Microbial Biocatalysts Research Articles

Production of lipopeptide biosurfactant by Kurthia gibsonii KH2 and their synergistic action in biodecolourisation of textile wastewater

Textile dyes are recalcitrant molecules and contain a high level of chemicals and colour which poses a serious challenge to surrounding environments. Therefore, this study aims to produce biosurfactant and investigate the synergistic action on decolourisation of textile dyes by the combination of bacteria and biosurfactant. An effective dye degrading strain and biosurfactant-producer, Kurthia gibsonii KH2, was isolated from textile wastewater using molasses as the only source of carbon and energy. The isolates were identified and screened for biosurfactant production using haemolytic activity, oil spreading technique, drop collapse test and emulsification index. Fourier Transform Infrared Spectrum (FTIR) and Thin Layer Chromatography (TLC) analyses were carried out to detect the type of biosurfactant. The effect of different physicochemical parameters on textile wastewater decolourisation was assessed within 24 h. The Kurthia gibsonii KH2 showed positive results for haemolytic activity, oil spreading technique, and drop collapse test. The emulsification test (E24) revealed that Kurthia gibsonii KH2 had a higher emulsification index of 63%. FTIR and TLC analyses indicated that the biosurfactant was a lipopeptide and was formed with a yield of 2 gL −1 . The synergistic activity of Kurthia gibsonii KH2 and lipopeptide biosurfactant resulted in decolourisation levels of 85% at 100 mg/L concentration and pH 7 was recorded at 168 h of incubation. The high attributes of these combinations and the phytotoxicity tests implied that the metabolites were less toxic, making it a promising option for the biodecolourisation and biodegradation of industrial textile wastewater and various environmental conditions. • Textile wastewater treatment using utilisation of microbial biocatalysts is vital. • The lipopeptide biosurfactant was derived from the bacteria Kurthia gibsonii KH2. • The strain showed synergistic action in textile dyes removal efficiency. • Decolorisation process was the dominant removal mechanism.

Metabolism of syringyl lignin-derived compounds in Pseudomonas putida enables convergent production of 2-pyrone-4,6-dicarboxylic acid

Valorization of lignin, an abundant component of plant cell walls, is critical to enabling the lignocellulosic bioeconomy. Biological funneling using microbial biocatalysts has emerged as an attractive approach to convert complex mixtures of lignin depolymerization products to value-added compounds. Ideally, biocatalysts would convert aromatic compounds derived from the three canonical types of lignin: syringyl (S), guaiacyl (G), and p-hydroxyphenyl (H). Pseudomonas putida KT2440 (hereafter KT2440) has been developed as a biocatalyst owing in part to its native catabolic capabilities but is not known to catabolize S-type lignin-derived compounds. Here, we demonstrate that syringate, a common S-type lignin-derived compound, is utilized by KT2440 only in the presence of another energy source or when vanAB was overexpressed, as syringate was found to be O-demethylated to gallate by VanAB, a two-component monooxygenase, and further catabolized via extradiol cleavage. Unexpectedly, the specificity (kcat/KM) of VanAB for syringate was within 25% that for vanillate and O-demethylation of both substrates was well-coupled to O2 consumption. However, the native KT2440 gallate-cleaving dioxygenase, GalA, was potently inactivated by 3-O-methylgallate. To engineer a biocatalyst to simultaneously convert S-, G-, and H-type monomers, we therefore employed VanAB from Pseudomonas sp. HR199, which has lower activity for 3MGA, and LigAB, an extradiol dioxygenase able to cleave protocatechuate and 3-O-methylgallate. This strain converted 93% of a mixture of lignin monomers to 2-pyrone-4,6-dicarboxylate, a promising bio-based chemical. Overall, this study elucidates a native pathway in KT2440 for catabolizing S-type lignin-derived compounds and demonstrates the potential of this robust chassis for lignin valorization.

Biotransformation of Methane and Carbon Dioxide Into High-Value Products by Methanotrophs: Current State of Art and Future Prospects.

