Hydrolysis Of Lignocellulosic Biomass Research Articles

Production of 5-Hydroxymethylfurfural from Hydrolysate of pretreated lignocellulosic biomass in a biphasic system reactor

Production of 5-Hydroxymethylfurfural from Hydrolysate of pretreated lignocellulosic biomass in a biphasic system reactor

Catalytic pyrolysis of hydrolyzed lignin using HZSM-5/MCM-41 supported transition-metal to produce monocyclic aromatic hydrocarbons

Hydrolyzed lignin (HL), one kind of organic solid wastes, comes from the production of hydrolysis of lignocellulosic biomass for high-value chemicals, unsuitable treatment of which not only meaning the waste of resources but also resulting environmental pollution. The pyrolysis of HL has the problem of high oxygen-containing compounds and low hydrocarbons in the products. The HZSM-5/MCM-41 (HM) meso-microporous composite catalyst, containing nickel and molybdenum, was synthesized through impregnation to enhance the production of monocyclic aromatic hydrocarbons (MAHs) during the in-situ catalytic pyrolysis of hydrolyzed lignin. Different transition metals loaded on composite molecular sieves were prepared for comparison. The relationship between catalyst structure and pyrolysis efficiency was studied based on characterization techniques including SEM, BET, and XRD. This study investigated the catalytic properties of modified composite molecular sieves with Ni and Mo at different loaded ratios on the pyrolysis of hydrolyzed lignin. Results indicated that HM loaded with different metals had different catalytic pyrolysis selectivity. The relative yield of MAHs on 3 %Ni-HM was the highest, at 27.78 %, while that of phenolic on 5 %Mo-HM was 51.02 %. Thus, by modifying the co-loading ratios between Ni and Mo, the combined catalytic pyrolysis of hydrolyzed lignin was performed, achieving a 39.24 % yield of MAHs on the catalyst Mo1Ni2-HM, markedly surpassing the 17.69 % yield from unaltered HZSM-5/MCM-41. In addition, the improvement of active sites was caused by transition metals loaded and the synergistic effect between bimetals enhanced the catalytic activity and stability. HZSM-5/MCM-41 loaded with metal oxides promoted the depolymerization of macromolecules in the intermediate products of pyrolysis, and thus improved the relative yield of MAHs among pyrolysis products of hydrolyzed lignin.

Microbial production of levulinic acid from glucose by engineered Pseudomonas putida KT2440

Levulinic acid(LA) is produced through acid-catalyzed hydrolysis and dehydration of lignocellulosic biomass. It is a key platform chemical used as an intermediate in various industries including biofuels, cosmetics, pharmaceuticals, and polymers. Traditional LA production uses chemical conversion, which requires high temperatures and pressures, strong acids, and produces undesirable side reactions, repolymerization products, and waste problems Therefore, we designed an integrated process to produce LA from glucose through metabolic engineering of Pseudomonas putida KT2440. As a metabolic engineering strategy, codon optimized phospho-2-dehydro-3-deoxyheptonate aldolase (AroG), 3-dehydroshikimate dehydratase (AsbF), and acetoacetate decarboxylase (Adc) were introduced to express genes of the shikimate and β-ketoadipic acid pathways, and the 3-oxoadipate CoA-transferase (pcaIJ) gene was deleted to prevent loss of biosynthetic intermediates. To increase the accumulation of the produced LA, the lva operon encoding levulinyl-CoA synthetase (LvaE) was deleted resulting in the high LA-producing strain P. putida HP203. Culture conditions such as medium, temperature, glucose concentration, and nitrogen source were optimized, and under optimal conditions, P. putida HP203 strain biosynthesized 36.3 mM (4.2 g/L) LA from glucose in a fed-batch fermentation system. When lignocellulosic biomass hydrolysate was used as the substrate, this strain produced 7.31 mM of LA. This is the first report of microbial production of LA from glucose by P. putida. This study suggests the possibility of manipulating biosynthetic pathway to produce biological products from glucose for various applications.

