Enzyme and Microbial Technology最新文献

Engineering the D-lactic acid responsive promoter/repressor system as dynamic metabolic engineering tool in Lactobacillus delbrueckii subsp. bulgaricus for controlled D-lactic acid biosynthesis

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-02-07 DOI: 10.1016/j.enzmictec.2025.110606

Payal Mukherjee , Senthilkumar Sivaprakasam

Dynamic metabolic engineering integrates synthetic logic circuits into cellular systems, optimizing metabolic fluxes and augmenting biosynthesis of target metabolites. This study evaluated a D-lactic acid (DLA)-responsive promoter-repressor system from Pseudomonas fluorescens A506, re-engineered for heightened sensitivity and functional efficacy in Lactobacillus delbrueckii subsp. bulgaricus VI104. The codon-optimized regulatory architecture exhibited peak performance at DLA inducer concentration range of 60–100 mM, validated by fluorometry and microscopy. As an application, overexpression of D-lactate dehydrogenase (dldh) downstream of the engineered promoter repressor system enabled finely tuned modulation of DLA biosynthesis, autonomously regulating the transition between growth and production phases, thereby attenuating overall metabolic load. Cross-species compatibility was confirmed by excising regulatory elements from the promoter-repressor system and functionally assessing them in recombinant L. bulgaricus. Molecular docking elucidated critical noncovalent interactions between D-LldR repressor and operator nucleotide sequence in absence of inducer DLA. The engineered promoter construct with high efficiency effectively elevated DLA biosynthesis by 2.15-folds and expanded the overall fermentation time relative to constitutive systems, attaining maximum DLA titre of 9.02 g L⁻¹ in bioreactor setup. These results substantially broaden the molecular cloning toolkit available for L. bulgaricus, fostering potential future applications in biotherapeutics and probiotics.

{"title":"Engineering the D-lactic acid responsive promoter/repressor system as dynamic metabolic engineering tool in Lactobacillus delbrueckii subsp. bulgaricus for controlled D-lactic acid biosynthesis","authors":"Payal Mukherjee , Senthilkumar Sivaprakasam","doi":"10.1016/j.enzmictec.2025.110606","DOIUrl":"10.1016/j.enzmictec.2025.110606","url":null,"abstract":"<div><div>Dynamic metabolic engineering integrates synthetic logic circuits into cellular systems, optimizing metabolic fluxes and augmenting biosynthesis of target metabolites. This study evaluated a D-lactic acid (DLA)-responsive promoter-repressor system from <em>Pseudomonas fluorescens</em> A506, re-engineered for heightened sensitivity and functional efficacy in <em>Lactobacillus delbrueckii</em> subsp. <em>bulgaricus</em> VI104. The codon-optimized regulatory architecture exhibited peak performance at DLA inducer concentration range of 60–100 mM, validated by fluorometry and microscopy. As an application, overexpression of D-lactate dehydrogenase (<em>dldh</em>) downstream of the engineered promoter repressor system enabled finely tuned modulation of DLA biosynthesis, autonomously regulating the transition between growth and production phases, thereby attenuating overall metabolic load. Cross-species compatibility was confirmed by excising regulatory elements from the promoter-repressor system and functionally assessing them in recombinant <em>L. bulgaricus</em>. Molecular docking elucidated critical noncovalent interactions between D-<em>LldR</em> repressor and operator nucleotide sequence in absence of inducer DLA. The engineered promoter construct with high efficiency effectively elevated DLA biosynthesis by 2.15-folds and expanded the overall fermentation time relative to constitutive systems, attaining maximum DLA titre of 9.02 g L⁻<sup>1</sup> in bioreactor setup. These results substantially broaden the molecular cloning toolkit available for <em>L. bulgaricus</em>, fostering potential future applications in biotherapeutics and probiotics.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"185 ","pages":"Article 110606"},"PeriodicalIF":3.4,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143377871","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

Identification of 2’-deoxyguanosine for an α-amylase inhibitor in the extracts of the earthworm Eisenia fetida and characterization of its inhibitory activity against porcine pancreatic α-amylase

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-02-04 DOI: 10.1016/j.enzmictec.2025.110604

Masako Ogasawara , Katsuhiro Yoshii , Jun Wada , Yoshihiro Yamamoto , Kuniyo Inouye

