Engineering topics
Publications and source records attributed to Kuatsjah, Eugene.
ALE dataset for Pseudomonas putida from https://www.osti.gov/biblio/2404295
White-rot fungi (WRF) are the most efficient lignin-degrading organisms in nature. However, their capacity to use lignin-related aromatic compounds, such as 4-hydroxybenzoate, as carbon sources has only been described recently. Previously, the hydroxyquinol pathway was proposed for the bioconversion of these compounds in fungi, but gene- and structure-function relationships of the full enzymatic pathway remain uncharacterized in any single fungal species. Here, we characterize seven enzymes from two WRF, Trametes versicolor and Gelatoporia subvermispora, which constitute a four-enzyme cascade from 4-hydroxybenzoate to β-ketoadipate via the hydroxyquinol pathway. Furthermore, we solve the crystal structure of four of these enzymes and identify mechanistic differences with the closest bacterial and fungal structural homologs. Overall, this research expands our understanding of aromatic catabolism by WRF and establishes an alternative strategy for the conversion of lignin-related compounds to the valuable molecule β-ketoadipate, contributing to the development of biological processes for lignin valorization.
In this study, biological conversion of lignin from biomass offers a promising strategy for sustainable production of fuels and chemicals. However, aromatic compounds derived from lignin commonly contain methoxy groups, and O-demethylation of these substrates is often a rate-limiting reaction that influences catabolic efficiency. Several enzyme families catalyze aromatic O-demethylation, but they are rarely compared in vivo to determine an optimal biocatalytic strategy. Here, two pathways for aromatic O-demethylation were compared in Pseudomonas putida KT2440. The native Rieske non-heme iron monooxygenase (VanAB) and, separately, a heterologous tetrahydrofolate-dependent demethylase (LigM) were constitutively expressed in P. putida, and the strains were optimized via adaptive laboratory evolution (ALE) with vanillate as a model substrate. All evolved strains displayed improved growth phenotypes, with the evolved strains harboring the native VanAB pathway exhibiting growth rates ~1.8x faster than those harboring the heterologous LigM pathway. Enzyme kinetics and transcriptomics studies investigated the contribution of selected mutations toward enhanced utilization of vanillate. The VanAB-overexpressing strains contained the most impactful mutations, including those in VanB, the reductase for vanillate O-demethylase, PP_3494, a global regulator of vanillate catabolism, and fghA, involved in formaldehyde detoxification. These three mutations were combined into a single strain, which exhibited approximately 5x faster vanillate consumption than the wild-type strain in the first 8 h of cultivation. Overall, this study illuminates the details of vanillate catabolism in the context of two distinct enzymatic mechanisms, yielding a platform strain for efficient O-demethylation of lignin-related aromatic compounds to value-added products.
Sphingobium sp. strain SYK-6 is an efficient aromatic catabolic bacterium that can consume all four stereoisomers of 1,2-diguaiacylpropane-1,3-diol (DGPD), which is a ring-opened β-1-type dimer. Recently, LdpA-mediated catabolism of erythro-DGPD was reported in SYK-6, but the catabolic pathway for threo-DGPD was as yet unknown. Here, in this study, we elucidated the catabolism of threo-DGPD, which proceeds through conversion to erythro-DGPD. When threo-DGPD was incubated with SYK-6, the Cα hydroxy groups of threo-DGPD (DGPD I and II) were initially oxidized to produce the Cα carbonyl form (DGPD-keto I and II). This initial oxidation step is catalyzed by Cα-dehydrogenases, which belong to the short-chain dehydrogenase/reductase (SDR) family and are involved in the catabolism of β-O-4-type dimers. Analysis of seven candidate genes revealed that NAD + -dependent LigD and LigL are mainly involved in the conversion of DGPD I and II, respectively. Next, we found that DGPD-keto I and II were reduced to erythro-DGPD (DGPD III and IV) in the presence of NADPH. Genes involved in this reduction were sought from Cα-dehydrogenase and ldpA-neighboring SDR genes. The gene products of SLG_12690 (ldpC) and SLG_12640 (ldpB) catalyzed the NADPH-dependent conversion of DGPD-keto I to DGPD III and DGPD-keto II to DGPD IV, respectively. Mutational analysis further indicated that ldpC and ldpB are predominantly involved in the reduction of DGPD-keto. Together, these results demonstrate that SYK-6 harbors a comprehensive catabolic enzyme system to utilize all four β-1-type stereoisomers through successive oxidation and reduction reactions of the Cα hydroxy group of threo-DGPD with a net stereoinversion using multiple dehydrogenases.
