Engineering Pseudomonas putida for improved utilization of syringyl aromatics

Abstract

Lignin is a largely untapped source for the bioproduction of value-added chemicals.Pseudomonas putida KT2440 has emerged as a strong candidate for bioprocessing of lignin feedstocks due to its resistance to several industrial solvents, broad metabolic capabilities, and genetic amenability. Here we demonstrate the engineering of P. putida for the ability to metabolize syringic acid, one of the major products that comes from the breakdown of the syringyl component of lignin. The rational design was first applied for the construction of strain Sy-1 by overexpressing a native vanillate demethylase. Subsequent adaptive laboratory evolution (ALE) led to the generation of mutations that achieved robust growth on syringic acid as a sole carbon source. The best mutant showed a 30% increase in the growth rate over the original engineered strain. Genomic sequencing revealed multiple mutations repeated in separate evolved replicates. Reverse engineering of mutations identified in agmR, gbdR, fleQ, and the intergenic region of gstB and yadG into the parental strain recaptured the improved growth of the evolved strains to varied extent. These findings thus reveal the ability of P. putida to utilize lignin more fully as a feedstock and make it a more economically viable chassis for chemical production.adaptive lab evolution (ALE), Pseudomonas putida KT2440, syringyl lignin-derived aromatics | INTRODUCTIONLignin is a complex organic polymer that makes up 20%-35% of plant cell walls. It is polymerized from three monolignols: p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, which are categorized as the corresponding hydroxy (H), guaiacyl (G), and syringyl (S) lignin components (Figure 1a;Feofilova & Mysyakina, 2016). Depending on the type of plants, the ratio of H, G, and S subunits in lignin varies, where the grass lignin consists of all subunits, the softwood lignin often has low S component

Application of functional genomics for domestication of novel non-model microbes

“…Genome-scale studies are an essential step in developing a host for industrial uses as they enable the identification of genetic factors associated with complex traits for strain optimization. There are a variety of methods that have been employed including adaptive laboratory evolution (Dragosits & Mattanovich, 2013 ; Lim et al., 2021 ; Mueller et al., 2022 ; Wang et al., 2023 ; Zhan et al., 2023 ), transposon-mediated gene disruption (Tn-Seq) (Bleem et al., 2023 ; Borchert et al., 2023 ; Price et al., 2018 ; Shields & Jensen, 2019 ; Wetmore et al., 2015 ), CRISPR-based modifications including interference (CRISPRi), CRISPR activation (CRISPRa), and base editing (Adli, 2018 ; Lee et al., 2019 ; Li et al., 2016 ; Liu et al., 2022 ; Saber Sichani et al., 2023 ; Silvis et al., 2021 ; Volke et al., 2023 ), and random mutagenesis (Freed et al., 2018 ; Riley & Guss, 2021 ). Herein we will focus on CRISPRi, a recently emerged robust functional genomic screening and selection method (Trivedi et al., 2023 ).…”

Biotransformation of lignin into 4-vinylphenol derivatives toward lignin valorization

“…58,59 The ability of the engineered P. putida KT2440 to utilize syringic acid had been demonstrated through chromosomal overexpression of VanAB and adaptive laboratory evolution. 60,61 Other aromatic derivatives, such as phenol and guaiacol, can enter the central metabolism through the generation of the platform compound catechol. Catechol is assimilated through ortho-cleavage and meta-cleavage pathway to support the cell growth (Fig.…”

Identification of a Phylogenetically Divergent Vanillate O-Demethylase from Rhodococcus ruber R1 Supporting Growth on Meta-Methoxylated Aromatic Acids

“…This enzyme comprises a Rieske domain-containing oxygenase subunit encoded by vanA , and a reductase subunit that encompasses FMN, NADPH, and [2Fe-2S] cluster binding domains encoded by vanB , and providing electron equivalents to enable the enzymatic conversion [ 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 ]. It was shown that the VanOD encoded by vanAB genes from different bacteria were able to catalyze two types of reaction: methoxy group demethylation at the meta position of VA and analogs such as 3-methoxybenzoate (3-MB), veratrate, or syringate, with concomitant release of formaldehyde; or methyl group hydroxylation in m -toluate, 4-hydroxy-3-methylbenzoate, or 4-hydroxy-3,5-dimethylbenzoate; although, with the exception of veratrate and syringate, none of the analogs have been reported to support cell growth employing this enzyme [ 16 , 18 , 19 , 20 , 21 ]. Alternatively, it has been reported that distinct tetrahydrofolate (H 4 folate)-dependent O-demethylases, analogous to aromatic O-demethylases from anaerobic bacteria, are responsible for VA O-demethylation in Sphingobium sp.…”