Enzyme and Microbial Technology最新文献

Chemical modification and immobilization of cellulase for simultaneous pretreatment and saccharification of lignocellulosic biomass in ternary deep eutectic solvent

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2025-03-23 DOI: 10.1016/j.enzmictec.2025.110638

Yue Zhang , Chi Lei , Liangzhi Li , Bin Zou , Tao Xu , Lishi Yan

{"title":"Chemical modification and immobilization of cellulase for simultaneous pretreatment and saccharification of lignocellulosic biomass in ternary deep eutectic solvent","authors":"Yue Zhang , Chi Lei , Liangzhi Li , Bin Zou , Tao Xu , Lishi Yan","doi":"10.1016/j.enzmictec.2025.110638","DOIUrl":"10.1016/j.enzmictec.2025.110638","url":null,"abstract":"<div><div>In this study, chemical modification coupled with immobilization of cellulase was developed for simultaneous pretreatment and saccharification (SPS) of lignocellulosic biomass in ternary deep eutectic solvent (DES). Cellulase was firstly modified with acid anhydrides to form citraconic anhydride modified cellulase (CA-Ce) and phthalic anhydride modified cellulase (PA-Ce), respectively. Then the modified cellulases were immobilized on amino-functionalized mesoporous silica nanoparticles (MSN-NH<sub>2</sub>) to fabricate CA-Ce@MSN-NH<sub>2</sub> and PA-Ce@MSN-NH<sub>2</sub>. A series of physiochemical characterizations were employed to characterize modified cellulases or immobilization carriers. Thermodynamic analysis and deactivation kinetics in ternary DES (NMMO/Bet/OA) revealed that CA-Ce@MSN-NH<sub>2</sub> had desired thermal stability and solvent tolerance, which maintained 80.5 % initial activity at 70 °C and 77.8 % initial activity in 50 % NMMO/Bet/OA. The SPS of bagasse in NMMO/Bet/OA via CA-Ce@MSN-NH<sub>2</sub> resulted in 84.1 % total reducing sugar (TRS) yield at optimal condition.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"188 ","pages":"Article 110638"},"PeriodicalIF":3.4,"publicationDate":"2025-03-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143725344","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Simultaneous production of linear α-olefins and 2,5-furandicarboxylic acid by combining two recombinant enzymes OleT-ELP and HMFO-ELP

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2025-03-22 DOI: 10.1016/j.enzmictec.2025.110637

Yaqi Fu , Siyu Mao , Tianyue Liao , Wei Feng

引用次数: 0

Alcalase immobilization in iota-carrageenan-matrix hydrogel beads derived from the macroalga Solieria filiformis 将钙化酶固定在由大型藻类丝状藻（Solieria filiformis）提取的藻胶基质水凝胶珠中

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2025-03-22 DOI: 10.1016/j.enzmictec.2025.110636

Alan Portal D'Almeida , Luciana Rocha Barros Gonçalves , Tiago Lima de Albuqueque da Silva , Roberto Fernandez-Lafuente , Ivanildo José da Silva Jr.

{"title":"Alcalase immobilization in iota-carrageenan-matrix hydrogel beads derived from the macroalga Solieria filiformis","authors":"Alan Portal D'Almeida , Luciana Rocha Barros Gonçalves , Tiago Lima de Albuqueque da Silva , Roberto Fernandez-Lafuente , Ivanildo José da Silva Jr.","doi":"10.1016/j.enzmictec.2025.110636","DOIUrl":"10.1016/j.enzmictec.2025.110636","url":null,"abstract":"<div><div>This study aims to immobilize <em>Bacillus licheniformis</em> (Alcalase) protease in iota-carrageenan (ιCAR) matrix hydrogels via adsorption. CAR was extracted from macroalgae <em>Solieria filiformis</em> and used to produce hydrogels using Al<sup>3 +</sup> as the gelling agent. Subsequently, enzyme immobilization was performed at 25ºC, for 120 min using particles of ∼2.0 mm diameter, varying the medium pH values (7.0, 8.0, and 9.0). The immobilization at pH 8.0 resulted in the biocatalyst with the highest immobilization yield (100 %), expressed activity (88.9 %), and mass activity (10.4 U/g) for 1.0 mg/g of enzyme loading. When using particles with different diameters (1.0, 2.0, and 3.0 mm), the best results were obtained using 1.0 mm particles. This permitted a 100 % immobilization yield, 95.8 % expressed activity, and high mass activity (11.2 U/g). The lyophilized biocatalyst presented varying macro-pore diameters, ranging from 21 to 126 µm. The immobilized biocatalyst was 11 times more stable than the soluble enzyme at 60ºC and pH 8.0 and presented > 80 % retained activity in the pH range 6.0–9.0.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"188 ","pages":"Article 110636"},"PeriodicalIF":3.4,"publicationDate":"2025-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143704153","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Catalytic tunnel engineering of thermostable endoglucanase of GH7 family (W356C) from Aspergillus fumigatus gains catalytic rate