Inhibitory activity (IA) against porcine pancreatic α-amylase (PPA) has been found in the water extract of Eisenia fetida. IA is recovered mostly in a fraction (U3EE) containing compounds of low molecular mass under 3 kDa. U3EE is treated with 85 % ethanol (EtOH) and the obtained EtOH extract is applied to the solid-phase extraction (SPE) system. An IA fraction named AI is separated from SPE by elution with water, and then a fraction AI* has been obtained by elution with 5 % EtOH. In the previous study, PPA inhibitors of guanine (Gua), guanosine (Guo), and inosine (Ino) have been isolated from AI. In the present study, IA of AI* was examined and a new inhibitor 2’-deoxyguanosine (2’-dGuo) was isolated by RP-HPLC. It was a mixed-type inhibitor showing IC₅₀ of 0.7 ± 0.1 mM and inhibitor constants K_i and K_i’ of 2.0 ± 0.5 and 0.9 ± 0.2 mM, respectively, at pH 6 and at 37 °C. PPA inhibitors of U3EE were arranged in the order according to the inhibitory efficiency as Gua> 2’-dGuo> Guo> Ino. This study reports that compounds related to nucleic acids could be PPA inhibitors. These inhibitors as well as U3EE might be suitable for functional foods to prevent obesity and type 2 diabetes.

{"title":"Identification of 2’-deoxyguanosine for an α-amylase inhibitor in the extracts of the earthworm Eisenia fetida and characterization of its inhibitory activity against porcine pancreatic α-amylase","authors":"Masako Ogasawara , Katsuhiro Yoshii , Jun Wada , Yoshihiro Yamamoto , Kuniyo Inouye","doi":"10.1016/j.enzmictec.2025.110604","DOIUrl":"10.1016/j.enzmictec.2025.110604","url":null,"abstract":"<div><div>Inhibitory activity (IA) against porcine pancreatic α-amylase (PPA) has been found in the water extract of <em>Eisenia fetida</em>. IA is recovered mostly in a fraction (U3EE) containing compounds of low molecular mass under 3 kDa. U3EE is treated with 85 % ethanol (EtOH) and the obtained EtOH extract is applied to the solid-phase extraction (SPE) system. An IA fraction named AI is separated from SPE by elution with water, and then a fraction AI* has been obtained by elution with 5 % EtOH. In the previous study, PPA inhibitors of guanine (Gua), guanosine (Guo), and inosine (Ino) have been isolated from AI. In the present study, IA of AI* was examined and a new inhibitor 2’-deoxyguanosine (2’-dGuo) was isolated by RP-HPLC. It was a mixed-type inhibitor showing IC<sub>50</sub> of 0.7 ± 0.1 mM and inhibitor constants <em>K</em><sub>i</sub> and <em>K</em><sub>i</sub>’ of 2.0 ± 0.5 and 0.9 ± 0.2 mM, respectively, at pH 6 and at 37 °C. PPA inhibitors of U3EE were arranged in the order according to the inhibitory efficiency as Gua> 2’-dGuo> Guo> Ino. This study reports that compounds related to nucleic acids could be PPA inhibitors. These inhibitors as well as U3EE might be suitable for functional foods to prevent obesity and type 2 diabetes.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"185 ","pages":"Article 110604"},"PeriodicalIF":3.4,"publicationDate":"2025-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143350651","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

Development of an enzymatic aptasensor for monitoring recombinant His-tagged proteins in microbial biotechnology

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-02-03 DOI: 10.1016/j.enzmictec.2025.110603

Mohammad Javad Jadidi , Rahman Emamzadeh , Mahboobeh Nazari , Sayed Rasoul Zaker

The utilization of polyhistidine tags (His-tag) for the purification and analysis of recombinant proteins is a widely adopted technique in biotechnology. Considering the high costs associated with antibody-based methods, the development of cost-effective techniques for protein identification following purification could significantly lower research expenses. This study developed a novel His-tag aptasensor, combining an anti-His tag aptamer with a G-quadruplex-based DNAzyme, which demonstrates limits of detection (LODs) of 0.29 μM and 0.73 μM for a His-tagged protein in calorimetric and point-of-care assays, respectively. These LODs are significantly lower than typical protein concentrations obtained through Ni-NTA affinity chromatography, indicating that the His-tag aptasensor provides an efficient solution for in vitro analysis and post-purification monitoring of His-tagged proteins.