Conventional chemical methods to transform methane and carbon dioxide into useful chemicals are plagued by the requirement for extreme operating conditions and expensive catalysts. Exploitation of microorganisms as biocatalysts is an attractive alternative to sequester these C1 compounds and convert them into value-added chemicals through their inherent metabolic pathways. Microbial biocatalysts are advantageous over chemical processes as they require mild-operating conditions and do not release any toxic by-products. Methanotrophs are potential cell-factories for synthesizing a wide range of high-value products via utilizing methane as the sole source of carbon and energy, and hence, serve as excellent candidate for methane sequestration. Besides, methanotrophs are capable of capturing carbon dioxide and enzymatically hydrogenating it into methanol, and hence qualify to be suitable candidates for carbon dioxide sequestration. However, large-scale production of value-added products from methanotrophs still presents an overwhelming challenge, due to gas-liquid mass transfer limitations, low solubility of gases in liquid medium and low titer of products. This requires design and engineering of efficient reactors for scale-up of the process. The present review offers an overview of the metabolic architecture of methanotrophs and the range of product portfolio they can offer. Special emphasis is given on methanol biosynthesis as a potential biofuel molecule, through utilization of methane and alternate pathway of carbon dioxide sequestration. In view of the gas-liquid mass transfer and low solubility of gases, the key rate-limiting step in gas fermentation, emphasis is given toward reactor design consideration essential to achieve better process performance.

Principles and practice of designing microbial biocatalysts for fuel and chemical production.

The finite nature of fossil fuels and the environmental impact of its use have raised interest in alternate renewable energy sources. Specifically, nonfood carbohydrates, such as lignocellulosic biomass, can be used to produce next generation biofuels, including cellulosic ethanol and other nonethanol fuels like butanol. However, currently there is no native microorganism that can ferment all lignocellulosic sugars to fuel molecules. Thus, research is focused on engineering improved microbial biocatalysts for production of liquid fuels at high productivity, titer, and yield. A clear understanding and application of the basic principles of microbial physiology and biochemistry are crucial to achieve this goal. In this review, we present and discuss the construction of microbial biocatalysts that integrate these principles with ethanol-producing Escherichia coli as an example of metabolic engineering. These principles also apply to fermentation of lignocellulosic sugars to other chemicals that are currently produced from petroleum.

Immobilized microbial cell biocatalysts: An annotated selection of World Wide Web sites relevant to the topics in microbial biotechnology.

Immobilized microbial cell biocatalysts: An annotated selection of World Wide Web sites relevant to the topics in microbial biotechnology.

Improved biodiesel from palm oil using lipase immobilized calcium alginate and Irvingia gabonensis matrices

BackgroundLipase is an important microbial enzyme and biocatalyst in biodiesel production. The study investigated fuel properties of biodiesel produced from palm oil (PO) using lipase immobilized on Irvingia gabonensis and calcium alginate.ResultsBiodiesel yield from PO using free and immobilized lipases was highest at 35 °C and pH 7, with product yield using calcium alginate-immobilized lipase, CAIL (94.42, 96.9%) higher than using Irvingia gabonensis-immobilized lipase, IGIL (92.54, 95.8%). Biodiesel produced using immobilized lipases had similar pour point, cloud point, and kinematic viscosity, and they possessed improved fuel properties compared to free lipase biodiesel in terms of densities at 15 °C and flash point. Pour points, flash point, and kinematic viscosity of biodiesel produced using CAIL and IGIL met American and European Standards but density at 15 °C and cloud points are below both standards. CAIL and IGIL biodiesel had similar fatty acid methyl ester (FAME) compounds and consisted more of unsaturated fatty acids (hexadecanoate, 9-octadecenoate, octadecanoate, dodecanoate, and 9,12-octadeca-dienoate) than obtained in biodiesel from free lipase. IGIL and CAIL were re-used in 8 and 12 cycles respectively, with > 90% biodiesel yield achieved in four and 11 cycles.ConclusionsThe study showed that lipase immobilized on Irvingia gabenensis and calcium alginate and used in biodiesel production retained high enzyme activity and biodiesel yield in repeated cycles.

Development of Clostridium tyrobutyricum as a Microbial Cell Factory for the Production of Fuel and Chemical Intermediates From Lignocellulosic Feedstocks

Microbial conversion of renewable, lignocellulosic substrates to fuel and platform chemical intermediates offers a sustainable route to establish a viable bioeconomy. However, such approaches face a series of key technical, economic, and sustainability hurdles, including: incomplete substrate utilization, lignocellulosic hydrolysate and/or end-product toxicity, inefficient product recovery, incompatible cultivation requirements, and insufficient productivity metrics. Development of a production host with native traits suitable for high productivity conversion of lignocellulosic substrates under process-relevant conditions offers a means to bypass the above-described hurdles and accelerate the development of microbial biocatalyst deployment. Clostridium tyrobutyricum, a native producer of short chain fatty acids, displays a series of characteristics that make it an ideal candidate for conversion of lignocellulosic substrates and thus represents a promising host for microbial production of diverse carboxylate-derived product suites. Herein, we review recent progress and future directions in the development of this bacterium as an industrial microbial cell factory, with emphases on the utilization of lignocellulosic substrates and metabolic engineering approaches.