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2025-03-21 DOI: 10.1016/j.enzmictec.2025.110632

Musaddique Hossain, Subba Reddy Dodda, Shalini Das, Kaustav Aikat, Sudit S. Mukhopadhyay

{"title":"Catalytic tunnel engineering of thermostable endoglucanase of GH7 family (W356C) from Aspergillus fumigatus gains catalytic rate","authors":"Musaddique Hossain, Subba Reddy Dodda, Shalini Das, Kaustav Aikat, Sudit S. Mukhopadhyay","doi":"10.1016/j.enzmictec.2025.110632","DOIUrl":"10.1016/j.enzmictec.2025.110632","url":null,"abstract":"<div><div>Tunnel engineering targets the access tunnels in enzymes, which is crucial for substrate binding and product release. Modifying the tunnels can lead to better biomass-degrading abilities of the lignocellulolytic enzymes. In this report, we have engineered the thermostable GH7 family endoglucanase from <em>Aspergillus fumigatus</em> (<em>Af</em>Egl7). The residues in the open tunnel having the highest bottleneck radius are mutated. Mutations are created (T229F, W356C) in the non-conserved region. The mutant W356C showed a 2-fold increase in product release rate (V<sub>max</sub> = 375.8 µM/min) and 2.5-fold higher catalytic activity (K<sub>cat</sub> = 75.1 min<sup>−1</sup>) compared to wild-type (V<sub>max</sub>= 232 µM/min; K<sub>cat</sub> = 30.9 min<sup>−1</sup>) using CM cellulose as substrate. The mutant T229F lost both catalytic activity and thermostability. Molecular dynamic simulations and docking studies of W356C revealed a change in structure near the product exit region, which may facilitate faster product release and account for the increased catalytic efficiency of the mutant. This study showed how redesigning the access pathways can be a promising strategy for protein engineering and <em>de novo</em> protein design by tailoring the open tunnel geometry to a ligand-specific manner.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"187 ","pages":"Article 110632"},"PeriodicalIF":3.4,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143697986","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Biodegradation of crude oil by newly enriched biosurfactant-producing bacterial consortium

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2025-03-21 DOI: 10.1016/j.enzmictec.2025.110635

Jinhui Liu , Yuke kong , Junchao Pan , Mengjiao Qiao , Xinling Ruan , Yangyang Wang