引用次数: 0

An all-in-one strategy for the simultaneous production of bioplastics and degrading enzymes in engineered Escherichia coli

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-01-31 DOI: 10.1016/j.enzmictec.2025.110593

Suwon Kim , Yebin Han , Gaeun Lim , See-Hyoung Park , Kyungmoon Park , Shashi Kant Bhatia , Yung-Hun Yang

Bioplastics are promising alternatives for traditional plastics, which contribute significantly to environmental pollution and have a detrimental impact on ecosystems. To advance their use, further research into bioplastic biodegradation is essential. In this study, we propose a novel approach for simultaneous polyhydroxybutyrate (PHB) and degrading enzyme production in a single-cell system using engineered Escherichia coli. Typically, PHB depolymerases, such as PhaZ, disrupt bioplastic synthesis in cells, leading to a self-defeating cycle of production and degradation. To counter this, we introduced synthetic PHB production genes and triacylglycerol lipase (TGL) from Bacillus sp. JY35, along with a native signal peptide for secretion. This enabled PHB accumulation inside the cells while TGL was secreted into the supernatant. The concentrations of PHB produced with and without TGL were similar (31.44 % PHB with TGL and 32.12 % PHB without TGL). TGL was efficiently secreted in E. coli, achieving specific esterase activities of 7.1 U/mg and 15.7 U/mg for p-Nitrophenyl butyrate and p-nitrophenyl octanoate, respectively, and degraded PHB film by 30.1 % over 14 d. Moreover, TGL retained 86 % and 91 % of its activities for the C4 and C8 substrates, respectively, after 30 d of storage at room temperature, suggesting potential use PHB degradation after use. Our study demonstrates a straightforward one-month circular cycle for bioplastic production and degradation by a single producer.

{"title":"An all-in-one strategy for the simultaneous production of bioplastics and degrading enzymes in engineered Escherichia coli","authors":"Suwon Kim , Yebin Han , Gaeun Lim , See-Hyoung Park , Kyungmoon Park , Shashi Kant Bhatia , Yung-Hun Yang","doi":"10.1016/j.enzmictec.2025.110593","DOIUrl":"10.1016/j.enzmictec.2025.110593","url":null,"abstract":"<div><div>Bioplastics are promising alternatives for traditional plastics, which contribute significantly to environmental pollution and have a detrimental impact on ecosystems. To advance their use, further research into bioplastic biodegradation is essential. In this study, we propose a novel approach for simultaneous polyhydroxybutyrate (PHB) and degrading enzyme production in a single-cell system using engineered <em>Escherichia coli</em>. Typically, PHB depolymerases, such as PhaZ, disrupt bioplastic synthesis in cells, leading to a self-defeating cycle of production and degradation. To counter this, we introduced synthetic PHB production genes and triacylglycerol lipase (TGL) from <em>Bacillus</em> sp. JY35, along with a native signal peptide for secretion. This enabled PHB accumulation inside the cells while TGL was secreted into the supernatant. The concentrations of PHB produced with and without TGL were similar (31.44 % PHB with TGL and 32.12 % PHB without TGL). TGL was efficiently secreted in <em>E. coli</em>, achieving specific esterase activities of 7.1 U/mg and 15.7 U/mg for p-Nitrophenyl butyrate and p-nitrophenyl octanoate, respectively, and degraded PHB film by 30.1 % over 14 d. Moreover, TGL retained 86 % and 91 % of its activities for the C4 and C8 substrates, respectively, after 30 d of storage at room temperature, suggesting potential use PHB degradation after use. Our study demonstrates a straightforward one-month circular cycle for bioplastic production and degradation by a single producer.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"185 ","pages":"Article 110593"},"PeriodicalIF":3.4,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143103487","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

Development of an enzymatic method for efficient production of DHA-enriched phospholipids through immobilized phospholipase A1 in AOT-water reverse micelles

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-01-30 DOI: 10.1016/j.enzmictec.2025.110600

Qin Gao , Xiaolin Yu , Jianming Wei , Xuechao Hu , Lujing Ren

The demand for omega-3 polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA), has been steadily increasing due to their significant health benefits. Traditional methods for producing DHA-enriched phospholipids often suffer from low efficiency and high costs. In this study, we developed an efficient enzymatic process to prepare phospholipid-DHA, which used immobilized phospholipase A1 to catalyze transesterification in AOT-water reverse micelle systems. Initially, high concentrations of free fatty acids were produced via acid hydrolysis of algae oil followed by crystallization. Among six evaluated reverse micelle systems, one was selected for further optimization. The substrate/enzyme ratio, temperature, reaction time, and water content were optimized using single-factor experiments and response surface methodology. To enhance cost-efficiency and eco-friendly practices, substrate recycling was implemented to maximize substrate utilization. This study established a comprehensive process chain for the preparation of phospholipid-DHA, promoting its industrial production and providing a reference for the production of other phospholipid products.

