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Abstract
BackgroundRalstonia eutropha is an important bacterium for the study of polyhydroxyalkanoates (PHAs) synthesis and CO2 fixation, which makes it a potential strain for industrial PHA production and attractive host for CO2 conversion. Although the bacterium is not recalcitrant to genetic manipulation, current methods for genome editing based on group II introns or single crossover integration of a suicide plasmid are inefficient and time-consuming, which limits the genetic engineering of this organism. Thus, developing an efficient and convenient method for R. eutropha genome editing is imperative.ResultsAn efficient genome editing method for R. eutropha was developed using an electroporation-based CRISPR-Cas9 technique. In our study, the electroporation efficiency of R. eutropha was found to be limited by its restriction-modification (RM) systems. By searching the putative RM systems in R. eutropha H16 using REBASE database and comparing with that in E. coli MG1655, five putative restriction endonuclease genes which are related to the RM systems in R. eutropha were predicated and disrupted. It was found that deletion of H16_A0006 and H16_A0008-9 increased the electroporation efficiency 1658 and 4 times, respectively. Fructose was found to reduce the leaky expression of the arabinose-inducible pBAD promoter, which was used to optimize the expression of cas9, enabling genome editing via homologous recombination based on CRISPR-Cas9 in R. eutropha. A total of five genes were edited with efficiencies ranging from 78.3 to 100%. The CRISPR-Cpf1 system and the non-homologous end joining mechanism were also investigated, but failed to yield edited strains.ConclusionsWe present the first genome editing method for R. eutropha using an electroporation-based CRISPR-Cas9 approach, which significantly increased the efficiency and decreased time to manipulate this facultative chemolithoautotrophic microbe. The novel technique will facilitate more advanced researches and applications of R. eutropha for PHA production and CO2 conversion.
Highlights
Ralstonia eutropha is an important bacterium for the study of polyhydroxyalkanoates (PHAs) synthesis and CO2 fixation, which makes it a potential strain for industrial PHA production and attractive host for C O2 conversion
It was found that the electroporation efficiency of pBBR1-rfp extracted from R. eutropha was 1677 times higher than that of its counterpart from E. coli S17-1 (Fig. 1a)
The two plasmids had the same DNA sequence, but were likely modified by different RM systems in E. coli and R. eutropha, which indicated that the R. eutropha RM systems may be the major cause of the low electroporation efficiency
Summary
Ralstonia eutropha is an important bacterium for the study of polyhydroxyalkanoates (PHAs) synthesis and CO2 fixation, which makes it a potential strain for industrial PHA production and attractive host for C O2 conversion. Ralstonia eutropha H16, known as Cupriavidus necator H16, is a Gram-negative β-proteobacterium that is ubiquitously present in soil and freshwater environments [1] It has attracted considerable research interest due to its significant economic potential [2] and CO2. R. eutropha grows to very high cell densities (281 g/L) under nutritionally rich conditions, and accumulates large amounts of PHAs (232 g/L) when the nitrogen or phosphate source is limited [2]. This characteristic makes it an attractive host for the synthesis of industrially relevant PHA materials
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