Jianping Wen

Enhanced FK506 production in Streptomyces tsukubaensis by rational feeding strategies based on comparative metabolic profiling analysis

FK506, a widely used immunosuppressant, is produced by industrial fermentation processes using various Streptomyces species. However, the low titer becomes a bottleneck for its application and industrialization. It urgently required a full understanding of the biological mechanisms for FK506 overproduction. Towards this end, comparative metabolomics approach was employed to analyze metabolite concentrations difference of Streptomyces tsukubaensis cultivated in two media with low and high productivities. Initially, 98 intracellular metabolites were identified and 13 metabolites involved in five pathways were determined to be directly correlated with FK506 biosynthesis. Then in‐depth analysis elucidated how those key factors exerted influence on FK506 biosynthesis. Many previously unreported metabolites were shown to play an important role in FK506 biosynthesis and provided potential regulation points for external manipulation. Based on such key information, rationally designed feeding strategy was carried out. Results showed that the FK506 yield increased from 251 to 405 mg/L, whereas, by‐products FK520 and 37,38‐dihydro‐FK506 decreased by 31% and 39%, respectively, compared with the values of control. To our knowledge, it is the first study to apply the comparative metabolomics method to identify key metabolites to promote the FK506 production. The strategies developed here can easily be extended to titer improvement of other important microbial natural products and process optimization. Biotechnol. Bioeng. 2013;110: 2717–2730.

Genome-scale metabolic network guided engineering of Streptomyces tsukubaensis for FK506 production improvement.

BackgroundFK506 is an important immunosuppressant, which can be produced by Streptomyces tsukubaensis. However, the production capacity of the strain is very low. Hereby, a computational guided engineering approach was proposed in order to improve the intracellular precursor and cofactor availability of FK506 in S. tsukubaensis.ResultsFirst, a genome-scale metabolic model of S. tsukubaensis was constructed based on its annotated genome and biochemical information. Subsequently, several potential genetic targets (knockout or overexpression) that guaranteed an improved yield of FK506 were identified by the recently developed methodology. To validate the model predictions, each target gene was manipulated in the parent strain D852, respectively. All the engineered strains showed a higher FK506 production, compared with D852. Furthermore, the combined effect of the genetic modifications was evaluated. Results showed that the strain HT-ΔGDH-DAZ with gdhA-deletion and dahp-, accA2-, zwf2-overexpression enhanced FK506 concentration up to 398.9 mg/L, compared with 143.5 mg/L of the parent strain D852. Finally, fed-batch fermentations of HT-ΔGDH-DAZ were carried out, which led to the FK506 production of 435.9 mg/L, 1.47-fold higher than the parent strain D852 (158.7 mg/L).ConclusionsResults confirmed that the promising targets led to an increase in FK506 titer. The present work is the first attempt to engineer the primary precursor pathways to improve FK506 production in S. tsukubaensis with genome-scale metabolic network guided metabolic engineering. The relationship between model prediction and experimental results demonstrates the rationality and validity of this approach for target identification. This strategy can also be applied to the improvement of other important secondary metabolites.

Immobilized smart RNA on graphene oxide nanosheets to specifically recognize and adsorb trace peptide toxins in drinking water

The contaminations of peptide toxins in drinking water lead directly to sickness and even death in both humans and animals. A smart RNA as aptamer is covalently immobilized on graphene oxide to form a polydispersed and stable RNA-graphene oxide nanosheet. RNA-graphene oxide nanosheets can resist nuclease and natural organic matter, and specifically adsorb trace peptide toxin (microcystin-LR) in drinking water. The adsorption data fit the pseudo-second-order kinetics and the Langmuir isotherm model. The adsorption capacity of RNA-graphene oxide nanosheets decreases at extreme pH, temperature, ionic strength and natural organic matter, but it is suitable to adsorb trance pollutants in contaminated drinking water. Compared with other chemical and biological sorbents, RNA-graphene oxide nanosheets present specific and competitive adsorption, and are easily synthesized and regenerated. Aptamer (RNA) covalently immobilized on graphene oxide nanosheets is a potentially useful tool in recognizing, enriching and separating small molecules and biomacromolecules in the purification of contaminated water and the preparation of samples.