{"title":"Development of an enzymatic method for efficient production of DHA-enriched phospholipids through immobilized phospholipase A1 in AOT-water reverse micelles","authors":"Qin Gao , Xiaolin Yu , Jianming Wei , Xuechao Hu , Lujing Ren","doi":"10.1016/j.enzmictec.2025.110600","DOIUrl":"10.1016/j.enzmictec.2025.110600","url":null,"abstract":"<div><div>The demand for omega-3 polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA), has been steadily increasing due to their significant health benefits. Traditional methods for producing DHA-enriched phospholipids often suffer from low efficiency and high costs. In this study, we developed an efficient enzymatic process to prepare phospholipid-DHA, which used immobilized phospholipase A1 to catalyze transesterification in AOT-water reverse micelle systems. Initially, high concentrations of free fatty acids were produced via acid hydrolysis of algae oil followed by crystallization. Among six evaluated reverse micelle systems, one was selected for further optimization. The substrate/enzyme ratio, temperature, reaction time, and water content were optimized using single-factor experiments and response surface methodology. To enhance cost-efficiency and eco-friendly practices, substrate recycling was implemented to maximize substrate utilization. This study established a comprehensive process chain for the preparation of phospholipid-DHA, promoting its industrial production and providing a reference for the production of other phospholipid products.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"185 ","pages":"Article 110600"},"PeriodicalIF":3.4,"publicationDate":"2025-01-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143074292","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

The combination of ultraviolet mutagenesis and PPX1 overexpression synergistically enhanced S-adenosyl-L-methionine synthesis in industrial Saccharomyces cerevisiae

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-01-27 DOI: 10.1016/j.enzmictec.2025.110591

Zhong-Ce Hu , Hong-Wei Dai , Bing-Qing Gu , Yuan-Shan Wang , Zhi-Qiang Liu , Yu-Guo Zheng

S-adenosyl-L-methionine (SAM) is the only injectable drug among the hepatoprotective and choleretic drugs, which has remarkable efficacy and is favored by hepatopaths. The demand for SAM is constantly increasing in clinical settings. Therefore, many efforts have been made to increase SAM biosynthesis from L-methionine and ATP in Saccharomyces cerevisiae. This study aimed to construct a stable and high-accumulating SAM industrial strain through successive ultraviolet irradiation (UV) mutations coupled with three resistant (ethionine, nystatin, and cordycepin, respectively) screening procedures and metabolic engineering strategies. Following multiple UV mutagenesis, a higher production mutant strain ZJT15–33 was successfully obtained. In addition, the recombinant strain spe2△-PPX1 was derived from ZJT15–33 by deleting the SPE2 and overexpressing the PPX1, resulting in a 2.5-fold enhanced ATP accumulation, which promoted the synthesis of 2.41 g/L SAM in the shake-flask, representing an 11.4-fold enhancement over the original strain (0.21 g/L). Furthermore, 11.65 g/L SAM was accumulated with 113 mg/g DCW SAM content in a 5-L fermenter at 96 h, marking a 36.57 % increase compared to strain ZJT15–33 (8.53 g/L). These results indicated that UV mutagenesis combined with PPX1 overexpression could effectively improve SAM synthesis in S. cerevisiae, providing a feasible approach for developing highly SAM industrial production.

