Improved vectors for transcriptional/translational signal screening in corynebacteria using the melC operon from Streptomyces glaucescens as reporter

Abstract

The tyrosinase operon ( melC) from Streptomyces glaucescens was cloned and functionally expressed in Brevibacterium lactofermentum and Corynebacterium glutamicum under the control of the promoter of the kan gene from Tn 5. Recombinant corynebacterial cells containing the tyrosinase operon produced melanin on agar plates and in liquid culture when supplemented with copper and tyrosine. A conjugative bifunctional replacement vector for transcriptional/translational signal screening (pEMel-1) was constructed using expression of the melC operon from S. glaucescens, which can be used for cloning promoter sequences as EcoRI- NdeI fragments. When the DNA fragments with promoter activity such as cspBp or trpp were inserted into pEMel-1, B. lactofermentum harboring the chimeric plasmids produced melanin at different stages of growth, allowing temporal detection of promoter activity. The vector was also used to detect the activity of a Streptomyces promoter ( xysAp), which was inactive in B. lactofermentum, after PCR mutagenesis. The melC operon can be used for the visual, inexpensive (compared to the high price of starch azure for amylase detection), and non-selective (in contrast to the kan or cat genes) screening of several thousand clones at high colony density without killing of the transformants due to the presence of iodine (as in the case of amylase assay).

Production process and characterization of extra cellular li-pids from bacterial strain from oil industries waste

“…The lipase was very stable over a broad pH range from 5 to 9 after incubation for 36 hour, indicating its potential for practical use in industrial purpose which requires stability over wide pH ranges. In this study, the pH maximized lipase production is similar to the results reported by previous investigators (Adham, 2003). In contrast, maximum production of lipase by strain of Bacillus sp.…”

“…The lipase has been produced form the range of temperatures from 25 °C to 40 °C from the bacterial strain Pseudomonas fragi (Schueppet al, 1997). In contrast, the lipase has been produced from Psychotropic bacteria Serration marcescens at 25 °C (Adham, 2003). The optimal pH for lipase production was determined to be around pH 6.0 (table 4).…”

Altered morphology produced by ftsZ expression in Corynebacterium glutamicum ATCC 13869

“…These primers were designed to replace the two final codons of ftsZ [CAG (Gln) and TAA (stop)] by an NdeI site (CAT ATG) (His and Met). All PCR reactions were performed as described previously (Adham et al, 2003) and the amplified fragment was digested with BamHI (present in the amplified fragment) + NdeI (CATATG) and cloned together with gfp (as a NdeI-XbaI fragment) into plasmid pET28a, creating pETGFP (Table 1). Therefore the last amino acid of FtsZ Cg will be His instead of Gln, and fused to GFP.…”

A semi-quantitative high-throughput screening method for microbial l-tyrosine production in microtiter plates

“…Another possible approach could be the coupling of Ltyrosine overproduction with melanin biosynthesis, where the conversion of L-tyrosine by a tyrosinase leads to the formation of a dark pigment. The cultivation conditions of recombinant E. coli in mineral salts medium for optimum melanin production has been described recently [14] and the use of the melC operon from Streptomyces glaucescens as reporter for an inexpensive high-throughput screening method in corynebacteria has been suggested [1].…”