Deletion of genomic islands in the Pseudomonas putida KT2440 genome can create an optimal chassis for synthetic biology applications

Abstract

Background: 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. A model strain Pseudomonas putida KT2440 has broad applications in biocatalysis, biotransformation and biodegradation. Results: In 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. Conclusions: These 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.

Engineering of a genome-reduced strain Bacillus amyloliquefaciens for enhancing surfactin production

“…Besides, an ideal chassis is expected to possess high heterologous protein expression capacity. In previous studies, green uorescent protein (GFP) was selected as a model heterologous protein in genome-reduced P. putida KT2440 mutants, and the expression capacity of heterologous protein was characterized by the GFP uorescence per biomass unit [9,33]. In this study, the production capability of GFP was evaluated in GR167 by transcriptional level and the uorescence intensity.…”

“…Moderate genome reduction can create synthetic biology chassis with optimized genomic sequences, e cient metabolic regulatory networks and superior cellular physiological characteristics [3][4][5]. So far, several model microorganisms, such as Escherichia coli [6], Bacillus subtilis [7,8] and Pseudomonas putida [9], have been intensively researched for minimal genome construction due to their clear genetic background and e cient genome editing approaches.…”

A combination of genome reduction and promoter engineering can enhance surfactin production by Bacillus amyloliquefaciens LL3

“…Besides, an ideal chassis is expected to possess high heterologous protein expression capacity. In previous studies, green uorescent protein (GFP) was selected as a model heterologous protein in genome-reduced P. putida KT2440 mutants, and the GFP uorescence per biomass unit was act as the expression capacity of heterologous protein [9,33]. In this study, GFP was also selected as a heterologous protein, and which expression was evaluated at transcriptional and production levels by quantitative real-time PCR (RT-qPCR) and the uorescence intensity.…”

Engineering Lactococcus lactis as a multi-stress tolerant biosynthetic chassis by deleting the prophage-related fragment

“…In general, deletion of non-essential genome fragments, such as prophages and transposons, could confer some advantages on bacterial strains [17], including enhanced growth, increased biomass, and higher level of proteins synthesis [18]. Besides, prophages can usually synthesize proteins like prophage lysin that affect the host cell wall/membrane [19], and cell wall/membrane is essential for maintaining cellular integrity and resisting environmental stress [20].…”