{"title":"The combination of ultraviolet mutagenesis and PPX1 overexpression synergistically enhanced S-adenosyl-L-methionine synthesis in industrial Saccharomyces cerevisiae","authors":"Zhong-Ce Hu , Hong-Wei Dai , Bing-Qing Gu , Yuan-Shan Wang , Zhi-Qiang Liu , Yu-Guo Zheng","doi":"10.1016/j.enzmictec.2025.110591","DOIUrl":"10.1016/j.enzmictec.2025.110591","url":null,"abstract":"<div><div>S-adenosyl-L-methionine (SAM) is the only injectable drug among the hepatoprotective and choleretic drugs, which has remarkable efficacy and is favored by hepatopaths. The demand for SAM is constantly increasing in clinical settings. Therefore, many efforts have been made to increase SAM biosynthesis from L-methionine and ATP in <em>Saccharomyces cerevisiae</em>. This study aimed to construct a stable and high-accumulating SAM industrial strain through successive ultraviolet irradiation (UV) mutations coupled with three resistant (ethionine, nystatin, and cordycepin, respectively) screening procedures and metabolic engineering strategies. Following multiple UV mutagenesis, a higher production mutant strain ZJT15–33 was successfully obtained. In addition, the recombinant strain <em>spe2△-PPX1</em> was derived from ZJT15–33 by deleting the <em>SPE2</em> and overexpressing the <em>PPX1</em>, resulting in a 2.5-fold enhanced ATP accumulation, which promoted the synthesis of 2.41 g/L SAM in the shake-flask, representing an 11.4-fold enhancement over the original strain (0.21 g/L). Furthermore, 11.65 g/L SAM was accumulated with 113 mg/g DCW SAM content in a 5-L fermenter at 96 h, marking a 36.57 % increase compared to strain ZJT15–33 (8.53 g/L). These results indicated that UV mutagenesis combined with <em>PPX1</em> overexpression could effectively improve SAM synthesis in <em>S. cerevisiae</em>, providing a feasible approach for developing highly SAM industrial production.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"185 ","pages":"Article 110591"},"PeriodicalIF":3.4,"publicationDate":"2025-01-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143078947","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

Carbohydrate-active enzyme-catalyzed stereoselective glycosylation of complex natural product glycosides

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-01-22 DOI: 10.1016/j.enzmictec.2025.110589

Daijing Wei , Jiawei Hu , Xudong Wu , Yi Li , Wenlin Wu , Ying Xu , Xuefei Wang , Yinggang Luo

Natural products and their derivatives are precious resources with extensive applications in various industrial fields. Enzymatic glycosylation is an efficient approach for chemical structure diversification and biological activity alternation of natural products. Herein, we reported a stereoselective glycosylation of complex natural product glycosides catalyzed by two carbohydrate-active enzymes (CAZys). ASP OleD, a mutant of glycosyltransferase OleD from Streptomyces antibioticus, catalyzed an explicit β-1,x-linkage glycosylation of the OH group of the glycosyl moiety of the representative plant-derived complex natural product glycosides, protodioscin (1) and epimedin C (2), producing two complex glycoside derivatives. The glycoside hydrolase Δ27ThCGT, a truncated cyclodextrin glucanotransferase from Thermoanaerobacter sp., exhibited a definite α-1,x-linkage glycosylation of the OH group of the glycosyl moiety of the glycosides 1, 2, and astragaloside IV (3), generating four complex glycoside derivatives. The chemical structures and absolute configurations of these enzymatic glycosylation products were determined by analysis of their HRMS and NMR data. The present study expands the enzymatic glycosylation diversification of complex glycosides catalyzed by the CAZys.

{"title":"Carbohydrate-active enzyme-catalyzed stereoselective glycosylation of complex natural product glycosides","authors":"Daijing Wei , Jiawei Hu , Xudong Wu , Yi Li , Wenlin Wu , Ying Xu , Xuefei Wang , Yinggang Luo","doi":"10.1016/j.enzmictec.2025.110589","DOIUrl":"10.1016/j.enzmictec.2025.110589","url":null,"abstract":"<div><div>Natural products and their derivatives are precious resources with extensive applications in various industrial fields. Enzymatic glycosylation is an efficient approach for chemical structure diversification and biological activity alternation of natural products. Herein, we reported a stereoselective glycosylation of complex natural product glycosides catalyzed by two carbohydrate-active enzymes (CAZys). ASP OleD, a mutant of glycosyltransferase OleD from <em>Streptomyces antibioticus</em>, catalyzed an explicit <em>β</em>-1,x-linkage glycosylation of the OH group of the glycosyl moiety of the representative plant-derived complex natural product glycosides, protodioscin (<strong>1</strong>) and epimedin C (<strong>2</strong>), producing two complex glycoside derivatives. The glycoside hydrolase Δ27ThCGT, a truncated cyclodextrin glucanotransferase from <em>Thermoanaerobacter</em> sp., exhibited a definite <em>α</em>-1,x-linkage glycosylation of the OH group of the glycosyl moiety of the glycosides <strong>1</strong>, <strong>2</strong>, and astragaloside IV (<strong>3</strong>), generating four complex glycoside derivatives. The chemical structures and absolute configurations of these enzymatic glycosylation products were determined by analysis of their HRMS and NMR data. The present study expands the enzymatic glycosylation diversification of complex glycosides catalyzed by the CAZys.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"185 ","pages":"Article 110589"},"PeriodicalIF":3.4,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143046030","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

