Plasmids were transformed by electroporation into various strains of Bacillus thuringiensis with frequencies of up to 10(5) transformants/micrograms. pC 194 transformed all strains tested at a high frequency and cells could be stably transformed with pC194 and pUB110 simultaneously by electroporation with a frequency of 10(2) pC194+ pUB110 transformants/micrograms DNA. Low transformation frequencies observed with some plasmids, especially those grown initially in Escherichia coli, could be increased by passage through B. thuringiensis, B. thuringiensis var. israelensis and in acrystalliferous mutant of the same strain transformed at frequencies of 10(4)-10(5)/micrograms DNA with most of the plasmids tested. A cloned israelensis 27-kDa delta-endotoxin gene was introduced into the israelensis acrystalliferous mutant and a kurstaki acrystalliferous mutant by electroporation. Both transformants were shown to express the endotoxin gene and to be toxic to Aedes aegypti larvae.

Five recombinant E. coli clones exhibiting toxicity to Aedes aegypti larvae were obtained from a library of 800 clones containing XbaI DNA fragments of 110 kb plasmid from B. thuringiensis var. israelensis. All the five clones (pMU 14/258/303/388/679) had the same 3.8-kb insert and encoded a major protein of 130 kDa which was highly toxic to A. aegypti larvae. Three clones (pMU 258/303/388) transcribed the 130 kD a gene in the same direction as that of lac Z promoter of pUC12 vector whereas the transcription of the other two (pMU 14/679) was in the opposite direction. A 1.9-kb fragment of the 3.8 kb insert coded for a protein of 65 kDa. Partial DNA sequence of the 3.8 kb insert, corresponding to the 5'-terminal of the 130 kDa gene, revealed a continuous reading frame, a Shine-Dalgarno sequence and a tentative 5'-regulatory region. These results demonstrated that the 3.8 kb insert is a minimal DNA fragment containing a regulatory region plus the coding sequence of the 130 kDa protein that is highly toxic to mosquito larvae.

The complete nucleotide sequence of a cloned gene encoding a 130-kDa crystal protein of Bacillus tlzuringiensis (B.t.) subspecies israelensis has been determined. The recombinant protein (Bt8) was purified and shown to be a mosquito-specific toxin with a LCso value of 43 ng/ml to third-instar larvae of Aedes aegypti. Bt8 is processed by proteases or midgut extracts of mosquito larvae into toxic fragments of 68 -78 kDa. Deletion mapping indicated that the active fragment of Bt8 is localized in the N-terminal half of the protoxin molecule. The deduced amino acid sequence of Bt8 has been compared with that of Bt2, a Lepidoptera-specific toxin, previously cloned from Bacillus thuringiensis berliner. Highly homologous amino acid stretches are present in the C-terminal half of the proteins. The N-terminal parts show much less sequence homology but they display a strikingly similar distribution of hydrophilic and hydrophobic amino acids. In addition, Bt8 and Bt2 show a significant immunological crossreaction. The data indicate that although these B.t. delta endotoxins exhibit a different insect-host specificity, they are structurally related and might use a similar mechanism to interact with insect cell membranes. B. thuringiensis subspecies israelensis produces a crystal that is highly toxic to the larvae of mosquitoes and blackflies [3, 51. The solubilized crystal proteins of B.t. isruelensis also exhibit hemolytic activity and non-specific cytotoxicity towards insect and mammalian cells in vitro [6]. The B.1. israelensis crystal is composed of at least three major polypeptides of 28 kDa, 65 kDa and 130 kDa [7], which differ in their antigenic structure [8].It is still unclear which crystal protein is responsible for the strong mosquitocidal activity. through cloning and expression of their genes. The availability of purified recombinant protein also allows one to investigate in detail the structural and functional characteristics of these insect-specific toxins.In this paper we present the complete nucleotide sequence of a gene (bt8), encoding a 130-kDa mosquitocidal protein of B.t. israelensis. The recombinant protein (Bt8) was purified from the E. coli clone. Its properties were compared to those of Bt2, a previously described [I 91 Lepidoptera-specific B.t. toxin. MATERIALS AND METHODS Strains and plasmidsB. thuringiensis subspecies israelensis strain 442-72 was obtained from the Bacillus Genetic Stock Center (Columbia, Ohio). E. coli JM107 carrying plasmid pMU388 has been generated previously [18]. E. coli K514 (pGI502), containing a B.t. herliner toxin gene and expressing a 130-kDa protein (Bt2), has been described [19]. Crystal purificationCrystals of B.t. israelensis strain 442-72 and B.t. berliner 171 5 were purified from sporulating cultures as described by Mahillon and Delcour [20]. Purification of' cloned B.t. toxinThe cell pellet from 1 1 saturated overnight culture of E. coli JM107 (pMU388) was suspended in 100 ml50 mM Tris/ HC1, pH 7.9/50 mM EDTA/15% (massivol.) sucrose and frozen at -20°C. After thaw...

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BackgroundMicroalgae are promising sources of lipid triacylglycerol (TAG) for biodiesel production. However, to date, microalgal biodiesel is technically feasible, but not yet economically viable. Increasing TAG content and productivity are important to achieve economic viability of microalgal biodiesel. To increase TAG content, oleaginous microalga Neochloris oleoabundans was genetically engineered with an endogenous key enzyme diacylglycerol acyltransferase 2 (NeoDGAT2) responsible for TAG biosynthesis.ResultsThe integration of NeoDGAT2 expression cassettes in N. oleoabundans transformant was confirmed by PCR. The neutral lipid accumulation in the transformant detected by Nile red staining was accelerated and 1.9-fold higher than in wild type; the lipid bodies in the transformant visualized under fluorescence microscope were also larger. The NeoDGAT2 transcript was two-fold higher in the transformant than wild type. Remarkably higher lipid accumulation was found in the transformant than wild type: total lipid content increased 1.6-to 2.3-fold up to 74.5 ± 4.0% dry cell weight (DCW) and total lipid productivity increased 1.6- to 3.2-fold up to 14.6 ± 2.0 mg/L/day; while TAG content increased 1.8- to 3.2-fold up to 46.1 ± 1.6% DCW and TAG productivity increased 1.6- to 4.3-fold up to 8.9 ± 1.3 mg/L/day. A significantly altered fatty acid composition was detected in the transformant compared to wild type; the levels of saturated fatty acid C16:0 increased double to 49%, whereas C18:0 was reduced triple to 6%. Long-term stability was observed in the transformant continuously maintained in solid medium over 100 generations in a period of about 4 years.ConclusionsOur results demonstrate the increased TAG content and productivity in N. oleoabundans by NeoDGAT2 overexpression that may offer the first step towards making microalgae an economically feasible source for biodiesel production. The strategy for genetically improved microalga presented in this study can be applied to other microalgal species possessing desired characteristics for industrial biofuel production.