Development and evaluation of a plant-based air filter system for bacterial growth control

We investigated a novel plant-based air filter system for bacterial growth control. The volatile components released from the experimental plant (Cupressus macrocarpa) were used as the basis of the bacterial growth control and inhibition. We monitored the effect of light on the gas exhausted from the system, and we found that the presence of light induced an increase in the O2 concentration and a decrease in the CO2 concentration in the exhaust gas. A variety of Gram-positive and -negative bacteria was used to elucidate the effect of the exhaust gas on bacterial growth. In the Bacillus subtilis cultivation aerated with the exhaust gas (batch mode), we observed a decrease in the specific growth rate (μ = 0.227 h-1) compared with the control experiments (0.257 h-1). The same result was observed for the Staphylococcus aureus cultivation aerated with the exhaust gas. In the case of Gram-negative bacterial cultivation aerated with the gas, no significant inhibitory effect of the exhaust gas on the bacterial growth was observed. When the number of bacteria (B. subtilis) in a continuous culture was varied at different aeration rates (between 50 to 200 mL/min) using the exhaust gas, a prominent inhibitory effect was observed. Preliminary gas analysis showed that the major inhibitory factors in the exhaust gas are α- and β-pinene and linalool. The results show that the air filter system used in this study could be applied not only as a methodological aspect for estimating antibacterial activity but also for bacteria control in a given system.Keywords: Plant-based biofilter, Cupressus macrocarpa, Bacillus subtilis, Staphylococcus aureus, α-pinene, β-pineneAfrican Journal of Biotechnology Vol. 12(16), pp. 2027-203

Molecular and biochemical characterization of a novel isoprene synthase from Metrosideros polymorpha

Abstract Background Isoprene is a five-carbon chemical that is an important starting material for the synthesis of rubber, elastomers, and medicines. Although many plants produce huge amounts of isoprene, it is very difficult to obtain isoprene directly from plants because of its high volatility and increasing environmental regulations. Over the last decade, microorganisms have emerged as a promising alternative host for efficient and sustainable bioisoprene production. Isoprene synthase (IspS) has received much attention for the conversion of isoprene from dimethylallyl diphosphate (DMAPP). Herein, we isolated a highly expressible novel IspS gene from Metrosideros polymorpha (MpIspS), which was cloned and expressed in Escherichia coli, using a plant cDNA library and characterized its molecular and biochemical properties. Results The signal sequence deleted MpIspS was cloned and expressed in E. coli as a 65-kDa monomer. The maximal activity of the purified MpIspS was observed at pH 6.0 and 55 °C in the presence of 5 mM Mn2+. The K m, k cat, and k cat/K m for DMAPP as a substrate were 8.11 mM, 21 min− 1, and 2.59 mM− 1 min− 1, respectively. MpIspS was expressed along with the exogenous mevalonate pathway to produce isoprene in E. coli. The engineered cells produced isoprene concentrations of up to 23.3 mg/L using glycerol as the main carbon source. Conclusion MpIspS was expressed in large amounts in E. coli, which led to increased enzymatic activity and resulted in isoprene production in vivo. These results demonstrate a new IspS enzyme that is useful as a key biocatalyst for bioisoprene production in engineered microbes

C1 Compound Biosensors: Design, Functional Study, and Applications

The microbial assimilation of one-carbon (C1) gases is a topic of interest, given that products developed using this pathway have the potential to act as promising substrates for the synthesis of valuable chemicals via enzymatic oxidation or C−C bonding. Despite extensive studies on C1 gas assimilation pathways, their key enzymes have yet to be subjected to high-throughput evolution studies on account of the lack of an efficient analytical tool for C1 metabolites. To address this challenging issue, we attempted to establish a fine-tuned single-cell−level biosensor system constituting a combination of transcription factors (TFs) and several C1-converting enzymes that convert target compounds to the ligand of a TF. This enzymatic conversion broadens the detection range of ligands by the genetic biosensor systems. In this study, we presented new genetic enzyme screening systems (GESSs) to detect formate, formaldehyde, and methanol from specific enzyme activities and pathways, named FA-GESS, Frm-GESS, and MeOH-GESS, respectively. All the biosensors displayed linear responses to their respective C1 molecules, namely, formate (1.0−250 mM), formaldehyde (1.0−50 μM), and methanol (5−400 mM), and they did so with high specificity. Consequently, the helper enzymes, including formaldehyde dehydrogenase and methanol dehydrogenase, were successfully combined to constitute new versatile combinations of the C1-biosensors

Long-Term Stable and Tightly Controlled Expression of Recombinant Proteins in Antibiotics-Free Conditions.

