Biosynthesis of polyhydroxybutyrate from methane and carbon dioxide using type II methanotrophs.

Methane (CH4) and carbon dioxide (CO2) are the dominant greenhouse gases (GHGs) that are increasing at an alarming rate. Methanotrophs have emerged as potential CH4 and CO2 biorefineries. This study demonstrated the synchronous incorporation of CH4 and CO2 into polyhydroxybutyrate (PHB) for the first time using 13C-labeling experiments in methanotrophs. By supplying substantial amounts of CO2, PHB content was enhanced in all investigated type II methanotrophic strains by 140 %, 146 %, and 162 %. The highest content of PHB from CH4 and CO2 in flask-scale cultivation reached 38 % dry cell weight in Methylocystis sp. MJC1, in which carbon percentage in PHB from CO2 was 45 %. Flux balance analysis predicted the critical roles of crotonyl-CoA carboxylase/reductase and phosphoenolpyruvate carboxylase in CO2 recycling. This study provided proof of the conversion of GHGs into a valuable and practical product using methanotrophic bacteria, contributing to addressing GHG emissions.

Enhancing Sesquiterpenoid Production from Methane via Synergy of the Methylerythritol Phosphate Pathway and a Short-Cut Route to 1-Deoxy-D-xylulose 5-Phosphate in Methanotrophic Bacteria.

Sesquiterpenoids are one of the most diverse classes of isoprenoids which exhibit numerous potentials in industrial biotechnology. The methanotrophs-based methane bioconversion is a promising approach for sustainable production of chemicals and fuels from methane. With intrinsic high carbon flux though the ribulose monophosphate cycle in Methylotuvimicrobium alcaliphilum 20Z, we demonstrated here that employing a short-cut route from ribulose 5-phosphate to 1-deoxy-d-xylulose 5-phosphate (DXP) could enable a more efficient isoprenoid production via the methylerythritol 4-phosphate (MEP) pathway, using α-humulene as a model compound. An additional 2.8-fold increase in α-humulene production yield was achieved by the fusion of the nDXP enzyme and DXP reductase. Additionally, we utilized these engineering strategies for the production of another sesquiterpenoid, α-bisabolene. The synergy of the nDXP and MEP pathways improved the α-bisabolene titer up to 12.24 ± 0.43 mg/gDCW, twofold greater than that of the initial strain. This study expanded the suite of sesquiterpenoids that can be produced from methane and demonstrated the synergistic uses of the nDXP and MEP pathways for improving sesquiterpenoid production in methanotrophic bacteria.

Developments of Riboswitches and Toehold Switches for Molecular Detection-Biosensing and Molecular Diagnostics.

Riboswitches and toehold switches are considered to have potential for implementation in various fields, i.e., biosensing, metabolic engineering, and molecular diagnostics. The specific binding, programmability, and manipulability of these RNA-based molecules enable their intensive deployments in molecular detection as biosensors for regulating gene expressions, tracking metabolites, or detecting RNA sequences of pathogenic microorganisms. In this review, we will focus on the development of riboswitches and toehold switches in biosensing and molecular diagnostics. This review introduces the operating principles and the notable design features of riboswitches as well as toehold switches. Moreover, we will describe the advances and future directions of riboswitches and toehold switches in biosensing and molecular diagnostics.

ß,ß-carotene 15,15'-monooxygenase and its substrate ß-carotene modulate migration and invasion in colorectal carcinoma cells.

BACKGROUND: ß,ß-Carotene 15,15'-monooxygenase (BCMO1) converts ß-carotene to retinaldehyde. Increased ß-carotene consumption is linked to antitumor effects. Retinoic acid reduces the invasiveness in cancer, through inhibition of matrix metalloproteinases (MMPs). In our studies of a mouse model that develops intestinal tumors after low dietary folate, we found reduced BCMO1 expression in normal preneoplastic intestine of folate-deficient tumor-prone mice. OBJECTIVE: Our goal was to determine whether BCMO1 expression could influence transformation potential in human colorectal carcinoma cells, by examining the effect of BCMO1 modulation on cellular migration and invasion, and on expression of MMPs. DESIGN: LoVo colon carcinoma cells were transfected with BCMO1 small interfering RNA (siRNA) or scrambled siRNA. Migration and invasion were measured, and the expression of BCMO1, MMP7, and MMP28 was assessed by quantitative reverse-transcriptase polymerase chain reaction. These variables were also measured after treatment of cells with retinoic acid, 5-aza-2'-deoxycytidine, folate-depleted/high-methionine medium, and ß-carotene. RESULTS: Retinoic acid decreased the migration, invasion, and expression of MMP28 mRNA. Transfection of cells with BCMO1 siRNA inhibited BCMO1 expression, enhanced migration and invasion, and increased expression of MMP7 and MMP28. 5-Aza-2'-deoxycytidine decreased, whereas folate-depleted/high-methionine medium increased invasiveness. ß-Carotene increased BCMO1 expression and reduced invasiveness with a decrease in expression of MMP7 and MMP28. CONCLUSIONS: Inhibition of BCMO1 expression is associated with increased invasiveness of colon cancer cells and increased expression of MMP7 and MMP28. ß-Carotene can upregulate BCMO1 and reverse these effects. These novel associations suggest a critical role for BCMO1 in cancer and provide a mechanism for the proposed antitumor effects of ß-carotene.

Reduced Ran expression in Ran(+/-) fibroblasts increases cytokine-stimulated nuclear abundance of the AP-1 subunits c-Fos and c-Jun.

Ran (Ras-related nuclear protein), a Ras family GTPase, is involved in multiple cellular functions, including the regulation of DNA replication, cell cycle progression, nuclear structure formation, RNA processing-exportation, and nuclear protein importation. Ran(+/-) embryonic stem (ES) cells were produced in an attempt to generate Ran null mutant mice. Even after an extremely large number of blastocyst injections, no Ran(+/-) chimeric mice could be generated. Ran(+/-) ES cell-derived fibroblasts showed reduced Ran protein expression, and manifested augmented nuclear abundance of AP-1 factors (c-Jun and c-Fos) upon cytokine stimulation. Our experiments demonstrated that intracellular Ran protein levels controlled the nuclear presence of certain transcription factors, such as c-Fos and c-Jun.

Ran overexpression leads to diminished T cell responses and selectively modulates nuclear levels of c-Jun and c-Fos.

Ras-related nuclear protein (Ran) is a Ras family GTPase, and its documented functions are the regulation of DNA replication, cell cycle progression, nuclear structure formation, RNA processing and exportation, and nuclear protein importation. In this study, we performed detailed mapping of Ran expression during mouse ontogeny using in situ hybridization. High Ran expression was found in various organs and tissues including the thymus cortex and spleen white pulp. Ran was induced in T cells 24 h after their activation. The function of Ran in the immune system was investigated using Ran transgenic (Tg) mice. In Ran Tg T cells, there was compromised activation marker expression, lymphokine secretion, and proliferation upon T cell receptor activation in vitro when compared with wild type T cells. Tg mice also manifested defective delayed type hypersensitivity in vivo. Upon PMA and ionomycin stimulation, Tg T cells were defective in nuclear accumulation of AP-1 factors (c-Jun and c-Fos) but not NF-kappaB family members. Our experiments showed that Ran had important regulatory function in T cell activation. One of the possible mechanisms is that intracellular Ran protein levels control the nuclear retention for selective transcription factors such as c-Jun and c-Fos of AP-1, which is known to be critical in T cell activation and proliferation and lymphokine secretion.