Metabolism of syringyl lignin-derived compounds in Pseudomonas putida enables convergent production of 2-pyrone-4,6-dicarboxylic acid

Notonier, Sandra; Werner, Allison Z.; Kuatsjah, Eugene; Dumalo, Linda; Abraham, Paul E.; Hatmaker, E. Anne; Hoyt, Caroline B.; Amore, Antonella; Ramirez, Kelsey J.; Woodworth, Sean P.; Klingeman, Dawn M.; Giannone, Richard J.; Guss, Adam M.; Hettich, Robert L.; Eltis, Lindsay D.; Johnson, Christopher W.; Beckham, Gregg T.

doi:10.1016/j.ymben.2021.02.005

Cited by 60 publications

(37 citation statements)

References 81 publications

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“…A calibration verification standard was run every 10–15 samples to ensure detector stability. Glucose was quantified by HPLC with refractive index detection coupled with an Aminex HPX-87H column (Bio-Rad) 61 , while xylose was similarly and simultaneously quantified. Pure standards were purchased from Sigma-Aldrich.…”

Section: Methodsmentioning

confidence: 99%

Muconic acid production from glucose and xylose in Pseudomonas putida via evolution and metabolic engineering

et al. 2022

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Muconic acid is a bioprivileged molecule that can be converted into direct replacement chemicals for incumbent petrochemicals and performance-advantaged bioproducts. In this study, Pseudomonas putida KT2440 is engineered to convert glucose and xylose, the primary carbohydrates in lignocellulosic hydrolysates, to muconic acid using a model-guided strategy to maximize the theoretical yield. Using adaptive laboratory evolution (ALE) and metabolic engineering in a strain engineered to express the D-xylose isomerase pathway, we demonstrate that mutations in the heterologous D-xylose:H+ symporter (XylE), increased expression of a major facilitator superfamily transporter (PP_2569), and overexpression of aroB encoding the native 3-dehydroquinate synthase, enable efficient muconic acid production from glucose and xylose simultaneously. Using the rationally engineered strain, we produce 33.7 g L−1 muconate at 0.18 g L−1 h−1 and a 46% molar yield (92% of the maximum theoretical yield). This engineering strategy is promising for the production of other shikimate pathway-derived compounds from lignocellulosic sugars.

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Section: Methodsmentioning

confidence: 99%

Muconic acid production from glucose and xylose in Pseudomonas putida via evolution and metabolic engineering

et al. 2022

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“…Recent studies on the enzymatic demethylation of lignin-derived compounds and their model compounds have primarily focused on white-rot, soft-rot, and brown-rot fungi and some bacteria [ 7 , 8 ]. Although some valuable enzymes have been identified for the demethylation of lignin and its derivatives, there is still a large demand for developing new O -demethylases for the demethylation of lignin and its derivatives either from natural sources or laboratory evolution [ 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ]. Previously, an artificial P450BM3 peroxygenase system, which has a dual-functional small molecule (DFSM) installed in the active pocket, was developed to perform an H 2 O 2 -dependent monooxygenation of non-native substrates [ 18 , 19 , 20 ].…”

Section: Introductionmentioning

confidence: 99%

Protein Engineering of an Artificial P450BM3 Peroxygenase System Enables Highly Selective O-Demethylation of Lignin Monomers

Miao

et al. 2022

Molecules

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The O-demethylation of lignin monomers, which has drawn substantial attention recently, is critical for the formation of phenols from aromatic ethers. The P450BM3 peroxygenase system was recently found to enable the O-demethylation of different aromatic ethers with the assistance of dual-functional small molecules (DFSM), but these prepared mutants only have either moderate O-demethylation activity or moderate selectivity, which hinders their further application. In this study, we improve the system by introducing different amino acids into the active site of P450BM3, and these amino acids with different side chains impacted the catalytic ability of enzymes due to their differences in size, polarity, and hydrophobicity. Among the prepared mutants, the combination of V78A/F87A/T268I/A264G and Im-C6-Phe efficiently catalyzed the O-demethylation of guaiacol (TON = 839) with 100% selectivity. Compared with NADPH-dependent systems, we offer an economical and practical bioconversion avenue.

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“…The analysis of lignin linkages supported the above results as the major linkages of lignin were further degraded by ligninolytic P. putida during fermentation ( Figure 4A ; Supplementary Figure 2 ). Interestingly, the S/G ratio was increased after fermentation because ligninolytic P. putida preferred to consume G and H units ( Notonier et al, 2021 ). After fermentation, the carboxyl group and p -hydroxyl phenyl OH group were decreased ( Figure 4B ), indicating the effective catabolism of more hydrophilic molecules.…”

Section: Resultsmentioning

confidence: 99%

Microbial Valorization of Lignin to Bioplastic by Genome-Reduced Pseudomonas putida

Zong

et al. 2022

Front. Microbiol.

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As the most abundant natural aromatic resource, lignin valorization will contribute to a feasible biobased economy. Recently, biological lignin valorization has been advocated since ligninolytic microbes possess proficient funneling pathways of lignin to valuable products. In the present study, the potential to convert an actual lignin stream into polyhydroxyalkanoates (PHAs) had been evaluated using ligninolytic genome-reduced Pseudomonas putida. The results showed that the genome-reduced P. putida can grow well on an actual lignin stream to successfully yield a high PHA content and titer. The designed fermentation strategy almost eliminated the substrate effects of lignin on PHA accumulation. Employing a fed-batch strategy produced the comparable PHA contents and titers of 0.35 g/g dried cells and 1.4 g/L, respectively. The molecular mechanism analysis unveiled that P. putida consumed more small and hydrophilic lignin molecules to stimulate cell growth and PHA accumulation. Overall, the genome-reduced P. putida exhibited a superior capacity of lignin bioconversion and promote PHA accumulation, providing a promising route for sustainable lignin valorization.

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Metabolism of syringyl lignin-derived compounds in Pseudomonas putida enables convergent production of 2-pyrone-4,6-dicarboxylic acid

Cited by 60 publications

References 81 publications

Muconic acid production from glucose and xylose in Pseudomonas putida via evolution and metabolic engineering

Muconic acid production from glucose and xylose in Pseudomonas putida via evolution and metabolic engineering

Protein Engineering of an Artificial P450BM3 Peroxygenase System Enables Highly Selective O-Demethylation of Lignin Monomers

Microbial Valorization of Lignin to Bioplastic by Genome-Reduced Pseudomonas putida

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