Rational improvement of the engineered isobutanol-producing Bacillus subtilis by elementary mode analysis

BackgroundIsobutanol is considered as a leading candidate for the replacement of current fossil fuels, and expected to be produced biotechnologically. Owing to the valuable features, Bacillus subtilis has been engineered as an isobutanol producer, whereas it needs to be further optimized for more efficient production. Since elementary mode analysis (EMA) is a powerful tool for systematical analysis of metabolic network structures and cell metabolism, it might be of great importance in the rational strain improvement.ResultsMetabolic network of the isobutanol-producing B. subtilis BSUL03 was first constructed for EMA. Considering the actual cellular physiological state, 239 elementary modes (EMs) were screened from total 11,342 EMs for potential target prediction. On this basis, lactate dehydrogenase (LDH) and pyruvate dehydrogenase complex (PDHC) were predicted as the most promising inactivation candidates according to flux flexibility analysis and intracellular flux distribution simulation. Then, the in silico designed mutants were experimentally constructed. The maximal isobutanol yield of the LDH- and PDHC-deficient strain BSUL05 reached 61% of the theoretical value to 0.36 ± 0.02 C-mol isobutanol/C-mol glucose, which was 2.3-fold of BSUL03. Moreover, this mutant produced approximately 70 % more isobutanol to the maximal titer of 5.5 ± 0.3 g/L in fed-batch fermentations.ConclusionsEMA was employed as a guiding tool to direct rational improvement of the engineered isobutanol-producing B. subtilis. The consistency between model prediction and experimental results demonstrates the rationality and accuracy of this EMA-based approach for target identification. This network-based rational strain improvement strategy could serve as a promising concept to engineer efficient B. subtilis hosts for isobutanol, as well as other valuable products.

Metabolic profiling of a Rhizopus oryzae fumaric acid production mutant generated by femtosecond laser irradiation

Femtosecond laser irradiation was employed to induce mutations in Rhizopus oryzae, leading to increases in fumaric acid production. Compared to the parental strain, mutant strain FM19 exhibited an increase in titer and yield of 56.3% and 36.6%, respectively, corresponding to a titer of 49.4 g/L and a yield of 0.56 g fumaric acid per g glucose. Metabolic profiling by gas chromatography-mass spectrometry revealed that higher levels of carbon (Embden-Meyerhof-Parnas and tricarboxylic acid cycle) and amino acid metabolism were operating in the high-yielding strain; particularly, 4-aminobutyric acid and 5-aminolevulinic acid were increased 10.33- and 7.22-fold, respectively, compared with parental strain during stationary phase. These findings provided new insights into metabolic characterization of high-yielding fumaric acid R. oryzae.

Rational medium optimization based on comparative metabolic profiling analysis to improve fumaric acid production.

To rationally guide fumaric acid production improvement, metabolic profiling approach was performed to analyze metabolite changes of Rhizopus oryzae FM19 under different fermentation conditions. A correlation between the metabolic profiling and fumaric acid production was revealed by principal component analysis as well as partial least squares. Citric acid, oxaloacetic acid, 2-oxoglutarate, lactic acid, proline, alanine, valine, leucine were identified to be mainly responsible for the metabolism difference, which were involved in the Embden-Meyerhof-Parnas, tricarboxylic acid cycle, amino acid metabolism and fatty acid metabolism. Through the further analysis of metabolites changes together with the above pathways, exogenous addition strategies were developed, which resulted in 14% increase of fumaric acid (up to 56.5 g/L) and less by-products. These results demonstrated that metabolic profiling analysis could be successfully applied to the rational guidance of medium optimization and the productivity improvement of value-added compounds.