Understanding the role of porous particle characteristics and water state distribution at multi-scale variation on cellulase hydrolysis of lignocellulosic biomass

Porous particle characteristics of lignocellulosic biomass and the water state distribution surrounding particles determine enzyme/solute transfer, while inherently affecting the liquefaction and hydrolysis reactions. For submillimeter biomass particles (<500 μm), deep eutectic solvent pretreatment tended to create additional macropores (∼4%, pore volume of 4.3 mL/g) and micropores (∼3%, pore area of 2.26 m2/g) at the extragranular and cell wall scales, simultaneously with reducing particle size by 15%. The pretreated biomass exhibited preferential rapid liquefaction over sugar production, leading to a ∼70% reduction in particle size only after 3 h hydrolysis. This further increased particle porosity (∼85% after 12 h hydrolysis) with the transition from large surface-pores to interior micropores. The increases in the tissue/cell-scale pore size and intraparticle area had greater significance for improving hydrolysis performance than particle size reduction. Additionally, particle liquefaction released constrained water in capillary pores and enhanced slurry fluidity; capillary water and free water became main transfer carriers during the hydrolysis. Free water activity was positively related to sugar production (r = 0.80–0.85), and integrating it with particle component and porosity allowed for accurate prediction of hydrolysis yields (R2 = 0.98–0.99). Inspired by particle liquefaction, a short-time feeding strategy was proposed to reconstruct high-solid hydrolysis.

Sulfonated carbon–based heterogeneous acid catalysts in direct biomass redox flow fuel cell: A review

AbstractThis review focused on the potential applications of sulfonated carbon–based heterogeneous acid catalysts for the hydrolysis of lignocellulosic biomass (LCB) fuel feedstock and the development of membrane electrode assembly (MEA) for the direct biomass redox flow fuel cell (DBRFFC). LCBs are hydrolysed to yield simple sugars, which are subsequently oxidized over catalysts in the anode tank of the DBRFFC to generate electricity. Ferric chloride used as a catalyst in the DBRFFC is not efficient for glucose production from LCB, such that the power performance of the DBRFFC is affected during glucose oxidation due to low glucose yield from LCB for electron generation. Sulfonated carbon–based solid acid catalysts (SCSACs) have been established as efficient catalysts for the hydrolysis of LCB and as metal catalyst supports for the fabrication of MEA for further oxidation of glucose and its oxidation by‐products. These capabilities of SCSACs can be explored and applied to significantly improve the power output of the DBRFFC through efficient hybrid catalyst design. There is still a scarcity of literature on this subject and their combination with ferric chloride to enhance glucose yield and oxidation in DBRFFC. This gap was filled by discussing the various types of sulfonated carbon–based catalysts, highlighting their synthesis routes, and their applications in organic compound synthesis, and membrane electrode development in DBRFFC. The knowledge derived will certainly be beneficial to researchers willing to improve the performance of DBRFFC through molecular catalyst design and electrode membrane development for application in DBRFFC.

Transgressive phenotypes from outbreeding between the Trichoderma reesei hyper producer RutC30 and a natural isolate.

The filamentous fungus Trichoderma reesei produces cellulolytic enzymes that are essential for the hydrolysis of lignocellulosic biomass into monomerics sugars. The filamentous fungus T. reesei produces cellulolytic enzymes that are essential for the hydrolysis of lignocellulosic biomass into monomerics sugars, which can in turn be fermented to produce second-generation biofuels and bioproducts. Production performance improvement, which is essential to reduce production cost, relies on classical mutagenesis and genetic engineering techniques. Although sexual reproduction is a powerful tool for improving domesticated species, it is often difficult to apply to industrial fungi since most of them are considered asexual. In this study, we demonstrated that outbreeding is an efficient strategy to optimize T. reesei. Crossing between a natural isolate and a mutagenized strain generated a biodiverse progeny with some offspring displaying transgressive phenotype for cellulase activities.

