Transformation of Thermoanaerobacterium sp. strain JW/SL-YS485 with plasmid pIKM1 conferring kanamycin resistance

Abstract

The industrial application of thermophilic (eu)bacteria is hampered by the lack of genetic systems for these bacteria. We report here the first unequivocal transformation of a Gram‐positive, thermophilic, anaerobic microorganism, Thermoanaerobacterium, with the kanamycin resistance‐mediating plasmid pIKM1. The construct pIKM1 is based on the Escherichia coli–Clostridium acetobutylicum shuttle vector pIMP1 and contains the thermostable kanamycin cassette from S. faecalis plasmid pKD102. Using electrotransformation, plasmid pIKM1 mediated kanamycin resistance in Thermoanaerobacterium sp. strain JW/SL‐YS485 up to 400 μg ml−1 at 48°C and 200 μg ml−1 at 60°C.

Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield

“…Transformation of T. saccharolyticum was performed as previously described (14,16) with modifications during selection on erythromycin. Cells transformed with pSGD8E were allowed to recover at 48°C for 4 h and subsequently plated on solid medium at pH 6.0 containing erythromycin at 5 g/ml and incubated at 48°C for 4 days.…”

“…Knockout plasmid pSGD9 was created by inserting the thermostable S. faecalis kanamycin resistance cassette (14) between the pta and ack homology regions. Knockout plasmid pSGD8E targeting L-ldh was constructed with the same 5Ј and 3Ј homology regions reported earlier (18) with a fusion of the kanamycin promoter region (primers K1, K2) and adenine methylase gene (primers E1, E2) conferring erythromycin resistance (15) inserted between them.…”

“…Extensive efforts using classical mutagenesis techniques to obtain stable strains exhibiting high-ethanol yields over a range of conditions have not been successful (13). Genetic systems suitable for engineering thermophiles have long limited strain development, but have started to emerge (14)(15)(16) along with the first reports of metabolic engineering in thermophilic, saccharolytic hosts (17,18). Here, we report engineering T. saccharolyticum JW/SL-YS485 to produce ethanol as the only significant organic product.…”

Establishment of an efficient transformation protocol and its application in marine-derived Bacillus strain

“…pHT3101 harbors the replication region of a resident plasmid of Bacillus thuringiensis. To further evaluate the optimized protocol, the other two plasmids carrying divergent replicons and conferring different antibiotic resistance were also tested, including pKSV7 with the temperature-sensitive origin of replication for B. subtilis [23] and pIKM1 with the replication region of B. subtilis plasmid pIM13 [24]. As shown in Table 3, the transformation efficiencies for unmethylated pKSV7 and pIKM1 were 4.1×10 2 and 1.0×10 2 cfu μg 1 plasmid DNA, respectively, which were much lower than that of unmethylated pHT3101; conversely, no transformant appeared for methylated pKSV7 and pIKM1, and only a few transformants were obtained for methylated pHT3101.…”

“…2095) [2] and Bacillus licheniforms strain EI-34-6 (BGSC catalog 5A37) [21] have been described previously. The replicative plasmids with different replicons and antibiotic resistance markers, including pHT3101 [22], pKSV7 [23] and pIKM1 [24], were used for method development and evaluation. Plasmids extractions and DNA purifications were carried out using commercial kits (Omega Bio-Tek, USA).…”