No abstract

• Blockade of NAE and bortezomib induces phosphatidylinositol 3-kinase/ mTOR inhibition.• NAE inhibition and bortezomib combined induce synergistic plasma cell apoptosis.The function and survival of normal and malignant plasma cells depends on the elaborately regulated ubiquitin proteasome system. Proteasome inhibitors such as bortezomib have proved to be highly effective in the treatment of multiple myeloma (MM), and their effects are related to normal protein homeostasis which is critical for plasma cell survival. Many ubiquitin ligases are regulated by conjugation with NEDD8. Therefore, neddylation may also impact survival and proliferation of malignant plasma cells. Here, we show that MLN4924, a potent NEDD8 activating enzyme (NAE) inhibitor, induced cytotoxicity in MM cell lines, and its antitumor effect is associated with suppression of the AKT and mammalian target of rapamycin (mTOR) signaling pathways through increased expression of REDD1. Combining MLN4924 with the proteasome inhibitor bortezomib induces synergistic apoptosis in MM cell lines which can overcome the prosurvival effects of growth factors such as interleukin-6 and insulin-like growth factor-1. Altogether, our findings demonstrate an important function for REDD1 in MLN4924-induced cytotoxicity in MM and also provide a promising therapeutic combination strategy for myeloma. (Blood. 2014;123(21):3269-3276)

Poly-γ-glutamic acid (γ-PGA) is an important biopolymer with greatly potential in industrial and medical applications. In the present study, we constructed a metabolically engineered glutamate-independent Bacillus amyloliquefaciens LL3 strain with considerable γ-PGA production, which was carried out by single, double, and triple markerless deletions of three degradation genes pgdS, ggt, and cwlO. The highest γ-PGA production (7.12 g/L) was obtained from the pgdS and cwlO double-deletion strain NK-pc, which was 93 % higher than that of wild-type LL3 strain (3.69 g/L). The triple-gene-deletion strain NK-pgc showed a 28 % decrease in γ-PGA production, leading to a yield of 2.69 g/L. Furthermore, the cell morphologies of the mutant strains were also characterized. The cell length of cwlO deletion strains NK-c and NK-pc was shorter than that of the wild-type strain, while the ggt deletion strains NK-g, NK-pg, NK-gc, and NK-pgc showed longer cell lengths. This is the first report concerning the markerless deletion of γ-PGA degradation genes to improve γ-PGA production in a glutamate-independent strain and the first observation that γ-glutamyltranspeptidase (encoded by ggt) could be involved in the inhibition of cell elongation.

Bioproduction of renewable chemicals is considered as an urgent solution for fossil energy crisis. However, despite tremendous efforts, it is still challenging to generate microbial strains that can produce target biochemical to high levels. Here, we report an example of biosynthesis of high-value and easy-recoverable derivatives built upon natural microbial pathways, leading to improvement in bioproduction efficiency. By leveraging pathways in solventogenic clostridia for co-producing acyl-CoAs, acids and alcohols as precursors, through rational screening for host strains and enzymes, systematic metabolic engineering-including elimination of putative prophages, we develop strains that can produce 20.3 g/L butyl acetate and 1.6 g/L butyl butyrate. Techno-economic analysis results suggest the economic competitiveness of our developed bioprocess. Our principles of selecting the most appropriate host for specific bioproduction and engineering microbial chassis to produce high-value and easy-separable end products may be applicable to other bioprocesses.

Oxygen is a limiting factor in the production of γ-PGA by the glutamic acid-independent strain Bacillus amyloliquefaciens LL3 because of the high viscosity of the culture broth. The vgb gene encoding Vitreoscilla hemoglobin (VHb) was introduced into LL3 to overcome the low concentration of dissolved oxygen (DO). First, recombinant plasmid pWHV was constructed by cloning vgb into the Bacillus expression vector pWH1520 and transformed into LL3. Carbon monoxide difference spectral analysis confirmed the expression of VHb. The γ-PGA yield of LL3 (pWHV) under the optimized fermentation conditions increased by 9.56%. To overcome the instability of pWH1520 and to establish stable expression of VHb, the engineered strain LL3-PVK was constructed by homologous recombination between the integration vector pKSVPVK and the 16S rRNA gene of LL3. The temperature-sensitive plasmid was used to perform the integration, which successfully circumvented the obstacle of the low transformation efficiency of B. amyloliquefaciens LL3. Bacillus amyloliquefaciens LL3-PVK showed an increase of 30% in γ-PGA production, while the biomass was increased by 7.9%. To our knowledge, this is the first report describing enhancement of γ-PGA production in a glutamic acid-independent strain as a result of vgb expression.