Li-Jen Lin

Systematic Approach To Engineer Escherichia coli Pathways for Co-utilization of a Glucose–Xylose Mixture

Glucose and xylose are two major sugars of lignocellulosic hydrolysate. The regulatory program of catabolite repression in Escherichia coli dictates the preferred utilization of glucose over xylose, which handicaps the development of the lignocellulose-based fermentation process. To co-utilize a glucose-xylose mixture, the E. coli strain was manipulated by pathway engineering in a systematic way. The approach included (1) blocking catabolite repression, (2) enhancing glucose transport, (3) increasing the activity of the pentose phosphate pathway, and (4) eliminating undesirable pathways. Moreover, the ethanol synthetic pathway from Zymomonas mobilis was introduced into the engineered strain. As a consequence, the resulting strain was able to simultaneously metabolize glucose and xylose and consume all sugars (30 g/L each) in 16 h, leading to 97% of the theoretical ethanol yield. Overall, this indicates that this approach is effective and straightforward to engineer E. coli for the desired trait.

Selective Delivery of Cargo Entities to Tumor Cells by Nanoscale Artificial Oil Bodies

Artificial oil bodies (AOBs) are oil droplets that result from self-assembly of a mixture containing triacylglycerols, phospholipids, and membrane proteins of plant seeds. Owing to their small size, stability, hydrophobic core, biocompatibility, and biodegradability, AOBs were explored to examine their feasibility as a drug delivery carrier. This was approached by fusion sesame oleosin (Ole), the primary membrane protein of seed oil bodies, with a small domain consisting of the arginine-glycine-aspartic acid (RGD) motif. The resulting Ole-RGD fusion protein was overproduced in Escherichia coli and then isolated for reconstitution of AOBs. At the optimal condition, the size of stable AOBs was within the range of 100-400 nm. Furthermore, AOBs entrapped with a hydrophobic fluorescence dye were incubated with human tumor cells. As visualized by fluorescent microscopy and confocal microscopy, the RGD-tagged AOBs were able to selectively target cells expressing the αvβ3 integrin. Moreover, these AOBs were effectively internalized and the fluorescence dye that they carried was subsequently released inside the cells. The percentage of cells internalized by AOBs could reach 80% as analyzed by flow cytometry. Taken together, it illustrates a great promise of this proposed approach for targeted delivery of cargo entities to tumor cells.

Selective internalization of self-assembled artificial oil bodies by HER2/neu-positive cells

A novel delivery carrier was developed using artificial oil bodies (AOBs). Plant seed oil bodies (OBs) consist of a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with the storage protein oleosin (Ole). Ole consists of a central hydrophobic domain with two amphiphatic arms that extrude from the surface of OBs. In this study, a bivalent anti-HER2/neu affibody domain (ZH2) was fused with Ole at the C terminus. After overproduction in Escherichia coli, the fusion protein (Ole-ZH2) was recovered to assemble AOBs. The size of self-assembled AOBs was tailored by varying the oil/Ole-ZH2 ratio and pH to reach a nanoscale. Upon co-incubation with tumor cells, the nanoscale AOBs encapsulated with a hydrophobic fluorescence dye were selectively internalized by HER2/neu-overexpressing cells and displayed biocompatibility with the cells. In addition, the ZH2-mediated endosomal entry of AOBs occurred in a time- and AOB dose-dependent manner. The internalization efficiency was as high as 90%. The internalized AOBs disintegrated at the non-permissive pH (e.g. in acidic endosomes) and the cargo dye was released. Results of in vitro study revealed a sustained and prolonged release profile. Taken together, our findings indicate the potential of AOBs as a delivery carrier.

Systematic Engineering of Escherichia coli for d-Lactate Production from Crude Glycerol.