Effects of Sisal Saponins on Enzymatic Hydrolysis and Fermentation of Lignocellulosic Biomass

Effects of Sisal Saponins on Enzymatic Hydrolysis and Fermentation of Lignocellulosic Biomass

Enhanced Saccharification Yield of Alkali Pretreated Sugarcane Bagasse Utilizing Customized Cellulase Cocktail from Trichoderma harzianum and Trichoderma viride

The experiment was conducted during 2023 at Bioenergy Laboratory, Department of RBEE, College of Agricultural Engineering and Technology, CCSHAU, Hisar, Haryana, India. Compositional changes in sugarcane bagasse subjected to varying concentrations of sodium hydroxide (0.3% to 1.2%), revealing significant increases in glucan content (from 37.13% to 53.81%) alongside decreases in xylan, acid-insoluble lignin, acid-soluble lignin, ash, and other extractives. These changes were validated using microscopic technique SEM, confirming the efficacy of the pretreatment process. The utilization of a customized cellulase cocktail derived from Trichoderma harzianum and Trichoderma viride holds significant promise in enhancing the saccharification from alkali-pretreated sugarcane bagasse. This study investigates the synergistic effects of cellulase enzymes produced by these fungi on the hydrolysis of lignocellulosic biomass. The enzymatic hydrolysis process is optimized by varying enzyme dosages, reaction conditions, and incubation times to maximize the release of fermentable sugars. Results indicate a substantial improvement in saccharification efficiency with the customized cellulase cocktail, highlighting its potential for sustainable biofuel production. The pretreated sugarcane bagasse, when saccharified with Trichoderma harzianum and Trichoderma viride individually, released 254.43 mg g-1 and 325.53 mg g-1 of reducing sugars, respectively, after 40 h of incubation. In contrast, the combined enzymatic cocktail achieved a substantial increase in glucose yields (345.12 mg g-1) at 40 h, showcasing the synergistic effect of the combined enzymatic activity. This research contributes to advancing bioconversion technologies for utilizing lignocellulosic biomass resources efficiently and economically, thus addressing key challenges in sustainable energy production.

Recombinant GH3 β-glucosidase stimulated by xylose and tolerant to furfural and 5-hydroxymethylfurfural obtained from Aspergillus nidulans

The β-glucosidase gene from Aspergillus nidulans FGSC A4 was cloned and overexpressed in the A. nidulans A773. The resulting purified β-glucosidase, named AnGH3, is a monomeric enzyme with a molecular weight of approximately 80 kDa, as confirmed by SDS-PAGE. Circular dichroism further validated its unique canonical barrel fold (β/α), a feature also observed in the 3D homology model of AnGH3. The most striking aspect of this recombinant enzyme is its robustness, as it retained 100% activity after 24 h of incubation at 45 and 50 ºC and pH 6.0. Even at 55 °C, it maintained 72% of its enzymatic activity after 6 h of incubation at the same pH. The kinetic parameters Vmax, KM, and Kcat/KM for ρ-nitrophenyl-β-D-glucopyranoside (ρNPG) and cellobiose were also determined. Using ρNPG, the enzyme demonstrated a Vmax of 212 U mg − 1, KM of 0.0607 mmol L − 1, and Kcat/KM of 4521 mmol L − 1 s − 1 when incubated at pH 6.0 and 65 °C. The KM, Vmax, and Kcat/KM using cellobiose were 2.7 mmol L − 1, 57 U mg − 1, and 27 mmol –1 s − 1, respectively. AnGH3 activity was significantly enhanced by xylose and ethanol at concentrations up to 1.5 mol L − 1 and 25%, respectively. Even in challenging conditions, at 65 °C and pH 6.0, the enzyme maintained its activity, retaining 100% and 70% of its initial activity in the presence of 200 mmol L − 1 furfural and 5-hydroxymethylfurfural (HMF), respectively. The potential of this enzyme was further demonstrated by its application in the saccharification of the forage grass Panicum maximum, where it led to a 48% increase in glucose release after 24 h. These unique characteristics, including high catalytic performance, good thermal stability in hydrolysis temperature, and tolerance to elevated concentrations of ethanol, D-xylose, furfural, and HMF, position this recombinant enzyme as a promising tool in the hydrolysis of lignocellulosic biomass as part of an efficient multi-enzyme cocktail, thereby opening new avenues in the field of biotechnology and enzymology.

