Construction of Recombinant Bacillus subtilis for Production of Polyhydroxyalkanoates
State Key Laboratory of Chinese Medicine and Molecular Pharmacology, Shenzhen, China. Applied Biochemistry and Biotechnology
(Impact Factor: 1.74).
02/2006; 129-132(1):1015-22. DOI: 10.1385/ABAB:132:1:1015
Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates synthesized by numerous bacteria as intracellular carbon and energy storage compounds and accumulated as granules in the cytoplasm of cells. In this work, we constructed two recombinant plasmids, pBE2C1 and pBE2C1AB, containing one or two PHA synthse genes, respectively. The two plasmids were inserted into Bacillus subtilis DB104 to generate modified strains, B. subtilis/pBE2C1 and B. subtilis/pBE2C1AB. The two recombinants strains were subjected to fermentation and showed PHA accumulation, the first reported example of mcl-PHA production in B. subtilis. Gas Chromatography analysis identified the compound produced by B. subtilis/pBE2C1 to be a hydroxydecanoate-co-hydroxydodecanoate (HD-co-HDD) polymer whereas that produced by B. subtilis/pBE2C1AB was a hydroxybutyrate-co-hydroxydecanoate-co-hydroxydodecanoate (HB-HD-HDD) polymer.
Available from: Mirul Shukor
- "One of the recombinant E . coli that produce high yield of P ( 3HB ) content is the E . coli JM109 transformant harbouring phaC Cs ( Bhubalan et al . , 2011a ) . The synthase was from Chromobacterium sp USM2 . P . aeruginosa phaC1AB and phaC1 from R . eutropha were used as additional synthase to B . subtilis DB104 ( Wang et al . , 2006 ) . The production of MCL - PHAs was found and the recombinant strain was the first of B . subtilis to show the production of PHA using the synthase from different PHA producers ."
Available from: Sergio Grimbs
- "We investigate the metabolic networks of three model organisms, namely Bacillus subtilis (Oh et al., 2007), Escherichia coli (Feist et al., 2007), and seeds of Hordeum vulgare (Grafahrend-Belau et al., 2009), for which growth was predicted in silico and experimentally validated. All considered organisms have several important agricultural or biotechnological applications: B. subtilis is used for food and enzyme production and has been genetically engineered for producing riboflavin and polyhydroxyalkanoates (Schallmey et al., 2004; Perkins et al., 1999; Wang et al., 2006); E. coli has a long history of biotechnological applications, such as: production of insulin, lycopene, and succinic acid (Goeddel et al., 1979; Alper et al., 2005; Lee et al., 2005), and is currently explored for its use in producing polymers and biofuels (Atsumi et al., 2008; Bond-Watts et al., 2011; Yim et al., 2011); H. vulgare has been genetically engineered for enhanced breeding properties, protein synthesis, food and cellulose production (Horvath et al., 2000, 2001; von Wettstein et al., 2000; Patel et al., 2000). We point out that our approach is not restricted to optimizing biomass yield, and thus allows for the detection of reactions which, when introduced into the respective network, improve any metabolic objective of interest. "
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ABSTRACT: Reconstruction of genome-scale metabolic networks has resulted in models capable of reproducing experimentally observed biomass yield/growth rates and predicting the effect of alterations in metabolism for biotechnological applications. The existing studies rely on modifying the metabolic network of an investigated organism by removing or inserting reactions taken either from evolutionary similar organisms or from databases of biochemical reactions (e.g., KEGG). A potential disadvantage of these knowledge-driven approaches is that the result is biased towards known reactions, as such approaches do not account for the possibility of including novel enzymes, together with the reactions they catalyze.
Here, we explore the alternative of increasing biomass yield in three model organisms, namely Bacillus subtilis, Escherichia coli, and Hordeum vulgare, by applying small, chemically feasible network modifications. We use the predicted and experimentally confirmed growth rates of the wild-type networks as reference values and determine the effect of inserting mass-balanced, thermodynamically feasible reactions on predictions of growth rate by using flux balance analysis.
While many replacements of existing reactions naturally lead to a decrease or complete loss of biomass production ability, in all three investigated organisms we find feasible modifications which facilitate a significant increase in this biological function. We focus on modifications with feasible chemical properties and a significant increase in biomass yield. The results demonstrate that small modifications are sufficient to substantially alter biomass yield in the three organisms. The method can be used to predict the effect of targeted modifications on the yield of any set of metabolites (e.g., ethanol), thus providing a computational framework for synthetic metabolic engineering.
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ABSTRACT: The kinetics of inactivation of Gram-positive strain, Bacillus subtilis in aquatic systems was investigated as function ozone aeration duration under varied conditions. Oxygen flow was in situ enriched with ozone using ozoniser, with [O(3)] ranging from (0.3 - 9.8) x 10(-5) moles per liter of oxygen. The inactivation kinetics of B. subtilis followed pseudo-first-order kinetics with respect to microbe, under excess [O(3)] conditions. The disinfection kinetics had first order dependence on ozone concentration and the overall second-order rate constant was (7.54 +/- 1.37) x 10(3) M(-1) min(-1). The effect initial temperature and pH of the system on the ozone initiated inactivation of microbe was also explored. Relative to hydroxyl radicals, molecular ozone was found more effective in microbial inactivation. Appropriate mechanism for ozone initiated inactivation is proposed. Ozone aeration significantly decreased the BOD levels of natural and B. subtilis spiked waters.
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