sulfoxide (DMSO) solubilized barley straw lignin fraction was used as a carbon source for Rhodococcus sp. YHY01 and resulted in 0.130 g dcw/L with 39% w/w lipid accumulation. Major fatty acids were palmitic acid (C16:0) 51.87%, palmitoleic acid (C16:l) 14.90%, and oleic acid (C18:1) 13.76%, respectively. Properties of biodiesel produced from barley straw lignin were as iodine value (IV) 27.25, cetane number (CN) 65.57, cold filter plugging point (CFPP) 14.36, viscosity (υ) 3.81, and density (ρ) 0.86.

Increased Tolerance to Furfural by Introduction of Polyhydroxybutyrate Synthetic Genes to Escherichia coli.

Polyhydroxybutyrate (PHB), the most well-known polyhydroxyalkanoate, is a bio-based, biodegradable polymer that has the potential to replace petroleum-based plastics. Lignocellulose hydrolysate, a non-edible resource, is a promising substrate for the sustainable, fermentative production of PHB. However, its application is limited by the generation of inhibitors during the pretreatment processes. In this study, we investigated the feasibility of PHB production in E. coli in the presence of inhibitors found in lignocellulose hydrolysates. Our results show that the introduction of PHB synthetic genes (bktB, phaB, and phaC from Ralstonia eutropha H16) improved cell growth in the presence of the inhibitors such as furfural, 4-hydroxybenzaldehyde, and vanillin, suggesting that PHB synthetic genes confer resistance to these inhibitors. In addition, increased PHB production was observed in the presence of furfural as opposed to the absence of furfural, suggesting that this compound could be used to stimulate PHB production. Our findings indicate that PHB production using lignocellulose hydrolysates in recombinant E. coli could be an innovative strategy for cost-effective PHB production, and PHB could be a good target product from lignocellulose hydrolysates, especially glucose.

L-Glycine Alleviates Furfural-Induced Growth Inhibition during Isobutanol Production in Escherichia coli.

Lignocellulose is now a promising raw material for biofuel production. However, the lignin complex and crystalline cellulose require pretreatment steps for breakdown of the crystalline structure of cellulose for the generation of fermentable sugars. Moreover, several fermentation inhibitors are generated with sugar compounds, majorly furfural. The mitigation of these inhibitors is required for the further fermentation steps to proceed. Amino acids were investigated on furfural-induced growth inhibition in E. coli producing isobutanol. Glycine and serine were the most effective compounds against furfural. In minimal media, glycine conferred tolerance against furfural. From the IC50 value for inhibitors in the production media, only glycine could alleviate growth arrest for furfural, where 6 mM glycine addition led to a slight increase in growth rate and isobutanol production from 2.6 to 2.8 g/l under furfural stress. Overexpression of glycine pathway genes did not lead to alleviation. However, addition of glycine to engineered strains blocked the growth arrest and increased the isobutanol production about 2.3-fold.

Functional Study of Lysine Decarboxylases from Klebsiella pneumoniae in Escherichia coli and Application of Whole Cell Bioconversion for Cadaverine Production.

Klebsiella pneumoniae is a gram-negative, non-motile, rod-shaped, and encapsulated bacterium in the normal flora of the intestines, mouth, skin, and food, and has decarboxylation activity, which results in generation of diamines (cadaverine, agmatine, and putrescine). However, there is no specific information on the exact mechanism of decarboxylation in K. pnuemoniae. Specifically lysine decarboxylases that generate cadaverine with a wide range of applications has not been shown. Therefore, we performed a functional study of lysine decarboxylases. Enzymatic characteristics such as optimal pH, temperature, and substrates were examined by overexpressing and purifying CadA and LdcC. CadA and LdcC from K. pneumoniae had a preference for L-lysine, and an optimal reaction temperature of 37°C and an optimal pH of 7. Although the activity of purified CadA from K. pneumoniae was lower than that of CadA from E. coli, the activity of K. pneumoniae CadA in whole cell bioconversion was comparable to that of E. coli CadA, resulting in 90% lysine conversion to cadaverine with pyridoxal 5'-phosphate L-lysine.