Alkaline pretreatment can depolymerize lignin into a diverse mixture of monomers and smaller compounds, enabling bioconversion of these compounds to value-added chemicals in engineered bacterial hosts such as Pseudomonas putida KT2440. Acetate and salts comprise approximately 8% and 32% of the lignin stream, respectively. High concentrations of these components can cause cell death in P. putida, so lignin-derived streams must be fed in lower amounts to decrease stress. Investigation of the mechanisms of bacterial tolerance to chemicals in lignin-rich substrates is therefore required to overcome this obstacle in lignin bioconversion. Previous work has shown that trehalose, an endogenously produced disaccharide, is involved in tolerance to heat, cold, and osmotic stress, as well as implicated in acid stress in other bacteria. As such, trehalose biosynthesis presents a useful target to increase acetate and osmotic tolerance in P. putida. This work investigates the native TreSA, TreSB, and TreY/TreZ trehalose biosynthesis pathways in this bacterial strain as well as the OtsAB pathway of Sphingobium sp. SYK-6. Overexpression of the OtsAB pathway in P. putida increased tolerance to acetate relative to wild-type. Additionally, functional knockouts of treSA, treSB, treY, and treZ in P. putida showed that interruption of any trehalose biosynthesis pathway, and, in the case of TreY/TreZ, interruption of the pathway at any point, significantly hindered growth in acetate as well as on glucose and LB broth. Each trehalose biosynthesis pathway was then engineered for overexpression in P. putida to determine whether overexpression of these genes conferred additional tolerance to acetate. The TreSA, TreSB, OtsA, and OtsB enzymes were also expressed and purified to determine the reaction rates and kinetics of these pathways. The hypothetical glycoside hydrolase family 15 protein, encoded in the gene located in between otsB and otsA in the Sphingobium operon, was purified as well to investigate its potential involvement in the OtsAB pathway. The findings of this work illuminated the mechanisms of trehalose biosynthesis in P. putida and identified genetic targets to improve strain tolerance to common components of alkaline-pretreated lignin streams. These findings could ultimately improve yields of desired products from lignin in engineered strains of P. putida.
Deciphering the mechanisms of bacterial fatty acid biosynthesis is crucial for both the engineering of bacterial hosts to produce fatty acid-derived molecules and the development of new antibiotics. However, gaps in our understanding of the initiation of fatty acid biosynthesis remain. Here, we demonstrate that the industrially relevant microbe Pseudomonas putida KT2440 contains three distinct pathways to initiate fatty acid biosynthesis. The first two routes employ conventional β-ketoacyl-ACP synthase III enzymes, FabH1 and FabH2, that accept short- and medium-chain-length acyl-CoAs, respectively. The third route utilizes a malonyl-ACP decarboxylase enzyme, MadB. A combination of exhaustive in vivo alanine-scanning mutagenesis, in vitro biochemical characterization, X-ray crystallography, and computational modeling elucidate the presumptive mechanism of malonyl-ACP decarboxylation via MadB. Given that functional homologs of MadB are widespread throughout domain Bacteria, this ubiquitous alternative fatty acid initiation pathway provides new opportunities to target a range of biotechnology and biomedical applications.
Lignin valorization is being intensely pursued via tandem catalytic depolymerization and biological funneling to produce single products. In many lignin depolymerization processes, aromatic dimers and oligomers linked by carbon–carbon bonds remain intact, necessitating the development of enzymes capable of cleaving these compounds to monomers. Recently, the catabolism of erythro -1,2-diguaiacylpropane-1,3-diol ( erythro -DGPD), a ring-opened lignin-derived β-1 dimer, was reported in Novosphingobium aromaticivorans . The first enzyme in this pathway, LdpA (formerly LsdE), is a member of the nuclear transport factor 2 (NTF-2)-like structural superfamily that converts erythro -DGPD to lignostilbene through a heretofore unknown mechanism. In this study, we performed biochemical, structural, and mechanistic characterization of the N. aromaticivorans LdpA and another homolog identified in Sphingobium sp. SYK-6, for which activity was confirmed in vivo. For both enzymes, we first demonstrated that formaldehyde is the C 1 reaction product, and we further demonstrated that both enantiomers of erythro -DGPD were transformed simultaneously, suggesting that LdpA, while diastereomerically specific, lacks enantioselectivity. We also show that LdpA is subject to a severe competitive product inhibition by lignostilbene. Three-dimensional structures of LdpA were determined using X-ray crystallography, including substrate-bound complexes, revealing several residues that were shown to be catalytically essential. We used density functional theory to validate a proposed mechanism that proceeds via dehydroxylation and formation of a quinone methide intermediate that serves as an electron sink for the ensuing deformylation. Overall, this study expands the range of chemistry catalyzed by the NTF-2-like protein family to a prevalent lignin dimer through a cofactorless deformylation reaction.