Optimization of Yarrowia lipolytica-based consolidated biocatalyst through synthetic biology approach: transcription units and signal peptides shuffling

Nowadays considerable effort is being pursued towards development of consolidated microbial biocatalysts that will be able to utilize complex, non-pretreated substrates and produce valuable compounds. In such engineered microbes, synthesis of extracellular hydrolases may be fine-tuned by different approaches, like strength of promoter, type of secretory tag, and gene copy number. In this study, we investigated if organization of a multi-element expression cassette impacts the resultant Yarrowia lipolytica transformants’ phenotype, presuming that different variants of the cassette are composed of the same regulatory elements and encode the same mature proteins. To this end, Y. lipolytica cells were transformed with expression cassettes bearing a pair of genes encoding exactly the same mature amylases, but fused to four different signal peptides (SP), and located interchangeably in either first or second position of a synthetic DNA construction. The resultant strains were tested for growth on raw and pretreated complex substrates of different plant origin for comprehensive examination of the strains’ acquired characteristics. Optimized strain was tested in batch bioreactor cultivations for growth and lipids accumulation. Based on the conducted research, we concluded that the positional order of transcription units (TU) and the type of exploited SP affect final characteristics of the resultant consolidated biocatalyst strains, and thus could be considered as additional factors to be evaluated upon consolidated biocatalysts optimization.Key Points• Y. lipolytica growing on raw starch was constructed and tested on different substrates.• Impact of expression cassette design and SP on biocatalysts’ phenotype was evidenced.• Consolidated biocatalyst process for lipids production from starch was conducted.

Comprehensive Optimization of Culture Conditions for Production of Biomass-Hydrolyzing Enzymes of Trichoderma SG2 in Submerged and Solid-State Fermentation.

Lignocellulose biomass contain large macromolecules especially cellulose and hemicelluloses that can be converted to fuel and chemicals using microbial biocatalysts. This study presents comprehensive optimization of production of biomass-hydrolyzing enzymes (BHE) by a high β-glucosidase-producing Trichoderma SG2 for bioconversion of lignocellulose biomass. Overall, a mixture of paper powder and switchgrass was most suited for production of BHE in submerged fermentation (SmF). BHE production was significantly different for various organic and inorganic nitrogen sources. The combination of peptone, yeast extract, and ammonium sulfate resulted in the highest activities (Units/mL) of BHE: 9.85 ± 0.55 cellulase, 38.91 ± 0.31 xylanase, 21.19 ± 1.35 β-glucosidase, and 7.63 ± 0.31 β-xylosidase. Surfactants comparably enhanced BHE production. The highest cellulase activity (4.86 ± 0.55) was at 25°C, whereas 35°C supported the highest activities of xylanase, β-glucosidase, and β-xylosidase. A broad initial culture pH (4-7) supported BHE production. The Topt for cellulase and xylanase was 50°C. β-xylosidase and β-glucosidase were optimally active at 40 and 70°C, respectively; pH 5 resulted in highest cellulase, β-glucosidase, and β-xylosidase activities; and pH 6 resulted in highest xylanase activity. Response surface methodology (RSM) was used to optimize major medium ingredients. BHE activities were several orders of magnitude higher in solid-state fermentation (SSF) than in SmF. Therefore, SSF can be deployed for one-step production of complete mixture of Trichoderma SG2 BHE for bioconversion of biomass to saccharide feedstock.

Functionalized Gold and Silver Bimetallic Nanoparticles Using Deinococcus radiodurans Protein Extract Mediate Degradation of Toxic Dye Malachite Green.

