Shan-Yu Chen

Improved Production of Biosurfactant with Newly Isolated Pseudomonas aeruginosa S2

An indigenous strain Pseudomonas aeruginosa S2 (P. aeruginosa S2), isolated from diesel‐contaminated soil, produced extracellular surface‐active material identified as rhamnolipid. Due to its excellent surface activity, rhamnolipid is known to be well‐suited for stimulating the bioremediation efficiency of oil contaminated sites. To improve production yield of rhamnolipid with P. aeruginosa S2, various carbon and nitrogen sources were screened to select favorable ones leading to better biosurfactant production yield. It was found that using 4% glucose could attain better rhamnolipid yield, while 50 mM NH4NO3 appeared to be the most preferable nitrogen source. Meanwhile, the effect of carbon to nitrogen ratio (C/N ratio) on rhamnolipid yield was also investigated, and the optimal C/N ratio was identified as approximately 11.4. Moreover, response surface methodology (RSM) was applied to optimize the trace element concentration for rhamnolipid production. Results from two‐level design indicate that concentrations of MgSO4 and FeSO4 were the most significant factors affecting rhamnolipid production. Using steepest ascent method and RSM analysis, an optimal medium composition was determined, giving a rhamnolipid production yield of 2.37 g/L in 100 h at 37 °C and 200 rpm agitation. Scale‐up production of rhamnolipid in a well‐controlled 5 L jar fermentor using the optimal medium and operating condition (at 37 °C and pH 6.8) further elevated the biosurfactant production yield to 5.31 g/L (in 97 h), which is over 2‐fold higher than the best results obtained from shake‐flask tests.

Production and Characterization of Fengycin by Indigenous Bacillus subtilis F29-3 Originating from a Potato Farm

Fengycin, a lipopeptide biosurfactant, was produced by indigenous Bacillus subtilis F29-3 isolated from a potato farm. Although inhibiting the growth of filamentous fungi, the fengycin is ineffective against yeast and bacteria. In this study, fengycin was isolated from fermentation broth of B. subtilis F29-3 via acidic precipitation (pH 2.0 with 5 N HCl) followed by purification using ultrafiltration and nanofiltration. The purified fengycin product was characterized qualitatively by using fast atom bombardment-mass spectrometer, Fourier transform infrared spectrometer, ultraviolet-visible spectrophotometer, 13C-nuclear magnetic resonance spectrometer and matrix assisted laser desorption ionization-time of flight, followed by quantitative analysis using reversed-phase HPLC system. This study also attempted to increase fengycin production by B. subtilis F29-3 in order to optimize the fermentation medium constituents. The fermentation medium composition was optimized using response surface methodology (RSM) to increase fengycin production from B. subtilis F29-3. According to results of the five-level four-factor central composite design, the composition of soybean meal, NaNO3, MnSO4·4H2O, mannitol-mannitol, soybean meal-mannitol, soybean meal-soybean meal, NaNO3-NaNO3 and MnSO4·4H2O-MnSO4·4H2O significantly affected production. The simulation model produced a coefficient of determination (R2) of 0.9043, capable of accounting for 90.43% variability of the data. Results of the steepest ascent and central composite design indicated that 26.2 g/L of mannitol, 21.9 g/L of soybean meal, 3.1 g/L of NaNO3 and 0.2 g/L of MnSO4·4H2O represented the optimal medium composition, leading to the highest production of fengycin. Furthermore, the optimization strategy increased the fengycin production from 1.2 g/L to 3.5 g/L.

Production and characterization of ectoine by Marinococcus sp. ECT1 isolated from a high-salinity environment.

A halophilic bacterium isolated from a salt environment in southern Taiwan was identified as a Marinococcus sp. ECT1. This bacterium could synthesize and accumulate intracellular ectoine as a compatible solute capable of resisting osmotic stress in a hyper-osmotic environment. This study also developed a semi-synthesized medium (YAMS medium), capable of facilitating the growth of this Marinococcus sp. ECT1 with 600 mg/L crude ectoine production. Moreover, Marinococcus sp. ECT1 was grown on YAMS medium containing different initial yeast extract concentrations (C(YE)) (0 to 60 g/L) to demonstrate how C(YE) affects crude ectoine production. While the maximum cell concentration was increased by 23-fold when the C(YE) was 40 g/L, the maximum crude ectoine production reached 2.5 g/L when C(YE) was 40 g/L. In addition to demonstrating the success of the fermentation strategy of ectoine in increasing the production and production yield, experimental results further demonstrated that the fermentation medium of ectoine is highly promising for commercialization. Furthermore, the molecular weight and chemical structure of ectoine were identified and characterized by FAB-MS and (1)H-NMR.

In vivo immobilization of D-hydantoinase in Escherichia coli.

