Biology subjects

Ebert, B. E.

Publications and source records attributed to Ebert, B. E..

12 recordsLinked to original sources

bioRxiv2025

Optimizing cytochrome P450 activity for quillaic acid biosynthesis in Saccharomyces cerevisiae

QS-21, a saponin extract from the Chilean tree Quillaja saponaria, is gaining popularity as a potent vaccine adjuvant, but its production is constrained due to its low natural abundance and complex chemical structure of the triterpenoid saponins, which hinder large-scale production through plant extraction or chemical synthesis. Microbial biosynthesis presents a promising alternative, with Saccharomyces cerevisiae emerging as a desirable host for triterpenoid production. A key step toward microbial QS-21 synthesis is the efficient biosynthesis of its aglycone core, the triterpenoid quillaic acid. However, its efficient production remains limited by challenges of cytochrome P450 enzyme (CYP450s) activity, including cofactor availability and electron transfer efficiency. To address this limitation, we applied a multi-faceted metabolic engineering strategy to optimise CYP450 activity, including CYP450 expression and cytochrome P450 reductase (CPR) selection. Additionally, aligning CYP450 expression with the ethanol phase, enhanced the metabolic flux toward quillaic acid synthesis, leading to an 85-fold increase in titre. Together, these strategies led to a quillaic acid titre of 385 {+/-} 14 mg/L in flask fermentation. Fed-batch bioreactor fermentations increased quillaic acid titre to 471 {+/-} 20 mg/L and significantly increased the selectivity for QA from 32.6% to 65.1% of the total triterpenoids produced. These findings demonstrate the effectiveness of enhancing CYP450 activity through targeted strategies and reveal potential bottlenecks in CYP450 expression, providing valuable insights for future optimization of triterpenoid production in yeast.

synthetic biology↗

bioRxiv2025

Co-Substrate Free Valorisation of Lignin Monomers by Assimilation of C1 and C2 By-Products

Lignin is an underutilised global resource with significant potential for the production of chemicals that are currently derived from fossil resources. However, biotechnological lignin valorisation faces various challenges including its recalcitrance and the toxicity of aromatic intermediates, products, and by-products like formaldehyde. While biochemical production from lignin-derived monomers has been demonstrated by disrupting native lignin degradation pathways, this approach required co-feeding additional carbon sources such as glucose for growth. This dependence on additional carbon sources can create competition with the food industry and undermine the economic sustainability of the bioprocess. Here, we report growth of a protocatechuate production strain of Pseudomonas putida EM42 on the by-products from p-coumarate and ferulate valorisation, achieving carbon efficiencies of up to 78 %. Additional flux balance analysis identified C1 assimilation pathways, including two novel pathways, beneficial for the growth on the formaldehyde by-product from ferulate degradation leading to improved carbon utilisation. This study demonstrates how by-product utilisation from lignin conversion can eliminate the need for co-feeding additional carbon sources, thereby potentially improving the efficiency of lignin valorisation.

bioengineering↗

bioRxiv2025

Systematic engineering of synthetic serine cycles in Pseudomonas putida uncovers emergent topologies for methanol assimilation

The urgent need for a circular carbon economy has driven research into sustainable substrates, including one-carbon (C1) compounds. The non-pathogenic soil bacterium Pseudomonas putida is a promising host for exploring synthetic methylotrophy due to its versatile metabolism. In this work, we implemented synthetic serine cycle variants in P. putida for methanol assimilation combining modular engineering and growth-coupled selection, whereby methanol assimilation supported biosynthesis of the essential amino acid serine. The serine cycle forms acetyl-coenzyme A from C1 molecules without carbon loss but has bottlenecks that hinder engineering efforts. We adopted three synthetic variants (serine-threonine cycle, homoserine cycle, and modified serine cycle) that yield serine in a methanol-dependent fashion to overcome these challenges. By dividing these metabolic designs into functional modules, we systematically compared their performance for implementation in vivo. Additionally, we harnessed native pyrroloquinoline quinone-dependent dehydrogenases for engineering methylotrophy. Recursive rewiring of synthetic and native activities revealed novel metabolic topologies for methanol utilization, termed enhanced serine-threonine cycle, providing a blueprint for engineering C1 assimilation in non-model heterotrophic bacteria. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/638773v1_ufig1.gif" ALT="Figure 1"> View larger version (79K): org.highwire.dtl.DTLVardef@1bfccd0org.highwire.dtl.DTLVardef@11f77deorg.highwire.dtl.DTLVardef@f6a904org.highwire.dtl.DTLVardef@1d24a34_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Genomic and metabolic plasticity drive alternative scenarios for adapting Pseudomonas putida to non-native substrate D-xylose.

