Enhanced rhamnolipid production of Pseudomonas aeruginosa SG by increasing copy number of rhlAB genes with modified promoter

Zhao, Feng; Cui, Qiu; Han, Shun; Dong, Hanping; Zhang, Jie; Ma, Fang; Zhang, Ying

doi:10.1039/c5ra13415c

Cited by 25 publications

(12 citation statements)

References 34 publications

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“…Although much effort has been invested in heterologous expression of rhl AB, the pathogenic strain P . aeruginosa PAO1 remains the best producer of rhamnolipids (Zhao et al., 2015). To evaluate the safety of an attenuated P .…”

Section: Resultsmentioning

confidence: 99%

Single-Stranded DNA-Binding Protein and Exogenous RecBCD Inhibitors Enhance Phage-Derived Homologous Recombination in Pseudomonas

et al. 2019

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Summary The limited efficiency of the available tools for genetic manipulation of Pseudomonas limits fundamental research and utilization of this genus. We explored the properties of a lambda Red-like operon (BAS) from Pseudomonas aeruginosa phage Ab31 and a Rac bacteriophage RecET-like operon (RecTE Psy ) from Pseudomonas syringae pv. syringae B728a. Compared with RecTE Psy , the BAS operon was functional at a higher temperature indicating potential to be a generic system for Pseudomonas . Owing to the lack of RecBCD inhibitor in the BAS operon, we added Redγ or Pluγ and found increased recombineering efficiencies in P . aeruginosa and Pseudomonas fluorescens but not in Pseudomonas putida and P . syringae . Overexpression of single-stranded DNA-binding protein enhanced recombineering in several contexts including RecET recombination in E . coli . The utility of these systems was demonstrated by engineering P. aeruginosa genomes to create an attenuated rhamnolipid producer. Our work enhances the potential for functional genomics in Pseudomonas .

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Section: Resultsmentioning

confidence: 99%

Single-Stranded DNA-Binding Protein and Exogenous RecBCD Inhibitors Enhance Phage-Derived Homologous Recombination in Pseudomonas

et al. 2019

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“…To determine whether an increase in the gene dosage discussed above was sufficient to confer the adenosine growth variant phenotype, we used the plasmid pBBR1MCS-5 (44) to increase the gene copy number of the 6-kb region common to all variants. Pseudomonas aeruginosa contains approximately 5 copies of this plasmid per cell (45). We cloned the region encompassing PA0143 to PA0148 into pBBR1MCS-5 (see Table S1 in the supplemental material).…”

Section: Resultsmentioning

confidence: 99%

Gene Duplication in Pseudomonas aeruginosa Improves Growth on Adenosine

et al. 2017

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The laboratory strain of , PAO1, activates genes for catabolism of adenosine using quorum sensing (QS). However, this strain is not well-adapted for growth on adenosine, with doubling times greater than 40 h. We previously showed that when PAO1 is grown on adenosine and casein, variants emerge that grow rapidly on adenosine. To understand the mechanism by which this adaptation occurs, we performed whole-genome sequencing of five isolates evolved for rapid growth on adenosine. All five genomes had a gene duplication-amplification (GDA) event covering several genes, including the quorum-regulated nucleoside hydrolase gene,, and PA0148, encoding an adenine deaminase. In addition, two of the growth variants also exhibited a promoter mutation. We recapitulated the rapid growth phenotype with a plasmid containing six genes common to all the GDA events. We also showed that and PA0148, the two genes at either end of the common GDA, were sufficient to confer rapid growth on adenosine. Additionally, we demonstrated that the variant promoter increased basal expression of but maintained its QS regulation. Therefore, GDA in confers the ability to grow efficiently on adenosine while maintaining QS regulation of nucleoside catabolism. thrives in many habitats and is an opportunistic pathogen of humans. In these diverse environments, must adapt to use myriad potential carbon sources. PAO1 cannot grow efficiently on nucleosides, including adenosine; however, it can acquire this ability through genetic adaptation. We show that the mechanism of adaptation is by amplification of a specific region of the genome and that the amplification preserves the regulation of the adenosine catabolic pathway by quorum sensing. These results demonstrate an underexplored mechanism of adaptation by , with implications for phenotypes such as development of antibiotic resistance.

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“…It had been shown that a simple increment in the copy number of rhlAB under the control of indigenous strong promoter of oprL in P. aeruginosa SG could raise the rhamnolipid titer to 20.98 g/l [ 47 ]. Similarly, the overexpression of rhlAB under tac promoter in B. kururiensis increased the rhamnolipid titer from 0.78 to 5.76 g/l [ 48 ].…”

Section: Application Of Various Strategies To Increase Rhamnolipid Yimentioning

confidence: 99%

Microbial production of rhamnolipids: opportunities, challenges and strategies

2017

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Rhamnolipids are a class of biosurfactants which contain rhamnose as the sugar moiety linked to β-hydroxylated fatty acid chains. Rhamnolipids can be widely applied in many industries including petroleum, food, agriculture and bioremediation etc. Pseudomonas aeruginosa is still the most competent producer of rhamnolipids, but its pathogenicity may cause safety and health concerns during large-scale production and applications. Therefore, extensive studies have been carried out to explore safe and economical methods to produce rhamnolipids. Various metabolic engineering efforts have also been applied to either P. aeruginosa for improving its rhamnolipid production and diminishing its pathogenicity, or to other non-pathogenic strains by introducing the key genes for safe production of rhamnolipids. The three key enzymes for rhamnolipid biosynthesis, RhlA, RhlB and RhlC, are found almost exclusively in Pseudomonas sp. and Burkholderia sp., but have been successfully expressed in several non-pathogenic host bacteria to produce rhamnolipids in large scales. The composition of mono- and di-rhamnolipids can also be modified through altering the expression levels of RhlB and RhlC. In addition, cell-free rhamnolipid synthesis by using the key enzymes and precursors from non-pathogenic sources is thought to not only eliminate pathogenic effects and simplify the downstream purification processes, but also to circumvent the complexity of quorum sensing system that regulates rhamnolipid biosynthesis. The pathogenicity of P. aeruginosa can also be reduced or eliminated through in vivo or in vitro enzymatic degradation of the toxins such as pyocyanin during rhamnolipid production. The rhamnolipid production cost can also be significantly reduced if rhamnolipid purification step can be bypassed, such as utilizing the fermentation broth or the rhamnolipid-producing strains directly in the industrial applications of rhamnolipids.

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Enhanced rhamnolipid production of Pseudomonas aeruginosa SG by increasing copy number of rhlAB genes with modified promoter

Abstract: Increasing the copy number of rhlAB genes with a modified promoter efficiently enhanced the production of rhamnolipid by P. aeruginosa.

Cited by 25 publications

References 34 publications

Single-Stranded DNA-Binding Protein and Exogenous RecBCD Inhibitors Enhance Phage-Derived Homologous Recombination in Pseudomonas

Single-Stranded DNA-Binding Protein and Exogenous RecBCD Inhibitors Enhance Phage-Derived Homologous Recombination in Pseudomonas

Gene Duplication in Pseudomonas aeruginosa Improves Growth on Adenosine

Microbial production of rhamnolipids: opportunities, challenges and strategies

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