Engineering topics
Publications and source records attributed to Werner, Allison Z..
Pseudomonas putida produces two distinct populations of membrane vesicles during growth on lignin - Proteomics, Targeted Quantitation, and Lipidomics
Growth and metabolite quantitation for bacterial cultivations
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ProteomeXchange version of Pseudomonas putida produces two distinct populations of membrane vesicles during growth on lignin - Proteomics, Targeted Quantitation, and Lipidomics
raw data for replicates for https://pubs.acs.org/doi/full/10.1021/acssynbio.5c00171
Table S1, putative gene knockout targets in P. putida KT2440 to enhance vesiculation; Table S2, protein sequence identity of OmpA from E. coli K12 to P. putida KT2440 genes; Table S3, strains utilized in this study and corresponding construction details; Table S4, oligonucleotides utilized in this study; Table S5, plasmids utilized in this study; Table S6, sequences for mNeonGreen, tags, and codon-optimized genes; Figure S1, particle count per gCDW for KT2440 and knockout strains corresponding to data presented in Figure 1B; Figure S2, OD600 measurements of extracted MVs from KT2440 and knockout strains; Figure S3, particle count per gCDW for WT, ΔPP_4669, and ΔPP_1502; Figure S4, particle count per gCDW for KT2440 and knockout strains corresponding to data presented in Figure 3C; Figure S5, sizes of MVs corresponding to particle counts in Figure S4; Figure S6, particle count per gCDW for KT2440 grown on 20 mM glucose alone or 20 mM glucose plus 12.5 mM p-coumarate and 12.5 mM ferulate; Figure S7 and Figure S8, principal component analysis of the cellular fractions; Figure S9, heatmap of outer membrane proteins with differential abundance; and Figure S10, mNeonGreen (mNG) fluorescence signal for the cellular fraction and the extracellular fraction
Cellular and membrane vesicle proteomes from Pseudomonas putida KT2440 and engineered derivative strains. Proteomic quantification by diDO-IPTL (Waldbauer et al. 2017 Analytical Chemistry).
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Bioconversion of a heterogeneous mixture of lignin-related aromatic compounds (LRCs) to a single product via microbial biocatalysts is a promising approach to valorize lignin. Here, Pseudomonas putida KT2440 was engineered to convert mixed p-coumaroyl– and coniferyl-type LRCs to β-ketoadipic acid, a precursor for performance-advantaged polymers. Expression of enzymes mediating aromatic O-demethylation, hydroxylation, and ring-opening steps was tuned, and a global regulator was deleted. β-ketoadipate titers of 44.5 and 25 grams per liter and productivities of 1.15 and 0.66 grams per liter per hour were achieved from model LRCs and corn stover-derived LRCs, respectively, the latter representing an overall yield of 0.10 grams per gram corn stover-derived lignin. Technoeconomic analysis of the bioprocess and downstream processing predicted a β-ketoadipate minimum selling price of $\$2.01$ per kilogram, which is cost competitive with fossil carbon-derived adipic acid ($\$1.10$ to 1.80 per kilogram). Overall, this work achieved bioproduction metrics with economic relevance for conversion of lignin-derived streams into a performance-advantaged bioproduct.
In 2014, Linger et al. presented a tandem process for lignin valorization by integrating chemical and biological catalysis. Chemical pretreatment of corn stover generated mixed lignocellulose-derived monomers that were converted to a single product, polyhydroxyalkanoates, by Pseudomonas putida. Tandem processes have since been developed for diverse feedstocks to support the bioeconomy.
