Biology subjects

Gurdo, N.

Publications and source records attributed to Gurdo, N..

6 recordsLinked to original sources

bioRxiv2026

Adaptive laboratory evolution rewires Pseudomonas putida for resource-efficient acetate assimilation

Acetate is an attractive renewable two-carbon substrate for microbial biotechnology, but its toxicity limits growth and carbon-use efficiency at process-relevant concentrations. Here, we used adaptive laboratory evolution to improve acetate tolerance in a genome-reduced strain of Pseudomonas putida and combined whole-genome sequencing, reverse engineering, transcriptomics, proteomics, and 13C-acetate fluxomics to resolve the underlying adaptation mechanisms. Evolution under increasing acetate concentrations selected recurrent mutations in gacA and fabB, which encode a global response regulator and a fatty acid biosynthesis enzyme, respectively. Reverse engineering of these mutations recovered most of the evolved phenotype, including shorter lag phase and substantially higher biomass yield from acetate. Multi-omic analyses showed repression of type VI secretion systems, carbohydrate storage functions, fatty acid metabolism, and oxidative stress-associated proteins, indicating resource reallocation away from costly stress and non-essential programs. Fluxomics further revealed reduced EDEMP cycling and increased glyoxylate shunt flux, consistent with improved acetate-carbon retention in biomass. These results establish acetate tolerance in P. putida as a resource-efficiency phenotype and identify gacA and fabB as actionable targets for acetate-based bioproduction.

synthetic biology↗

bioRxiv2026

Improving acetate metabolism of Pseudomonas putida KT2440 by evolutionary and rational engineering

Acetate is a promising carbon source for microbial biotechnology as it can be produced sustainably from lignocellulosic biomass or C1 gases. Since acetate is directly activated to acetyl-CoA, it is especially suitable for producing acetyl-CoA-derived products, showcased here with the production of 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAAs). P. putida KT2440 can natively metabolize acetate, but the weak acid has also inhibitory effects on microbial growth. We present an in-depth study on the physiology of P. putida KT2440 using acetate as carbon and energy source and evaluate acetate as feedstock for the biosynthesis of HAAs. Initially, a rational engineering approach to overexpress acetyl-CoA synthetase for acetate activation resulted in an improved growth rate of 16% and reduced lag phase by six hours. To further increase the performance of P. putida KT2440 on acetate, adaptive laboratory evolution was performed. This resulted in an improvement in the growth rate from 0.4 h-1 to 0.6 h-1 and enabled growth on up to 12.5 g L-1 acetate with a shortened lag phase compared to the wild type. Whole-genome sequencing revealed mutations in proteins involved in gene expression regulation and signal transduction. This evolutionary engineering approach informed the deletions of gacS and crc, which resulted in a reduction in the lag phase from seven hours to one hour and an improvement of the growth rate by 25 %, matching the growth properties of the evolved clones. Using the evolved strains for the production of HAAs resulted in faster biomass and product formation with product titers reaching up to 94 % of that of the wild type. In conclusion, we identified mechanisms in the acetate metabolism of P. putida KT2440 and improved the growth performance of the strain by rational and evolutionary engineering, demonstrating the potential of the promising, but challenging 3rd generation feedstock acetate.

microbiology↗

bioRxiv2025

Integrated control of redox and energy metabolism by the membrane-bound and soluble transhydrogenases of Pseudomonas putida across metabolic regimes

Redox homeostasis is central to microbial physiology and stress adaptation, yet the functional roles of transhydrogenases remain poorly understood beyond a few organisms. In this study, we systematically explored how Pseudomonas putida, a model soil bacterium, integrates two distinct transhydrogenases (membrane-bound PntAB and soluble SthA) into a flexible and reversible redox-balancing system that supports metabolic robustness across diverse metabolic regimes. While single deletions of either enzyme had minimal impact on the overall fitness, the double {Delta}pntAB {Delta}sthA mutant exhibited growth defects, disrupted energy charge, and redox imbalance. Unexpectedly, SthA proved essential for acetate-dependent growth, a phenotype traced to a transcriptional regulator involved in glyoxylate metabolism. Transhydrogenases also mediated tolerance to formate, a key one-carbon (C1) substrate for biotechnology, by channeling reducing equivalents released during feedstock oxidation. Synergistic activity with native formate dehydrogenases enabled redox buffering, even under stressful conditions. Functional complementation with native and engineered NAD+- or NADP+-dependent dehydrogenases validated SthA as the main sink for excess NADH. Comparative genomics linked transhydrogenase gene neighborhoods to stress and membrane processes, highlighting their evolutionary significance. These findings redefine transhydrogenases as dynamic regulators of redox metabolism, not passive cofactor shuttles. Furthermore, this work positions P. putida as a prime host for redox-intensive applications, informing design principles for C1-based metabolic engineering. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/686620v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@17c0e06org.highwire.dtl.DTLVardef@17cd094org.highwire.dtl.DTLVardef@161b5b9org.highwire.dtl.DTLVardef@1e1897e_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2023