D-Xylose, a major constituent of plant biomass and second most abundant sugar on Earth, holds a considerable potential as a substrate for sustainable bio-production. Pseudomonas putida KT2440 is an attractive bacterial host for valorizing biogenic feedstocks but lacks a xylose utilization pathway. While several attempts to engineer P. putida for growth on xylose have been reported, a comprehensive understanding of xylose metabolism in this bacterium is lacking, hindering its further improvement and rational tailoring for specific biotechnological purposes. In this study, we elucidated the xylose metabolism in the genome-reduced P. putida strain, EM42, endowed with xylose isomerase pathway (xylAB) and transporter (xylE) from Escherichia coli and used the obtained knowledge in combination with adaptive laboratory evolution to accelerate the bacteriums growth on the pentose sugar. Carbon flux analyses, targeted gene knock-outs, and in vitro enzyme assays portrayed xylose assimilation in P. putida and confirmed a partially cyclic upper xylose metabolism. Deletion of the local transcriptional regulator gene hexR de-repressed genes of several key catabolic enzymes and reduced the lag phase on xylose. Guided by metabolic modeling, we augmented P. putida with additional heterologous pentose phosphate pathway genes and subjected rationally prepared strains to adaptive laboratory evolution (ALE) on xylose. The descendants showed accelerated growth and reduced growth lag. Genomic and proteomic analysis of engineered and evolved mutants revealed the importance of a large genomic re-arrangement, transaldolase overexpression, and balancing gene expression in the synthetic xylABE operon. Importantly, omics analyses found that similar growth characteristics of two superior mutants were achieved through distinct evolutionary paths. This work provides a unique insight into how cell metabolism adjusts to a non-native substrate; it highlights the remarkable genomic and metabolic plasticity of P. putida and demonstrates the power of combining knowledge-driven engineering with ALE in generating desirable microbial phenotypes. HighlightsO_LIElucidated xylose catabolism via exogenous isomerase pathway in P. putida EM42. C_LIO_LIDeletion of transcriptional regulator HexR improved growth on xylose. C_LIO_LIKnowledge-guided interventions and adaptive evolution accelerated growth. C_LIO_LIOmics analyses of selected mutants highlighted the genomic and metabolic plasticity of P. putida. C_LIO_LITwo mutants with superior characteristics emerged from distinct evolutionary paths. C_LI

bioengineering↗

bioRxiv2022

Triterpenoid production with a minimally engineered Saccharomyces cerevisiae chassis

Triterpenoids, one of the most diverse classes of natural products, have been used for centuries as active ingredients in essential oils and Chinese medicines and are of interest for many industrial applications ranging from low-calorie sweeteners to cosmetic ingredients and vaccine adjuvants. However, not only can the extraction from plant material be cumbersome due to low concentrations of the specific triterpenoid, but concerns are also increasing regarding the sustainability of wild plant harvest while meeting market demands. The alternative is to produce triterpenoids with engineered microbes. Here, we present a generally applicable strategy for triterpenoid production in the yeast Saccharomyces cerevisiae based on a modified oxidosqualene cyclase Erg7. The modification reduces the flux into the sterol pathway while increasing the precursor supply for triterpenoid production. The minimally engineered strain was exploited for the exemplary production of the lupane triterpenoids betulin, betulin aldehyde, and betulinic acid at a total titer above 6 g/L, the highest reported so far. To further highlight the chassis concept, squalene, oleanane- and dammarane-type triterpenoids were synthesized to titers at a similar gram scale. We propose the developed bakers yeast as a host for the thousands of triterpenoid synthesis pathways from plants, reducing the pressure on the natural resources.

