Experimental Investigation of Electrospraying Properties Based on Ring Electrode Modification.

Electrospraying uses a high-voltage potential difference to create fine droplets. This study conducts a comparative analysis of the spray pattern and droplet properties using ring electrode parameters. The spray pattern and droplet characteristics are analyzed based on the experimental parameters of the ring electrode. The results show that the cone-jet mode forms quickly for the ring electrode. In addition, as the ring diameter decreases, the ring voltage increases and an increase in the distance between the ring and the nozzle in the bottom direction decreases the Sauter mean diameter and its standard deviation. The optimal conditions for the formation of fine and uniform droplets include a ring diameter of 15 mm, a ring voltage of 7 kV, and a nozzle-to-ring distance of (+) 20 mm.

Repurposing type III polyketide synthase as a malonyl-CoA biosensor for metabolic engineering in bacteria.

Malonyl-CoA is an important central metabolite for the production of diverse valuable chemicals including natural products, but its intracellular availability is often limited due to the competition with essential cellular metabolism. Several malonyl-CoA biosensors have been developed for high-throughput screening of targets increasing the malonyl-CoA pool. However, they are limited for use only in Escherichia coli and Saccharomyces cerevisiae and require multiple signal transduction steps. Here we report development of a colorimetric malonyl-CoA biosensor applicable in three industrially important bacteria: E. coli, Pseudomonas putida, and Corynebacterium glutamicum RppA, a type III polyketide synthase producing red-colored flaviolin, was repurposed as a malonyl-CoA biosensor in E. coli Strains with enhanced malonyl-CoA accumulation were identifiable by the colorimetric screening of cells showing increased red color. Other type III polyketide synthases could also be repurposed as malonyl-CoA biosensors. For target screening, a 1,858 synthetic small regulatory RNA library was constructed and applied to find 14 knockdown gene targets that generally enhanced malonyl-CoA level in E. coli These knockdown targets were applied to produce two polyketide (6-methylsalicylic acid and aloesone) and two phenylpropanoid (resveratrol and naringenin) compounds. Knocking down these genes alone or in combination, and also in multiple different E. coli strains for two polyketide cases, allowed rapid development of engineered strains capable of enhanced production of 6-methylsalicylic acid, aloesone, resveratrol, and naringenin to 440.3, 30.9, 51.8, and 103.8 mg/L, respectively. The malonyl-CoA biosensor developed here is a simple tool generally applicable to metabolic engineering of microorganisms to achieve enhanced production of malonyl-CoA-derived chemicals.

Metabolic engineering of Escherichia coli for high-level astaxanthin production with high productivity.

Astaxanthin is a reddish keto-carotenoid classified as a xanthophyll found in various microbes and marine organisms. As a powerful antioxidant having up to 100 times more potency than other carotenoids such as ß-carotene, lutein, and lycopene, astaxanthin is a versatile compound utilized in animal feed, food pigment, health promotion and cosmetic industry. Here, we report development of metabolically engineered Escherichia coli capable of producing astaxanthin to a high concentration with high productivity. First, the heterologous crt genes (crtE, crtY, crtI, crtB, and crtZ) from Pantoea ananatis and the truncated BKT gene (trCrBKT) from Chlamydomonas reinhardtii were introduced to construct the astaxanthin biosynthetic pathway. Then, eight different fusion tags were examined by attaching them to the N- or C-terminus of the trCrBKT membrane protein to allow stable expression and to efficiently guide trCrBKT to the E. coli membrane. When the signal peptide of OmpF and TrxA were tagged to the N-terminus and C-terminus of trCrBKT, respectively, astaxanthin production reached 12.90 mg/L (equivalent to 3.84 mg/gDCW), which was 2.08-fold higher than that obtained without tagging. Upon optimization of culture conditions, this engineered strain WLGB-RPP harboring pAX15 produced 332.23 mg/L (5.38 mg/gDCW) of astaxanthin with the productivity of 3.79 mg/L/h by fed-batch fermentation. In order to further increase astaxanthin production, in silico flux variability scanning based on enforced objective flux (FVSEOF) was performed to identify gene overexpression targets. The engineered strain WLGB-RPP (pAX15, pTrc-ispDF) which simultaneously overexpressing the ispD and ispF genes identified by FVSEOF produced astaxanthin to a higher concentration of 377.10 mg/L (6.26 mg/gDCW) with a productivity of 9.20 mg/L/h upon induction with 1 mM IPTG. When cells were induced with 0.5 mM IPTG to reduce the metabolic burden, astaxanthin concentration further increased to 432.82 mg/L (7.12 mg/gDCW) with a productivity of 9.62 mg/L/h. To more stably maintain plasmid during the fed-batch fermentation of WLGB-RPP (pAX15, pTrc-ispDF), the post-segregational killing hok/sok system was introduced. This strain produced 385.04 mg/L (6.98 mg/gDCW) of astaxanthin with a productivity of 7.86 mg/L/h upon induction with 0.5 mM IPTG. The strategies reported here will be useful for the enhanced production of astaxanthin and related carotenoid products by engineered E. coli strains.

CuiD is a crucial gene for survival at high copper environment in Salmonella enterica serovar Typhimurium.

Copper ion is an essential micronutrient but it is also extremely cytotoxic when it exists in excess. Our studies have shown that Salmonella enterica serovar Typhimurium can survive potentially lethal copper exposures by the way of copper efflux system. A copper ion inducible gene was identified in virulent S. typhimurium by using the technique of MudJ (Km, lac)-directed lacZYA operon fusions. A copper ion inducible strain LF153 (cuiD::MudJ) has been identified. The cuiD mutant exhibits a copper sensitive phenotype but possesses normal resistance to other metal ions, and lost DMP oxidase activity. Therefore, we suggest that cuiD is an important gene for copper homeostasis and the copper resistance response. The copper sensitive phenotype was complemented by pYL3.0 carrying cuiD+. Sequence analysis showed cuiD contains 1,614 bp encoding a 536 amino acid with a 27 amino acid signal peptide and a 509 amino acid residues comprising the mature peptide. The CuiD shows 81% homology to YacK, a putative multicopper oxidases which extrudes copper in Escherichia coli. This ORF contains four conserved regions that contain 12 copper ligands (types 1, 2, and 3) present in various copper homeostasis responsible proteins. The H2O2 sensitive phenotype of the cuiD mutant indicates that cuiD may be involved in oxidative stress response.