Allison Z. Werner

Bio: Allison Z. Werner is an academic researcher from National Renewable Energy Laboratory. The author has contributed to research in topics: Pseudomonas putida & Medicine. The author has an hindex of 5, co-authored 10 publications receiving 122 citations. Previous affiliations of Allison Z. Werner include Colorado State University & Oak Ridge National Laboratory.

Outer membrane vesicles catabolize lignin-derived aromatic compounds in Pseudomonas putida KT2440

Abstract: Lignin is an abundant and recalcitrant component of plant cell walls. While lignin degradation in nature is typically attributed to fungi, growing evidence suggests that bacteria also catabolize this complex biopolymer. However, the spatiotemporal mechanisms for lignin catabolism remain unclear. Improved understanding of this biological process would aid in our collective knowledge of both carbon cycling and microbial strategies to valorize lignin to value-added compounds. Here, we examine lignin modifications and the exoproteome of three aromatic–catabolic bacteria: Pseudomonas putida KT2440, Rhodoccocus jostii RHA1, and Amycolatopsis sp. ATCC 39116. P. putida cultivation in lignin-rich media is characterized by an abundant exoproteome that is dynamically and selectively packaged into outer membrane vesicles (OMVs). Interestingly, many enzymes known to exhibit activity toward lignin-derived aromatic compounds are enriched in OMVs from early to late stationary phase, corresponding to the shift from bioavailable carbon to oligomeric lignin as a carbon source. In vivo and in vitro experiments demonstrate that enzymes contained in the OMVs are active and catabolize aromatic compounds. Taken together, this work supports OMV-mediated catabolism of lignin-derived aromatic compounds as an extracellular strategy for nutrient acquisition by soil bacteria and suggests that OMVs could potentially be useful tools for synthetic biology and biotechnological applications.

Mixed plastics waste valorization through tandem chemical oxidation and biological funneling

Abstract: Mixed plastics waste represents an abundant and largely untapped feedstock for the production of valuable products. The chemical diversity and complexity of these materials, however, present major barriers to realizing this opportunity. In this work, we show that metal-catalyzed autoxidation depolymerizes comingled polymers into a mixture of oxygenated small molecules that are advantaged substrates for biological conversion. We engineer a robust soil bacterium, Pseudomonas putida, to funnel these oxygenated compounds into a single exemplary chemical product, either β-ketoadipate or polyhydroxyalkanoates. This hybrid process establishes a strategy for the selective conversion of mixed plastics waste into useful chemical products. Description Funneling mixed waste with microbes Current plastic recycling methods require sorting by chemical composition, a method that is expensive and results in products that are of lower quality and value than the starting plastic. If plastic waste could instead be converted to valuable chemical intermediates, then economical use of mixed waste as a feedstock might be feasible. Sullivan et al. developed a two-stage oxidation and biological funneling approach that can break down and reform mixtures of common consumer plastics (see the Perspective by Yan). The end products can be adjusted by metabolic engineering of the microbes in the second step, which should enable tailored conversion into various platform or specialty chemicals. —MAF Autoxidation and biological funneling converts mixed plastics to a single product.

NOREVA: enhanced normalization and evaluation of time-course and multi-class metabolomic data.

Abstract: Biological processes (like microbial growth & physiological response) are usually dynamic and require the monitoring of metabolic variation at different time-points. Moreover, there is clear shift from case-control (N=2) study to multi-class (N>2) problem in current metabolomics, which is crucial for revealing the mechanisms underlying certain physiological process, disease metastasis, etc. These time-course and multi-class metabolomics have attracted great attention, and data normalization is essential for removing unwanted biological/experimental variations in these studies. However, no tool (including NOREVA 1.0 focusing only on case-control studies) is available for effectively assessing the performance of normalization method on time-course/multi-class metabolomic data. Thus, NOREVA was updated to version 2.0 by (i) realizing normalization and evaluation of both time-course and multi-class metabolomic data, (ii) integrating 144 normalization methods of a recently proposed combination strategy and (iii) identifying the well-performing methods by comprehensively assessing the largest set of normalizations (168 in total, significantly larger than those 24 in NOREVA 1.0). The significance of this update was extensively validated by case studies on benchmark datasets. All in all, NOREVA 2.0 is distinguished for its capability in identifying well-performing normalization method(s) for time-course and multi-class metabolomics, which makes it an indispensable complement to other available tools. NOREVA can be accessed at https://idrblab.org/noreva/.

