A pathway for chitin oxidation in marine bacteria

Abstract

Oxidative degradation of chitin, initiated by lytic polysaccharide monooxygenases (LPMOs), contributes to microbial bioconversion of crystalline chitin, the second most abundant biopolymer in nature. However, our knowledge of oxidative chitin utilization pathways, beyond LPMOs, is very limited. Here, we describe a complete pathway for oxidative chitin degradation and its regulation in a marine bacterium, Pseudoalteromonas prydzensis. The pathway starts with LPMO-mediated extracellular breakdown of chitin into C1-oxidized chitooligosaccharides, which carry a terminal 2-(acetylamino)-2-deoxy-D-gluconic acid (GlcNAc1A). Transmembrane transport of oxidized chitooligosaccharides is followed by their hydrolysis in the periplasm, releasing GlcNAc1A, which is catabolized in the cytoplasm. This pathway differs from the known hydrolytic chitin utilization pathway in enzymes, transporters and regulators. In particular, GlcNAc1A is converted to 2-keto-3-deoxygluconate 6-phosphate, acetate and NH₃ via a series of reactions resembling the degradation of D-amino acids rather than other monosaccharides. Furthermore, genomic and metagenomic analyses suggest that the chitin oxidative utilization pathway may be prevalent in marine Gammaproteobacteria.

Fig. 1. The proposed catabolic pathway for GlcNAc1A in this study and reported catabolic pathways for GlcNAc and N-acetyl-D-serine.

Catabolism of GlcNAc is initiated by phosphorylation by a GlcNAc kinase (e.g. in Shewanella oneidensis) or a PTS (e.g. in Vibrionaceae^,), which is subsequently deacetylated and deaminated to produce fructose-6-P. Catabolism of N-acetyl-D-serine in some bacteria starts with deacetylation followed by deamination and formation of pyruvate^,. In the proposed catabolic pathway for GlcNAc1A in this study, GlcNAc1A is deacetylated and deaminated directly to produce KDG without activation by phosphorylation. As such, this GlcNAc1A degradation pathway resembles N-acetyl-D-amino acid catabolism through deacetylation and deamination. Enzymes involved in catabolic pathways for GlcNAc1A, GlcNAc and N-acetyl-D-serine are shown in red, blue and green, respectively. The protein families of enzymes involved in each pathway are indicated in parentheses. PTS phosphotransferase, GlcN D-glucosamine, PLP pyridoxal 5-phosphate, KDG 2-keto-3-deoxygluconate.

Fig. 2. Growth of chitinolytic marine Pseudoalteromonas strains on GlcNAc1A and functional analysis of the recombinant AA10 LPMO from P. prydzensis ACAM 620.

a Growth of chitinolytic Pseudoalteromonas spp. in the minimal medium supplemented with 0.2% (w/v) GlcNAc1A. The y-axis represents log₂ transformation of the OD₆₀₀ value. Data are presented as mean ± standard deviations (SD) (n = 2 independent experiments). b Genetic organization of the cdc cluster of P. prydzensis ACAM 620. c Heat map for the top ten most abundant proteins in the secretome of strain ACAM 620 grown on 0.5% (w/v) colloidal chitin as the sole carbon source. The colors in the heat map indicate relative protein abundance, ranging from high (red) to low abundance (blue). The data are presented as log₂ transformation of the mean values of two biological replicates for each protein. These ten proteins account for 83.86% of the total protein abundance. The locus tag, protein annotation, type of signal peptides and CAZy family (glycosyl hydrolase (GH), carbohydrate-binding module (CBM) and auxiliary activity (AA)) are shown. Chitinolytic enzymes encoded by the cdc cluster are marked by solid circles and the other one by an empty circle. SpI, signal peptidase I cleavage site; SpII, signal peptidase II cleavage site. d Positive-mode Q-TOF-MS spectrum of products generated by the AA10 LPMO from strain ACAM 620 acting on 0.2% (w/v) squid pen β-chitin in the presence of 1 mM AscA. The insets show the negative control reactions without either LPMO or AscA, which did not generate detectable amounts of oxidized chitooligosaccharides. 100% relative intensity in the inserts represents 9.1 ×10³ (control reaction without LPMO) and 7.0 ×10³ (control reaction without AscA) arbitrary units (a.u.), respectively. Theoretical masses of relevant products are listed in Supplementary Table 2. DP, degree of polymerization; subscript OX, C1-oxidized chitooligosaccharides with a GlcNAc1A moiety; DP3_ox-Ac, DP3_ox lacking one acetyl group; DP4_ox-Ac, DP4_ox lacking one acetyl group. The graph shows a representative MS spectrum of at least three independent replicates. Source data are provided as a Source Data file.