Time-resolved, deuterium-based fluxomics uncovers the hierarchy and dynamics of sugar processing by Pseudomonas putida

Pseudomonas putida, a soil bacterium widely used for synthetic biology and metabolic engineering, processes glucose through convergent peripheral pathways that ultimately yield 6-phosphogluconate. Such a periplasmic gluconate shunt (PGS), composed by glucose and gluconate dehydrogenases, sequentially transforms glucose into gluconate and 2-ketogluconate. Although the secretion of these organic acids by P. putida has been extensively recognized, the mechanism and spatiotemporal regulation of the PGS remained elusive thus far. To address this challenge, we have developed a novel methodology for metabolic flux analysis, D-fluxomics, based on deuterated sugar substrates. D-Fluxomics demonstrated that the PGS underscores a highly dynamic metabolic architecture in glucose-dependent batch cultures of P. putida, characterized by hierarchical carbon uptake by the PGS throughout the cultivation. Additionally, we show that gluconate and 2-ketogluconate accumulation and consumption can be solely explained as a result of the interplay between growth rate-coupled and decoupled metabolic fluxes. As a consequence, the formation of these acids in the PGS is inversely correlated to the bacterial growth rate--unlike the widely studied overflow metabolism of Escherichia coli and yeast. Our findings, which underline survival strategies of soil bacteria thriving in their natural environments, open new avenues for engineering P. putida towards efficient, sugar-based bioprocesses.

systems biology↗

bioRxiv2022

A synthetic C2 auxotroph of Pseudomonas putida for evolutionary engineering of alternative sugar catabolic routes

Acetyl-coenzyme A (AcCoA) is a metabolic hub in virtually all living cells, serving as both a key precursor of essential biomass components and a metabolic sink for catabolic pathways of a large variety of substrates. Owing to this dual role, tight growth-production coupling schemes can be implemented around the AcCoA node. Inspired by this concept, a synthetic C2 auxotrophy was implemented in the platform bacterium Pseudomonas putida through an in silico-guided engineering approach. A growth-coupling strategy, driven by AcCoA demand, allowed for direct selection of an alternative sugar assimilation route--the phosphoketolase (PKT) shunt from bifidobacteria. Adaptive laboratory evolution forced the synthetic auxotroph to integrate the PKT shunt to restore C2 prototrophy. Large-scale structural chromosome rearrangements were identified as possible mechanisms for adjusting the network-wide proteome profile, resulting in improved PKT-dependent growth phenotypes. 13C-based metabolic flux analysis revealed an even split between the native Entner-Doudoroff and the synthetic PKT pathway for glucose processing, leading to enhanced carbon conservation. These results demonstrate that the P. putida metabolism can be radically rewired to incorporate a synthetic C2 metabolism, creating novel network connectivities and highlighting the importance of unconventional engineering strategies to support efficient microbial production.

synthetic biology↗

bioRxiv2022

An automated workflow for multi-omics screening of microbial model organisms

Multi-omics datasets are becoming of key importance to drive discovery in fundamental research as much as generating knowledge for applied biotechnology. However, the construction of such large datasets is usually time-consuming and expensive. Automation is needed to overcome these issues by streamlining workflows from sample generation to data analysis. Here, we describe the construction of a complex workflow for the generation of high-throughput microbial multi-omics datasets. The workflow comprises a custom-built platform for automated cultivation and sampling of microbes, sample preparation protocols, analytical methods for sample analysis and automated scripts for raw data processing. We demonstrate possibilities and limitations of such workflow in generating data for three biotechnologically relevant model organisms, namely Escherichia coli, Saccharomyces cerevisiae, and Pseudomonas putida.

systems biology↗