Aryl-O-demethylation is a common rate-limiting step in the catabolism of lignin-related compounds, including guaiacol. In this work, we used randomly barcoded transposon insertion sequencing (RB-TnSeq) in the bacterium Novosphingobium aromaticivorans to identify a Rieske-type guaiacol O-demethylase, GdmA. Similarity searches identified GdmA homologs in other bacteria, along with candidate reductase partners, denoted GdmB. GdmAB combinations were biochemically characterized for activity with several lignin-related substrates. Structural and sequence comparisons of vanillate- and guaiacol-specific O-demethylase active sites revealed conserved hallmarks of substrate specificity. GdmAB combinations were also evaluated in Pseudomonas putida KT2440, which does not natively utilize guaiacol. GdmAB from Cupriavidus necator N-1 demonstrated the highest rate of guaiacol turnover in vitro and in engineered P. putida strains and notably higher catalytic efficiency than a cytochrome P450 system (GcoAB) and the vanillate Rieske-type O-demethylase from P. putida (VanAB). The GdmAB O-demethylases described here expand the suite of options for microbial conversion of a model lignin-derived substrate.
The application of microbes to valorize aromatic compounds derived from the abundant plant biopolymer lignin is a rapidly developing area of research that may ultimately enable viable conversion of this recalcitrant and heterogeneous resource to valuable bio-based chemical products. Starting from the three canonical lignin building blocks, which differ in the extent of aromatic ring methoxylation, several common classes of enzymatic reaction occur in the upper pathways of aromatic catabolism to prepare aromatic compounds for assimilation into central carbon metabolism, including aromatic O-demethylation, hydroxylation and decarboxylation. These critical enzymatic steps can often be rate-limiting for efficient biological funnelling of aromatic compounds. Here we review the known enzymatic mechanisms for these reactions that are relevant for aerobic aromatic catabolism of lignin-related monomers, highlighting opportunities at the intersection of biochemistry, enzyme engineering and metabolic engineering for applications in the expanding field of microbial lignin valorization.
The transformation of 4-hydroxybenzoate (4-HBA) to protocatechuate (PCA) is catalyzed by flavoprotein oxygenases known as para-hydroxybenzoate-3-hydroxylases (PHBHs). In Pseudomonas putida KT2440 (P. putida) strains engineered to convert lignin-related aromatic compounds to muconic acid (MA), PHBH activity is rate-limiting, as indicated by the accumulation of 4-HBA, which ultimately limits MA productivity. Here, we hypothesized that replacement of PobA, the native P. putida PHBH, with PraI, a PHBH from Paenibacillus sp. JJ-1b with a broader nicotinamide cofactor preference, could alleviate this bottleneck. Biochemical assays confirmed the strict preference of NADPH for PobA, while PraI can utilize either NADH or NADPH. Kinetic assays demonstrated that both PobA and PraI can utilize NADPH with comparable catalytic efficiency and that PraI also efficiently utilizes NADH at roughly half the catalytic efficiency. The X-ray crystal structure of PraI was solved and revealed absolute conservation of the active site architecture to other PHBH structures despite their differing cofactor preferences. To understand the effect in vivo, we compared three P. putida strains engineered to produce MA from p-coumarate (pCA), showing that expression of praI leads to lower 4-HBA accumulation and decreased NADP+/NADPH ratios relative to strains harboring pobA, indicative of a relieved 4-HBA bottleneck due to increased NADPH availability. In bioreactor cultivations, a strain exclusively expressing praI achieved a titer of 40 g/L MA at 100% molar yield and a productivity of 0.5 g/L/h. Altogether, this study demonstrates the benefit of sampling readily available natural enzyme diversity for debottlenecking metabolic flux in an engineered strain for microbial conversion of lignin-derived compounds to value-added products.
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.
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