Xylitol production by a Wickerhamomyces anomalus strain adapted for enhanced tolerance to sugarcane bagasse hemicellulosic hydrolysate with high content of fermentation inhibitors

BackgroundXylitol, a five-carbon polyalcohol, is used in the food and pharmaceutical industries and as a building block in the synthesis of high-value chemicals. It can be sustainably produced from renewable sources through xylose assimilating microbe fermentation. ResultsWe screened microbial strains for xylitol production and identified Wickerhamomyces anomalus Z1 as a key xylitol producer. Utilizing lignocellulosic biomass hydrolysates for xylitol production poses challenges due to microbial sensitivity to inhibitors from biomass pre-treatment. In this study, an adaptive laboratory evolution (ALE) of W. anomalus Z1 was performed by culturing the yeast in a mineral medium supplemented with gradual increases of sugarcane bagasse hemicellulosic hydrolysate (SCHH) obtained by intensified steam explosion pretreatment. The performance of the adapted yeast, named Wickerhamomyces anomalus ALE, was assessed in comparison to the wild-type strain regarding its capacity to produce xylitol using SCHH. The evolved yeast reached a xylitol yield of 0.11 g xylitol/g xylose whereas the wild-type strain could not produce xylitol. Removing acetic acid from SCHH enhanced W. anomalus ALE performance, with optimal results at 75% hydrolyzed hemicellulose, yielding 0.44 g xylitol/g xylose and 13.41 g/L xylitol. ConclusionsThis study demonstrates the potential of W. anomalus ALE in successfully valorizing the hemicellulosic fraction of sugarcane bagasse for sustainable xylitol production.How to cite: Bonfiglio F, Cagno M, Nuñez L, et al. Xylitol production by a Wickerhamomyces anomalus strain adapted for enhanced tolerance to sugarcane bagasse hemicellulosic hydrolysate with high content of fermentation inhibitors. Electron J Biotechnol 2024;71. https://doi.org/10.1016/j.ejbt.2024.05.004.

Enzyme immobilization with nanomaterials for hydrolysis of lignocellulosic biomass: Challenges and future Perspectives

Enzyme immobilization has emerged as a prodigious strategy in the enzymatic hydrolysis of lignocellulosic biomass (LCB) promising enhanced efficacy and stability of the enzymes. Further, enzyme immobilization on magnetic nanoparticles (MNPs) facilitates the easy recovery and reuse of biocatalysts. This results in the development of a nanobiocatalytic system, that serves as an eco-friendly and inexpensive LCB deconstruction approach. This review provides an overview of nanomaterials used for immobilization with special emphasis on the nanomaterial-enzyme interactions and strategies of immobilization. After the succinct outline of the immobilization procedures and supporting materials, a comprehensive assessment of the catalysis enabled by nanomaterial-immobilized biocatalysts for the conversion and degradation of lignocellulosic biomasses is provided by gathering state-of-the-art examples. The challenges and future directions associated with this technique providing a potential solution in the present article. Insight on the recent advancements in the process of nanomaterial-based immobilization for the hydrolysis of lignocellulosic biomass has also been highlighted in the article.

