Enhance Bacitracin Production By Increasing The TCA Cycle In Bacillus Licheniformis

The dodecapeptide antibiotic bacitracin,produced by several strains of Bacillus licheniformis and Bacillus subtilis,is widely used as an antibacterial animal feed additive.Several genetic strategies were explored to enhance its production.The availability of building block amino acids for bacitracin production was found to play an important role in its synthesis.As the central metabolic pathway,the TCA cycle is a major source for energy supply and intermediates for anabolism.In this study,the TCA cycle in the industrial strain B.licheniformis DW2 was strengthened by overexpression of the key enzymes pyruvate carboxylase,citrate synthase and isocitrate dehydrogenase(ICDH).This study unveiled the metabolic responses of the increased TCA cycle in B.licheniformis,and provided a novel strategy for enhancing bacitracin production.There are three enzymes that play key roles in the TCA cycle,citrate synthase,isocitrate dehydrogenase and a-ketoglutarate dehydrogenase.The catalytic reaction of a-ketoglutarate dehydrogenase may reduce the intracelluar content of a-ketoglutaric acid which was the important precursor for amino acid biosynthesis.Therefore,citrate synthase encoded by citZ and mmgD,and isocitrate dehydrogenase encoded by icd were chosen to be overexpressed in this study.The fermentation results showed that the bacitracin yield of lcd episomal-expression strain increased 7.7%compare with the wild-type strain DW2,and the yield of bacitracin decreased in citZ and mmgD episomal-expression strains.The bacitracin yields of icd and citZ episomal-expression strains increased 25.9%and 13.9%compare with the control strain DW2/pHY300PLK,while the yield of bacitracin decreased in mmgD episomal-expression strain.This phenomenon indicates that the over-expression of icd gene can increase the yield of bacitracin significantly.In order to build a stable strain and eliminate the negative effect of the plasmid,the icd gene was integrated into the prophage area on the strain genome.The fermentation results showed that the bacitracin yield of icd integrative-expression strain increased 11.5%compare with the wild-type strain DW2 and 4%higher than icd episomal-expressing strain.The enzyme activity assay of isocitrate dehydrogenase(ICDH)showed that the ICDH enzyme activity in icd integrative-expressing strain was 2.7 folds higher than the original strain.In order to explore the effect on the metabolism of icd integrative-expressed strain,the ATP and NADPH supply were detected.The results showed that the contents of ATP and NADPH in icd integrative-expressing strain were significantly increased.Gene transcription levels were compared between the icd integrative-expressing strain and the original strain by qRT-PCR.The transcription levels of several genes involved TCA cycle were incresed,and the transcription levels of ccpC and ccpA which encoded the transcriptional regulators CcpC and CcpA were suppressed in the icd overexpression strains.And compared with the original strain,the transcription levels of the glutamate synthase encoding gene glt A and the glutamate dehydrogenase encoding gene rocG were increased by 3.4 and 3.5 folds.Finally,the concentrations of the intracellular and extracellular amino acidsin the icd integrative-expression strain and the original strain were detected.At the end of the fermentation,higher extracellular concentrations of bacitracin-containing amino acids were observed for original strain,and intracellular concentrations of these amino acids were higher in icd integrative-expressing strain.The results showed that intracellular biosynthesis and extracellular uptake of the bacitracin-containing amino acids increased due to icd overexpression.In conclusion,increased TCA cycle flux was achieved by overexpressing the key enzyme ICDH to generate more energy and precursor intermediates for amino acid biosynthesis,resulting in enhanced bacitracin production.This study also obtained TCA cycle-enhanced strains by enhancing the main anaplerotic reaction.The main anaplerotic reaction is a reaction catalyzed by pyruvate carboxylase which catalyze the carboxylation of pyruvate to form oxaloacetate.The pyruvate carboxylase overexpression strain DW2/pHY-pyccA was constructed.The fermentation results showed that the bacitracin yield of DW2/pHY-pycA increased 14.5%compared with the control strain DW2/pHY300PLK.,and the biomass of the pycA episomal-expressing strain strain increased significantly.Based on analysis by qRT-PCR,the transcription levels of all genes involved the TCA cycle were increased.The results showed that the content of oxaloacetate in DW2/pHY-pycA was also increased.These results indicated that pycA overexpression can enhance the TCA cycle and increase the content of oxaloacetate which provides more precursors for the synthesise of bacitracin-containing amino acids,such as Asp,Asn,Lys,and Ile.In this study,the increased TCA cycle flux was achieved to generate more energy and precursor intermediates for amino acid biosynthesis.Thus,the availability of bacitracin-containing amino acids increased,resulting in enhanced bacitracin production.