D-P-Hydroxyphenylglycine (D-HPG) is a precursor required for the synthesis of semi-synthetic antibiotics. This unnatural amino acid can be produced by a transformation reaction mediated by D-hydantoinase (D-HDT) and d-amidohydrolase. In this study, a method was developed to integrate production and immobilization of recombinant D-HDT in vivo. This was approached by first fusion of the gene encoding D-HDT with phaP (encoding phasin) of Ralstonia eutropha H16. The fusion gene was then expressed in the Escherichia coli strain that carried a heterologous synthetic pathway for polyhydroxyalkanoate (PHA). As a result, d-HDT was found to associate with isolated PHA granules. Further characterization illustrated that D-HDT immobilized on PHA exhibited the maximum activity at pH 9 and 60°C and had a half-life of 95 h at 40°C. Moreover, PHA-bound d-HDT could be reused for 8 times with the conversion yield exceeding 90%. Overall, it illustrates the feasibility of this approach to facilitate in vivo immobilization of enzymes in heterologous E. coli strain, which may open a new avenue of enzyme application in industry.

Functional Expression of phaCAB Genes from Cupriavidus taiwanensis Strain 184 in Escherichia coli for Polyhydroxybutyrate Production

Polyhydroxyalkanoates are polyesters synthesized by numerous microorganisms. These polyesters are biodegradable and have similar properties to those of conventional plastics. Cupriavidus taiwanensis strain 184 is phylogenetically related to the well-known polyhydroxybutyrate (PHB) producer Ralstonia eutropha (Cupriavidus necator) and is also shown to be able to accumulate significant amounts of PHB. In this study, we cloned the PHB synthesis genes (phaCAB) from C. taiwanensis 184 into Escherichia coli for biosynthesis of PHB. The recombinant E. coli strains were able to synthesize significant amounts of PHB. The PHB amounted to about 66∼70% of total cell material of these recombinant strains.

Engineering of Escherichia coli for targeted delivery of transgenes to HER2/neu‐positive tumor cells

Targeting of non‐phagocytic tumor cells and prompt release of gene cargos upon entry into tumors are two limiting steps in the bacterial gene delivery path. To tackle these problems, the non‐pathogenic Escherichia coli strain BL21(DE3) was engineered to display the anti‐HER2/neu affibody on the surface. After co‐incubation with tumor cells for 3 h, the anti‐HER2/neu affibody‐presenting E. coli strain was selectively internalized into HER2/neu‐positive SKBR‐3 cells. The invasion efficiency reached as high as 30%. Furthermore, the bacteria were equipped with the phage ϕX174 lysin gene E‐mediated autolysis system. Carrying the transgene (e.g., eukaryotic green fluorescent protein, GFP), the tumor‐targeting bacteria were subjected to the thermal shock to trigger the autolysis system upon entry into HER2/neu‐positive cells. Flow cytometric analysis revealed that 3% of infected cells expressed GFP 24 h post thermal induction. Overall, the results show a promise of the proposed approach for developing bacteria as a delivery carrier. Biotechnol. Bioeng. 2011; 108:1662–1672.

Functional cis-expression of phaCAB genes for poly(3-hydroxybutyrate) production by Escherichia coli.

Aims:  To develop a microbial strain producing poly(3‐hydroxybutyrate) [P(3HB)], in the absence of antibiotic supplementation (normally required to stabilize a recombinant plasmid), by constructing a recombinant Escherichia coli strain with phaCAB and vgb integrated into the chromosome.

Effects of Different Substrate Composition on Biosynthesis of Polyhydroxybutyrate-co-hydroxyvalerate by Recombinant Escherichia coli

Cupriavidus necator is well known for its ability to accumulate polyhydroxybutyrate (PHB). When supplemented with propionic acid (or sodium propionate) in the growth medium, the bacterium is also able to synthesize polyhydroxybutyrate-co-hydroxyvalerate (PHBV). In order to increase the fraction of 3-hydroxyvalerate (3HV) in PHBV, we cloned the propionate permease gene prpP from C. necator and the propionyl-CoA synthase gene prpE from Cupriavidus taiwanensis and transformed into an Escherichia coli containing phaCAB operon of C. necator. The effects on PHBV accumulation in cells co-expressed with phaCAB and prpE or prpP in the media contained mixed carbon sources (glucose and sodium propionate) were evaluated. The HV fraction in PHBV increased when prpE or prpP was overexpressed in the cells. Concentrations of yeast extracts could also affect the fraction of HV. In addition, when glucose was replaced by sodium pyruvate, sodium succinate, or sodium gluconate, only PHB were detected in the recombinant strains.

Marker-free chromosomal expression of foreign and native genes in Escherichia coli.

Genetic manipulation of Escherichia coli strains for desired traits is the most applied strain engineering approach in industrial applications. For chromosomal insertion of genes and controlled expression of genomic genes in E. coli, the replicon-free and markerless method is described based on a series of conditional-replication plasmids called phage-integration vectors. They mainly carry the multiple cloning site and the prophage attachment site, which are sandwiched by two FRT sites. With the aid of the phage integrase from conditional-replication helper plasmids, the passenger genes of either foreign or native type incorporated into the integration vectors can be specifically integrated into bacterial genome at the prophage attachment site. Finally, the inserted DNA containing replicon and/or selective markers in integrants can be eliminated by the act of the FLP recombinase provided from a conditional-replication helper plasmid.