Engineered Pseudomonas putida KT2440 co-utilizes galactose and glucose

Peabody, George L.; Elmore, Joshua R.; Martinez-Baird, Jessica; Guss, Adam M.

doi:10.1186/s13068-019-1627-0

Cited by 17 publications

(19 citation statements)

References 35 publications

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“…Introducing the whole heterogenous catabolic pathway has been considered an effective strategy for improving substrate fermentation. The DLD pathway, requiring only four enzymes to convert galactose to pyruvate, has been successfully introduced into the organism that does not natively utilize galactose and allowed for rapid growth with galactose as the sole carbon source ( Peabody et al, 2019 ). Therefore, heterologous integration of the complete DLD pathway into M. thermophila was performed.…”

Section: Resultsmentioning

confidence: 99%

“…2-Keto-3-deoxygalactonate (KDG) aldolase has a wide substrate range, including KDG and 2-keto-3-deoxy-6-phosphogalactonate (KDGP). The genes Pfkdgk encoding KDG kinase from P. fluorescens have been successfully heterologously overexpressed for improved galactose utilization previously ( Peabody et al, 2019 ). Herein, Pfkdgk was overexpressed in strain HW2607 to catalyze the phosphorylation reaction of KDG to KDGP before aldolase reactions.…”

Section: Resultsmentioning

confidence: 99%

“…Furthermore, KDG is phosphorylated by kinase and cleaved to release pyruvate and glyceraldehyde-3-phosphate by 2-keto-3-deoxygluconate (KDG) aldolase. The DLD pathway, requiring four enzymes, is a short and simple route of galactose catabolism and has been introduced into organisms to enhance galactose metabolism ( Peabody et al, 2019 ).…”

Section: Introductionmentioning

confidence: 99%

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Transcriptional Profiling of Myceliophthora thermophila on Galactose and Metabolic Engineering for Improved Galactose Utilization

et al. 2021

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Efficient biological conversion of all sugars from lignocellulosic biomass is necessary for the cost-effective production of biofuels and commodity chemicals. Galactose is one of the most abundant sugar in many hemicelluloses, and it will be important to capture this carbon for an efficient bioconversion process of plant biomass. Thermophilic fungus Myceliophthora thermophila has been used as a cell factory to produce biochemicals directly from renewable polysaccharides. In this study, we draw out the two native galactose utilization pathways, including the Leloir pathway and oxido-reductive pathway, and identify the significance and contribution of them, through transcriptional profiling analysis of M. thermophila and its mutants on galactose. We find that galactokinase was necessary for galactose transporter expression, and disruption of galK resulted in decreased galactose utilization. Through metabolic engineering, both galactokinase deletion and galactose transporter overexpression can activate internal the oxido-reductive pathway and improve the consumption rate of galactose. Finally, the heterologous galactose-degradation pathway, De Ley–Doudoroff (DLD) pathway, was successfully integrated into M. thermophila, and the consumption rate of galactose in the engineered strain was increased by 57%. Our study focuses on metabolic engineering for accelerating galactose utilization in a thermophilic fungus that will be beneficial for the rational design of fungal strains to produce biofuels and biochemicals from a variety of feedstocks with abundant galactose.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Transcriptional Profiling of Myceliophthora thermophila on Galactose and Metabolic Engineering for Improved Galactose Utilization

et al. 2021

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“…As a saprotrophic, rhizosphere bacterium, P. putida might encounter many different plant‐derived carbohydrates, including d ‐glucose, d ‐galactose and l ‐arabinose, which are found predominantly in cellulosic and hemicellulosic hydrolysates, and lignocellulosic biomasses (Derrien et al ., 2004; Peabody et al ., 2019; Wang et al ., 2019). Some of these sugars were not taken up for intracellular metabolism such as d ‐galactose or l ‐arabinose, but were rather oxidized in the periplasm in BES (Fig.…”