Enhancing hydrolysis of lignocellulosic biomass through molecular modification of lytic polysaccharide monooxygenase from Aspergillus niger

Enhancing hydrolysis of lignocellulosic biomass through molecular modification of lytic polysaccharide monooxygenase from Aspergillus niger

Elucidation of enhanced cellulase immobilization onto synthetic magnetic nickel nanomaterials for lignocellulosic biomass hydrolysis

The immobilization of cellulase enzymes for the conversion of lignocellulosic biomass into sustainable biochemical products is essential for the stability and recovery of the enzymes. In this study, nickel nanoparticles (NiNPs) were synthesized and coated with 3-aminopropyl triethoxysilane (APTES) to serve as cellulase enzyme carriers. Cellulase enzyme immobilization on the prepared NiNPs was achieved using glutaraldehyde as a cross-linker. The physicochemical properties of the carrier were determined before and after enzyme immobilization using X-ray Powder Diffraction (XRD), Transmission Electron Microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), Vibrating-Sample Magnetometer (VSM), and Field Emission Scanning Electron Microscopy (FESEM). The impacts of different experimental factors on the performance of the cellulase enzymes immobilized on the synthesized NiNPs and commercial nickel nanoparticles were compared. The results showed comparable optimal cellulase enzyme immobilization conditions on both substrates in terms of the immobilization time, pH, and cellulase enzyme concentration. However, the recommended temperatures for cellulase enzyme immobilization on the synthesized and commercial NiNPs were 50 °C and 40 °C, respectively. Under these optimum conditions, the immobilized cellulase enzyme on the synthesized NiNPs had an activity of about 99.1% (in comparison to the activity of free cellulase enzyme), while the activity of the enzyme upon immobilization on commercial NiNPs was about 93%. The particle size of the NiNPs was found to be crucial for enzyme immobilization efficiency and its magnetic strength. Therefore, cellulase enzyme immobilization on tunable NiNPs could be a sustainable and eco-friendly approach towards high recovery of cellulose from lignocellulosic materials.

Effect of Hemicelluloses Fractionated by Graded Ethanol Precipitation from Corn Stover on the Enzymatic Hydrolysis of Lignocellulosic Biomass

Effect of Hemicelluloses Fractionated by Graded Ethanol Precipitation from Corn Stover on the Enzymatic Hydrolysis of Lignocellulosic Biomass

Recovery of sulfuric acid during sugar purification from pretreated wheat straw hydrolysate using electro-electrodialysis system for 2G bioethanol production

Concentrated acid hydrolysis (CAH) of lignocellulosic biomass (LCB) is an established method to obtain a near theoretical yield of monomeric sugars from the biomass. The CAH process produced 83.23 % of glucose from pretreated wheat straw (PWS) at optimum conditions (i.e., stage I = 74 % w/w acid, 1:2 biomass loading, and 50 °C for 1 h; and stage II = 30 % w/w acid, 90 °C for 2 h). However, the separation of sugar from acidic hydrolysate is a major constraint of this technology. In this study, an electro-electrodialysis (EED) process using a novel anion exchange membrane (AEM) has been developed to recover sulfuric acid from biomass hydrolysate. The synthetic hydrolysate (i.e., feed acid concentration = 2 M; glucose = 80 g L−1) was used to optimize the EED process parameters. The acid was successfully recovered from alkali pretreated wheat straw (PWS) hydrolysate using the most effective EED process parameters. This resulted in an 82.72 % acid recovery in the permeate under the following conditions: current density of 88 mA cm−2, flow rate of 3 L h−1, and feed acid content of 2 M. During the EED process, the highest glucose recovery in the feed was 90.98 %, achieved with a maximum current efficiency of 93.57 % and the lowest energy consumption of 0.052 kW h mol−1 at optimal conditions using PWS Hydrolysate. Saccharomyces cerevisiae fermented the acid-free PWS hydrolysate, resulting in a maximum ethanol yield of 92.3 %. The EED process cost of $0.049 kg H2SO4−1 and total revenue was $0.02 kg biomass−1 from the second batch onwards suggest the feasibility of the EED process for large-scale 2G bioethanol productions.