Industrial biotechnology of Pseudomonas putida: advances and prospects

Abstract: Pseudomonas putida is a Gram-negative, rod-shaped bacterium that can be encountered in diverse ecological habitats. This ubiquity is traced to its remarkably versatile metabolism, adapted to withstand physicochemical stress, and the capacity to thrive in harsh environments. Owing to these characteristics, there is a growing interest in this microbe for industrial use, and the corresponding research has made rapid progress in recent years. Hereby, strong drivers are the exploitation of cheap renewable feedstocks and waste streams to produce value-added chemicals and the steady progress in genetic strain engineering and systems biology understanding of this bacterium. Here, we summarize the recent advances and prospects in genetic engineering, systems and synthetic biology, and applications of P. putida as a cell factory. KEY POINTS: • Pseudomonas putida advances to a global industrial cell factory. • Novel tools enable system-wide understanding and streamlined genomic engineering. • Applications of P. putida range from bioeconomy chemicals to biosynthetic drugs.

Passive membrane transport of lignin-related compounds.

Abstract: Lignin is an abundant aromatic polymer found in plant secondary cell walls. In recent years, lignin has attracted renewed interest as a feedstock for bio-based chemicals via catalytic and biological approaches and has emerged as a target for genetic engineering to improve lignocellulose digestibility by altering its composition. In lignin biosynthesis and microbial conversion, small phenolic lignin precursors or degradation products cross membrane bilayers through an unidentified translocation mechanism prior to incorporation into lignin polymers (synthesis) or catabolism (bioconversion), with both passive and transporter-assisted mechanisms postulated. To test the passive permeation potential of these phenolics, we performed molecular dynamics simulations for 69 monomeric and dimeric lignin-related phenolics with 3 model membranes to determine the membrane partitioning and permeability coefficients for each compound. The results support an accessible passive permeation mechanism for most compounds, including monolignols, dimeric phenolics, and the flavonoid, tricin. Computed lignin partition coefficients are consistent with concentration enrichment near lipid carbonyl groups, and permeability coefficients are sufficient to keep pace with cellular metabolism. Interactions between methoxy and hydroxy groups are found to reduce membrane partitioning and improve permeability. Only carboxylate-modified or glycosylated lignin phenolics are predicted to require transporters for membrane translocation. Overall, the results suggest that most lignin-related compounds can passively traverse plant and microbial membranes on timescales commensurate with required biological activities, with any potential transport regulation mechanism in lignin synthesis, catabolism, or bioconversion requiring compound functionalization.

Depolymerization and conversion of lignin to value-added bioproducts by microbial and enzymatic catalysis

Abstract: Lignin, the most abundant renewable aromatic compound in nature, is an excellent feedstock for value-added bioproducts manufacturing; while the intrinsic heterogeneity and recalcitrance of which hindered the efficient lignin biorefinery and utilization. Compared with chemical processing, bioprocessing with microbial and enzymatic catalysis is a clean and efficient method for lignin depolymerization and conversion. Generally, lignin bioprocessing involves lignin decomposition to lignin-based aromatics via extracellular microbial enzymes and further converted to value-added bioproducts through microbial metabolism. In the review, the most recent advances in degradation and conversion of lignin to value-added bioproducts catalyzed by microbes and enzymes were summarized. The lignin-degrading microorganisms of white-rot fungi, brown-rot fungi, soft-rot fungi, and bacteria under aerobic and anaerobic conditions were comparatively analyzed. The catalytic metabolism of the microbial lignin-degrading enzymes of laccase, lignin peroxidase, manganese peroxidase, biphenyl bond cleavage enzyme, versatile peroxidase, and β-etherize was discussed. The microbial metabolic process of H-lignin, G-lignin, S-lignin based derivatives, protocatechuic acid, and catechol was reviewed. Lignin was depolymerized to lignin-derived aromatic compounds by the secreted enzymes of fungi and bacteria, and the aromatics were converted to value-added compounds through microbial catalysis and metabolic engineering. The review also proposes new insights for future work to overcome the recalcitrance of lignin and convert it to value-added bioproducts by microbial and enzymatic catalysis.

Mechanism-Based Design of Efficient PET Hydrolases

Abstract: Polyethylene terephthalate (PET) is the most widespread synthetic polyester, having been utilized in textile fibers and packaging materials for beverages and food, contributing considerably to the global solid waste stream and environmental plastic pollution. While enzymatic PET recycling and upcycling have recently emerged as viable disposal methods for a circular plastic economy, only a handful of benchmark enzymes have been thoroughly described and subjected to protein engineering for improved properties over the last 16 years. By analyzing the specific material properties of PET and the reaction mechanisms in the context of interfacial biocatalysis, this Perspective identifies several limitations in current enzymatic PET degradation approaches. Unbalanced enzyme–substrate interactions, limited thermostability, and low catalytic efficiency at elevated reaction temperatures, and inhibition caused by oligomeric degradation intermediates still hamper industrial applications that require high catalytic efficiency. To overcome these limitations, successful protein engineering studies using innovative experimental and computational approaches have been published extensively in recent years in this thriving research field and are summarized and discussed in detail here. The acquired knowledge and experience will be applied in the near future to address plastic waste contributed by other mass-produced polymer types (e.g., polyamides and polyurethanes) that should also be properly disposed by biotechnological approaches.