Abstract
Simple SummaryTerpenoid phenazines generally produced in Streptomyces exhibit potential antitumor and antibacterial activities. In this study, we designed and constructed an artificial biosynthetic pathway for the synthesis of terpenoid phenazines in Pseudomonas chlororaphis P3. We successfully synthesized endophenazine A and endophenazine A1 for the first time in Pseudomonas by introducing the prenyltransferase PpzP. Moreover, we revealed the biosynthetic pathway of endophenazine A1 in P. chlororaphis P3. This study enriches the diversity of phenazines in P. chlororaphis P3 and provides a reference for the heterologous synthesis of terpenoid phenazines.Endophenazine A is a terpenoid phenazine with phenazine-1-carboxylic acid (PCA), and dimethylallyl diphosphate (DMAPP) derived from the 2-methyl-D-erythritol-4-phosphate (MEP) pathway as the precursor, which shows good antimicrobial activity against several Gram-positive bacteria and fungi. However, the highest yield of endophenazine A was about 20 mg/L in Streptomyces, limiting its large-scale industrial development. Pseudomonas chlororaphis P3, possessing an efficient PCA synthesis and MEP pathways, is a suitable chassis to synthesize endophenazine A. Herein, we designed an artificial biosynthetic pathway for the synthesis of endophenazine A in P. chlororaphis P3. Primarily, the prenyltransferase PpzP from Streptomyces anulatus 9663 was introduced into P. chlororaphis P3 and successfully synthesized endophenazine A. Another phenazine compound, endophenazine A1, was discovered and identified as a leakage of the intermediate 4-hydroxy-3-methyl-2-butene pyrophosphate (HMBPP). Finally, the yield of endophenazine A reached 279.43 mg/L, and the yield of endophenazine A1 reached 189.2 mg/L by metabolic engineering and medium optimization. In conclusion, we successfully synthesized endophenazine A and endophenazine A1 in P. chlororaphis P3 for the first time and achieved the highest titer, which provides a reference for the heterologous synthesis of terpenoid phenazines.
Highlights
Phenazines are a class of important nitrogen-containing heterocyclic compounds that are mainly secreted by Pseudomonas and Streptomyces [1]
The concentration of endophenazine A and endophenazine A1 did not decrease significantly after 60 h of incubation (Figure 4A,B). These results showed that endophenazine A1 and endophenazine A cannot interconvert in P. chlororaphis P3
We found that endophenazine A1 and endophenazine A are relatively stable in the culture of P. chlororaphis P3 and cannot be converted to each other, indicating that endophenazine A1 is not an intermediate in the synthesis of endophenazine A and endophenazine A cannot be oxidized to form endophenazine A1 (Figure 4A,B)
Summary
Phenazines are a class of important nitrogen-containing heterocyclic compounds that are mainly secreted by Pseudomonas and Streptomyces [1]. They have attracted significant attention due to their widespread applications in agriculture, medicine, and industry [2,3,4,5]. Endophenazine A is a C-prenylation phenazine of PCA, catalyzed by prenyltransferase PpzP, and possesses good antibacterial activity [12,13] (Figure 1). Co-overexpression of ppzP and ispG in P3-A1. Co-overexpression of ppzP and ispH in P3-A1. Overexpression of ispG in P3-A8 Lab stock This study. Plasmids pK18mobsacB pK18-phzH pK18-idi pBBR1MCS pBBR-ppzP pBBR-ppzP-idi pBBR-ppzP-ispG pBBR- ppzP-ispH pBBR-ppzP-idi-ispH
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