Engineering and systems-level analysis of Pseudomonas chlororaphis for production of phenazine-1-carboxamide using glycerol as the cost-effective carbon source

Abstract

BackgroundGlycerol, an inevitable byproduct of biodiesel, has become an attractive feedstock for the production of value-added chemicals due to its availability and low price. Pseudomonas chlororaphis HT66 can use glycerol to synthesize phenazine-1-carboxamide (PCN), a phenazine derivative, which is strongly antagonistic to fungal phytopathogens. A systematic understanding of underlying mechanisms for the PCN overproduction will be important for the further improvement and industrialization.ResultsWe constructed a PCN-overproducing strain (HT66LSP) through knocking out three negative regulatory genes, lon, parS, and prsA in HT66. The strain HT66LSP produced 4.10 g/L of PCN with a yield of 0.23 (g/g) from glycerol, which was of the highest titer and the yield obtained among PCN-producing strains. We studied gene expression, metabolomics, and dynamic 13C tracer in HT66 and HT66LSP. In response to the phenotype changes, the transcript levels of phz biosynthetic genes, which are responsible for PCN biosynthesis, were all upregulated in HT66LSP. Central carbon was rerouted to the shikimate pathway, which was shown by the modulation of specific genes involved in the lower glycolysis, the TCA cycle, and the shikimate pathway, as well as changes in abundances of intracellular metabolites and flux distribution to increase the precursor availability for PCN biosynthesis. Moreover, dynamic 13C-labeling experiments revealed that the presence of metabolite channeling of 3-phosphoglyceric acid to phosphoenolpyruvate and shikimate to trans-2,3-dihydro-3-hydroxyanthranilic acid in HT66LSP could enable high-yielding synthesis of PCN.ConclusionsThe integrated analysis of gene expression, metabolomics, and dynamic 13C tracer enabled us to gain a more in-depth insight into complex mechanisms for the PCN overproduction. This study provides important basis for further engineering P. chlororaphis for high PCN production and efficient glycerol conversion.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1123-y) contains supplementary material, which is available to authorized users.

Building microbial factories for the production of aromatic amino acid pathway derivatives: From commodity chemicals to plant-sourced natural products

“…Moving downstream beyond DHS and SA, chorismate becomes the next hub, from which various benzoic acids can be synthesized. Many health-related products, such as folate (vitamin B 9 ), CoQ 9 , and aspirin, need benzoic acid as the precursor of their synthesis (Noda et al, 2016;Yao et al, 2018).…”

“…Arbutin is a plant-derived glycosylated hydroquinone and has been widely used in cosmetic, healthcare, and medical industries because of its ability to block melanin synthesis for skin lightening, as well as its other anti-microbial, anti-inflammatory, and anti-oxidant activities (Shen et al, 2017;Yao et al, 2018). Extended from 4-HBA, the de novo biosynthesis of arbutin can be achieved by the expression of 4-HBA 1-hydroxylase (Mnx1) from Candida parapsilosis and glucosyltransferase (AS) from Rauvolfia serpentina at 4.19 g/L in E. coli strains engineered to accumulate 4-HBA (Shen et al, 2017).…”

“…oryzae), and PhzC (the P. chlororaphis version of DAHP synthase), and with deletions of pykA (pyruvate kinase converting PEP to pyruvate), phzE (converting chorismate for phenazine biosynthesis), and three negative regulatory genes (lon, rpeA, and rsmE) produced arbutin at 6.79 g/L and 0.094 g/L/h with mixed fed-batch fermentation of glucose and 4-HBA. Previously, the disruption of another set of pathway-specific transcriptional repressors encoded by lon, parS, and psrA enabled P. chlororaphis to enhance its production of phenazine-1-carboxamide (PCN) at 4.1 g/L, with a yield of 0.23 g/g glycerol (Yao et al, 2018). PCN is naturally produced by Pseudomonas strains and displays broad-spectrum antibiotic properties.…”

Rewiring glycerol metabolism for enhanced production of poly-γ-glutamic acid in Bacillus licheniformis

“…To gain a further insight into mechanisms for the γ-PGA overproduction, the intracellular metabolite concentrations in WX-02 and BC4 were measured by GC–MS-based metabolome approach [ 28 , 29 ]. Based on our results, the metabolites (containing intermediates of glycolysis, PPP, TCA cycle, and amino acids pools) with significant differences among the two strains were listed in Fig.…”

Enhanced biosynthesis of arbutin by engineering shikimate pathway in Pseudomonas chlororaphis P3

“…The samples were analyzed by the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS) as reported earlier [ 17 , 36 ]. Mobile phase A (water containing 1‰ formic acid) and B (methanol containing 1‰ formic acid) were used as the following gradient: 0–2 min, 95% A; 2–10 min, 95–85% A; 10–15 min, 95% A, with a constant flow rate of 0.2 mL/min.…”