{"title":"Biodegradation of crude oil by newly enriched biosurfactant-producing bacterial consortium","authors":"Jinhui Liu , Yuke kong , Junchao Pan , Mengjiao Qiao , Xinling Ruan , Yangyang Wang","doi":"10.1016/j.enzmictec.2025.110635","DOIUrl":"10.1016/j.enzmictec.2025.110635","url":null,"abstract":"<div><div>Crude oil contamination in different environmental media is a global environmental problem, biodegradation is a potential, environmentally friendly method for remediating this pollutant. In the present study, a biosurfactant-producing and crude oil degrading bacterial consortium (S1) was enriched from a contaminated soil, and its degradation efficiency of crude oil in solution and soil under the optimum conditions was studied. The results showed that the predominant species of S1 were <em>Pseudomonadaceae</em> and <em>Alcaligenaceae</em>. S1 could produce surfactant, with the maximum content of 2.27 g/L, which was identified as rhamnolipids. The optimal pH, temperature, and (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> concentration for crude oil degradation were 7.0, 40 °C, and 3 g/L, respectively, with the maximum degradation efficiency of 51.51 % after 7 days incubation. Plackett-Burman experiment and response surface methodology demonstrated that Cu, Co, and Zn could significantly promote the degradation of crude oil, with their optimum concentration of 0.36, 0.88, and 0.60 mg/L, respectively. Under the optimum conditions, the highest crude oil degradation efficiency reached 53.23 % within 7 days. Kinetic analysis showed that the first-order reaction kinetic was suitable for describing the degradation of crude oil by S1, with a half-life of 4.57 days. Furthermore, S1 also could degrade the crude oil in soil efficiently, with the maximum degradation efficiency of 60.34 % within 56 days. These results indicate that S1 has great potential for practical application in remediation of crude oil contamination.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"187 ","pages":"Article 110635"},"PeriodicalIF":3.4,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143697984","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Establishing a novel pathway for the biosynthesis of nicotinamide mononucleotide.

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2025-03-21 DOI: 10.1016/j.enzmictec.2025.110633

Rongchen Feng, Ziting Yan, Guoguang Wei, Chaoqiang Wu, Feifei Chen, Alei Zhang, Sheng Xu, Xin Wang, Kequan Chen

{"title":"Establishing a novel pathway for the biosynthesis of nicotinamide mononucleotide.","authors":"Rongchen Feng, Ziting Yan, Guoguang Wei, Chaoqiang Wu, Feifei Chen, Alei Zhang, Sheng Xu, Xin Wang, Kequan Chen","doi":"10.1016/j.enzmictec.2025.110633","DOIUrl":"https://doi.org/10.1016/j.enzmictec.2025.110633","url":null,"abstract":"<p><p>Nicotinamide mononucleotide (NMN) is a pivotal molecule within the realm of metabolic health, serving as a precursor to nicotinamide adenine dinucleotide (NAD<sup>+</sup>), a critical coenzyme in cellular energy metabolism. In recent years, the biological production of NMN has garnered significant interest. In this study, we developed the novel NRK-dependent synthesis routes for NMN production. Two strategies were designed to supply D-ribose-1-phosphate (R-1-P): (1) phosphorylation of exogenous D-ribose to ribose-5-phosphate (R-5-P) using engineered ribokinase (RK), followed by isomerization to R-1-P; (2) R-5-P synthesis from glucose through the pentose phosphate pathway. An optimized in vitro multi-enzyme cascade (XapA/PNP/NRK, PPM, NRK) identified NRK as the most efficient catalyst for NMN biosynthesis from D-ribose and niacinamide. In Escherichia coli, overexpression of this cascade, knockout of competing pathways, and secretion enhancement via a pelB signal peptide-fused PnuC transporter achieved an NMN titer of 62.0 mg L<sup>-</sup>¹ .This work provides a viable alternative for the biosynthesis of NMN.</p>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":" ","pages":"110633"},"PeriodicalIF":3.4,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143709236","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Characterization of a novel short-chain dehydrogenase from Sugiyamaella lignohabitans and its application in the degradation of patulin

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2025-03-20 DOI: 10.1016/j.enzmictec.2025.110630

Wei Xu , Jiayi Yao , Chenyu Song , Ting Xue , Wanmeng Mu

{"title":"Characterization of a novel short-chain dehydrogenase from Sugiyamaella lignohabitans and its application in the degradation of patulin","authors":"Wei Xu , Jiayi Yao , Chenyu Song , Ting Xue , Wanmeng Mu","doi":"10.1016/j.enzmictec.2025.110630","DOIUrl":"10.1016/j.enzmictec.2025.110630","url":null,"abstract":"<div><div>Patulin (PAT) is a widespread and hazardous fungal toxin that is commonly found in fruits and grain crops, threatening global food safety. Increasing attention has been focused on biological approaches for PAT decontamination in recent years. This study successfully identified a short-chain dehydrogenase with PAT-degrading activity derived from <em>Sugiyamaella lignohabitans</em> and made a preliminary characterization of the recombinant enzyme <em>Suli</em>-SDR. The <em>Suli</em>-SDR displayed the optimal activity at pH 7.0 and 80 °C, indicating a good activity when applied in the pasteurization procedure of the apple juice. <em>Suli</em>-SDR with a final concentration of 1 μg/mL could degrade 88 % of PAT (50 μg/mL) within 12 h, revealing its superior degradation performance. After measuring the parameters related to the quality of apple juice, including vitamin C, titratable acids, total phenols, etc., it was found that there was no significant effect on the parameters of apple juice after enzymatic treatment by <em>Suli</em>-SDR. In a short summary, this study evaluated the catalytic ability of <em>Suli</em>-SDR and explored the feasibility of enzyme on PAT control in practical production.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"187 ","pages":"Article 110630"},"PeriodicalIF":3.4,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143680587","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