Crude glycerol resulting from biodiesel production is an abundant and renewable resource. However, the impurities in crude glycerol usually make microbial fermentation problematic. This issue was addressed by systematic engineering of Escherichia coli for the production of d-lactate from crude glycerol. First, mgsA and the synthetic pathways of undesired products were eliminated in E. coli, rendering the strain capable of homofermentative production of optically pure d-lactate. To direct carbon flux toward d-lactate, the resulting strain was endowed with an enhanced expression of glpD-glpK in the glycerol catabolism and of a heterologous gene encoding d-lactate dehydrogenase. Moreover, the strain was evolved to improve its utilization of cruder glycerol and subsequently equipped with the FocA channel to export intracellular d-lactate. Finally, the fed-batch fermentation with two-phase culturing was carried out with a bioreactor. As a result, the engineered strain enabled production of 105 g/L d-lactate (99.9% optical purity) from 121 g/L crude glycerol at 40 h. The result indicates the feasibility of our approach to engineering E. coli for the crude glycerol-based fermentation.

A synthetic consortium of Escherichia coli for n-butanol production by fermentation of the glucose-xylose mixture

The microbial production of n-butanol using glucose and xylose, the major components of plant biomass, can provide a sustainable and renewable fuel as crude oil replacement. However, Escherichia coli prefers glucose to xylose as programmed by carbohydrate catabolite repression (CCR). In this study, a synthetic consortium consisting of two strains was developed by transforming the CCR-insensitive strain into a glucose-selective strain and a xylose-selective strain. Furthermore, the dual culture was reshaped by distribution of the synthetic pathway of n-butanol into two strains. Consequently, the co-culture system enabled effective co-utilization of both sugars and production of 5.2 g/L n-butanol at 30 h. The result leads to the conversion yield and productivity accounting for 63% of the theoretical yield and 0.17 g L-1 h-1, respectively. Overall, the technology platform as proposed is useful for production of other value-added chemicals, which require complicated pathways for their synthesis by microbial fermentation of a sugar mixture.

Antioxidant molecular targets of wheat bran fermented by white rot fungi and its potential modulation of antioxidative status in broiler chickens

ABSTRACT 1. The study focused on antioxidant molecular targets of wheat bran fermented by white rot fungi (WRF) in poultry. After solid-state fermentation of wheat bran by WRF for 12 d, scanning electron microscopy found that the lignocellulose structure showed degradation and fragmentation. 2. A total of 300 1-d-old broilers were evenly divided by gender and randomly allocated into the following treatments: (1) maize–soybean meal (control group), (2) 10% of wheat bran replacing maize (10% WB group) or (3) 10% of fermented wheat bran replacing maize (10% FWB group). 3. The results indicated that the antioxidant gene expression, such as haem oxygenase-1 and glutathione-S-transferase of chicken peripheral blood mononuclear cells, of the 10% FWB group was significantly higher than that of the control group at d 35. For genes of Nicotinamide adenine dinucleotide phosphate oxygenase 1 and reactive oxygen species modulator protein 1, the expression of the 10% FWB group was lower than that of the control group at d 21 and 35. 4. In conclusion, wheat bran fermented by WRF could increase lignocellulolytic enzyme activities and the levels of active components that further regulate the expression of antioxidant molecular targets in poultry.

Evaluation of potential antioxidant and anti-inflammatory effects of Antrodia cinnamomea powder and the underlying molecular mechanisms via Nrf2- and NF-κB-dominated pathways in broiler chickens