Application of Thermomyces lanuginosus polygalacturonase produced in Komagataella phaffii in biomass hydrolysis and textile bioscouring

In this work, the polygalacturonase (TL-PG1) from the thermophilic fungus Thermomyces lanuginosus was heterologously produced for the first time in the yeast Komagataella phaffii. The TL-PG1 was successfully expressed under the control of the AOX1 promoter and sequentially purified by His-tag affinity. The purified recombinant pectinase exhibited an activity of 462.6 U/mL toward polygalacturonic acid under optimal conditions (pH 6 and 55 ˚C) with a 2.83 mg/mL and 0.063 μmol/minute for Km and Vmax, respectively. When used as supplementation for biomass hydrolysis, TL-PG1 demonstrated synergy with the enzymatic cocktail Ctec3 to depolymerize orange citrus pulp, releasing 1.43 mg/mL of reducing sugar. In addition, TL-PG1 exhibited efficiency in fabric bioscouring, showing potential usage in the textile industry. Applying a protein dosage of 7 mg/mL, the time for the fabric to absorb water was 19.77 seconds (ten times faster than the control). Adding the surfactant Triton to the treatment allowed the reduction of the enzyme dosage by 50% and the water absorption time to 6.38 seconds. Altogether, this work describes a new versatile polygalacturonase from T. lanuginosus with the potential to be employed in the hydrolysis of lignocellulosic biomass and bioscouring.

Engineering yeast to convert lignocellulose into vanillin

Valorizing lignocellulosic biomass (LCB) into platform aromatic chemicals could contribute to a sustainable biomass-based bioeconomy. However, lacking simultaneous bioconversion route of lignin and carbohydrates hinders the LCB utilization. The artificial biological funneling pathway (ABFP) was hence designed to convert full components of LCB into vanillin. The results showed that combinational engineering strategies enabled the successful accumulation of vanillin in Saccharomyces cerevisiae. The funneling pathways of lignin-derived monomers to vanillin were constructed. Enzyme fusion and S-adenosylmethionine metabolism regulation of heterologous rate-limiting enzyme further promoted the vanillin production to 2.49 and 1.94 mmol/L from ferulic acid and p-coumaric acid, respectively. The construction of funneling pathways of hexose and pentose promoted the carbon flux towards vanillin biosynthesis. Together with the regulation of branch and shikimic acid pathways, the microbial cell factory with a novel ABFP produced 1.97 mmol/L vanillin from real LCB hydrolysates with a recorded yield of 10.5 mg vanillin/g carbon source. Overall, the engineered S. cerevisiae simultaneously valorized full components into fine aromatic chemicals, holding the promise to transform biorefinery mode and contribute to the biomass-based economy.

Mixotrophic Syngas Conversion Enables the Production of meso-2,3-butanediol with Clostridium autoethanogenum

Providing simultaneously autotrophic and heterotrophic carbon sources is a promising strategy to overcome the limits of autotrophic syngas fermentations. D-xylose and L-arabinose are particularly interesting as they can be obtained by the hydrolysis of lignocellulosic biomass. The individual conversion of varying initial concentrations of these pentoses and D-fructose as reference was studied with C. autoethanogenum in fully controlled stirred-tank reactors with a continuous syngas supply. All mixotrophic batch processes showed increased biomass and product formation compared to an autotrophic reference process. Simultaneous CO and D-xylose or L-arabinose conversion was observed in contrast to D-fructose. In the mixotrophic batch processes with L-arabinose or D-xylose, the simultaneous CO and sugar conversion resulted in high final alcohol-to-acid ratios of up to 58 g g−1. L-arabinose was superior as a mixotrophic carbon source because biomass and alcohol concentrations (ethanol and 2,3-butanediol) were highest, and significant amounts of meso-2,3-butanediol (&gt;1 g L−1) in addition to D-2,3-butanediol (&gt;2 g L−1) were solely produced with L-arabinose. Furthermore, C. autoethanogenum could not produce meso-2,3 butanediol under purely heterotrophic conditions. The mixotrophic production of meso-2,3-butanediol from L-arabinose and syngas, both available from residual lignocellulosic biomass, is very promising for use as a monomer for bio-based polyurethanes or as an antiseptic agent.