bioengineering↗

bioRxiv2020

High titer methyl ketone production with tailored Pseudomonas taiwanensis VLB120

Methyl ketones present a group of highly reduced platform chemicals industrially produced from petroleum-derived hydrocarbons. They find applications in the fragrance, flavor, pharmacological, and agrochemical industries, and are further discussed as biodiesel blends. In recent years, intense research has been carried out to achieve sustainable production of these molecules by re-arranging the fatty acid metabolism of various microbes. One challenge in the development of a highly productive microbe is the high demand for reducing power. Here, we engineered Pseudomonas taiwanensis VLB120 for methyl ketone production as this microbe has been shown to sustain exceptionally high NAD(P)H regeneration rates. The implementation of published strategies resulted in 2.1 g Laq-1 methyl ketones in fed-batch fermentation. We further increased the production by eliminating competing reactions suggested by metabolic analyses. These efforts resulted in the production of 9.8 g Laq-1 methyl ketones (corresponding to 69.3 g Lorg-1 in the in situ extraction phase) at 53 % of the maximum theoretical yield. This represents a 4-fold improvement in product titer compared to the initial production strain and the highest titer of recombinantly produced methyl ketones reported to date. Accordingly, this study underlines the high potential of P. taiwanensis VLB120 to produce methyl ketones and emphasizes model-driven metabolic engineering to rationalize and accelerate strain optimization efforts.Competing Interest StatementJay D. Keasling has a financial interest in Amyris, Lygos, Demetrix, Napigen, Maple Bio, Apertor Labs, Ansa Biotechnologies, and Berkeley Brewing Sciences.Abbreviationsaqaqueous phaseCDWcell dry weightDOdissolved oxygenGCgas chromatographyGLCglucoseHPLChigh-performance liquid chromatographyMiMBlMinimization of Metabolites BalancesMKmethyl ketoneMSmass spectrometryOD600nmoptical density determined at 600 nmorgorganic phase(p)FBA(parsimonious) flux balance analysisView Full Text

synthetic biology↗

bioRxiv2020

Protein allocation and enzymatic constraints explain Escherichia coli wildtype and mutant phenotypes

Proteins have generally been recognized to constitute the key cellular component in shaping microbial phenotypes. Due to limited cellular resources and space, optimal allocation of proteins is crucial for microbes to facilitate maximum proliferation rates while allowing a flexible response to environmental changes. Regulatory patterns of protein allocation were utilized to account for the condition-dependent proteome in a genome-scale metabolic reconstruction of Escherichia coli by linearly linking mass concentrations of protein sectors and single metabolic enzymes to flux variables. The resulting protein allocation model (PAM) correctly approximates wildtype phenotypes and flux distributions for various substrates, even under data scarcity. Moreover, we showed the ability of the PAM to predict metabolic responses of single gene deletion mutants by additionally assuming growth-limiting, transcriptional restrictions. Thus, we promote the integration of protein allocation constraints into classical constraint-based models to foster their predictive capabilities and application for strain analysis and metabolic engineering purposes.

systems biology↗

bioRxiv2019

Pseudomonas mRNA 2.0: Boosting Gene Expression Through Enhanced mRNA Stability and Translational Efficiency

High gene expression of enzymes partaking in recombinant production pathways is a desirable trait among cell factories belonging to all different kingdoms of life. High enzyme abundance is generally aimed for by utilizing strong promoters, which ramp up gene transcription and mRNA levels. Increased protein abundance can alternatively be achieved by optimizing the expression on the post-transcriptional level. Here, we evaluated protein synthesis with a previously proposed optimized gene expression architecture, in which mRNA stability and translation initiation are modulated by genetic parts such as self-cleaving ribozymes and a bicistronic design, which have initially been described to support the standardization of gene expression. The optimized gene expression architecture was tested in Pseudomonas taiwanensis VLB120, a promising, novel microbial cell factory. The expression cassette was employed on a plasmid basis and after single genomic integration. We used three constitutive and two inducible promoters to drive the expression of two fluorescent reporter proteins and a short acetoin biosynthesis pathway. The performance was confronted with that of a traditional expression cassette harboring the same promoter and gene of interest but lacking the genetic parts for increased expression efficiency. The optimized expression cassette granted higher protein abundance independently of the expression basis or promoter used proving its value for applications requiring high protein abundance.

synthetic biology↗

bioRxiv2019

The metabolic response of Pseudomonas taiwanensis to NADH dehydrogenase deficiency

Obligate aerobic organisms rely on a functional electron transport chain for energy generation and NADH oxidation. Because of this essential requirement, the genes of this pathway are likely constitutively and highly expressed to avoid a cofactor imbalance and energy shortage under fluctuating environmental conditions.\n\nWe here investigated the essentiality of the three NADH dehydrogenases of the respiratory chain of the obligate aerobe Pseudomonas taiwanensis VLB120 and the impact of the knockouts of corresponding genes on its physiology and metabolism. While a mutant lacking all three NADH dehydrogenases seemed to be nonviable, the generated single or double knockout strains displayed none or only a marginal phenotype. Only the mutant deficient in both type 2 dehydrogenases showed a clear phenotype with biphasic growth behavior and strongly reduced growth rate in the second phase. In-depth analyses of the metabolism of the generated mutants including quantitative physiological experiments, transcript analysis, proteomics and enzyme activity assays revealed distinct responses to type II and type I dehydrogenase deletions. An overall high metabolic flexibility enables P. taiwanensis to cope with the introduced genetic perturbations and maintain stable phenotypes by rerouting of metabolic fluxes.\n\nThis metabolic adaptability has implications for biotechnological applications. While the phenotypic robustness is favorable in large-scale applications with inhomogeneous conditions, versatile redirecting of carbon fluxes upon genetic interventions can frustrate metabolic engineering efforts.\n\nImportanceWhile Pseudomonas has the capability for high metabolic activity and the provision of reduced redox cofactors important for biocatalytic applications, exploitation of this characteristic might be hindered by high, constitutive activity of and consequently competition with the NADH dehydrogenases of the respiratory chain. The in-depth analysis of NADH dehydrogenase mutants of Pseudomonas taiwanensis VLB120 presented here, provides insight into the phenotypic and metabolic response of this strain to these redox metabolism perturbations. The observed great metabolic flexibility needs to be taken into account for rational engineering of this promising biotechnological workhorse towards a host with controlled and efficient supply of redox cofactors for product synthesis.

