Abstract
BackgroundGenome streamlining is a feasible strategy for constructing an optimum microbial chassis for synthetic biology applications. Genomic islands (GIs) are usually regarded as foreign DNA sequences, which can be obtained by horizontal gene transfer among microorganisms. A model strain Pseudomonas putida KT2440 has broad applications in biocatalysis, biotransformation and biodegradation.ResultsIn this study, the identified GIs in P. putida KT2440 accounting for 4.12% of the total genome size were deleted to generate a series of genome-reduced strains. The mutant KTU-U13 with the largest deletion was advantageous over the original strain KTU in several physiological characteristics evaluated. The mutant KTU-U13 showed high plasmid transformation efficiency and heterologous protein expression capacity compared with the original strain KTU. The metabolic phenotype analysis showed that the types of carbon sources utilized by the mutant KTU-U13 and the utilization capabilities for certain carbon sources were increased greatly. The polyhydroxyalkanoate (PHA) yield and cell dry weight of the mutant KTU-U13 were improved significantly compared with the original strain KTU. The chromosomal integration efficiencies for the γ-hexachlorocyclohexane (γ-HCH) and 1,2,3-trichloropropane (TCP) biodegradation pathways were improved greatly when using the mutant KTU-U13 as the recipient cell and enhanced degradation of γ-HCH and TCP by the mutant KTU-U13 was also observed. The mutant KTU-U13 was able to stably express a plasmid-borne zeaxanthin biosynthetic pathway, suggesting the excellent genetic stability of the mutant.ConclusionsThese desirable traits make the GIs-deleted mutant KTU-U13 an optimum chassis for synthetic biology applications. The present study suggests that the systematic deletion of GIs in bacteria may be a useful approach for generating an optimal chassis for the construction of microbial cell factories.
Highlights
Genome streamlining is a feasible strategy for constructing an optimum microbial chassis for synthetic biology applications
43 Genomic islands (GIs) were predicted by using a gas chromatography (GC)-Profile from the Tianjin University Bioinformatics Center (TUBIC), and the GIs with a 5% difference from the average GC content of the KT2440 genome were selected as the deletion targets
Compared with the original strain KTU, the mutant KTU-U13 showed a 9.4- and 2.7-fold improvement in the relative fluorescence intensity in M9G and LB media, respectively. These results suggest that the deletion of GIs may improve the expression level of heterologous proteins in P. putida KT2440
Summary
Genome streamlining is a feasible strategy for constructing an optimum microbial chassis for synthetic biology applications. Genomic islands (GIs) are usually regarded as foreign DNA sequences, which can be obtained by horizontal gene transfer among microorganisms. Studies on genome sequencing and functional genes of many model organisms have found that microorganisms absorb exogenous DNA sequences and retain them in their own genome through gene horizontal transfer and other means in the long evolutionary process, resulting in increased genome capacity. Genome streamlining is an important strategy for constructing synthetic chassis. Genome-streamlined strains provide a basic platform for the introduction of functional modules. Choosing an optimum chassis can make the modules work efficiently, resulting in an ideal cell factory. The minimum genome factory has broad application prospects in the fields of metabolite production, environmental waste degradation, cytotoxin detection and biofuel production [4,5,6]
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