Disruption of phenazine synthesis in Lzh-T5
According to the research from Blankenfeldt, DHHA undergoes an isomerization reaction facilitated by PhzF and converts to 6-amino–5-oxocyclohex–2-ene–1-carboxylic acid in Pseudomonas spp [16]. In order to accumulate DHHA, phzF was disrupted in P. chlororaphis Lzh-T5. As shown in Fig. 2, after 48 hours of culture, the P. chlororaphis LDA–1 strain on an agar plate turns milky; however, the colony color of Lzh-T5 remains orange. This suggests that the phenazine derivatives could not be synthesized in LDA1. After fermentation and analyses by HPLC, phenazines (including 2-hydroxyphenazine, PCA, and 2-OH-PCA) disappeared in the broth of P. chlororaphis LDA1 (Fig. 3)..
Similar to previous research, a new absorption peak appears in the HPLC at a wavelength of 278 nm, which is the maximum absorption wavelength of DHHA (Fig. S1) [17]. According to the analysis of LC-MS, the compound was 156.0658 for [C7H10NO3]+ (the mass of DHHA is 155.0655; Fig. S1).. When phzF is overexpressed in the LDA1 strain, production of phenazine was recovered (Fig. 3).. According to these results, it suggests that disruption of phzF in Lzh-T5 causes accumulation of DHHA. Using an HPLC detection, the DHHA yield of LDA1 reached 2.15 g/L in 48 hours (Fig. 4a).. Our results suggest that disruption of phzF had little effect on Lzh-T5 cell growth (Fig. 4b)..
Knockout of negative regulatory genes to boost DHHA production
According to previous research, interruption the pykF enhances the production of 2-hydroxyphenazine [18]. In this study, pykF from LDA1 was initially chosen to inactivate, so we obtained a mutant strain LDA2 (Figs. 5). After analyses of the fermentation broth by HPLC, the DHHA yield of strain LDA2 increased from 2.15 g/L to 4.17 g/L (Fig. 5a). The growth condition of the strain was detected to have little effect after deletion of pykFFig. 5b).
PsrA, a sigma regulator, was first reported in P. chlororaphis PCL1391 [19]. According to Chin-A-Woeng et al, PsrA played a negative regulatory role in the production of the antifungal metabolite PCA in P. chlororaphis PCL1391 [19]. Similar results were obtained from psrA disruption in P. chlororaphis HT66 [20]. According to our research, the sigma regulator psrA also exists in P. chlororaphis Lzh-T5. The strain LDA–3 was obtained after the gene psrA was disrupted in LDA–2, and the production of DHHA increased from 4.17 to 4.92g/L (Fig. 5a)..
RpeA, a negative regulator of Phenazine, was mutated by insertion in P. chlororaphis GP72 and results in a production increase of 2-hydroxyphenazine [18, 21]. RpeA is part of the two-component signal transduction system (TCST) RpeA/RpeB, and is present in other Pseudomonas strains. For example, in P. chlororaphis 30–84, an RpeA homologue, negatively regulates the yield of PCA, indicating a conserved mechanism of Pseudomonas spp in phenazine synthesis regulation [22, 23]. In this study, rpeA was disrupted in the LDA–3 genome to construct LDA–4. Similar to the insertional mutagenesis of P. chlororaphis GP72, DHHA yield of LDA–4 increased from 4.92 g to 5.52 g/L (Figs. 5a)..
Enhanced DHHA production by key gene overexpression
We disrupted pykF to improve the yield of DHHA from 2.15 g/L to 4.17 g/L by diverting more metabolic flux into the shikimate pathway from other pathways (Fig. 5a).. This indicates that enhancing the lead synthesis pathway, we could improve yield of DHHA in P. chlororaphis Lzh-T5. Compared with knocking out negative regulatory genes, gene overexpression is another effective strategy often used to increase the yield of biologic products in microorganisms. According to previous research, overexpression of key genes in the shikimate pathway enhanced the yield of 2-OH-PHZ[18]. In order to increase the production of DHHA, aroB, aroD, aroE, phzC, tktA, and ppsA from Lzh-T5 were overexpressed from the shikimate, pentose phosphate, and gluconeogenesis pathways. We used a previously employed kind of modular vector, the BglBrick plasmid [24]. A recombinant plasmid containing six genes, pBbB5K-aroE-aroD-aroB-phzC-tktA-ppsA, was constructed. Strain LDA–5 was created after transformation into the strain LDA–4 by electrotansformation. After fermentation, the DHHA production of LDA–5 increased to 7.89 g/L after 48 hours (Fig. 5a).. This indicates that overexpression of key genes is an effective strategy to enhance the production of DHHA.
Enhanced DHHA production with Fe3+
Environmental factors have important effects on secondary metabolite production in Pseudomonas strains, especial ion concentration in the medium [25, 26].There is no universal medium suitable for Pseudomonas strains which can produce phenazines due to different nutritional requirements [25]. According to previous research, the DHHA production has a 30% increase after adding 3 mM of Fe3+ [17]. To improve the production of DHHA, the effect of different concentrations of Fe3+ in the medium was investigated. After fermentation, DHHA production was detected by HPLC. Low concentration of iron ions promoted DHHA production. High concentrations of iron ions inhibited DHHA production (Fig. 6).. Different from our previous research, in the fermentation of LDA–5, the optimum concentration which enhanced the production of DHHA was 2 mM. We obtained a maximum DHHA yield of 10.45 g/L with 2 mM Fe3+ (Fig. 6a)..