Section: Discussionmentioning

confidence: 99%

The anoxic electrode‐driven fructose catabolism of Pseudomonas putida KT2440

Nguyễn

Lai

Adrian

et al. 2021

Microbial Biotechnology

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Pseudomonas putida (P. putida) is a microorganism of interest for various industrial processes, yet its strictly aerobic nature limits application. Despite previous attempts to adapt P. putida to anoxic conditions via genetic engineering or the use of a bioelectrochemical system (BES), the problem of energy shortage and internal redox imbalance persists. In this work, we aimed to provide the cytoplasmic metabolism with different monosaccharides, other than glucose, and explored the physiological response in P. putida KT2440 during bioelectrochemical cultivation. The periplasmic oxidation cascade was found to be able to oxidize a wide range of aldoses to their corresponding (keto-)aldonates. Unexpectedly, isomerization of the ketose fructose to mannose also enabled oxidation by glucose dehydrogenase, a new pathway uncovered for fructose metabolism in P. putida KT2440 in BES. Besides the isomerization, the remainder of fructose was imported into the cytoplasm and metabolized. This resulted in a higher NADPH/NADP + ratio, compared to glucose. Comparative proteomics further revealed the upregulation of proteins in the lower central carbon metabolism during the experiment. These findings highlight that the choice of a substrate in BES can target cytosolic and periplasmic oxidation pathways, and that electrode-driven redox balancing can drive these pathways in P. putida under anaerobic conditions.

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“…These strains can utilize D-galactose or D-galactonic acid as carbon source for growth. Nevertheless, this is not the case for P. putida ATCC 47054, which is devoid of enzymes involved in De Ley-Doudoroff pathway [19]. In the case of HMFCA, it can be converted to FDCA by full oxidation of the hydroxymethyl group on furan ring by Cupriavidus basilensis HMF14 and Raoultella ornithinolytica BF60, but the strains with such characteristic is rather rare [20,21].…”

Section: Oxidation Of D-galactose and Hmf Using Pseudomonas Sp Strainsmentioning

confidence: 99%

Valorization of Gelidium amansii for dual production of D-galactonic acid and 5-hydroxymethyl-2-furancarboxylic acid by chemo-biological approach

et al. 2020

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Background Marine macroalgae Gelidium amansii is a promising feedstock for production of sustainable biochemicals to replace petroleum and edible biomass. Different from terrestrial lignocellulosic biomass, G. amansii is comprised of high carbohydrate content and has no lignin. In previous studies, G. amansii biomass has been exploited to obtain fermentable sugars along with suppressing 5-hydroxymethylfurfural (HMF) formation for bioethanol production. In this study, a different strategy was addressed and verified for dual production of D-galactose and HMF, which were subsequently oxidized to D-galactonic acid and 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) respectively via Pseudomonas putida. Results G. amansii biomass was hydrolyzed by dilute acid to form D-galactose and HMF. The best result was attained after pretreatment with 2% (w/w) HCl at 120 °C for 40 min. Five different Pseudomonas sp. strains including P. putida ATCC 47054, P. fragi ATCC 4973, P. stutzeri CICC 10402, P. rhodesiae CICC 21960, and P. aeruginosa CGMCC 1.10712, were screened for highly selective oxidation of D-galactose and HMF. Among them, P. putida ATCC 47054 was the outstanding suitable biocatalyst converting D-galactose and HMF to the corresponding acids without reduced or over-oxidized products. It was plausible that the pyrroloquinoline quinone-dependent glucose dehydrogenase and undiscovered molybdate-dependent enzyme(s) in P. putida ATCC 47054 individually played pivotal role for d-galactose and HMF oxidation. Taking advantage of its excellent efficiency and high selectivity, a maximum of 55.30 g/L d-galactonic acid and 11.09 g/L HMFCA were obtained with yields of 91.1% and 98.7% using G. amansii hydrolysates as substrate. Conclusions Valorization of G. amansii biomass for dual production of D-galactonic acid and HMFCA can enrich the product varieties and improve the economic benefits. This study also demonstrates the perspective of making full use of marine feedstocks to produce other value-added products.

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Engineered Pseudomonas putida KT2440 co-utilizes galactose and glucose

Cited by 17 publications

References 35 publications

Transcriptional Profiling of Myceliophthora thermophila on Galactose and Metabolic Engineering for Improved Galactose Utilization

Transcriptional Profiling of Myceliophthora thermophila on Galactose and Metabolic Engineering for Improved Galactose Utilization

The anoxic electrode‐driven fructose catabolism of Pseudomonas putida KT2440

Valorization of Gelidium amansii for dual production of D-galactonic acid and 5-hydroxymethyl-2-furancarboxylic acid by chemo-biological approach

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