Fig. 3. Identification of key genes in strain ACAM 620 involved in utilizing oxidized chitooligosaccharides based on transcriptomic and genetic analyses.

a Genetic organization of the ong cluster of strain ACAM 620. TBDR, TonB-dependent receptor; SSS, sodium solute symporter. b RNA-seq assay of the transcriptions of genes from the ong cluster and ongOT-2 in strain ACAM 620 in the minimal medium supplemented with 0.2% (w/v) GlcNAc1A. Values are expressed as fold change (log₂) compared to cultures in the minimal medium supplemented with 0.2% (w/v) glucose. Data are presented as mean ± SD (n = 2 independent experiments). c–i The growth phenotype of strain ACAM 620 with a single gene deletion in the ong cluster on GlcNAc1A or GlcNAc-GlcNAc1A as the sole carbon source. Wild-type strain (WT), mutant strains and complemented strains of mutants were grown at 25 °C in the minimal medium supplemented with 10 mM GlcNAc1A or 10 mM GlcNAc-GlcNAc1A. Deletion mutant strains with the empty plasmid pEV were used a control. The y-axes in c-i represent log₂ transformation of the OD₆₀₀ value. Data are presented as mean ± SD (n = 2 independent experiments). j, RT-qPCR assay of the transcriptions of lpmo, ongOT-2 and genes from the ong cluster in the WT and △ongR mutant strains in response to 0.2% (w/v) glucose (upper) or 0.2% (w/v) GlcNAc1A (lower) in the minimal medium. Values are expressed as fold change compared to precultures of the WT strain in the minimal medium supplemented with 0.2% (w/v) glucose. The rpoD gene was used as an internal reference. Data are presented as mean ± SD (n = 3 independent experiments). Source data are provided as a Source Data file.

Fig. 4. In vitro biochemical analyses of enzymes OngA, OngB, OngC and KdgK.

a Negative-mode Q-TOF-MS spectrum of products generated by the recombinant OngA acting on GlcNAc-GlcNAc1A. Q-TOF-MS spectrum of GlcNAc-GlcNAc1A only as the control was shown in Supplementary Fig. 12a. DP2_ox, GlcNAc-GlcNAc1A. b Negative-mode Q-TOF-MS spectrum of products generated by the recombinant OngB acting on GlcNAc1A. Q-TOF-MS spectrum of GlcNAc1A only as the control was shown in Supplementary Fig. 12b. c Negative-mode Q-TOF-MS spectrum of products generated by the recombinant enzymes, OngB and OngC, acting on GlcNAc1A. Q-TOF-MS spectrum of KDG only as the control was shown in Supplementary Fig. 12c. d Negative-mode Q-TOF-MS spectrum of products generated by OngB, OngC and KdgK successively acting on GlcNAc1A. Q-TOF-MS spectrum of KDG-6-P only as the control was shown in Supplementary Fig. 12d. In a-d all labeled MS peaks refer to [M-H]^- ions unless noted otherwise (e.g. [GlcNAc+Cl]^- ion). Theoretical masses of relevant products are listed in Supplementary Table 4. In a-d, the graphs show a representative MS spectrum of at least three independent replicates. e Maximum-likelihood tree of OngB and its homologs and other de-N-acetylases. All homologs to OngB (colored in red) are from the ong clusters of their respective bacterial strains. CE carbohydrate esterase, DA deacetylase, PGN peptidoglycan, Acetyl-D-Glu, acetyl-D-glutamate. f Neighbor-joining tree of OngC and its homologs and characterized pyridoxal 5-phosphate (PLP)-dependent enzymes. All homologs to OngC (colored in red) are from the ong clusters of their respective bacterial strains. Source data are provided as a Source Data file.

