pBLAM1-x: standardized transposon tools for high-throughput screening DOI Creative Commons
Lorea Alejaldre, Ana Mariya Anhel, Ángel Goñi‐Moreno

et al.

Synthetic Biology, Journal Year: 2023, Volume and Issue: 8(1)

Published: Jan. 1, 2023

The engineering of pre-defined functions in living cells requires increasingly accurate tools as synthetic biology efforts become more ambitious. Moreover, the characterization phenotypic performance genetic constructs demands meticulous measurements and extensive data acquisition for sake feeding mathematical models matching predictions along design-build-test lifecycle. Here, we developed a tool that eases high-throughput transposon insertion sequencing (TnSeq): pBLAM1-x plasmid vectors carrying Himar1 Mariner transposase system. These plasmids were derived from mini-Tn5 vector pBAMD1-2 built following modular criteria Standard European Vector Architecture (SEVA) format. To showcase their function, analyzed results 60 clones soil bacterium Pseudomonas putida KT2440. new has already been included latest SEVA database release, here describe its using laboratory automation workflows. Graphical Abstract.

Language: Английский

Model-guided dynamic control of essential metabolic nodes boosts acetyl-coenzyme A–dependent bioproduction in rewired Pseudomonas putida DOI Creative Commons
Ekaterina Kozaeva, Svetlana Volkova, Marta R. A. Matos

et al.

Metabolic Engineering, Journal Year: 2021, Volume and Issue: 67, P. 373 - 386

Published: July 31, 2021

Pseudomonas putida is evolutionarily endowed with features relevant for bioproduction, especially under harsh operating conditions. The rich metabolic versatility of this species, however, comes at the price limited formation acetyl-coenzyme A (CoA) from sugar substrates. Since acetyl-CoA a key precursor number added-value products, in work we deployed an silico-guided rewiring program central carbon metabolism upgrading P. as host acetyl-CoA-dependent bioproduction. An updated kinetic model, integrating fluxomics and metabolomics datasets addition to manually-curated information enzyme mechanisms, identified targets that would lead increased levels. Based on these predictions, set plasmids based clustered regularly interspaced short palindromic repeats (CRISPR) dead CRISPR-associated protein 9 (dCas9) was constructed silence genes by CRISPR interference (CRISPRi). Dynamic reduction gene expression two (gltA, encoding citrate synthase, essential accA gene, subunit carboxylase complex) mediated 8-fold increase content rewired putida. Poly(3-hydroxybutyrate) (PHB) adopted proxy availability, synthetic pathways were engineered biopolymer accumulation. By including cell morphology extra target CRISPRi approach, fully strains programmed PHB accumulation had 5-fold titers bioreactor cultures using glucose. Thus, strategy described herein allowed rationally redirecting fluxes towards product biosynthesis-especially when deletion not option.

Language: Английский

Citations

65
Lignin conversion to β-ketoadipic acid by Pseudomonas putida via metabolic engineering and bioprocess development DOI Creative Commons
Allison Z. Werner, William T. Cordell, Ciaran W. Lahive

et al.

Science Advances, Journal Year: 2023, Volume and Issue: 9(36)

Published: Sept. 6, 2023

Bioconversion of a heterogeneous mixture lignin-related aromatic compounds (LRCs) to single product via microbial biocatalysts is promising approach valorize lignin. Here, Pseudomonas putida KT2440 was engineered convert mixed p-coumaroyl- and coniferyl-type LRCs β-ketoadipic acid, precursor for performance-advantaged polymers. Expression enzymes mediating O-demethylation, hydroxylation, ring-opening steps tuned, global regulator deleted. β-ketoadipate titers 44.5 25 grams per liter productivities 1.15 0.66 hour were achieved from model corn stover-derived LRCs, respectively, the latter representing an overall yield 0.10 gram Technoeconomic analysis bioprocess downstream processing predicted minimum selling price $2.01 kilogram, which cost competitive with fossil carbon-derived adipic acid ($1.10 1.80 kilogram). Overall, this work bioproduction metrics economic relevance conversion lignin-derived streams into bioproduct.

Language: Английский

Citations

36
Perspective on Lignin Conversion Strategies That Enable Next Generation Biorefineries DOI
Shilva Shrestha, Shubhasish Goswami, Deepanwita Banerjee

et al.

ChemSusChem, Journal Year: 2024, Volume and Issue: 17(16)

Published: April 26, 2024

Abstract The valorization of lignin, a currently underutilized component lignocellulosic biomass, has attracted attention to promote stable and circular bioeconomy. Successful approaches including thermochemical, biological, catalytic lignin depolymerization have been demonstrated, enabling opportunities for lignino‐refineries biorefineries. Although significant progress in made, this review describes unexplored chemical biological routes thereby contributes economically environmentally sustainable lignin‐utilizing This also highlights the integration identifies research gaps while recommending future directions scaling processes establish lignino‐chemical industry.

Language: Английский

Citations

11
Genome-scale and pathway engineering for the sustainable aviation fuel precursor isoprenol production in Pseudomonas putida DOI Creative Commons
Deepanwita Banerjee, Ian Sofian Yunus, Xi Wang

et al.

