Abstract
Valorization of lignin, an abundant component of plant cell walls, is critical to enabling the lignocellulosic bioeconomy. Biological funneling using microbial biocatalysts has emerged as an attractive approach to convert complex mixtures of lignin depolymerization products to value-added compounds. Ideally, biocatalysts would convert aromatic compounds derived from the three canonical types of lignin: syringyl (S), guaiacyl (G), and p-hydroxyphenyl (H). Pseudomonas putida KT2440 (hereafter KT2440) has been developed as a biocatalyst owing in part to its native catabolic capabilities but is not known to catabolize S-type lignin-derived compounds. Here, we demonstrate that syringate, a common S-type lignin-derived compound, is utilized by KT2440 only in the presence of another energy source or when vanAB was overexpressed, as syringate was found to be O-demethylated to gallate by VanAB, a two-component monooxygenase, and further catabolized via extradiol cleavage. Unexpectedly, the specificity (kcat/KM) of VanAB for syringate was within 25% that for vanillate and O-demethylation of both substrates was well-coupled to O2 consumption. However, the native KT2440 gallate-cleaving dioxygenase, GalA, was potently inactivated by 3-O-methylgallate. To engineer a biocatalyst to simultaneously convert S-, G-, and H-type monomers, we therefore employed VanAB from Pseudomonas sp. HR199, which has lower activity for 3MGA, and LigAB, an extradiol dioxygenase able to cleave protocatechuate and 3-O-methylgallate. This strain converted 93% of a mixture of lignin monomers to 2-pyrone-4,6-dicarboxylate, a promising bio-based chemical. Overall, this study elucidates a native pathway in KT2440 for catabolizing S-type lignin-derived compounds and demonstrates the potential of this robust chassis for lignin valorization.
| Original language | English |
|---|---|
| Pages (from-to) | 111-122 |
| Number of pages | 12 |
| Journal | Metabolic Engineering |
| Volume | 65 |
| DOIs | |
| State | Published - May 2021 |
Funding
This work was authored in part by Alliance for Sustainable Energy, LLC, the manager and operator of the National Renewable Energy Laboratory for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. DOE under contract DE-AC05-00OR22725. The metabolic engineering and systems biology work was funded by The Center for Bioenergy Innovation , a U.S. Department of Energy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science . We also thank the U.S. Department of Energy Office of Energy Efficiency and Renewable Energy Bioenergy Technologies Office for funding for preliminary enzyme purifications, analytics, and strain evaluation. EK, LD, and LDE were supported by Natural Sciences and Engineering Research Council of Canada ( NSERC ) Discovery Grants 171359 to LDE. LDE is the recipient of a Tier 1 Canada Research Chair in Microbial Catabolism and Biocatalysis. We thank Jie Liu for preparing pVP91-Ht-PcaHG and pET41GalA, Nicholas Rorrer for assistance with NMR analyses, Jason C. Grigg for proteomic LC-MS analyses, Lin Wang and Costas Maranas for metabolic pathway insights, and Rhiannon McGeehan for a critical read of the manuscript. The views expressed in the article do not necessarily represent the views of the DOE or the U.S. Government. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes. As reported by Sonoki et al., we observed that wild-type KT2440 did not utilize SA (Fig. 2A) but grew on VA (Fig. 2B) as the sole substrate (Sonoki et al., 2018). However, SA was demethylated while VA was present, with a substantial accumulation of 3MGA (Fig. 2C). A slight excess of VA was required to completely consume SA (Fig. 2D). These observations are in agreement with the hypothesis presented by Sonoki et al. that the native VanAB enzyme in KT2440 demethylates SA (Sonoki et al., 2018). Based on the cessation of SA utilization upon VA depletion, we further hypothesized that an additional growth substrate suffices to support SA catabolism. In SA cultivations supplemented with 20 mM glucose as an auxiliary source of carbon and energy, SA was demethylated to produce 3MGA (Fig. 2E), which ceased upon depletion of glucose (Fig. S1A). With periodic glucose supplementation, SA was completely utilized albeit with accumulation of 3MGA (Fig. 2F). SN166 (KT2440 ?vanAB) did not utilize SA with glucose feeding (Fig. S1B), further indicating that VanAB is the enzyme that catalyzes SA O-demethylation.We next sought to