BackgroundBiodegradation of toxic organic dye using nanomaterial-based microbial biocatalyst is an ecofriendly and promising technique.Materials and MethodsHere, we have investigated the novel properties of functionalized Au-Ag bimetallic nanoparticles using extremophilic Deinococcus radiodurans proteins (Drp-Au-AgNPs) and their degradation efficiency on the toxic triphenylmethane dye malachite green (MG).Results and DiscussionThe prepared Drp-Au-AgNPs with an average particle size of 149.8 nm were capped by proteins through groups including hydroxyl and amide. Drp-Au-AgNPs demonstrated greater degradation ability (83.68%) of MG than D. radiodurans cells and monometallic AuNPs. The major degradation product was identified as 4-(dimethylamino) benzophenone, which is less toxic than MG. The degradation of MG was mainly attributed to the capping proteins on Drp-Au-AgNPs. The bimetallic NPs could be reused and maintained MG degradation ability (>64%) after 2 cycles.ConclusionThese results suggest that the easily prepared Drp-Au-AgNPs have potential applications as novel nanomedicine for MG detoxification, and nanomaterial for biotreatment of a toxic polyphenyl dye-containing wastewater.

Highly regio- and stereoselective hydroxylation of vitamin D2 by CYP109E1

Vitamin D2 is a form of vitamin D derived from mushrooms and plants which is structurally modified in the body due to the action of several enzymes. The resulting metabolites represent important compounds with potential bioactive properties. However, they are poorly studied and their availability is mostly limited. In order to identify new enzymes capable of producing vitamin D2 metabolites, we investigated a bacterial P450 monooxygenase, CYP109E1, which was previously shown to be a vitamin D3 hydroxylase. It was found that CYP109E1 catalyzes a vitamin D2 two-step hydroxylation at positions C24 and C25 resulting in the generation of 24(R),25-diOH VD2. Interestingly, the enzyme showed high selectivity towards vitamin D2, whereas it showed an unselective product pattern for the structurally similar vitamin D3. Our docking results for vitamin D2 and D3 revealed favorable hydroxylation positions for both substrates and suggested an explanation for the high selectivity of CYP109E1 towards vitamin D2. In addition, we established a whole-cell biocatalyst expressing CYP109E1 in Bacillus megaterium to produce 24(R),25-diOH VD2 and a production yield of 12.3 ± 1.2 mg/L was obtained after 48 h. To the best of our knowledge, this is the first report on the generation of 24(R),25-diOH VD2 by a microbial biocatalyst allowing a low-cost and eco-friendly production of this pharmaceutically interesting and expensive metabolite from the relatively cheap substrate, VD2.

RETRACTED: Lignocellulosic bio-refinery approach for microbial 2,3-Butanediol production

Bio-refinery approach using agricultural and industrial waste material as feedstock is becoming a preferred area of interest in biotechnology in the current decades. The reasons for this trend are mainly because of the declining petroleum resources, greenhouse gas emission risks and fluctuating market price of crude oil. Most chemicals synthesized petro chemically, can be produced using microbial biocatalysts. 2,3-Butanediol (BDO) is such an important platform bulk chemical with numerous industrial applications including as a fuel additive. Although microbial production of BDO is well studied, strategies that could successfully upgrade the current lab-scale researches to an industrial level have to be developed. This review presents an overview of the recent trends and developments in the microbial production of BDO from different lignocellulose biomass.

Connecting Microbial Genotype with Phenotype in the Omics Era.

Although rational design-based metabolic engineering has been applied widely to obtain promising microbial biocatalysts, conventional strategies such as adaptive laboratory evolution (ALE) and mutagenesis are still efficient approaches to improve microorganisms for exceptional features such as a broad spectrum of substrate utilization, robustness of cell growth, as well as high titer, yield, and productivity of the target products. In this chapter, we describe the procedure to generate mutant strains with desired phenotypes using ALE and a new mutagenesis approach of Atmosphere and Room Temperature Plasma (ARTP). In addition, we discuss the methodology to combine next-generation sequencing (NGS)-based genome-resequencing and RNA-Seq transcriptomics approaches to characterize the mutant strains and connect the phenotypes with their corresponding genotypic changes.

Genetic engineering for enhanced productivity in bioelectrochemical systems.

A shift from petrochemical processes toward a bio-based economy is one of the most advocated developments for a sustainable future. To achieve this will require the biotechnological production of platform chemicals that can be further processed by chemical engineering. Bioelectrochemical systems (BESs) are a novel tool within the biotechnology field. In BESs, microbes serve as biocatalysts for the production of biofuels and value-added compounds, as well as for the production of electricity. Although the general feasibility of bioelectrochemical processes has been demonstrated in recent years, much research has been conducted to develop biocatalysts better suited to meet industrial demands. Initially, mainly natural exoelectrogenic organisms were investigated for their performance in BESs. Driven by possibilities of recent developments in genetic engineering and synthetic biology, the spectrum of microbial catalysts and their versatility (substrate and product range) have expanded significantly. Despite these developments, there is still a tremendous gap between currently achievable space-time yields and current densities on the one hand and the theoretical limits of BESs on the other. It will be necessary to move the performance of the biocatalysts closer to the theoretical possibilities in order to establish viable production routines. This review summarizes the status quo of engineering microbial biocatalysts for anode-applications with high space-time yields. Furthermore, we will address some of the theoretical limitations of these processes exemplarily and discuss which of the present strategies might be combined to achieve highly synergistic effects and, thus, meet industrial demands.

