Preparation, characteristics and cytotoxicity of green synthesized selenium nanoparticles using Paenibacillus motobuensis LY5201 isolated from the local specialty food of longevity area.

Selenium is an essential micronutrient element. For the extremely biotoxic of selenite, Selenium nanoparticles (SeNPs) is gaining increasing interest. In this work, a selenium-enriched strain with highly selenite-resistant (up to 173 mmol/L) was isolated from the local specialty food of longevity area and identified as Paenibacillus motobuensis (P. motobuensis) LY5201. Most of the SeNPs were accumulated extracellular. SeNPs were around spherical with a diameter of approximately 100 nm. The X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy showed that the purified SeNPs consisted of selenium and proteins. Our results suggested that P. motobuensis LY5201could be a suitable and robust biocatalyst for SeNPs synthesis. In addition, the cytotoxicity effect and the anti-invasive activity of SeNPs on the HepG2 showed an inhibitory effect on HepG2, indicating that SeNPs could be used as a potential anticancer drug.

Sodium formate redirects carbon flux and enhances heterologous mevalonate production in Methylobacterium extorquens AM1.

Methylobacterium extorquens AM1 (AM1), a model strain of methylotrophic cell factories (MeCFs) could be used to produce fine chemicals from methanol. Synthesis of heterologous products usually needs reducing cofactors, but AM1 growing on methanol lack reducing power. Formate could be used as a reducing agent. In this study, mevalonic acid (MEV) yield of 0.067 gMEV/g methanol was reached by adding 10 mmol L-1 sodium formate in MEV accumulating stage (at 72 h). The yield was improved by 64.57%, and represented the highest yield reported to date. 13 C-labeling experiments revealed global effects of sodium formate on metabolic pathways in engineered Methylobacterium extorquens AM1. Sodium formate significantly increased the ratios of reducing equivalents, enhanced the metabolic rate of pathways demanding reducing cofactors and redirected the carbon flux to MEV synthesis. As a result, coupling formate to methanol-based production provide a promising way for converting C1 substances to useful chemical products.

Breeding of Methanol-Tolerant Methylobacterium extorquens AM1 by Atmospheric and Room Temperature Plasma Mutagenesis Combined With Adaptive Laboratory Evolution.

Methylobacterium extorquens AM1, which can be used as a methylotrophic cell factory (MeCF) for the production of fine chemicals from methanol, is the most extensively studied model methylotrophic strain. However, its low tolerance for methanol limits the development of bioprocesses and there have been no reports of improved methanol tolerance of M. extorquens AM1. In this study, atmospheric and room temperature plasma (ARTP) mutagenesis, in combination with adaptive laboratory evolution (ALE), is used to generate a mutant with high methanol tolerance (referred to as CLY-2533). The final cell density of CLY-2533 is 7.10 times higher than that of the wild-type strain in medium containing 5% (v/v) methanol. Through comparative genomics analysis and overexpression of the exploited putative genes, seven mutated genes are identified as being closely related to the higher methanol tolerance of CLY-2533. Additionally, the mvt operon, which contains genes related to the biosynthesis of mevalonate acid (MEV), is introduced into CLY-2533. This recombinant strain shows significant improvements in both MEV production and cell growth in 5% methanol medium. These findings will be helpful in rational design of methanol-utilizing strain for an improved host platform for methanol based biomanufacturing.

Cre/loxP-Mediated Multicopy Integration of the Mevalonate Operon into the Genome of Methylobacterium extorquens AM1.

Methylobacterium extorquens AM1 is the model strain for methylotrophic bacteria that metabolize methanol as the sole carbon and energy source. Genetically modified M. extorquens AM1 is used as a methylotrophic cell factory (MeCF) for high value-added chemical production. We tested the Cre-loxP recombination system for its ability to mediate multicopy gene integration of the mvt3 operon (mvt3) in M. extorquens AM1. mvt3 controls the expression of the first three enzymes of the mevalonate synthesis pathway. We assayed for Cre-mediated multigene integration by screening for multicopy mutants via their survival in culture with a high kanamycin concentration (600 µg/mL). We identified mutant strains in which the mevalonate titer was increased by up to 1.9-fold compared with M2 (M. extorquens AM1ΔcelABCΔattTn7::mvt3::loxP) and confirmed mvt3 integration at 2-3 copies per genome. This result demonstrates the feasibility of multicopy integration in M. extorquens AM1 mediated by Cre-loxP recombination and its potential for improving the output of M. extorquens AM1 metabolic pathways, e.g., optimization of terpenoid synthesis.

