Design of 1-OH-PHZ biosynthetic pathway in P. chlororaphis H18

We have isolated a strain P. chlororaphis H18 that can secrete reddish-brown pigments from the plant rhizosphere soil, and its colorful metabolites were purified and their structures elucidated to be PCA (1), 2-OH-PHZ (2), and 2-hydroxy-phenazine-1-carboxylic acid (3). Using genome-mining and sequence alignment, their biosynthetic gene cluster was confirmed (Fig. 1A). Since the basic biosynthetic pathway of phenazines has already been clearly resolved, proteins PhzABCDEFGO catalyze the shikimate pathway end product chorismate to form the phenazine common precursor PCA (Fig. 1B). Then, a monooxygenase PhzO converts PCA into 2-OH-PHZ, and 2-hydroxy-phenazine-1-carboxylic acid is the intermediate of this process [20]. A previous study showed that FAD-dependent monooxygenase PhzS of P. aeruginosa PAO1 can catalyze the oxidative decarboxylation of PCA to generate 1-OH-PHZ [28]. So we planned to integrate this phzS gene into P. chlororaphis H18 genome to construct 1-OH-PHZ biosynthetic pathway.

Biosynthesis and identification of 1-OH-PHZ

When cultured in KB medium for 48 h, P. chlororaphis H18 produces 86.6 mg/L PCA, 71.8 mg/L 2-OH-PHZ, and 14.8 mg/L 2-hydroxy-phenazine-1-carboxylic acid. The formation of 2-OH-PHZ and 2-hydroxy-phenazine-1-carboxylic acid catalyzed by PhzO consumed a significant amount of PhzS substrate PCA. Therefore, we synthesized gene phzS from P. aeruginosa PAO1 and replaced phzO with homologous recombination to obtain mutant PH18-1 (Additional file 1: Figs. S3 and S4). In the PH18-1 fermentation broth, 72.3 mg/L of 1-OH-PHZ (4) generation was detected and the production of 2-OH-PHZ (2) and 2-hydroxy-phenazine-1-carboxylic acid (3) was abolished (Fig. 2A, B). 1-OH-PHZ shows outstanding biological activities against crop pathogens, for instance, Bipolaris maydis, Alternaria solani, and Fusarium graminearum (Additional file 1: Table S5).

Fig. 2
figure2

1-Hydroxyphenazine producing strain fermentation and detection. A HPLC chromatograms of fermentation broth of (i) P. chlororaphis H18, (ii) P. chlororaphis H18-1. B The HRMS analysis of 1-hydroxyphenazine. 1, phenazine-1-carboxylic acid; 2, 2-hydroxyphenazine; 3, 2-hydroxy-phenazine-1-carboxylic acid; 4, 1-hydroxyphenazine

Semi-rational design and remodeling to improve the catalytic activity of PhzS

The concentration of 1-OH-PHZ in PH18-1 fermentation broth is relatively low (72.3 mg/L), which limited its industrial application. To address this issue, the promotion of the 1-OH-PHZ conversion rate is an essential job. We know that enzymes are the most critical factors in the biosynthesis process of natural products, especially those rate-limiting step enzymes. In the 1-OH-PHZ biosynthetic pathway, the flavin-dependent hydroxylase PhzS catalyzes the crucial step of decarboxylation and hydroxylation at PCA 1-position. We believe that optimizing PhzS will be an effective way to improve pathway efficiency. Semi-rational design and molecular remodeling are feasible methods for promoting protein catalytic performance. First, PCA was docked into the crystal structure of PhzS (PDB: 2RGJ) as a ligand to predict the PCA-binding hydrophobic pocket or activity site dominated by N48, L76, M205, V207, R215, V217, Y219, M238, P317, M318, G319 [21] (Fig. 4A). To facilitate the detection of PhzS mutants’ activity, we expressed PhzS protein in E. coli BL21 Rosetta (DE3) and verified its activity by whole-cell catalysis (Fig. 3). Then, alanine scanning was employed to identify the function of these residues by detecting mutant enzyme activities on PCA in vitro. N48A, L76A, Y219A, and M238A mutants completely lost their catalytic abilities, only M318A presented a 24% activity increase. Next, amino acids M318 and V217 were subjected to saturation mutagenesis, M318T, M318I, and V217I resulted in 40%, 5%, and 35% higher activity than the wild type enzyme (Additional file 1: Fig. S1). More site-directed mutants in M205, V207, R215, P317, and G319 were constructed using residues I, S, T, and V respectively, among which better activity (1.4-fold of the WT strain) was observed in PhzSV207I (Additional file 1: Fig. S2). Afterward, we used M318A, M318T, V217I, and V207I to construct double and triple mutants. In these mutants, the resulting PhzSM318A/V207I achieved the highest reactivity (an increase of 70% compared with PhzS) to catalyze PCA (Fig. 4B). These mutations are likely to remodel PhzS conformation and consequently influence the substrate-binding pocket, which enhances PhzS catalytic activity. When protein PhzSM318A/V207I was introduced into P. chlororaphis H18, the obtained strain PH18-1–2 presented 151.8 mg/L 1-OH-PHZ concentration, which is 1.1 times higher than PH18-1 (Fig. 4C). This indicates that the engineering of PhzS is an effective way to increase the fermentation yield of 1-OH-PHZ.

