Transcriptome analysis of a phenol-producing Pseudomonas putida S12 construct: genetic and physiological basis for improved production

Abstract

The unknown genetic basis for improved phenol production by a recombinant Pseudomonas putida S12 derivative bearing the tpl (tyrosine-phenol lyase) gene was investigated via comparative transcriptomics, nucleotide sequence analysis, and targeted gene disruption. We show upregulation of tyrosine biosynthetic genes and possibly decreased biosynthesis of tryptophan caused by a mutation in the trpE gene as the genetic basis for the enhanced phenol production. In addition, several genes in degradation routes connected to the tyrosine biosynthetic pathway were upregulated. This either may be a side effect that negatively affects phenol production or may point to intracellular accumulation of tyrosine or its intermediates. A number of genes identified by the transcriptome analysis were selected for targeted disruption in P. putida S12TPL3. Physiological and biochemical examination of P. putida S12TPL3 and these mutants led to the conclusion that the metabolic flux toward tyrosine in P. putida S12TPL3 was improved to such an extent that the heterologous tyrosine-phenol lyase enzyme had become the rate-limiting step in phenol biosynthesis.

FIG. 1.

Genealogy of phenol-producing mutants of P. putida S12. The left column shows how each successive mutant was obtained. The right column indicates the yield (mol phenol/mol glucose) attained in a typical shake flask culture (51). NTG, N-methyl-N′-nitro-N-nitrosoguanidine; MFP^r: resistant to 100 mg m-fluoro-dl-phenylalanine·liter⁻¹; MFT^r, resistant to 100 mg m-fluoro-l-tyrosine·liter⁻¹.

FIG. 2.

Schematic overview of expression profiles of genes involved in relevant pathways of phenol production, as derived from transcriptome analysis of P. putida S12TPL3. Genes are indicated by locus tags from P. putida KT2440, followed by their gene name in parentheses where applicable. Green lettering indicates downregulation, and red indicates upregulation. Genes that were selected for targeted disruption are underlined.

FIG. 3.

Production and growth of P. putida S12TPL3 (A), P. putida S12TPL3r (B), P. putida STΔhpd (C), and P. putida STΔphhA (D). □, cell dry weight; ▪, phenol concentration; ▴, estimated 4-hydroxyphenylpyruvate (hpp) concentration. The profiles for strains STΔpobA and STΔhpd were similar; therefore, only results for STΔhpd are presented here. Cultures were performed in triplicate in mineral salts medium with 10 mM glucose as the carbon source. The variation between replicates was less than 10%.

FIG. 4.

Phenol and 4-hydroxyphenylpyruvate production in chemostat cultures of P. putida S12TPL3 after a tyrosine pulse. ▪, phenol concentration; ▵, tyrosine concentration; ▴, estimated 4-hydroxyphenylpyruvate (hpp) concentration. Values are the averages from duplicate experiments; variation between duplicates was less than 10%.

FIG. 5.

Phenol production by P. putida S12TPL3 in mineral salts medium with 20 mM glucose as the carbon source in the presence of different concentrations of phenol in the culture medium. ▪, maximum growth rate (h⁻¹); ▴, Q_p (average specific activity during the first 12 h of cultivation) [μmol (g cell dry weight)⁻¹ min⁻¹].