Isolation and Characterization of Levoglucosan-Metabolizing Bacteria

Abstract

Bacteria were isolated from wastewater and soil containing charred wood remnants based on their ability to use levoglucosan as a sole carbon source and on their levoglucosan dehydrogenase (LGDH) activity. On the basis of their 16S rRNA gene sequences, these bacteria represented the diverse genera Microbacterium, Paenibacillus, Shinella, and Klebsiella. Genomic sequencing of the isolates verified that two isolates represented novel species, Paenibacillus athensensis MEC069^T and Shinella sumterensis MEC087^T, while the remaining isolates were closely related to Microbacterium lacusdiani or Klebsiella pneumoniae. The genetic sequence of LGDH, lgdA, was found in the genomes of these four isolates as well as Pseudarthrobacter phenanthrenivorans Sphe3. The identity of the P. phenanthrenivorans LGDH was experimentally verified following recombinant expression in Escherichia coli. Comparison of the putative genes surrounding lgdA in the isolate genomes indicated that several other gene products facilitate the bacterial catabolism of levoglucosan, including a putative sugar isomerase and several transport proteins. IMPORTANCE Levoglucosan is the most prevalent soluble carbohydrate remaining after high-temperature pyrolysis of lignocellulosic biomass, but it is not fermented by typical production microbes such as Escherichia coli and Saccharomyces cerevisiae. A few fungi metabolize levoglucosan via the enzyme levoglucosan kinase, while several bacteria metabolize levoglucosan via levoglucosan dehydrogenase. This study describes the isolation and characterization of four bacterial species that degrade levoglucosan. Each isolate is shown to contain several genes within an operon involved in levoglucosan degradation, furthering our understanding of bacteria that metabolize levoglucosan.

Keywords: Klebsiella; Microbacterium; Paenibacillus; Pseudarthrobacter phenanthrenivorans; Shinella; levoglucosan dehydrogenase; pyrolysis.

FIG 1

Metabolism of levoglucosan by levoglucosan kinase (enzyme 1) and proposed levoglucosan dehydrogenase (2) pathway leading to d-glucose (enzymes 2 to 5). Presumably glucokinase (6) converts intracellular d-glucose generated from levoglucosan to d-glucose-6-phosphate. The levoglucosan pathway is drawn as previously proposed for B. smithii (17).

FIG 2

Phylogenetic tree of Paenibacillus 16S rRNA gene sequences constructed using the maximum-likelihood method. Members of the Bacillus were used as an outgroup. Paenibacillus presented were chosen from a tree of all Paenibacillus 16S rRNAs to represent major clades within the genus as well as to show the species with genome sequences closely related to P. athensensis MEC069. The tree with the highest log likelihood (−11,701.73) is drawn to scale, with the bar with units of the number of substitutions per site and bootstrap values presented next to tree nodes. There were a total of 1,623 positions in the final data set. Evolutionary analyses were conducted in MEGA X.

FIG 3

Phylogenetic tree of Shinella 16S rRNA gene sequences constructed using the maximum-likelihood method. Ensifer adhaerens LMG 20216^T, Rhizobium giardinii, and Rhizobium herbae were used as an outgroup. All 16S rRNA genes available from officially named Shinella organisms were used. The tree with the highest log likelihood (−3,378.77) is drawn to scale, with branch lengths measured in the number of substitutions per site and the bootstrap values shown next to the branches. There were a total of 1,535 positions in the final data set. Evolutionary analyses were conducted in MEGA X.

FIG 4

Phylogenetic tree of Microbacterium 16S rRNA gene sequences constructed using the maximum-likelihood method. Rarobacter faecitabidus DSM 4813^T was used as an outgroup. Microbacterium organisms presented were chosen from a tree of all Microbacterium 16S rRNAs to represent major clades within the genus as well as the species with genome sequences closely related to Microbacterium MEC084. The tree with the highest log likelihood (−4,487.68) is drawn to scale, with the bar with units of the number of substitutions per site and bootstrap values shown next to the branches. There were a total of 1,546 positions in the final data set. Evolutionary analyses were conducted in MEGA X.

FIG 5

Growth of K. pneumoniae subsp. pneumoniae DSM 30104^T (A) and K. pneumoniae subsp. pneumoniae MEC097 (B) on 10 mM cellobiosan as the sole carbon source. Symbols: cellobiosan (■), levoglucosan (▴), and OD (○). Means and standard deviations from triplicates are indicated.

FIG 6

Alignment of LGDH from the bacterial isolates and P. phenanthrenivorans Sphe3. No highlighting indicates a residue conserved in at least 60% of the sequences, gray highlighting indicates similar residues, and black highlighting indicates residues that are chemically different from the majority at that position.

FIG 7

Conserved genes neighboring lgdA in the genomes of levoglucosan-utilizing isolates. These genes are annotated as a substrate-binding component (red), ATP-binding component (orange), and membrane component (yellow) of a monosaccharide ABC transporter of the CUT2 family, a sugar phosphate isomerase/epimerase (lgdB, green), and a putative dehydrogenase (lgdA, blue, shown in this work to be levoglucosan dehydrogenase).

FIG 8

Consumption of levoglucosan by E. coli BL21(DE3) cells expressing the P. phenanthrenivorans lgdA (●), lgdB (▵), or both (■) genes following growth on PA-5052 autoinduction medium with 2 g/liter levoglucosan at 37°C and 250 rpm. Means and standard deviations from triplicate cultures are shown.