Metabolic Engineering, Journal Year: 2024, Volume and Issue: 82, P. 157 - 170

Published: Feb. 16, 2024

Sustainable aviation fuel (SAF) will significantly impact global warming in the sector, and important SAF targets are emerging. Isoprenol is a precursor for promising compound DMCO (1,4-dimethylcyclooctane) has been produced several engineered microorganisms. Recently, Pseudomonas putida gained interest as future host isoprenol bioproduction it can utilize carbon sources from inexpensive plant biomass. Here, we engineer metabolically versatile P. production. We employ two computational modeling approaches (Bilevel optimization Constrained Minimal Cut Sets) to predict gene knockout optimize "IPP-bypass" pathway maximize Altogether, highest production titer was achieved at 3.5 g/L under fed-batch conditions. This combination of strain engineering on an advanced biofuels vital significance enabling process that use renewable streams.

Language: Английский

Citations

10
Microbial assimilation of lignin-derived aromatic compounds and conversion to value-added products DOI Creative Commons
Christopher Chibueze Azubuike, Marco N. Allemann, Joshua K. Michener

et al.

Current Opinion in Microbiology, Journal Year: 2021, Volume and Issue: 65, P. 64 - 72

Published: Nov. 11, 2021

Language: Английский

Citations

46
Role of the CrcB transporter of Pseudomonas putida in the multi‐level stress response elicited by mineral fluoride DOI

Patricia Calero,

Nicolás Gurdo, Pablo I. Nikel

et al.

Environmental Microbiology, Journal Year: 2022, Volume and Issue: 24(11), P. 5082 - 5104

Published: June 21, 2022

Abstract The presence of mineral fluoride (F − ) in the environment has both a geogenic and anthropogenic origin, halide been described to be toxic virtually all living organisms. While evidence gathered different microbial species supports this notion, systematic exploration effects F salts on metabolism physiology environmental bacteria remained underexplored thus far. In work, we studied characterized tolerance mechanisms deployed by model soil bacterium Pseudomonas putida KT2440 against NaF. By adopting systems‐level omic approaches, including functional genomics metabolomics, gauged impact anion at regulatory levels under conditions that impair bacterial growth. Several genes involved were isolated genome‐wide Tn ‐Seq screening—among which crcB , encoding an ‐specific exporter, was shown play predominant role detoxification. High‐resolution combined with assessment intracellular extracellular pH values quantitative experiments, underscored key nodes central carbon affected . Taken together, our results indicate P undergoes general, multi‐level stress response when challenged NaF significantly differs from caused other saline stressors. responses oxidative challenges have extensively literature, very little is known about metabolism. This state affairs contrasts fact more abundant than halides Earth crust (e.g. some soils, concentration can reach up 1 mg g −1 ). Understanding global treatment not only relevant unveil distinct detoxification but it could also guide engineering approaches for target incorporation fluorine into value‐added organofluorine molecules. regard, constitutes ideal explore such scenarios, since particularly its high level resistance variety physicochemical perturbations.

Language: Английский

Citations

30
RB-TnSeq identifies genetic targets for improved tolerance of Pseudomonas putida towards compounds relevant to lignin conversion DOI Creative Commons
Andrew J. Borchert, Alissa Bleem, Gregg T. Beckham

et al.

Metabolic Engineering, Journal Year: 2023, Volume and Issue: 77, P. 208 - 218

Published: April 13, 2023

Language: Английский

Citations

19
Maximizing microbial bioproduction from sustainable carbon sources using iterative systems engineering DOI Creative Commons
Thomas Eng, Deepanwita Banerjee, Javier Menasalvas

et al.

Cell Reports, Journal Year: 2023, Volume and Issue: 42(9), P. 113087 - 113087

Published: Sept. 1, 2023

Maximizing the production of heterologous biomolecules is a complex problem that can be addressed with systems-level understanding cellular metabolism and regulation. Specifically, growth-coupling approaches increase product titers yields also enhance rates. However, implementing these methods for non-canonical carbon streams challenging due to gaps in metabolic models. Over four design-build-test-learn cycles, we rewire Pseudomonas putida KT2440 growth-coupled indigoidine from para-coumarate. We explore 4,114 potential solutions refine one design through laboratory evolution ensemble data-driven methods. The final strain produces 7.3 g/L at 77% maximum theoretical yield para-coumarate minimal medium. iterative use designs functional genomics experimental validation was highly effective agnostic specific hosts, streams, products thus generalizable across many systems.

Language: Английский

Citations

15
Milligrams to kilograms: making microbes work at scale DOI Creative Commons
William T. Cordell, Gennaro Avolio, Ralf Takors

et al.

Trends in biotechnology, Journal Year: 2023, Volume and Issue: 41(11), P. 1442 - 1457

Published: June 3, 2023

Language: Английский

Citations

13
Advances in engineering microbial biosynthesis of aromatic compounds and related compounds DOI Creative Commons
Roman M. Dickey, Amanda M. Forti, Aditya M. Kunjapur

et al.

Bioresources and Bioprocessing, Journal Year: 2021, Volume and Issue: 8(1)

Published: Sept. 27, 2021

Abstract Aromatic compounds have broad applications and been the target of biosynthetic processes for several decades. New biomolecular engineering strategies applied to improve production aromatic in recent years, some which are expected set stage next wave innovations. Here, we will briefly complement existing reviews on microbial by focusing a few trends where considerable work has performed last 5 years. The highlight pathway modularization compartmentalization, co-culturing, non-traditional host engineering, polymer feedstock utilization, engineered ring cleavage, aldehyde stabilization, biosynthesis non-standard amino acids. Throughout this review article, also touch unmet opportunities that future research could address.

Language: Английский

Citations

31