Abstract
Thermobifida fusca is a thermophilic actinobacterium. T. fusca muC obtained by adaptive evolution preferred yeast extract to ammonium sulfate for accumulating malic acid and ammonium sulfate for cell growth. We did transcriptome analysis of T. fusca muC on Avicel and cellobiose with addition of ammonium sulfate or yeast extract, respectively by RNAseq. The transcriptional results indicate that ammonium sulfate induced the transcriptions of the genes related to carbohydrate metabolisms significantly more than yeast extract. Importantly, Tfu_2487, encoding histidine-containing protein (HPr), didn’t transcribe on yeast extract at all, while it transcribed highly on ammonium sulfate. In order to understand the impact of HPr on malate production and cell growth of the muC strain, we deleted Tfu_2487 to get a mutant strain: muCΔ2487, which had 1.33 mole/mole-glucose equivalent malate yield, much higher than that on yeast extract. We then developed an E. coli-T. fusca shuttle plasmid for over-expressing HPr in muCΔ2487, a strain without HPr background, forming the muCΔ2487S strain. The muCΔ2487S strain had a much lower malate yield but faster cell growth than the muC strain. The results of both mutant strains confirmed that HPr was the key regulatory protein for T. fusca’s metabolisms on nitrogen sources.
Highlights
Thermobifida fusca is a thermophilic actinobacterium, which is an efficient degrader of plant cell walls[1]
The histidine phosphocarrier protein (Hpr or Tfu_2487) didn’t express at all in the muC strain on ammonium sulfate, with faster cell growth and less malic acid yield compared to the one on yeast extract
The muC strain was grown on four conditions with different carbon and nitrogen sources: 1) CB1: 2 g/L yeast extract, 5 g/L cellobiose; 2) CB2: 2 g/L yeast extract, 5 g/L Avicel; 3) CC1: 2 g/L ammonium sulfate, 5 g/L cellobiose; 4) CC2: 2 g/L ammonium sulfate, 5 g/L Avicel
Summary
Thermobifida fusca is a thermophilic actinobacterium, which is an efficient degrader of plant cell walls[1]. T. fusca has been heavily studied for more than 30 years because it can produce a variety of cellulases, hemicellulases and other enzymes. These enzymes especially cellulases are thermostable and their activities retain high from pH 4 to 102–9. We did transcriptome analysis of T. fusca muC on Avicel and cellobiose with addition of ammonium sulfate and yeast extract, respectively. The histidine phosphocarrier protein (Hpr or Tfu_2487) didn’t express at all in the muC strain on ammonium sulfate, with faster cell growth and less malic acid yield compared to the one on yeast extract. Tfu_2487 was over-expressed in muCΔ2 487, forming muCΔ2487S, which was grown on ammonium sulfate and yeast extract
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