Biosensor-assisted transcriptional regulator engineering for Methylobacterium extorquens AM1 to improve mevalonate synthesis by increasing the acetyl-CoA supply.

Acetyl-CoA is not only an important intermediate metabolite for cells but also a significant precursor for production of industrially interesting metabolites. Methylobacterium extorquens AM1, a model strain of methylotrophic cell factories using methanol as carbon source, is of interest because it produces abundant coenzyme A compounds capable of directing to synthesis of different useful compounds from methanol. However, acetyl-CoA is not always efficiently accumulated in M. extorquens AM1, as it is located in the center of three cyclic central metabolic pathways. Here we successfully demonstrated a strategy for sensor-assisted transcriptional regulator engineering (SATRE) to control metabolic flux re-distribution to increase acetyl-CoA flux from methanol for mevalonate production in M. extorquens AM1 with introduction of mevalonate synthesis pathway. A mevalonate biosensor was constructed and we succeeded in isolating a mutated strain (Q49) with a 60% increase in mevalonate concentration (an acetyl-CoA-derived product) following sensor-based high-throughput screening of a QscR transcriptional regulator library. The mutated QscR-49 regulator (Q8*,T61S,N72Y,E160V) lost an N-terminal α-helix and underwent a change in the secondary structure of the RD-I domain at the C terminus, two regions that are related to its interaction with DNA. 13C labeling analysis revealed that acetyl-CoA flux was improved by 7% and transcriptional analysis revealed that QscR had global effects and that two key points, NADPH generation and fumC overexpression, might contribute to the carbon flux re-distribution. A fed-batch fermentation in a 5-L bioreactor for QscR-49 mutant yielded a mevalonate concentration of 2.67g/L, which was equivalent to an overall yield of 0.055mol acetyl-CoA/mol methanol, the highest yield among engineered strains of M. extorquens AM1. This work was the first attempt to regulate M. extorquens AM1 on transcriptional level and provided molecular insights into the mechanism of carbon flux regulation.

Bioconversion of methanol to value-added mevalonate by engineered Methylobacterium extorquens AM1 containing an optimized mevalonate pathway.

Methylotrophic biosynthesis using methanol as a feedstock is a promising and attractive method to solve the over-dependence of the bioindustry on sugar feedstocks derived from grains that are used for food. In this study, we introduced and engineered the mevalonate pathway into Methylobacterium extorquens AM1 to achieve high mevalonate production from methanol, which could be a platform for terpenoid synthesis. We first constructed a natural operon (MVE) harboring the mvaS and mvaE genes from Enterococcus faecalis as well as an artificial operon (MVH) harboring the hmgcs1 gene from Blattella germanica and the tchmgr gene from Trypanosoma cruzi that encoded enzymes with the highest reported activities. We achieved mevalonate titers of 56 and 66 mg/L, respectively, in flask cultivation. Introduction of the phaA gene from Ralstonia eutropha into the operon MVH increased the mevalonate titer to 180 mg/L, 3.2-fold higher than that of the natural operon MVE. Further modification of the expression level of the phaA gene by regulating the strength of the ribosomal binding site resulted in an additional 20 % increase in mevalonate production to 215 mg/L. A fed-batch fermentation of the best-engineered strain yielded a mevalonate titer of 2.22 g/L, which was equivalent to an overall yield and productivity of 28.4 mg mevalonate/g methanol and 7.16 mg/L/h, respectively. The production of mevalonate from methanol, which is the initial, but critical step linking methanol with valuable terpenoids via methylotrophic biosynthesis, represents a proof of concept for pathway engineering in M. extorquens AM1.