Vanillin Production in Pseudomonas: Whole-Genome Sequencing of Pseudomonas sp. Strain 9.1 and Reannotation of Pseudomonas putida CalA as a Vanillin Reductase

Abstract

Microbial degradation of lignin and its related aromatic compounds has great potential for the sustainable production of chemicals and bioremediation of contaminated soils. We previously isolated Pseudomonas sp. strain 9.1 from historical waste deposits (forming so-called fiber banks) released from pulp and paper mills along the Baltic Sea coast. The strain accumulated vanillyl alcohol during growth on vanillin, and while reported in other microbes, this phenotype is less common in wild-type pseudomonads. As the reduction of vanillin to vanillyl alcohol is an undesired trait in Pseudomonas strains engineered to accumulate vanillin, connecting the strain 9.1 phenotype with a genotype would increase the fundamental understanding and genetic engineering potential of microbial vanillin metabolism. The genome of Pseudomonas sp. 9.1 was sequenced and assembled. Annotation identified oxidoreductases with homology to Saccharomyces cerevisiae alcohol dehydrogenase ScADH6p, known to reduce vanillin to vanillyl alcohol, in both the 9.1 genome and the model strain Pseudomonas putida KT2440. Recombinant expression of the Pseudomonas sp. 9.1 FEZ21_09870 and P. putida KT2440 PP_2426 (calA) genes in Escherichia coli revealed that these open reading frames encode aldehyde reductases that convert vanillin to vanillyl alcohol, and that P. putida KT2440 PP_3839 encodes a coniferyl alcohol dehydrogenase that oxidizes coniferyl alcohol to coniferyl aldehyde (i.e., the function previously assigned to calA). The deletion of PP_2426 in P. putida GN442 engineered to accumulate vanillin resulted in a decrease in by-product (vanillyl alcohol) yield from 17% to ∼1%. Based on these results, we propose the reannotation of PP_2426 and FEZ21_09870 as areA and PP_3839 as calA-IIIMPORTANCE Valorization of lignocellulose (nonedible plant matter) is of key interest for the sustainable production of chemicals from renewable resources. Lignin, one of the main constituents of lignocellulose, is a heterogeneous aromatic biopolymer that can be chemically depolymerized into a heterogeneous mixture of aromatic building blocks; those can be further converted by certain microbes into value-added aromatic chemicals, e.g., the flavoring agent vanillin. We previously isolated a Pseudomonas sp. strain with the (for the genus) unusual trait of vanillyl alcohol production during growth on vanillin. Whole-genome sequencing of the isolate led to the identification of a vanillin reductase candidate gene whose deletion in a recombinant vanillin-accumulating P. putida strain almost completely alleviated the undesired vanillyl alcohol by-product yield. These results represent an important step toward biotechnological production of vanillin from lignin using bacterial cell factories.

Keywords: NAD(P)H-dependent oxidoreductases; Pseudomonas; calA; de novo assembly; gene reannotation; vanillyl alcohol.

FIG 1

Phylogram of nonclinical Pseudomonas genomes in relation to strain 9.1, rooted at the model strain P. putida KT2440. Whole-genome phylogeny was calculated with the RealPhy pipeline, and trees were generated with RAxML (a maximum likelihood method). One hundred bootstrap iterations were used to construct the final tree. Bootstrap values are given at each branch. Strain 9.1 clearly clusters with other strains in the so-called P. fragi lineage (33). Pseudomonas sp. strain Lz4W clustered with Pseudomonas sp. 9.1 and P. fragi P121 throughout the analysis, but it had to be removed from the data set due to its high similarity to these strains resulting in bootstrap values below the 70% threshold (35).

FIG 2

Whole-cell oxidoreductase assays with E. coli BL21(DE3) cells expressing the three candidate genes FEZ21_09870 (red squares), PP_3839 (green triangles), and calA (purple crosses) and the negative-control pNIC28-Bsa4KpnI (blue diamonds). (A to C) Three assays with different substrates were performed and evaluated with HPLC, as follows: A1 and A2 show concentrations of vanillin (substrate) and vanillyl alcohol (product), respectively; B1 and B2 show concentrations of coniferyl alcohol (substrate) and coniferyl aldehyde (product), respectively; and C1 and C2 show concentrations of coniferyl aldehyde (substrate) and coniferyl alcohol (product), respectively;. Experiments were performed in duplicates, and the standard deviations are displayed with an error bar.

FIG 3

In vitro enzyme assays with intracellular extracts of E. coli BL21(DE3) cells expressing the three candidate genes FEZ21_09870 (red lines), PP_3839 (green lines), and calA (PP_2426) (purple lines) and the negative-control pNIC28-Bsa4KpnI (blue lines), with two different substrates (vanillyl alcohol and coniferyl alcohol) and two different redox cofactors (NAD⁺ and NADP⁺) in order to determine the preference of each enzyme for substrates and cofactors. Due to overlap in the absorbance (Abs) of products and reduced cofactors, the monitored absorbance was shifted from 340 to 365 nm, and no activity units were calculated. Experiments were performed in duplicate, and the standard deviations are displayed with error bars.

FIG 4

Fermentations of P. putida KT2440 and deletion strains with coniferyl alcohol 5 mM as the sole carbon source. (A) OD₆₂₀ measurements of P. putida KT2440 (blue diamonds), P. putida KT2440 ΔPP_3839 (red squares), and P. putida KT2440 ΔPP_2426 (green triangles). (B) HPLC-determined concentration of coniferyl alcohol with P. putida KT2440 (dark-blue diamonds), P. putida KT2440 ΔPP_3839 (red squares), and P. putida KT2440 ΔPP_2426 (green triangles) and concentration of ferulate detected with P. putida KT2440 (purple crosses), P. putida KT2440 ΔPP_3839 (light-blue line), and P. putida KT2440 ΔPP_2426 (orange circles). Experiments were performed in duplicate, and the standard deviations are displayed with error bars.

FIG 5

Bioconversion of ferulate into vanillin with the engineered P. putida strains GN442 (dashed lines) and GN442 ΔPP_2426 (solid lines). Concentrations of ferulate (blue circles), vanillin (red squares), and vanillyl alcohol (green triangles) were determined by HPLC. Experiments were performed in duplicate, and the standard deviations are displayed with error bars.

FIG 6

In silico predictions for the aromatic funneling pathway genes in Pseudomonas sp. 9.1 and P. putida KT2440. Growth has previously been demonstrated for strain 9.1 on ferulic acid, p-coumaric acid, benzoic acid and vanillin (19), which suggests that known P. putida funneling pathways are also likely to be found in 9.1. The suggested reannotations of the three candidate genes evaluated in this study (FEZ21_09870, PP_2426, and PP_3839) are referred to by the suggested reannotations (cf. the Discussion). The reaction in strain 9.1 to consume the accumulated vanillyl alcohol is unknown, and thus, the proposed reaction to oxidize it back to vanillin is denoted by a question mark. CoA, coenzyme A; TCA, tricarboxylic acid.