Hydrodynamic studies of innovative membrane reactor for enzymatic hydrolysis of lignocellulosic waste.

This paper presents the study concerning the impact of the basic operational parameters on the performance of an innovative microfiltration membrane reactor applied for enzymatic hydrolysis of lignocellulosic biomass. The concept and basic hydrodynamics of the reactor with tubular ceramic membranes and a propeller agitator were shown. Besides, the efficiency of enzymatic hydrolysis of corn straw was studied to check reactor functionality. It has been proven that the proposed reactor construction can improve the microfiltration of lignocellulosic suspension by reducing the cake layer on the membrane surface. Increasing the rotational speed of the propeller agitator also improved the filtration efficiency. The permeate flux during the microfiltration experiments was lower for smaller lignocellulose biomass fraction (D<425 μm) when compared to the less fragmented corn straw (425<D<900 μm). For larger solid fractions, a stirring speed increase enhanced the separation efficiency regardless of the differences in biomass concentration. In contrast, this trend for the finer biomass fraction was only noticeable for the highest used biomass concentration (C=2.0%). Considering the enzymatic hydrolysis of corn straw, membrane separation of reaction products positively influenced the process yield, and the results depended on the applied operational parameters.

Simultaneous glucose and xylose utilization by an Escherichia coli catabolite repression mutant.

As advances are made toward the industrial feasibility of mass-producing biofuels and commodity chemicals with sugar-fermenting microbes, high feedstock costs continue to inhibit commercial application. Hydrolyzed lignocellulosic biomass represents an ideal feedstock for these purposes as it is cheap and prevalent. However, many microbes, including Escherichia coli, struggle to efficiently utilize this mixture of hexose and pentose sugars due to the regulation of the carbon catabolite repression (CCR) system. CCR causes a sequential utilization of sugars, rather than simultaneous utilization, resulting in reduced carbon yield and complex process implications in fed-batch fermentation. A mutant of the gene encoding the cyclic AMP receptor protein, crp*, has been shown to disable CCR and improve the co-utilization of mixed sugar substrates. Here, we present the strain construction and characterization of a site-specific crp* chromosomal mutant in E. coli BL21 star (DE3). The crp* mutant strain demonstrates simultaneous consumption of glucose and xylose, suggesting a deregulated CCR system. The proteomics further showed that glucose was routed to the C5 carbon utilization pathways to support both de novo nucleotide synthesis and energy production in the crp* mutant strain. Metabolite analyses further show that overflow metabolism contributes to the slower growth in the crp* mutant. This highly characterized strain can be particularly beneficial for chemical production by simultaneously utilizing both C5 and C6 substrates from lignocellulosic biomass.IMPORTANCEAs the need for renewable biofuel and biochemical production processes continues to grow, there is an associated need for microbial technology capable of utilizing cheap, widely available, and renewable carbon substrates. This work details the construction and characterization of the first B-lineage Escherichia coli strain with mutated cyclic AMP receptor protein, Crp*, which deregulates the carbon catabolite repression (CCR) system and enables the co-utilization of multiple sugar sources in the growth medium. In this study, we focus our analysis on glucose and xylose utilization as these two sugars are the primary components in lignocellulosic biomass hydrolysate, a promising renewable carbon feedstock for industrial bioprocesses. This strain is valuable to the field as it enables the use of mixed sugar sources in traditional fed-batch based approaches, whereas the wild-type carbon catabolite repression system leads to biphasic growth and possible buildup of non-preferential sugars, reducing process efficiency at scale.