A fast spheroplast formation procedure in some 2,5-diketo-D-gluconate- and 2-keto-L-gulonate- producing bacteria.
Calcium 2-keto-L-gulonate (Ca-2-KLG, a key intermediate in vitamin C synthesis) is produced from calcium 2,5-diketo D-gluconate (Ca-2,5-DKG) by a variety of bacteria. A few bacterial species which efficiently convert glucose to Ca-2,5-DKG have been isolated in our laboratory. Our bacterial collection included species that possess the genes for production of Ca-2-KLG from Ca-2,5-DKG; however, the yield of the former is poor. A procedure for the preparation of spheroplasts in Ca-2,5-DKG- and Ca-2-KLG-producing bacteria was developed for the construction of recombinants (fusants), combining the genes for conversion of glucose to Ca-2-KLG efficiently by protoplast fusion. The standard procedure for spheroplast formation in Gram negative bacteria by the Tris-sucrose-EDTA-lysozyme system did not work in the organisms under investigation. The need for an alternative method was necessary. Our results show that, while the Tris-NaCl-EDTA-lysozyme system (pH 8.3) worked very well with bacterial strains of Gluconobacter oxydans (ATCC9937) and Acetobacter melanogenus (NCIM2259), the Tris-sucrose-EDTA-lysozyme system worked well for Erwinia herbicola (ATCC21998), Pseudomonas chlororaphis (NCIM2041) and Corynebacterium species (ATCC31090). However, none of these systems produced spheroplasts in Brevibacterium ketosoreductum (ATCC21914), for which a separate system is under development.
Verma
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Genetic Engineering Unit, Regional Research Laboratory, Jammu Tawi, India.
Qazi
G N
GN
Parshad
R
R
Chopra
C L
CL
D001419:Bacteria / Q000302:isolation & purification / Q000378:metabolism*; D001431:Bacteriological Techniques; D001709:Biotechnology; D004492:Edetic Acid; D005942:Gluconates / Q000378:metabolism*; D009113:Muramidase; D012965:Sodium Chloride; D013104:Spheroplasts / Q000302:isolation & purification / Q000378:metabolism*; D013395:Sucrose; D013400:Sugar Acids / Q000378:metabolism*; D014325:Tromethamine
D005942:Gluconates; D013400:Sugar Acids; D014325:Tromethamine; D012965:Sodium Chloride; C063875:2,5-diketogluconate; D013395:Sucrose; D004492:Edetic Acid; D009113:Muramidase; C011491:provitamin C