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Boosting Heterologous Phenazine Production in Pseudomonas putida KT2440 Through the Exploration of the Natural Sequence Space.

ABSTRACT: Phenazine-1-carboxylic acid (PCA) and its derivative pyocyanin (PYO) are natural redox mediators in bioelectrochemical systems and have the potential to enable new bioelectrochemical production strategies. The native producer Pseudomonas aeruginosa harbors two identically structured operons in its genome, which encode the enzymes responsible for PCA synthesis [phzA1-G1 (operon 1), phzA2-G2 (operon 2)]. To optimize heterologous phenazines production in the biotech host Pseudomonas putida KT2440, we compared PCA production from both operons originating from P. aeruginosa strain PAO1 (O1.phz1 and O1.phz2) as well as from P. aeruginosa strain PA14 (14.phz1 and 14.phz2). Comparisons of phenazine synthesis and bioelectrochemical activity were performed between heterologous constructs with and without the combination with the genes phzM and phzS required to convert PCA to PYO. Despite a high amino acid homology of all enzymes of more than 97%, P. putida harboring 14.phz2 produced 4-times higher PCA concentrations (80 ?g/mL), which resulted in 3-times higher current densities (12 ?A/cm2) compared to P. putida 14.phz1. The respective PCA/PYO producer containing the 14.phz2 operon was the best strain with 80 ?g/mL PCA, 11 ?g/mL PYO, and 22 ?A/cm2 current density. Tailoring phenazine production also resulted in improved oxygen-limited metabolic activity of the bacterium through enhanced anodic electron discharge. To elucidate the reason for this superior performance, a detailed structure comparison of the PCA-synthesizing proteins has been performed. The here presented characterization and optimization of these new strains will be useful to improve electroactivity in P. putida for oxygen-limited biocatalysis.

SUBMITTER: Askitosari TD

PROVIDER: S-EPMC6722869 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Boosting Heterologous Phenazine Production in Pseudomonas putida KT2440 Through the Exploration of the Natural Sequence Space.

Askitosari Theresia D TD Boto Santiago T ST Blank Lars M LM Rosenbaum Miriam A MA

Frontiers in microbiology 20190828

Phenazine-1-carboxylic acid (PCA) and its derivative pyocyanin (PYO) are natural redox mediators in bioelectrochemical systems and have the potential to enable new bioelectrochemical production strategies. The native producer Pseudomonas aeruginosa harbors two identically structured operons in its genome, which encode the enzymes responsible for PCA synthesis [phzA1-G1 (operon 1), phzA2-G2 (operon 2)]. To optimize heterologous phenazines production in the biotech host Pse ...[more]

PMID: 31555229

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Project description:BackgroundAlthough a transition toward sustainable production of chemicals is needed, the physiochemical properties of certain biochemicals such as biosurfactants make them challenging to produce in conventional bioreactor systems. Alternative production platforms such as surface-attached biofilm populations could potentially overcome these challenges. Rhamnolipids are a group of biosurfactants highly relevant for industrial applications. However, they are mainly produced by the opportunistic pathogen Pseudomonas aeruginosa using hydrophobic substrates such as plant oils. As the biosynthesis is tightly regulated in P. aeruginosa a heterologous production of rhamnolipids in a safe organism can relive the production from many of these limitations and alternative substrates could be used.ResultsIn the present study, heterologous production of biosurfactants was investigated using rhamnolipids as the model compound in biofilm encased Pseudomonas putida KT2440. The rhlAB operon from P. aeruginosa was introduced into P. putida to produce mono-rhamnolipids. A synthetic promoter library was used in order to bypass the normal regulation of rhamnolipid synthesis and to provide varying expression levels of the rhlAB operon resulting in different levels of rhamnolipid production. Biosynthesis of rhamnolipids in P. putida decreased bacterial growth rate but stimulated biofilm formation by enhancing cell motility. Continuous rhamnolipid production in a biofilm was achieved using flow cell technology. Quantitative and structural investigations of the produced rhamnolipids were made by ultra performance liquid chromatography combined with high resolution mass spectrometry (HRMS) and tandem HRMS. The predominant rhamnolipid congener produced by the heterologous P. putida biofilm was mono-rhamnolipid with two C10 fatty acids.ConclusionThis study shows a successful application of synthetic promoter library in P. putida KT2440 and a heterologous biosynthesis of rhamnolipids in biofilm encased cells without hampering biofilm capabilities. These findings expands the possibilities of cultivation setups and paves the way for employing biofilm flow systems as production platforms for biochemicals, which as a consequence of physiochemical properties are troublesome to produce in conventional fermenter setups, or for production of compounds which are inhibitory or toxic to the production organisms.

