Abstract
Background1-Hydroxyphenazine (1-OH-PHZ) is a phenazine microbial metabolite with broad-spectrum antibacterial activities against a lot of plant pathogens. However, its use is hampered by the low yield all along. Metabolic engineering of microorganisms is an increasingly powerful method for the production of valuable organisms at high levels. Pseudomonas chlororaphis is recognized as a safe and effective plant rhizosphere growth-promoting bacterium, and faster growth rate using glycerol or glucose as a renewable carbon source. Therefore, Pseudomonas chlororaphis is particularly suitable as the chassis cell for the modification and engineering of phenazines.ResultsIn this study, enzyme PhzS (monooxygenase) was heterologously expressed in a phenazine-1-carboxylic acid (PCA) generating strain Pseudomonas chlororaphis H18, and 1-hydroxyphenazine was isolated, characterized in the genetically modified strain. Next, the yield of 1-hydroxyphenazine was systematically engineered by the strategies including (1) semi-rational design remodeling of crucial protein PhzS, (2) blocking intermediate PCA consumption branch pathway, (3) enhancing the precursor pool, (4) engineering regulatory genes, etc. Finally, the titer of 1-hydroxyphenazine reached 3.6 g/L in 5 L fermenter in 54 h.ConclusionsThe 1-OH-PHZ production of Pseudomonas chlororaphis H18 was greatly improved through systematically engineering strategies, which is the highest, reported to date. This work provides a promising platform for 1-hydroxyphenazine engineering and production.Graphical
Highlights
Phenazines are a large class of nitrogen-containing heterocyclic naturally or chemically synthesized colored aromatic products [1, 2]
There have been more than 150 reported phenazine natural products [8]
We found that, PpsA, TktA, AroB, and AroE homologous proteins genes all exist in the in P. chlororaphis H18 genome
Summary
Phenazines are a large class of nitrogen-containing heterocyclic naturally or chemically synthesized colored aromatic products [1, 2]. One of the important natural phenazine compounds, phenazine1-carboxylic acid (PCA, 1), has been developed as a new biopesticide “Shenqinmycin” in 2011 and is widely used [5, 6]. Chemical synthesis of phenazines is feasible [7], the conversion rate is low, and the organic solvent or toxic byproducts in the reaction process may cause environmental pollution problems. Most naturally occurring phenazines’ yield is extremely low, and only a handful of phenazine derivatives, such as PCA [5], 2-hydroxyphenazine (2-OH-PHZ, 2) [9], and phenazine-1-carboxamide (PCN) [10] have been explored to enhance the fermentation performance. Research on the improving of phenazine production is urgently needed
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