Fig. 5. The proposed oxidative chitin utilization pathway in strain ACAM 620 and comparison with the well-characterized hydrolytic chitin utilization pathway in Gammaproteobacteria.

a The proposed oxidative chitin utilization pathway in strain ACAM 620. Strain ACAM 620 secrets a LPMO (as well as chitinases) to cleave chitin into C1-oxidized chitooligosaccharides which are imported across the outer membrane (OM) by two TBDRs, OngOT-1 and OngOT-2. OngA in the periplasm hydrolyzes oxidized chitooligosaccharides to generate GlcNAc1A which is transported across the inner membrane (IM) by a SSS family transporter, OngIT, and converted to KDG-6-P, NH₃ and acetate by OngB, OngC and KdgK in the cytoplasm. Enzyme/transporter symbols are colored according to the colors of their genes. Green solid arrows denote enzymatic reactions, and green dotted arrows denote transport. Black arrows represent the transcriptional directions of the cdc cluster as one operon and predicted operons of the ong cluster. The minus sign surrounded by a circle represents negative regulation. b Comparison of the proposed oxidative chitin utilization pathway in strain ACAM 620 and reported hydrolytic chitin utilization pathway in other Gammaproteobacteria. In the hydrolytic chitin utilization pathway, chitinases degrade chitin into chitooligosaccharides which are imported across OM by a specific porin in Vibrionaceae^,, or a predicted TBDR in Shewanella oneidensis. Non-OngA-type β-hexosaminidases in the periplasm hydrolyze chitooligosaccharides into GlcNAc which is transported into the cytoplasm by non-SSS-type transporters and converted to fructose-6-P, NH₃ and acetate by a GlcNAc kinase (or a PTS), a GlcNAc-6-P deacetylase and a GlcN-6-P deaminase successively. Enzymes involved in oxidative and hydrolytic chitin utilization pathways are shown in grey. Solid arrows denote enzymatic reactions, and dotted arrows denote transport.

Fig. 6. Distribution and ecological function of the oxidative chitin utilization pathway in bacteria.

a Distribution of the oxidative chitin utilization pathway in marine and terrestrial bacterial isolates. Except for terrestrial bacterium Achromobacter piechaudii ATCC 43553 belonging to Betaproteobacteria, all other bacterial strains with the complete oxidative chitin utilization pathway are from Gammaproteobacteria. For some representative strains containing more than one lpmo genes, only one lpmo gene was shown. b Comparison of the chitin-degrading abilities and related chitin-degrading genes of Pseudoalteromonas strains. Strains were cultivated in the minimal medium supplemented with 0.2% (w/v) shrimp shell α-chitin as the sole carbon source at 25 °C for 7 days. No bacterial growth was detectable for nine Pseudoalteromonas strains including P. aliena DSM 16473, P. aurantia DSM 6057, P. issachenkonii DSM 15925, P. lipolytica JCM 15903, P. luteoviolacea DSM 6061, P. rubra DSM 6842, P. tunicata DSM 14096, P. undina DSM 6065 and P. sp. SM9913, even though they were cultivated on α-chitin for 14 days at 25 °C. ND, undetectable growth; +, presence; -, absence. Growth data are presented as mean ± SD (n = 3 independent experiments). Source data are provided as a Source Data file.