Effect of Li diffusion on the composition of LiNbO3 at high temperature

Abstract LiNbO 3 crystals with a variety of compositions were prepared by the vapor transport equilibration (VTE) technique. Crystals were characterized through the measurements of fundamental absorption edge, lattice parameters and the electric field required for ferroelectric domain switching. The techniques employed for characterization served as tools for the determination of the composition and homogeneity of the crystals. We discussed the Li diffusion mechanisms in LiNbO 3 crystals at high temperature and found that the Li vacancy model is consistent with the experimental results for the VTE-grown LiNbO 3 crystal.

ssDNA Aptamer-Based Column for Simultaneous Removal of Nanogram Per Liter Level of Illicit and Analgesic Pharmaceuticals in Drinking Water

Aptamers are a new class of single-stranded DNA/RNA molecules selected from synthetic nucleic acid libraries for molecular recognition. Our group reports a novel aptamer column for the removal of trace (ng/L) pharmaceuticals in drinking water. In this study, cocaine and diclofenac were chosen as model molecules to test the aptamer column which presented high removal capacity, selectivity, and stability. The removal of pharmaceuticals was as high as 88-95%. The data of adsorption were fitted with Langmuir isotherm and a pseudo-second-order kinetic model. A thermodynamic experiment proved the adsorption processes were exothermic in spontaneity. The kinetics of aptamer was composed of three steps: activation, binding, and hybridization. The first step was the rate-controlling step. The adsorption system was divided into three parts: kinetic, mixed, and thermodynamic zones from 0% to 100% binding fraction of aptamer. Furthermore, the aptamer column was reusable and achieved strong removal efficiency from 4 to 30 °C at normal cation ion concentration (5-100 mg/L) for multipollutants without cross effects and secondary pollution. This work indicates that aptamer, as a new sorbent, can be used in the removal of persistent organic pollutants, biological toxins, and pathogenic bacteria from surface, drinking, and ground water.

Mutation of Candida tropicalis by Irradiation with a He-Ne Laser To Increase Its Ability To Degrade Phenol

ABSTRACT Candida tropicalis isolated from acclimated activated sludge was used in this study. Cell suspensions with 5 × 107 cells ml−1 were irradiated by using a He-Ne laser. After mutagenesis, the irradiated cell suspension was diluted and plated on yeast extract-peptone-dextrose (YEPD) medium. Plates with approximately 20 individual colonies were selected, and all individual colonies were harvested for phenol biodegradation. The phenol biodegradation stabilities for 70 phenol biodegradation-positive mutants, mutant strains CTM 1 to 70, ranked according to their original phenol biodegradation potentials, were tested continuously during transfers. Finally, mutant strain CTM 2, which degraded 2,600 mg liter−1 phenol within 70.5 h, was obtained on the basis of its capacity and hereditary stability for phenol biodegradation. The phenol hydroxylase gene sequences were cloned in wild and mutant strains. The results showed that four amino acids were mutated by irradiation with a laser. In order to compare the activity of phenol hydroxylase in wild and mutant strains, their genes were expressed in Escherichia coli BL21(DE3) and enzyme activities were spectrophotometrically determined. It was clear that the activity of phenol hydroxylase was promoted after irradiation with a He-Ne laser. In addition, the cell growth and intrinsic phenol biodegradation kinetics of mutant strain CTM 2 in batch cultures were also described by Haldanes kinetic equation with a wide range of initial phenol concentrations from 0 to 2,600 mg liter−1. The specific growth and degradation rates further demonstrated that the CTM 2 mutant strain possessed a higher capacity to resist phenol toxicity than wild C. tropicalis did.

Metabolic flux analysis and principal nodes identification for daptomycin production improvement by Streptomyces roseosporus.

In the present work, a comprehensive metabolic network of Streptomyces roseosporus LC-54-20 was proposed for daptomycin production. The analysis of extracellular metabolites throughout the batch fermentation was evaluated in addition to daptomycin and biomass production. Metabolic flux distributions were based on stoichiometrical reaction as well as the extracellular metabolites fluxes. Experimental and calculated values for both the specific growth rate and daptomycin production rate indicated that the in silico model proved a powerful tool to analyze the metabolic behaviors based on the analysis under different initial glucose concentrations throughout the fermentation. Through manipulating different pH values, the production rates of various extracellular metabolites were also presented in this paper. Flux distribution variations revealed that the daptomycin production could be significantly influenced by the branch points of glucose 6-phosphate, 3-phosphoglycerate, phosphoenolpyruvate, pyruvate, and oxaloacetate. The five principal metabolites were certified as the flexible nodes and could form potential bottlenecks for a further enhancement of daptomycin production. Furthermore, various concentrations of the five precursors were added into the batch fermentation and led to the enhancement of daptomycin concentration and production rate.