Multidimensional engineering of Saccharomyces cerevisiae for efficient synthesis of medium-chain fatty acids

Medium-chain fatty acids (MCFAs; C6–C12) are valuable molecules used for biofuel and oleochemical production; however, it is challenging to synthesize these fatty acids efficiently using microbial biocatalysts due to the cellular toxicity of MCFAs. In this study, both the endogenous fatty acid synthase (FAS) and an orthogonal bacterial type I FAS were engineered for MCFA production in the yeast Saccharomyces cerevisiae. To improve cellular tolerance to toxic MCFAs, we performed directed evolution of the membrane transporter Tpo1 and strain adaptive laboratory evolution, which elevated the MCFA production by 1.3 ± 0.3- and 1.7 ± 0.2-fold, respectively. We therefore further engineered the highly resistant strain to augment the metabolic flux towards MCFAs. This multidimensional engineering of the yeast at the single protein/enzyme level, the pathway level and the cellular level, combined with an optimized cultivation process, resulted in the production of >1 g l−1 extracellular MCFAs—a more than 250-fold improvement over the original strain. Efficient microbial production of medium-chain fatty acids (MCFAs; C6–C12)—valuable molecules in the oleochemical and biofuel industry—is challenging due to their cellular toxicity. Now, this work improves the production of extracellular MCFAs to over 1 g l−1 by systematically engineering yeast at multiple levels.

Biocatalytic characteristics of chitosan nanoparticle-immobilized alginate lyase extracted from a novel Arthrobacter species AD-10

Abstract The potential applications of alginate lyase in pharmaceutical, food, agriculture, bioenergy and medical diagnostic industries and the high cost of commercially available alginate lyase necessitate searching for microbial biocatalyst for sustainable and economical production of this enzyme. In the current study, a potent alginate lyase-producing bacterium was isolated from decomposed Sargassum seaweed waste off Barbados’ coast and identified as Arthrobacter sp. AD-10 (GenBank accession number MG983495) via 16S rRNA gene sequencing. Biocatalytic efficacy of this novel bacterium for cleaving alginate was assessed by immobilizing its extracted alginate lyase onto functionalized chitosan nanoparticles. Glutaraldehyde cross-linked chitosan nanoparticles could able to retain 79.3% of alginate lyase activity. Chitosan-nanoparticle immobilized alginate lyase exhibited pH and temperature optimum at pH 8 and 45 °C, respectively, and had a strong affinity toward alginate by displaying a low Km (1.21 mg/mL) and a high Vmax (11.3 U/mg-protein) value. Activation energy, enthalpy and entropy of catalysis values were recorded as 42.2 kJ/mol, 39.7 kJ/mol and −105.2 J/mol/K, respectively. Additionally, the immobilized alginate lyase was not affected by 0.34 M NaCl and retained 66.3% of its maximal activity after seven consecutive reuses. These promising biocatalytic characteristics suggest the industrial potential of chitosan-nanoparticle immobilized alginate lyase for bioprocessing of alginate.

Oxyfunctionalization of nonsteroidal anti-inflammatory drugs by filamentous-fungi.