Microbial synthesis of m-tyrosine via whole-cell biocatalysis

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-01-22 DOI: 10.1016/j.enzmictec.2025.110590

Vanna Nguyen, Ashley Tseng, Cui Guo, Mary Adwer, Yuheng Lin

Meta-tyrosine (m-tyrosine), a nonproteinogenic amino acid, has shown significant potential for applications as an herbicide in agriculture and for various medical uses. However, the natural abundance of m-tyrosine is very low, limiting its widespread use. In this study, we successfully achieved microbial production of m-tyrosine by establishing the in vivo enzyme activity of phenylalanine 3-hydroxylase (PacX from Streptomyces coeruleoribudus) in E. coli, which catalyzes the meta-hydroxylation of phenylalanine to produce m-tyrosine. Remarkably, PacX is capable of utilizing the native E. coli cofactor tetrahydromonapterin (MH4) for its hydroxylation activity. The integration of a non-native MH4 regeneration system significantly improved the bioconversion efficiency, resulting in the accumulation of m-tyrosine at a concentration of up to 368 mg/L. Additionally, we attempted to modify a well-characterized phenylalanine 4-hydroxylase (P4H) from Xanthomonas campestris to alter its regioselectivity through protein engineering. Remarkably, a double mutant (F184C/G199T) successfully shifted the enzyme’s hydroxylation specificity from the para- to the meta-position, demonstrating the feasibility of altering the regioselectivity of aromatic amino acid hydroxylases (AAAHs). To the best of our knowledge, this is the first report of microbial production of m-tyrosine through whole-cell biocatalysis.

{"title":"Microbial synthesis of m-tyrosine via whole-cell biocatalysis","authors":"Vanna Nguyen, Ashley Tseng, Cui Guo, Mary Adwer, Yuheng Lin","doi":"10.1016/j.enzmictec.2025.110590","DOIUrl":"10.1016/j.enzmictec.2025.110590","url":null,"abstract":"<div><div><em>Meta</em>-tyrosine (<em>m</em>-tyrosine), a nonproteinogenic amino acid, has shown significant potential for applications as an herbicide in agriculture and for various medical uses. However, the natural abundance of <em>m</em>-tyrosine is very low, limiting its widespread use. In this study, we successfully achieved microbial production of <em>m</em>-tyrosine by establishing the <em>in vivo</em> enzyme activity of phenylalanine 3-hydroxylase (PacX from <em>Streptomyces coeruleoribudus</em>) in <em>E. coli</em>, which catalyzes the <em>meta</em>-hydroxylation of phenylalanine to produce <em>m</em>-tyrosine. Remarkably, PacX is capable of utilizing the native <em>E. coli</em> cofactor tetrahydromonapterin (MH4) for its hydroxylation activity. The integration of a non-native MH4 regeneration system significantly improved the bioconversion efficiency, resulting in the accumulation of <em>m</em>-tyrosine at a concentration of up to 368 mg/L. Additionally, we attempted to modify a well-characterized phenylalanine 4-hydroxylase (P4H) from <em>Xanthomonas campestris</em> to alter its regioselectivity through protein engineering. Remarkably, a double mutant (F184C/G199T) successfully shifted the enzyme’s hydroxylation specificity from the <em>para-</em> to the <em>meta-</em>position, demonstrating the feasibility of altering the regioselectivity of aromatic amino acid hydroxylases (AAAHs). To the best of our knowledge, this is the first report of microbial production of <em>m</em>-tyrosine through whole-cell biocatalysis.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"185 ","pages":"Article 110590"},"PeriodicalIF":3.4,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143058402","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

Development of translationally active cell lysates from different filamentous fungi for application in cell-free protein synthesis

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-01-21 DOI: 10.1016/j.enzmictec.2025.110588

Stephanie Friedrich , Marina Schramm , Jan Kiebist , Kai-Uwe Schmidtke , Katrin Scheibner

There is an enormous potential for cell-free protein synthesis (CFPS) systems based on filamentous fungi in view of their simple, fast and mostly inexpensive cultivation with high biomass space-time yields and in view of their catalytic capacity.