Plasmid-based gene expression is a fundamental tool in the field of biotechnology. However, overexpression of genes of interest with multi-copy plasmids often causes detrimental effects on host cells. To overcome this problem, chromosomal integration of target genes has been used for decades; however, insufficient protein expression occurred with this method. In this study, we developed a novel cloning and expression system named the chromosomal vector (ChroV) system, that has features of stable and high expression of target genes on the F' plasmid in the Escherichia coli JM109(DE3) strain. We used an RMT cluster (KCTC 11994BP) containing a silent cat gene from a previous study to clone a gene into the F' plasmid. The ChroV system was applied to clone two model targets, GFPuv and carotenoids gene clusters (4 kb), and successfully used to prove the inducible tightly regulated protein expression in the F' plasmid. In addition, we verified that the expression of heterologous genes in ChroV system maintained stably in the absence of antibiotics for 1 week, indicating ChroV system is applicable to antibiotics-free production of valuable proteins. This protocol can be widely applied to recombinant protein expression for antibiotics-free, stable, and genome-based expression, providing a new platform for recombinant protein synthesis in E. coli. Overall, our approach can be widely used for the economical and industrial production of proteins in E. coli

Controlled Aggregation and Increased Stability of β-Glucuronidase by Cellulose Binding Domain Fusion

<div><p>Cellulose-binding domains (CBDs) are protein domains with cellulose-binding activity, and some act as leaders in the localization of cellulosomal scaffoldin proteins to the hydrophobic surface of crystalline cellulose. In this study, we found that a CBD fusion enhanced and improved soluble β-glucuronidase (GusA) enzyme properties through the formation of an artificially oligomeric state. First, a soluble CBD fused to the C-terminus of GusA (GusA-CBD) was obtained and characterized. Interestingly, the soluble GusA-CBD showed maximum activity at higher temperatures (65°C) and more acidic pH values (pH 6.0) than free GusA did (60°C and pH 7.5). Moreover, the GusA-CBD enzyme showed higher thermal and pH stabilities than the free GusA enzyme did. Additionally, GusA-CBD showed higher enzymatic activity in the presence of methanol than free GusA did. Evaluation of the protease accessibility of both enzymes revealed that GusA-CBD retained 100% of its activity after 1 h incubation in 0.5 mg/ml protease K, while free GusA completely lost its activity. Simple fusion of CBD as a single domain may be useful for tunable enzyme states to improve enzyme stability in industrial applications.</p></div

Expression of GFPuv using the ChroV system.

<p>A. The <i>E</i>. <i>coli</i> cells expressing GFPuv in the Fʹ plasmid (ChroV-GFPuv) and GFPuv in the pET plasmid (pRMT-GFPuv) was observed by colony fluorescent image analysis. B. Growth curves of ChroV and pRMT cells in LB media containing 0.5 mM IPTG and 25 μg/mL kanamycin. C. ChroV and pRMT cells were inoculated in M9 media containing 0.5 mM IPTG. The cell growths were monitored in 96 well plates for 24 h at 37°C with shaking at 200 rpm using the Infinite® 200 PRO microplate reader (Tecan Group Ltd, Switzerland) following the manufacturer’s instructions. Error bars indicate a deviation in the triplicate experiments.</p

Stable maintenance of GFPuv-expression levels in conditions without antibiotics.

<p>A. The expression of GFPuv was analyzed by fluorescent intensity at 510 nm after excitation at 385 nm. B. SDS-PAGE analysis of pRMT-GFPuv and ChroV-GFPuv. N: Negative control. 1: <i>E</i>. <i>coli</i> culture time for 4 h; 2: culture time for 33 h; 3: culture time for 67 h; 4: culture time for 100 h; 5: culture time for 134 h; 6: culture time for 168 h. GFPuv indicated by arrow.</p

Cloning of the β-carotene synthetic gene cluster in the ChroV system.

<p>Shown are <i>E</i>. <i>coli</i> colonies expressing the β-carotene synthetic genes, which produced yellow-pigmented colonies in the pRMT system [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166890#pone.0166890.ref019" target="_blank">19</a>] and ChroV system (in this study). The ChroV system showed more homogeneous sizes, shapes, and colors for the colonies.</p