We aimed to expand the microbial biocatalyst platform to generate essential oxyfunctionalized standards for pharmaceutical, toxicological and environmental research. In particular, we examined the production of oxyfunctionalized nonsteroidal anti-inflammatory drugs (NSAIDs) by filamentous-fungi. Four NSAIDs; diclofenac, ibuprofen, naproxen and mefenamic acid were used as substrates for oxyfunctionalization in a biocatalytic process involving three filamentous-fungi strains; Beauveriabassiana, Clitocybenebularis and Mucorhiemalis. Oxyfunctionalized metabolites that are major degradation intermediates formed by Cytochrome P450 monooxygenases in human metabolism were produced in isolated yields of up to 99% using 1gl-1 of substrate. In addition, a novel compound, 3',4'-dihydroxydiclofenac, was produced by B.bassiana. Proteomic analysis identified CYP548A5 that might be responsible for diclofenac oxyfunctionalization in B.bassiana. Efficient fungi catalysed oxyfunctionalization was achieved when using NSAIDs as substrates. High purities and isolated yields of the produced metabolites were achieved. The lack of current efficient synthetic strategies for oxyfunctionalization of NSAIDs is a bottleneck to perform pharmacokinetic, pharmacodynamic and toxicological analysis for the pharmaceutical industry. Additionally, oxyfunctionalized derivatives are needed for tracking the fate and impact of such metabolites in the environment. Herein, we described a fungi catalysed process that surpasses previously reported strategies in terms of efficiency, to synthesize oxyfunctionalized NSAIDs.

Comparison of Multi-Objective Evolutionary Algorithms to Solve the Modular Cell Design Problem for Novel Biocatalysis

A large space of chemicals with broad industrial and consumer applications could be synthesized by engineered microbial biocatalysts. However, the current strain optimization process is prohibitively laborious and costly to produce one target chemical and often requires new engineering efforts to produce new molecules. To tackle this challenge, modular cell design based on a chassis strain that can be combined with different product synthesis pathway modules has recently been proposed. This approach seeks to minimize unexpected failure and avoid task repetition, leading to a more robust and faster strain engineering process. In our previous study, we mathematically formulated the modular cell design problem based on the multi-objective optimization framework. In this study, we evaluated a library of state-of-the-art multi-objective evolutionary algorithms (MOEAs) to identify the most effective method to solve the modular cell design problem. Using the best MOEA, we found better solutions for modular cells compatible with many product synthesis modules. Furthermore, the best performing algorithm could provide better and more diverse design options that might help increase the likelihood of successful experimental implementation. We identified key parameter configurations to overcome the difficulty associated with multi-objective optimization problems with many competing design objectives. Interestingly, we found that MOEA performance with a real application problem, e.g., the modular strain design problem, does not always correlate with artificial benchmarks. Overall, MOEAs provide powerful tools to solve the modular cell design problem for novel biocatalysis.

Direct Writing of Tunable Living Inks for Bioprocess Intensification.

Critical to the success of three-dimensional (3D) printing of living materials with high performance is the development of new ink materials and 3D geometries that favor long-term cell functionality. Here we report the use of freeze-dried live cells as the solid filler to enable a new living material system for direct ink writing of catalytically active microorganisms with tunable densities and various self-supporting porous 3D geometries. Baker's yeast was used as an exemplary live whole-cell biocatalyst, and the printed structures displayed high resolution, large scale, high catalytic activity and long-term viability. An unprecedented high cell loading was achieved, and cell inks showed unique thixotropic behavior. In the presence of glucose, printed bioscaffolds exhibited increased ethanol production compared to bulk counterparts due largely to improved mass transfer through engineered porous structures. The new living materials developed in this work could serve as a versatile platform for process intensification of an array of bioconversion processes utilizing diverse microbial biocatalysts for production of high-value products or bioremediation applications.

Growth of engineered Pseudomonas putida KT2440 on glucose, xylose, and arabinose: Hemicellulose hydrolysates and their major sugars as sustainable carbon sources

AbstractLignocellulosic biomass is the most abundant bioresource on earth containing polymers mainly consisting of d‐glucose, d‐xylose, l‐arabinose, and further sugars. In order to establish this alternative feedstock apart from applications in food, we engineered Pseudomonas putida KT2440 as microbial biocatalyst for the utilization of xylose and arabinose in addition to glucose as sole carbon sources. The d‐xylose‐metabolizing strain P. putida KT2440_xylAB and l‐arabinose‐metabolizing strain P. putida KT2440_araBAD were constructed by introducing respective operons from Escherichia coli. Surprisingly, we found out that both recombinant strains were able to grow on xylose as well as arabinose with high cell densities and growth rates comparable to glucose. In addition, the growth characteristics on various mixtures of glucose, xylose, and arabinose were investigated, which demonstrated the efficient co‐utilization of hexose and pentose sugars. Finally, the possibility of using lignocellulose hydrolysate as substrate for the two recombinant strains was verified. The recombinant P. putida KT2440 strains presented here as flexible microbial biocatalysts to convert lignocellulosic sugars will undoubtedly contribute to the economic feasibility of the production of valuable compounds derived from renewable feedstock.