Comparative transcriptomics and proteomics of p-hydroxybenzoate producing Pseudomonas putida S12: novel responses and implications for strain improvement

Abstract

A transcriptomics and proteomics approach was employed to study the expression changes associated with p-hydroxybenzoate production by the engineered Pseudomonas putida strain S12palB1. To establish p-hydroxybenzoate production, phenylalanine-tyrosine ammonia lyase (pal/tal) was introduced to connect the tyrosine biosynthetic and p-coumarate degradation pathways. In agreement with the efficient p-hydroxybenzoate production, the tyrosine biosynthetic and p-coumarate catabolic pathways were upregulated. Also many transporters were differentially expressed, one of which--a previously uncharacterized multidrug efflux transporter with locus tags PP1271-PP1273--was found to be associated with p-hydroxybenzoate export. In addition to tyrosine biosynthesis, also tyrosine degradative pathways were upregulated. Eliminating the most prominent of these resulted in a 22% p-hydroxybenzoate yield improvement. Remarkably, the upregulation of genes contributing to p-hydroxybenzoate formation was much higher in glucose than in glycerol-cultured cells.

Fig. 1

Genealogy of aromatics producing mutants of P. putida S12. The right hand column shows how each successive mutant was obtained. oprB-1 porin B, aroF-1 class-I DAHP synthase, pobA p-hydroxybenzoate hydroxylase, hpd 4-hydroxyphenylpyruvate dioxygenase, PP1271-PP1273 multidrug efflux MFS transporter, pal/tal phenylalanine/tyrosine ammonia lyase, tpl tyrosine-phenol lyase, NTG N-methyl-N’-nitro-N-nitrosoguanidine, MFP ^r resistant to 100 mg/l m-fluoro-DL-phenylalanine, MFT ^r resistant to 100 mg/l m-fluoro-L-tyrosine

Fig. 2

Global response on transcriptome and proteome level between the control strain and P. putida S12palB1 on glucose (a) and glycerol (b). Percentages of differentially expressed genes (fold changes of at least 2.5) and proteins (fold change of at least 1.5) are given per COG group

Fig. 3

Schematic overview of differentially expressed genes involved in relevant pathways of p-hydroxybenzoate biosynthesis, as derived from transcriptomics analysis of P. putida S12palB1. Gene names are indicated in italic or when absent, indicated by locus tags from P. putida KT2440. Red lines indicate upregulation and green lines indicate downregulation in P. putida S12palB1. The text boxes describe the proposed role in the enhanced p-hydroxybenzoate production of the differentially expressed genes

Fig. 4

Growth and production of p-hydroxybenzoate of P. putida S12palB1 (a), P. putida S12palB2 (b), P. putida S12palB3 (c), P. putida S12B3 (d), P. putida S12pal_mfsB3 (e) and P. putida S12pal_mfsB2 (f) in mineral medium with 20 mM glucose as carbon source in shake flask cultures. p-hydroxybenzoate (square), p-coumarate (triangle) and CDW (cell dry weight; diamond). The data are averages of triplicate experiments. The maximum variation between the triplicates was less than 10%