Fig. 3
figure3

The protein expression of PhzS and enzymatic catalytic reactions in vitro. A Schematic representation of PCA oxidative decarboxylation by PhzS. B SDS-PAGE of PhzS (43 kD). M, marker; 1, unbound proteins; 2, washed proteins with 20 mM of imidazole; and 3, eluted proteins with 300 mM of imidazole. C HPLC chromatograms of PhzS catalyzed reaction with PCA as substrate at 250 nm. Reaction conditions: phenazine-1-carboxylic acid (0.5 mM), NADH (50 μM), PhzS whole-cell in 50 mM HEPES (pH 7.5) at 30 °C for 1 h. (i) phenazine-1-carboxylic acid standard; (ii) Boiled PhzS whole-cell and NADH with phenazine-1-carboxylic acid; (iii) PhzS whole-cell and NADH with phenazine-1-carboxylic acid; (iv) 1-hydroxyphenazine standard. 1, phenazine-1-carboxylic acid; 4, 1-hydroxyphenazine

Fig. 4
figure4

Rational design of the PhzS to improve its activity to PCA. A Docking of the ligand PCA into the PhzS crystal structure. PCA is shown in yellow. B Relative activity of wild type PhzS and mutants to PCA. C HPLC chromatograms of fermentation broth of PH18-1 and PH18-1–2 at 250 nm. The pink line: PH18-1–2; the black line: PH18-1. The data shown in B are from three experiment replicates, and are expressed as the mean value ± SD

The PH18-1–2 fermentation result showed that there is still a certain amount of precursor PCA in the fermentation broth. Afterward, we try to enhance the conversion of PCA to 1-OH-PHZ by adding PhzS copy numbers. When we inserted two copies of the PhzS M318A/V207I gene to replace PhzS in PH18-1, the constructed strain PH18-1–3 produced 202.4 mg/L 1-OH-PHZ and more PCA is consumed.

Disruption of negative regulatory genes and enrichment of precursor supply to enhance 1-OH-PHZ production

In order to improve 1-OH-PHZ yield, we first chose to increase the supply of phenazine substrate pool in P. chlororaphis H18. Chorismate, a shikimate pathway end product is the key phenazine derivative precursor. Several enzymes such as phosphoenolpyruvate (PEP) synthetase PpsA, transketolase TktA, quinate/shikimate dehydrogenase AroE, and dehydroquinic acid synthase AroB have been studied to enhance the shikimate pathway and chorismate [9, 24]. Through sequence alignment, we found that, PpsA, TktA, AroB, and AroE homologous proteins genes all exist in the in P. chlororaphis H18 genome. Hence, we planned to introduce one more copy of ppsA, tktA, aroB, and aroE genes into P. chlororaphis H18. Besides, RpeA, RsmE, PsrA, and Lon have been reported as negative regulators in Pseudomonas chlororaphis strains [29,30,31,32]. The disruption of these regulatory genes has been proven to effectively improve phenazines titer. Thus, we cloned aroE, tktA, aroB, and ppsA genes from P. chlororaphis H18 to replace the possible negative regulatory genes repA, rsmE, psrA, and lon to obtain strains PH18-1-4, PH18-1-5, PH18-1-6, PH18-1-7 respectively (Fig. 5A). According to the HPLC detection results, the 1-OH-PHZ concentration in the constructed final strain PH18-1-7 was 361.4 mg/L, a fourfold increase over PH18-1 when fermented in KB medium.