In-vitro optimization and active-site mutagenesis of CYP105D18 peroxygenase enhance the production of indigo

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2025-03-18 DOI: 10.1016/j.enzmictec.2025.110634

Bashu Dev Pardhe , HyunA Park , Prakash Paudel , Jaeho Jeong , Tae-Jin Oh , Kwon-Young Choi , Jungoh Ahn

{"title":"In-vitro optimization and active-site mutagenesis of CYP105D18 peroxygenase enhance the production of indigo","authors":"Bashu Dev Pardhe , HyunA Park , Prakash Paudel , Jaeho Jeong , Tae-Jin Oh , Kwon-Young Choi , Jungoh Ahn","doi":"10.1016/j.enzmictec.2025.110634","DOIUrl":"10.1016/j.enzmictec.2025.110634","url":null,"abstract":"<div><div>Practical implementation of efficient biocatalysts for large-scale production of indigo remains challenging. Microbial cytochrome P450s may be useful for indigo production, but this has been rarely reported. We discovered that CYP105D18 catalysed H<sub>2</sub>O<sub>2</sub>-mediated C-3 hydroxylation of indole to synthesize indigo. A cell-free lysate from <em>Escherichia coli</em> containing CYP105D18 peroxygenase obtained after cell disruption was optimized for <em>in vitro</em> reaction. Next, 250 µM hydroxylamine was added to the cell-free lysate to inhibit other H<sub>2</sub>O<sub>2</sub>-utilizing enzymes that interfere with the CYP105D18 function. Furthermore, the active-site residues of CYP105D18, namely L87, A235, A282, and I386, involved in indole binding were mutated. L87F resulted in an approximately 12-fold increase in CYP105D18 activity. The catalytic efficiencies of the wild-type and L87F mutant were 0.01 and 0.12 mM<sup>−1</sup>min<sup>−1</sup>, respectively. Fed-batch fermentation using enriched autoinduction medium was used for higher production of <em>E. coli</em> cells containing CYP105D18 peroxygenase. The Cell-free lysate of disrupted cells yielded 710 mg/L of indigo in 20 min. This represents a simple enzymatic approach for indigo biosynthesis using cell-free lysate of <em>E. coli</em> overexpressing CYP105D18, H<sub>2</sub>O<sub>2</sub>, and catalase inhibitor without the need for multi enzyme systems and expensive cofactors. This single-enzyme system, used in a rapid process for indigo formation, could serve as an efficient approach for commercial bio-indigo production.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"187 ","pages":"Article 110634"},"PeriodicalIF":3.4,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143680646","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A novel GH12 xyloglucanase from the white rot fungus Abortiporus biennis, synergistically enhances lignocellulose saccharification by commercial cellulases

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2025-03-12 DOI: 10.1016/j.enzmictec.2025.110628

Despoina Panoraia Bakouli , Elisavet Pedi , Nikolaos Labrou , Evangelos Topakas , Anastasia Zerva

