Ty1-fused protein-body formation for spatial organization of metabolic pathways in Saccharomyces cerevisiae.

Metabolite production through a multistep metabolic pathway can often be increased by efficient substrate channeling created by spatial sequestration of the metabolic reactions. Here, Tya, a structural component in the Ty1 retrotransposon element that forms virus-like particles (VLPs) in Saccharomyces cerevisiae, was used to spatially organize enzymes involved in a metabolic pathway into a multi-enzyme protein body in yeast. As a proof of principle, Tya fusion to three key enzymes involved in biosynthesis of the isoprenoids farnesene and farnesol was tested to assess its potential to improve productivity. The Tya-fusion protein resulted in three and fourfold increases in farnesene and farnesol production, respectively, as compared with that observed in a non-fused control. Specifically, two-phase partitioning fed-batch fermentations of S. cerevisiae ATCC200589 overexpressing Tya-fused enzymes (tHmg1, IspA, and α-farnesene synthase) yielded 930 ± 40 mg/L of farnesene after 7 days. Additionally, we observed that the Tya-fusion proteins tended to partition into particulate fractions upon 100,000g ultracentrifugation, suggesting the formation of large aggregates of protein bodies, with their particulate structure also observed by transmission electron microscopy. The dramatic increase in the biosynthetic productivity of metabolites via use of a Tya-fusion protein suggested that this approach might be useful for the creation of multi-enzyme complexes to improve metabolic engineering in yeast.

High-level recombinant production of squalene using selected Saccharomyces cerevisiae strains.

For recombinant production of squalene, which is a triterpenoid compound with increasing industrial applications, in microorganisms generally recognized as safe, we screened Saccharomyces cerevisiae strains to determine their suitability. A strong strain dependence was observed in squalene productivity among Saccharomyces cerevisiae strains upon overexpression of genes important for isoprenoid biosynthesis. In particular, a high level of squalene production (400 ± 45 mg/L) was obtained in shake flasks with the Y2805 strain overexpressing genes encoding a bacterial farnesyl diphosphate synthase (ispA) and a truncated form of hydroxyl-3-methylglutaryl-CoA reductase (tHMG1). Partial inhibition of squalene epoxidase by terbinafine further increased squalene production by up to 1.9-fold (756 ± 36 mg/L). Furthermore, squalene production of 2011 ± 75 or 1026 ± 37 mg/L was obtained from 5-L fed-batch fermentations in the presence or absence of terbinafine supplementation, respectively. These results suggest that the Y2805 strain has potential as a new alternative source of squalene production.

Control of membrane biofouling in MBR for wastewater treatment by quorum quenching bacteria encapsulated in microporous membrane.

Recently, enzymatic quorum quenching has proven its potential as an innovative approach for biofouling control in the membrane bioreactor (MBR) for advanced wastewater treatment. However, practical issues on the cost and stability of enzymes are yet to be solved, which requires more effective quorum quenching methods. In this study, a novel quorum quenching strategy, interspecies quorum quenching by bacterial cell, was elaborated and proved to be efficient and economically feasible biofouling control in MBR. A recombinant Escherichia coli which producing N-acyl homoserine lactonase or quorum quenching Rhodococcus sp. isolated from a real MBR plant was encapsulated inside the lumen of microporous hollow fiber membrane, respectively. The porous membrane containing these functional bacteria (i.e., "microbial-vessel") was put into the submerged MBR to alleviate biofouling on the surface of filtration membrane. The effect of biofouling inhibition by the microbial-vessel was evaluated over 80 days of MBR operation. Successful control of biofouling in a laboratory scale MBR suggests that the biofouling control through the interspecies quorum quenching could be expanded to the plant scale of MBR and various environmental engineering systems with economic feasibility.

Identification of the Regulators Binding to the Upstream Region of glxR in Corynebacterium glutamicum.

GlxR is considered as a global transcriptional regulator controlling a large number of genes having broad physiological aspects in Corynebacterium glutamicum. However, the expression profile revealing the transcriptional control of glxR has not yet been studied in detail. DNA affinity chromatography experiments revealed the binding of transcriptional regulators SucR, RamB, GlxR, and a GntR-type protein (hereafter denoted as GntR3) to the upstream region of glxR. The binding of different regulators to the glxR promoter was confirmed by EMSA experiments. The expression of glxR was analyzed in detail under various carbon sources in the wild-type and different mutant strains. The sucR and gntR3 deletion mutants showed decreased glxR promoter activities, when compared with the wild type, irrespective of the carbon sources. The promoter activity of glxR was derepressed in the ramB deletion mutant under all the tested carbon sources. These results indicate that SucR and GntR3 are acting as activators of GlxR, while RamB plays a repressor. As expected, the expression of glxR in the cyaB and glxR deletion mutants was derepressed under different media conditions, indicating that GlxR is autoregulated.