Project description:BackgroundRhamnolipids are potent biosurfactants with high potential for industrial applications. However, rhamnolipids are currently produced with the opportunistic pathogen Pseudomonas aeruginosa during growth on hydrophobic substrates such as plant oils. The heterologous production of rhamnolipids entails two essential advantages: Disconnecting the rhamnolipid biosynthesis from the complex quorum sensing regulation and the opportunity of avoiding pathogenic production strains, in particular P. aeruginosa. In addition, separation of rhamnolipids from fatty acids is difficult and hence costly.ResultsHere, the metabolic engineering of a rhamnolipid producing Pseudomonas putida KT2440, a strain certified as safety strain using glucose as carbon source to avoid cumbersome product purification, is reported. Notably, P. putida KT2440 features almost no changes in growth rate and lag-phase in the presence of high concentrations of rhamnolipids (> 90 g/L) in contrast to the industrially important bacteria Bacillus subtilis, Corynebacterium glutamicum, and Escherichia coli. P. putida KT2440 expressing the rhlAB-genes from P. aeruginosa PAO1 produces mono-rhamnolipids of P. aeruginosa PAO1 type (mainly C(10):C(10)). The metabolic network was optimized in silico for rhamnolipid synthesis from glucose. In addition, a first genetic optimization, the removal of polyhydroxyalkanoate formation as competing pathway, was implemented. The final strain had production rates in the range of P. aeruginosa PAO1 at yields of about 0.15 g/g(glucose) corresponding to 32% of the theoretical optimum. What's more, rhamnolipid production was independent from biomass formation, a trait that can be exploited for high rhamnolipid production without high biomass formation.ConclusionsA functional alternative to the pathogenic rhamnolipid producer P. aeruginosa was constructed and characterized. P. putida KT24C1 pVLT31_rhlAB featured the highest yield and titer reported from heterologous rhamnolipid producers with glucose as carbon source. Notably, rhamnolipid production was uncoupled from biomass formation, which allows optimal distribution of resources towards rhamnolipid synthesis. The results are discussed in the context of rational strain engineering by using the concepts of synthetic biology like chassis cells and orthogonality, thereby avoiding the complex regulatory programs of rhamnolipid production existing in the natural producer P. aeruginosa.

Project description:Pseudomonas putida KT2440 has become an attractive chassis for heterologous expression with the development of effective genetic manipulation tools. Improving the level of transcriptional regulation is particularly important for extending the potential of P. putida KT2440 in heterologous expression. Although many strategies have been applied to enhance the heterologous expression level in P. putida KT2440, it was still at a relatively low level. Herein we constructed a T7-like expression system in P. putida KT2440, mimicking the pET expression system in Escherichia coli, which consisted of T7-like RNA polymerase (MmP1) integrated strain and the corresponding expression vector for the heterologous expression enhancement. With the optimization of the insertion site and the copy number of RNA polymerase (RNAP), the relative fluorescence intensity (RFI) of the super-folder green fluorescent protein (sfGFP) was improved by 1.4-fold in MmP1 RNAP integrated strain. The induction point and IPTG concentration were also optimized. This strategy was extended to the gene-reduced strain EM42 and the expression of sfGFP was improved by 2.1-fold. The optimal RNAP integration site was also used for introducing T7 RNAP in P. putida KT2440 and the expression level was enhanced, indicating the generality of the integration site for the T7 expression system. Compared to other inducible expression systems in KT2440, the heterologous expression level of the Mmp1 system and T7 system were more than 2.5 times higher. Furthermore, the 3.6-fold enhanced expression level of a difficult-to-express nicotinate dehydrogenase from Comamonas testosteroni JA1 verified the efficiency of the T7-like expression system in P. putida KT2440. Taken together, we constructed and optimized the T7-like and T7 expression system in P. putida, thus providing a set of applicable chassis and corresponding plasmids to improve recombinant expression level, expecting to be used for difficult-to-express proteins.

Dataset Information

Boosting Heterologous Phenazine Production in Pseudomonas putida KT2440 Through the Exploration of the Natural Sequence Space.

Publications

Boosting Heterologous Phenazine Production in <i>Pseudomonas putida</i> KT2440 Through the Exploration of the Natural Sequence Space.

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