A metabolic pathway for catabolizing levulinic acid in bacteria

Abstract

Microorganisms can catabolize a wide range of organic compounds and therefore have the potential to perform many industrially relevant bioconversions. One barrier to realizing the potential of biorefining strategies lies in our incomplete knowledge of metabolic pathways, including those that can be used to assimilate naturally abundant or easily generated feedstocks. For instance, levulinic acid (LA) is a carbon source that is readily obtainable as a dehydration product of lignocellulosic biomass and can serve as the sole carbon source for some bacteria. Yet, the genetics and structure of LA catabolism have remained unknown. Here, we report the identification and characterization of a seven-gene operon that enables LA catabolism in Pseudomonas putida KT2440. When the pathway was reconstituted with purified proteins, we observed the formation of four acyl-CoA intermediates, including a unique 4-phosphovaleryl-CoA and the previously observed 3-hydroxyvaleryl-CoA product. Using adaptive evolution, we obtained a mutant of Escherichia coli LS5218 with functional deletions of fadE and atoC that was capable of robust growth on LA when it expressed the five enzymes from the P. putida operon. This discovery will enable more efficient use of biomass hydrolysates and metabolic engineering to develop bioconversions using LA as a feedstock.

Figure 1. Genetic characterization and proposed catabolic activity of the P. putida lva operon

a, Organization of the lvaRABCDEFG (9,323 bp) operon. b, Reverse Transcriptase (RT) PCR demonstrates that each gene is expressed in cells grown on LA. Samples were compared with the negative control (-RT) where reverse transcriptase was omitted from the reaction (n=1). c, RT-PCR of cDNA created with primer JMR237 demonstrates that the operon is polycistronic. Note that a product spanning each intergenic region was observed (n=1). d, lva operon induction assay. GFP fluorescence was measured from LB-cultures supplemented with various organic acids (20 mM) (n=3, biological). Error bars represent s.d. Insert shows the schematic of transcriptional GFP fusion used to test induction of the lva operon. lvaR was cloned onto a plasmid containing its native constitutive promoter and the native promoter region for lvaA. The fluorescent protein sfGFP was cloned in place of lvaA. e, Proposed pathway for LA metabolism. LA, levulinic acid; 4HV, 4-hydroxyvalerate; 3HV, 3-hydroxyvalerate; LA-CoA, levulinyl-CoA; 4HV-CoA, 4-hydroxyvaleryl-CoA; CoA, coenzyme-A; ATP, adenosine triphosphate; 4PV-CoA, 4-phosphovaleryl-CoA; 3KV-CoA, 3-ketovaleryl-CoA; NAD(P)H, Nicotinamide adenine dinucleotide (phosphate) reduced; GFP, green fluorescent protein.

Figure 2. Enzymatic activity and pathway characterization for lva operon

a, CoA-ligase activity assay schematic. Using the Enzchek^® Pyrophosphatase Assay kit, the amount of pyrophosphate released during the CoA ligase reaction was measured as an increase of absorbance at 360 nm. b, Activity of LvaE towards short and medium chain acids (n=3, technical). Baseline subtraction was performed on all samples with a control reaction containing no substrate, indicated by Δ absorbance. c, CoA species abundance in LC/MS analysis of in vitro enzyme combinations following 30 min incubation. Reactions contained LA, CoA, ATP, and NAD(P)H with varying enzyme combinations (n=3, technical). ABDE—C indicates that the LvaABDE reaction was performed first, metabolites were separated from LvaABDE, and the resulting solution was supplemented with LvaC. The reaction confirms that LvaC is capable of converting 4PV-CoA to 3HV-CoA. d, MS/MS spectra for 4HV-CoA. Assignment of selected fragments from 4HV-CoA below. e, MS/MS spectra for 4PV-CoA. Assignments of selected fragments from 4PV-CoA below. The masses between the selected fragments of 4PV-CoA and 4HV-CoA differ by the mass of PO₃H (79.967), indicating 4PV-CoA contains a phosphate group not found in 4HV-COA. Bold values indicate the mass of the parent ion. Peaks identified with the symbol (*) are fragments resulting from coenzyme A. See Supplementary Figure 6 and Supplementary Table 2 for additional fragmentation information. f, Abundance of pentenoyl-CoA and 3HV-CoA over a 60 min timecourse for a mixture of LvaABCDE, LA, CoA, ATP, and NAD(P)H (n=3, technical). AMP, adenosine monophosphate; PP_i, pyrophosphate; P_i, phosphate; MESG, 2-amino-6-mercapto-7-methylpurine ribonucleoside; PNP, purine nucleoside phosphorylase; Abs, absorbance; a.u., arbitrary units. Error bars represent s.d.

Figure 3. E. coli growth on propionate and LA

a, E. coli utilization of propionate (n=3, biological). We performed a growth study to evaluate E. coli growth on various concentrations of propionate, with and without acetate as a secondary carbon source. The maximum allowable concentration that stimulated growth was 20 mM propionate, both in the presence and absence of acetate. Using this information, the LA concentration was limited to 20 mM for growth and induction studies. b, Growth curve of E. coli strains on LA. All strains harbor lvaABCDE on pJMR32 (n=3, biological). Error bars represent s.d.

Figure 4. Predicted LA catabolism gene clusters in other genomes

a, Representation of lva operon enzymatic genes. b, Comparison of lva operon from P. putida KT2440 with homologous, predicted LA degradation gene clusters found in other organisms. *Cupriavidus necator had less than 30% homology to LvaA, below the homology cutoff set for species isolation listed in Supplementary Tables 5 and 6