ABSTRACT Antrodia cinnamomea, a precious and unique medical fungus existing exclusively in Taiwan, exhibits antioxidant and immunomodulatory properties. This study was conducted to evaluate the beneficial effects of A. cinnamomea powder (ACP) and to further illuminate its underlying antioxidant and immunomodulation molecular mechanisms in broilers. The functional compounds of ACP—crude triterpenoids, crude polysaccharides, and total phenolic content—were assayed, respectively. Two‐hundred‐forty one‐day‐old broilers (Ross 308) were assigned to 4 treatment groups receiving dietary supplementation with ACP at 0, 0.1, 0.2, and 0.4% for 35 days. Each group had 4 replicate pens, with 15 birds per pen. During 1 to 21‐ and 22 to 35‐day periods, chickens on ACP‐supplemented diet demonstrated increased body weight gain, compared to those on the control diet, resulting in increased weight gain throughout the entire experimental period with an increased tendency in feed consumption yet no significant difference in FCR. Blood antioxidant potentiality, superoxide dismutase (SOD), increased in birds fed the supplemented diet at both 21 and 35 d, accompanied by higher catalase (CAT) activity at 21 days. In vivo peripheral blood mononuclear cells (PBMC) exposed to lipopolysaccharide (LPS) and 2,2′‐Azobis(2‐amidinopropane) dihydrochloride (AAPH) capability showed that the diminished cell viability caused by both challenge factors was improved in ACP‐supplemented groups. Antioxidant genes dominated by Nrf2 genes, such as HO‐1 and GCLC, were up‐regulated in 35‐day‐old birds. Inflammatory‐related genes, such as IL‐1&bgr; and IL‐6, ruled mainly by NF‐&kgr;B, were rather down‐regulated by 0.2% ACP addition at 21 and 35 days. Protein expression of Nrf2 and NF‐&kgr;B in the liver supported the mRNA results, demonstrating that all ACP‐supplemented groups showed significantly higher Nrf2 expression, whereas the NF‐&kgr;B was inhibited. In conclusion, preferable microbial balance may putatively indicate the improvement of immunomodulatory‐related capacity by ACP. Furthermore, ACP could induce the Nrf2‐dependent pathway and decrease the NF‐&kgr;B‐dominated inflammatory signaling pathway. Antioxidant and immune capacity in terms of antioxidant enzymes and cell tolerance also was elevated by ACP. Concomitantly, body weight increasing with ACP supplementation as compared to the corresponding control group further implied the promising effects exerted by ACP.

Development of nanoscale oil bodies for targeted treatment of lung cancer

Lung cancer is the most widespread disease and is frequently associated with a high level of epidermal growth factor receptor (EGFR). This study was thus conducted to provide a proof-of-concept approach for targeted therapy of lung cancer by development of nanoscale oil bodies (NOBs). This was carried out by fusion of anti-EGFR affibody (ZEGFR2) with oleosin (Ole), a structure protein of plant seed oils. The fusion protein (Ole-ZEGFR2) was produced in Escherichia coli. NOBs were spontaneously assembled from plant oil, phospholipids, and Ole-ZEGFR2. Consequently, Ole-ZEGFR2-based NOBs were selectively internalized by EGFR-positive lung cancer cells with an efficiency exceeding 90%. Furthermore, the hydrophobic anticancer drug, camptothecin (CPT), was encapsulated into Ole-ZEGFR2-based NOBs. The administration of the CPT formulation based on NOBs resulted in a strong antitumor activity both in vitro and in vivo.

Ethanol Extract of Tripterygium wilfordii Hook. F. Induces G0/G1 Phase Arrest and Apoptosis in Human Leukemia HL-60 Cells Through c-myc and Mitochondria-Dependent Caspase Signaling Pathways

Tripterygium wilfordii Hook. f. is a traditional Chinese herb (Murphy, 2006; Qiu et al., 2003). The extract of Tripterygium wilfordii Hook. f. has been widely applied to the treatment of immune-related diseases, such as rheumatoid arthritis (RA), nephritis, and systemic lupus erythematosus (SLE) (Chang et al., 1999; Wang et al., 2000). Extracts of Tripterygium wilfordii Hook. f. have been shown to inhibit lymphocyte proliferation induced by mitogentic stimulation in-vitro (Wu et al., 2003). Triptolide (PG490, one of the most active components in Tripterygium wilfordii Hook. f. extract, possesses immunosuppressive, anti-inflammatory and anti-fertility actions in vivo and in vitro (Zhao et al., 2005; Leuenroth et al., 2005). Many reports have demonstrated that triptolide has anti-proliferate activity against L1210, U937, K562, HL60, and P388 leukemia cells (Lou et al., 2004; Chan et al., 2001; Wei et al., 1991). However, the cellular and molecular mechanisms underlying mediating Tripterygium wilfordii Hook. f.-induced differentiation and/or apoptosis in leukemia cells have not been well studied.