{"title":"A novel GH12 xyloglucanase from the white rot fungus Abortiporus biennis, synergistically enhances lignocellulose saccharification by commercial cellulases","authors":"Despoina Panoraia Bakouli , Elisavet Pedi , Nikolaos Labrou , Evangelos Topakas , Anastasia Zerva","doi":"10.1016/j.enzmictec.2025.110628","DOIUrl":"10.1016/j.enzmictec.2025.110628","url":null,"abstract":"<div><div>Xyloglucan is a complex, highly substituted plant biomass polysaccharide, which is largely overlooked in the design of enzyme cocktails for lignocellulose saccharification, due to its presence in specific plant tissues only, and its low content. Thus, the microbial mechanisms for its degradation have not been thoroughly studied. However, in the frame of the biorefinery concept, xyloglucan monomers also have to be utilized for the design of efficient bioprocesses. Moreover, in plant tissues, xyloglucan often covers cellulose fibrils, impeding the access of cellulases. In order to shed light on the enzymatic degradation of xyloglucan, a novel GH12 family xyloglucanase was studied, from the basidiomycete <em>Abortiporus biennis</em>. The enzyme was heterologously produced in <em>Pichia pastoris</em>, purified and characterized. <em>Abi</em>Xeg12a is a 28 kDa glycoprotein, with relatively strict substrate specificity, since it is only active in xyloglucan and β-glucan. The main hydrolysis products are the oligomers XXXG, XLXG/XXLG, XLLG and the optimum activity conditions are pH 4.5 and 55 °C. The enzyme contributes to the saccharification of corn bran and apple pulp by a commercial cellulase preparation, increasing the release of reducing sugars by up to 39 % and 18 %, respectively, while the addition of <em>Abi</em>Xeg12a can minimize the enzyme load of the reaction, at least for apple pulp, without loss in reducing sugar yield. Overall, the importance of xyloglucanases on the saccharification of xyloglucan-containing substrates was demonstrated in this study. The results could contribute to the design of more efficient, tailor-made enzyme cocktails for the saccharification and subsequent valorization of lignocellulose.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"187 ","pages":"Article 110628"},"PeriodicalIF":3.4,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143642329","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A metabolic-engineering framework approach via fed-batch fermentation for enhancing glucaric acid production in Komagataella phaffii

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2025-03-12 DOI: 10.1016/j.enzmictec.2025.110627

Jayachandran Krishna , Kabilan Subash Chandra Bose , Sindhu Varadharaj , Meenakshisundaram Sankaranarayanan

{"title":"A metabolic-engineering framework approach via fed-batch fermentation for enhancing glucaric acid production in Komagataella phaffii","authors":"Jayachandran Krishna , Kabilan Subash Chandra Bose , Sindhu Varadharaj , Meenakshisundaram Sankaranarayanan","doi":"10.1016/j.enzmictec.2025.110627","DOIUrl":"10.1016/j.enzmictec.2025.110627","url":null,"abstract":"<div><div>Glucaric acid (D-saccharic acid) is an organic compound belonging to glucuronic acid derivatives, whose commercial synthesis involves the use of hazardous solvents. Biosynthetic production in <em>Saccharomyces cerevisiae</em> has limitations, such as ethanolic fermentation, redox strategy limitations, and low pH toxicity. <em>Komagataella phaffii</em> (<em>K. phaffii</em>) formly known <em>Pichia pastoris</em>, an alternative and robust engineerable organism, is a promising biotransformation agent for glucaric acid production. However, <em>K. phaffii</em> lacks native biosynthetic pathways for glucaric acid synthesis at the industrial scale. There is no proof-of-concept glucaric acid production system. Therefore, gene expression profiling-based metabolic engineering of glucaric acid producing gene cassette was performed using in-fusion cloning. Product production was enhanced using fed-batch fermentation of the key metabolite, myo-inositol; this improved the yield of glucaric acid. The expression was optimized through cofactor recycling and codon optimization for the UDH gene. Fed-batch fermentation with mixed supplementation (Myo-inositol + Monosodium glutamate) as substrate in engineered <em>K. phaffii</em> (X33-GA) enhanced glucaric acid synthesis to 17.6 g/L. In addition, we present simple HPLC and LC-MS techniques for quantifying glucaric acid and its precursors in the fermentation samples. The proof-of-concept results from both shake flask and bioreactor studies provide a unique perspective on sustainable, cost-effective, and green technological alternatives for glucaric acid synthesis.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"187 ","pages":"Article 110627"},"PeriodicalIF":3.4,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143642330","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0