physiology↗

bioRxiv2018

Memote: A community-driven effort towards a standardized genome-scale metabolic model test suite

Several studies have shown that neither the formal representation nor the functional requirements of genome-scale metabolic models (GEMs) are precisely defined. Without a consistent standard, comparability, reproducibility, and interoperability of models across groups and software tools cannot be guaranteed.\n\nHere, we present memote (https://github.com/opencobra/memote) an open-source software containing a community-maintained, standardized set of metabolic model tests. The tests cover a range of aspects from annotations to conceptual integrity and can be extended to include experimental datasets for automatic model validation. In addition to testing a model once, memote can be configured to do so automatically, i.e., while building a GEM. A comprehensive report displays the models performance parameters, which supports informed model development and facilitates error detection.\n\nMemote provides a measure for model quality that is consistent across reconstruction platforms and analysis software and simplifies collaboration within the community by establishing workflows for publicly hosted and version controlled models.

systems biology↗

bioRxiv2018

Determination of growth-coupling strategies and their underlying principles

Metabolic coupling of product synthesis and microbial growth is a prominent approach for maximizing production performance. Growth-coupling (GC) also helps stabilizing target production and allows the selection of superior production strains by adaptive laboratory evolution. We have developed the computational tool gcOpt, which identifies knockout strategies leading to the best possible GC by maximizing the minimally guaranteed product yield. gcOpt implicitly favors solutions resulting in strict coupling of product synthesis to growth and metabolic activity while avoiding solutions inferring weak, conditional coupling.\n\nGC intervention strategies identified by gcOpt were examined for GC generating principles under diverse conditions. Curtailing the metabolism to render product formation an essential carbon drain was identified as one major strategy generating strong coupling of metabolic activity and target synthesis. Impeding the balancing of cofactors and protons in the absence of target production was the underlying principle of all other strategies and further increased the GC strength of the aforementioned strategies. Thus, generating a dependency between supply of global metabolic cofactors and product synthesis appears to be advantageous in enforcing strong GC.\n\nAbbreviations

systems biology↗

bioRxiv2017

Discovery and Evaluation of Biosynthetic Pathways for the Production of Five Methyl Ethyl Ketone Precursors

The limited supply of fossil fuels and the establishment of new environmental policies shifted research in industry and academia towards sustainable production of the 2nd generation of biofuels, with Methyl Ethyl Ketone (MEK) being one promising fuel candidate. MEK is a commercially valuable petrochemical with an extensive application as a solvent. However, as of today, a sustainable and economically viable production of MEK has not yet been achieved despite several attempts of introducing biosynthetic pathways in industrial microorganisms. We used BNICE.ch as a retrobiosynthesis tool to discover all novel pathways around MEK. Out of 1325 identified compounds connecting to MEK with one reaction step, we selected 3-oxopentanoate, but-3-en-2-one, but-1-en-2-olate, butylamine, and 2-hydroxy-2-methyl-butanenitrile for further study. We reconstructed 3679610 novel biosynthetic pathways towards these 5 compounds. We then embedded these pathways into the genome-scale model of E. coli, and a set of 18622 were found to be most biologically feasible ones based on thermodynamics and their yields. For each novel reaction in the viable pathways, we proposed the most similar KEGG reactions, with their gene and protein sequences, as candidates for either a direct experimental implementation or as a basis for enzyme engineering. Through pathway similarity analysis we classified the pathways and identified the enzymes and precursors that were indispensable for the production of the target molecules. These retrobiosynthesis studies demonstrate the potential of BNICE.ch for discovery, systematic evaluation, and analysis of novel pathways in synthetic biology and metabolic engineering studies.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC=\"FIGDIR/small/209569_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (27K):\norg.highwire.dtl.DTLVardef@15bb5corg.highwire.dtl.DTLVardef@bfc12borg.highwire.dtl.DTLVardef@1230500org.highwire.dtl.DTLVardef@1d74d2_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

synthetic biology↗