determine if increased expression of vanAB might improve SA utilization. In agreement with results presented by Sonoki et al. (2018), expression of vanAB on a plasmid (strain SN183) did not enable SA utilization as the sole carbon source (Fig. 3A). However, in the presence of glucose, SN183 rapidly catabolized SA (Fig. 3B). Integration of a second copy of vanAB into the chromosome of wild-type KT2440 driven by the strong and constitutive tac promoter (Elmore et al., 2017) (strain CJ486) resulted in catabolism of SA as the sole carbon source (Fig. 3C) which was enhanced by the presence of glucose (Fig. 3D). As with wild-type KT2440, the addition of formate also improved CJ486 growth and SA catabolism (Figs. S2C?D). The phenotypic discrepancy between plasmid-bearing SN183 and genome-integrated CJ486 suggests that the additional metabolic burden of either maintaining the vanAB overexpression plasmid, or additional protein synthesis due to a higher gene dosage associated with plasmid expression, precludes SA catabolism, consistent with the energy limitation described above. In support of this, SA utilization by CJ486 was significantly decreased when the strain harbored an empty pBTL-2 vector (Fig. S3, Fig. 3C). Despite the apparent energetic limitation, overexpression of vanAB in CJ486 resulted in growth with SA depletion (Fig. 3C) whereas no SA utilization nor biomass generation was observed in KT2440 (Fig. 2A) unless formate was supplied (Fig. S2A). These results demonstrate that chromosomal over-expression of vanAB is sufficient for catabolism of SA as the sole carbon source and that this activity is enhanced by supplementation with an auxiliary source of energy, such as VA, glucose, or formate.Pathways for the catabolism of compounds derived from H- and G-lignin have been well studied in pseudomonads including KT2440 (Jim?nez et al., 2010; Nogales et al., 2017). In contrast, there are few descriptions of these bacteria degrading monomers derived from S-lignin. In the present study, we demonstrate and characterize an S-lignin catabolic pathway of KT2440. The in vivo and in vitro data support the pathway proposed by Sonoki et al. (2018), and extends the pathway for GA catabolism described by Nogales et al., 2005, 2011. Remarkably, VanAB O-demethylates both SA and 3MGA in addition to VA, with strong preference for VA and SA compared to 3MGA. The specificity of VanABKT2440 is consistent with the activity reported for homologs from other organisms. For example, VanAs from Streptomyces sp. NL15-2K and Pseudomonas sp. HR199 transform SA, VA, m-anisate, and veratrate (Nishimura et al., 2014) (Lanfranchi et al., 2019). Structural insights into VanAHR199 and VanAKT2440 could potentially inform enzyme engineering to improve O-demethylation of SA and 3MGA, which will be pursued in future work. Nevertheless, the relatively high specificity of VanABKT2440 for SA and the coupling of the reaction to O2 and NADH consumption suggest that this activity is physiologically relevant and that the bacterium grows on mixtures of VA and SA in its natural environment.This work was authored in part by Alliance for Sustainable Energy, LLC, the manager and operator of the National Renewable Energy Laboratory for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. DOE under contract DE-AC05-00OR22725. The metabolic engineering and systems biology work was funded by The Center for Bioenergy Innovation, a U.S. Department of Energy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. We also thank the U.S. Department of Energy Office of Energy Efficiency and Renewable Energy Bioenergy Technologies Office for funding for preliminary enzyme purifications, analytics, and strain evaluation. EK, LD, and LDE were supported by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants 171359 to LDE. LDE is the recipient of a Tier 1 Canada Research Chair in Microbial Catabolism and Biocatalysis. We thank Jie Liu for preparing pVP91-Ht-PcaHG and pET41GalA, Nicholas Rorrer for assistance with NMR analyses, Jason C. Grigg for proteomic LC-MS analyses, Lin Wang and Costas Maranas for metabolic pathway insights, and Rhiannon McGeehan for a critical read of the manuscript. The views expressed in the article do not necessarily represent the views of the DOE or the U.S. Government. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes.
Keywords
- 2-Pyrone-4,6-dicarboxylic acid
- Aromatic catabolism
- Biological funneling
- S-Lignin
- Syringic acid