Fig. 5
figure5

The strategies for increasing 1-hydroxyphenazine production. A A summary of the steps in the genetic and metabolic engineering of P. chlororaphis H18 for 1-OH-PHZ production. B HPLC chromatograms of fermentation broth of PH18-1 and knocking out gacA gene in P. chlororaphis H18 at 250 nm. (i) PH18-1 fermentation broth; (ii) Fermentation broth of knocking out gacA gene in P. chlororaphis H18. 1, phenazine-1-carboxylic acid; 4, 1-hydroxyphenazine

Further improvement of 1-OH-PHZ production

Research suggested that GacA is a positive global regulator for phenazine biosynthesis in Pseudomonas chlororaphis 30–84 [32], but negatively regulated phenazines production in Pseudomonas sp. M18 and Pseudomonas chlororaphis GP72 [33]. When we knocked out the gacA gene from P. chlororaphis H18, the phenazine generation was almost abolished, which means GacA plays a role in positive regulation in P. chlororaphis H18 on PCA synthesis (Fig. 5B). Afterward, an additional copy of gacA was introduced into PH18-1–7 to replace the secondary metabolite pyrrolnitrin biosynthetic gene prnA. The resultant mutant PH18-1–8 had a 20% increase in 1-OH-PHZ titer (Fig. 5A and Additional file 1: Fig. S4), which is fivefold more than mutant PH18-1 (Fig. 6A).

Fig. 6
figure6

Improvement of 1-OH-PHZ production. A HPLC chromatograms of fermentation broth of PH18-1 and PH18-1–8 at 250 nm. (i) Fermentation broth of PH18-1; (ii) Fermentation broth of PH18-1–8. 1, phenazine-1-carboxylic acid; 4, 1-hydroxyphenazine. B PH18-1–8 production of 1-hydroxyphenazine in different media. C The yield of PH18-1–8 1-hydroxyphenazine by adding different concentrations of shikimic acid to the medium. The data are from three experiment replicates, and are expressed as the mean value ± SD

To further optimize 1-OH-PHZ titer, we tried different kinds of fermentation medium, for instance, KB, PPM, PCM, PPN, and SCM. The HPLC detection data illustrated that PPM is the best for 1-OH-PHZ production, in which the 1-OH-PHZ level is 70% higher than in KB broth (Fig. 6B).

Moreover, shikimic acid is a chief precursor of chorismate in the shikimate pathway, and all known phenazines are converted from chorismate. Hence, we suppose that directly increasing the supply of upstream intermediate or precursor substrate may affect the enhancement of 1-OH-PHZ production. Given the high price of chorismate on the market, the economic shikimic acid is suitable for feeding. When 100 mg/L, 200 mg/L, 400 mg/L, or 800 mg/L of shikimic acid was fed into 50 mL PH18-1–8 strain fermentation broth respectively in 12 h. HPLC detection demonstrates that the addition of shikimic acid can indeed improve the 1-OH-PHZ level (Fig. 6C). Among them, 200 mg/L shikimic acid feeding showed the best effect (20% increase over no feeding). Although higher concentrations of shikimic acid can also increase 1-OH-PHZ titer, the burden of metabolism or feedback inhibition of the synthetic pathway probably influences the yield. Otherwise, engineering feedback resistance of DHAP synthase is another effective approach to improve the yield of shikimate derived products, which could be used in our future work.

Optimization of fed-batch 1-OH-PHZ fermentation

To combine all the above engineering modifications, mutant strain PH18-1-8 (Fig. 5A and Additional file 1: Fig. S4) could generate 938.5 mg/L of 1-OH-PHZ in 50 mL shake flasks. Further improvement of the 1-OH-PHZ production level was carried out by fed-batch fermentation. Strain PH18-1-8 was first employed in a 5 L fermenter containing 2 L PPM liquid medium. During the fermentation process, glucose was also added to meet the demand of the carbon source. Production of 1-OH-PHZ continued to increase until 54 h, and the highest production was 3.6 g/L. The time-course analysis of glucose consumption, OD value, and 1-OH-PHZ concentration during fermentation are illustrated in Fig. 7, respectively.

Fig. 7
figure7

Production of 1-hydroxyphenazine by P. chlororaphis H18-1–8 via fed-batch fermentation. Time course of cell growth, glucose consumption and 1-hydroxyphenazine production during fed-batch fermentation. The data are from three experiment replicates, and are expressed as the mean value ± SD

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