We report Pseudomonas putida KT2440 is a well-studied bacterium for the conversion of lignin-derived aromatic compounds to bioproducts. The development of advanced genetic tools in P. putida has reduced the turnaround time for hypothesis testing and enabled the construction of strains capable of producing various products of interest. Here, we evaluate an inducible CRISPR-interference (CRISPRi) toolset on fluorescent, essential, and metabolic targets. Nuclease-deficient Cas9 (dCas9) expressed with the arabinose (8K)-inducible promoter was shown to be tightly regulated across various media conditions and when targeting essential genes. In addition to bulk growth data, single cell time lapse microscopy was conducted, which revealed intrinsic heterogeneity in knockdown rate within an isoclonal population. The dynamics of knockdown were studied across genomic targets in exponentially-growing cells, revealing a universal 1.75 ± 0.38 hour quiescent phase after induction where 1.5 ± 0.35 doublings occur before a phenotypic response is observed. To demonstrate application of this CRISPRi toolset, β-ketoadipate, a monomer for performance-advantaged nylon, was produced at a 4.39 ± 0.5 g/L and yield of 0.76 ± 0.10 mol/mol from p-coumarate, a hydroxycinnamic acid that can be derived from grasses. These cultivation metrics were achieved by using the higher strength IPTG (1K)-inducible promoter to knockdown the pcaIJ operon in the βKA pathway during early exponential phase. This allowed the majority of the carbon to be shunted into the desired product while eliminating the need for a supplemental carbon and energy source to support growth and maintenance.
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.
Lignin biosynthesis typically results in a polymer with several inter-monomer bond linkages, and the heterogeneity of linkages presents a challenge for depolymerization processes. While several enzyme classes have been shown to cleave common dimer linkages in lignin, the pathway of bacterial β-1 spirodienone linkage cleavage has not been elucidated. In this study, we identified a pathway for cleavage of 1,2-diguaiacylpropane-1,3-diol (DGPD), a β-1 linked biaryl representative of a ring-opened spirodienone linkage, in Novosphingobium aromaticivorans DSM12444. In vitro assays using cell lysates demonstrated that RS14230 (LsdE) converts DGPD to a lignostilbene intermediate, which the carotenoid oxygenase, LsdA, then converts to vanillin. A Pseudomonas putida KT2440 strain engineered with lsdEA expression catabolizes erythro-DGPD, but not threo-DGPD. We further engineered P. putida to convert DGPD to a product, cis,cis-muconic acid. Overall, this work demonstrates the potential to identify new enzymatic reactions in N. aromaticivorans and expands the biological funnel of P. putida for microbial lignin valorization.
Pseudomonas putida KT2440 (hereafter KT2440) is a well-studied platform bacterium for the production of industrially valuable chemicals from heterogeneous mixtures of aromatic compounds obtained from lignin depolymerization. KT2440 can grow on lignin-related monomers, such as ferulate (FA), 4-coumarate (4CA), vanillate (VA), 4-hydroxybenzoate (4HBA), and protocatechuate (PCA). Genes associated with their catabolism are known, but knowledge about the uptake systems remains limited. In this work, we studied the KT2440 transporters of lignin-related monomers and their substrate selectivity. Based on the inhibition by protonophores, we focused on five genes encoding aromatic acid/H+ symporter family transporters categorized into major facilitator superfamily that uses the proton motive force. Furthermore, the mutants of PP_1376 (pcaK) and PP_3349 (hcnK) exhibited significantly reduced growth on PCA/4HBA and FA/4CA, respectively, while no change was observed on VA for any of the five gene mutants. At pH 9.0, the conversion of these compounds by hcnK mutant (FA/4CA) and vanK mutant (VA) was dramatically reduced, revealing that these transporters are crucial for the uptake of the anionic substrates at high pH. Uptake assays using 14 C-labeled substrates in Escherichia coli and biosensor-based assays confirmed that PcaK, HcnK, and VanK have ability to take up PCA, FA/4CA, and VA/PCA, respectively. Additionally, analyses of the predicted protein structures suggest that the size and hydropathic properties of the substrate-binding sites of these transporters determine their substrate preferences. Overall, this study reveals that at physiological pH, PcaK and HcnK have a major role in the uptake of PCA/4HBA and FA/4CA, respectively, and VanK is a VA/PCA transporter. This information can contribute to the engineering of strains for the efficient conversion of lignin-related monomers to value-added chemicals.
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