Xiaoqiang Jia

Strain Improvement of Streptomyces roseosporus for Daptomycin Production by Rational Screening of He–Ne Laser and NTG Induced Mutants and Kinetic Modeling

To improve the yield of daptomycin by Streptomyces roseosporus, a method of rational screening of He–Ne Laser and N-methyl-N-nitro-N-nitrosoguanidine (NTG)-induced mutants was employed in this work. Under the optimal mutagenesis conditions (NTG of 0.3xa0mg/L, 40xa0min; irradiation at 15xa0mW, 15xa0min), two steps of combined mutations were conducted. Screening of mutants was done according to the individual resistance to n-decanoic acid and daptomycin. A mutant strain LC-54-16, with the highest daptomycin production ability of 616xa0mg/L was obtained, which was over 5 times higher than the wild strain LC-52-6. The transcription levels of the main dpt genes in the mutant were approximately five times higher than those in the wild. The superiority of the mutant to the wild strain was further proved by the comparative studies on the kinetics of the mutant and the wild. The decrease of the inhibition of substrate and product was further confirmed, as well. It was concluded that the method of rational screening of He–Ne Laser and NTG-induced mutants could efficiently improve the daptomycin production ability of S. roseosporus.

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.

Engineering a Metabolic Pathway for Isobutanol Biosynthesis in Bacillus subtilis

Isobutanol can be biosynthesized via α-ketoisovalerate catalyzed by heterologous keto acid decarboxylase (KDC) and alcohol dehydrogenase (ADH). In this work, isobutanol biosynthesis pathway was designed in Bacillus subtilis, a notable solvent-tolerant host. In order to do that, a plasmid pPKA expressing KDC and ADH under the control of a B. subtilis strong promoter P43 was constructed. Isobutanol was detected in the products of the recombinant B. subtilis harboring pPKA plasmid, whereas none was detected by the wild-type strain. Effects of the medium ingredients such as glucose concentration and valine addition, and operating parameters such as initial pH, inoculation volume, and medium work volume on isobutanol production were also investigated. Isobutanol production reached to the maximum of 0.607xa0g/L after 35-h cultivation under the conditions: glucose concentration of 3%, valine addition of 2%, initial pH of 7.0, inoculum of 1%, and work volume of 50xa0mL/250xa0mL. Though the isobutanol production by the recombinant was low, it was the first successful attempt to produce isobutanol in engineered B. subtilis, and the results showed its great potential as an isobutanol-producing cell factory.

Enhanced biodegradation of alkane hydrocarbons and crude oil by mixed strains and bacterial community analysis.

In this study, two strains, Acinetobacter sp. XM-02 and Pseudomonas sp. XM-01, were isolated from soil samples polluted by crude oil at Bohai offshore. The former one could degrade alkane hydrocarbons (crude oil and diesel, 1:4 (v/v)) and crude oil efficiently; the latter one failed to grow on alkane hydrocarbons but could produce rhamnolipid (a biosurfactant) with glycerol as sole carbon source. Compared with pure culture, mixed culture of the two strains showed higher capability in degrading alkane hydrocarbons and crude oil of which degradation rate were increased from 89.35 and 74.32u2009±u20094.09 to 97.41 and 87.29u2009±u20092.41xa0%, respectively. In the mixed culture, Acinetobacter sp. XM-02 grew fast with sufficient carbon source and produced intermediates which were subsequently utilized for the growth of Pseudomonas sp. XM-01 and then, rhamnolipid was produced by Pseudomonas sp. XM-01. Till the end of the process, Acinetobacter sp. XM-02 was inhibited by the rapid growth of Pseudomonas sp. XM-01. In addition, alkane hydrocarbon degradation rate of the mixed culture increased by 8.06 to 97.41xa0% compared with 87.29xa0% of the pure culture. The surface tension of medium dropping from 73.2u2009×u200910−3 to 28.6u2009×u200910−3xa0N/m. Based on newly found cooperation between the degrader and the coworking strain, rational investigations and optimal strategies to alkane hydrocarbons biodegradation were utilized for enhancing crude oil biodegradation.

Biodegradation of Crude Oil by a Newly Isolated Strain Rhodococcus sp. JZX-01

A highly efficient oil-degrading bacteria JZX-01 was isolated from the oil-contaminated soil of the seacoast near the Boxi Offshore Oil Field of China. Morphological, physiological, and 16S rDNA gene sequence analyses indicated that JZX-01 was assigned to the genus Rhodococcus sp. This strain decomposed 65.27u2009±u20095.63xa0% of the crude oil in 9xa0days. Gas chromatography–mass spectrometry analysis showed that even the long-chain hydrocarbons (C31–C38) and branched alkanes (pristine and phytane), which were regarded as the stubborn ones, could be degraded. Further study showed that the bacteria still has good oil degradation ability at low temperatures as well as under high salt conditions. Moreover, JZX-01 was found to have a biosurfactant-producing capacity, which significantly favors the surface tension reduction and crude oil degradation. The promising isolated strain Rhodococcus sp. JZX-01 could be further used for the bioremediation of oil-polluted soil or seawater in a wide range of temperatures and high salt conditions.