In 12 of the 22 different filamentous fungi examined, in vitro translation of at least one of the two reporter proteins GFP and firefly luciferase was detected. The lysates showing translation of a reporter protein usually were able to synthesize a functional cell-free expressed unspecific peroxygenase (UPO) from the basidiomycete Cyclocybe (Agrocybe) aegerita.

For the most promising candidate Neurospora crassa, the influence of different conditions of cultivation and lysate preparation on in vitro translation of the reporter proteins was investigated and optimized. In general, the greatest improvements in the translational activity were achieved by the choice of the growth medium, the addition of organic nitrogen being most beneficial. Optimizing the culture and preparation conditions of the N. crassa platform improved protein yield of the original lysate by a factor of 25 for firefly luciferase and 17 for GFP, respectively. In addition to the reporter proteins, the aforementioned UPO as well as a functional UPO from Aspergillus niger were cell-free expressed using the different lysates from N. crassa.

CFPS with fungal lysates opens the door to expressing UPOs in high throughput and in parallel, for example to optimize synthesis conditions or adapt catalyst properties. The presented method proves the general potential of fungal lysates for application in cell-free syntheses.

基于丝状真菌的无细胞蛋白质合成（CFPS）系统具有巨大的潜力，因为丝状真菌的培养简单、快速，而且大多成本低廉，具有很高的生物质时空产量和催化能力。在所研究的 22 种不同丝状真菌中，有 12 种真菌体外翻译了两种报告蛋白 GFP 和萤火虫荧光素酶中的至少一种。出现报告蛋白翻译的裂解液通常能够合成一种功能性无细胞表达的非特异性过氧化物酶（UPO），这种酶来自基枝菌 Cyclocybe (Agrocybe) aegerita。对于最有希望的候选菌冠突孢属（Neurospora crassa），研究人员研究并优化了培养和裂解物制备的不同条件对报告蛋白体外翻译的影响。一般来说，生长培养基的选择对翻译活性的提高最大，而添加有机氮则最为有益。通过优化 N. crassa 平台的培养和制备条件，萤火虫荧光素酶和 GFP 原始裂解物的蛋白质产量分别提高了 25 倍和 17 倍。除报告蛋白外，上述 UPO 以及黑曲霉的功能性 UPO 也利用 N. crassa 的不同裂解液进行了无细胞表达。使用真菌裂解物的 CFPS 为高通量和平行表达 UPO 打开了大门，例如，可用于优化合成条件或调整催化剂特性。该方法证明了真菌裂解物在无细胞合成中的普遍应用潜力。

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引用次数: 0

Pylb-based overexpression of cytochrome P450 in Bacillus subtilis 168

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-01-20 DOI: 10.1016/j.enzmictec.2025.110587

Thanaporn Wichai , Sarintip Sooksai , Sajee Noitang , Alisa S. Vangnai , Panaya Kotchaplai

Inducer-free expression systems are promising tools for biorefinery because they can reduce the reliance on inducers, reducing production costs and simplifying processes. Owing to their broad range of substrate structures and catalytic reactions, cytochrome P450s are promising biocatalysts to produce value-added compounds. However, unsuitable levels of cytochrome P450 expression could result in cell stress, affecting the efficiency of the biocatalyst. Here, we assessed the potential of Pylb, a reported growth-phase-dependent promoter derived from Bacillus subtilis 168, to develop an inducer-free expression system, especially cytochrome P450 expression, in B. subtilis, a key workhorse strain. Utilizing a green fluorescent protein (GFP) reporter, we observed differential expression patterns under the control of Pylb and the constitutive promoter P43 in recombinant Escherichia coli and B. subtilis. Recombinant B. subtilis cultivated at 37 °C showed 2.8-fold higher bacterial fluorescence compared to cultivation at 30 °C. Codon-optimized engineered P450-BM3, which can convert octane to octanols, was selected as a model cytochrome P450 in this study. In the Pylb-based system, the expression of cytochrome P450 in recombinant B. subtilis can be detected at 24 h and increases over time as shown by the purpald assay. The activity of the overexpressed P450 was confirmed by the conversion of octane to octanols. Within one hour, the resting cells of recombinant B. subtilis produced 0.15 ± 0.04 mM of 1-octanol and 0.31 ± 0.08 mM of 2-octanol. Overall, the inducer-free Pylb-based system developed here is a potential biocatalyst for biorefinery applications.

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引用次数: 0