Enzyme and Microbial Technology最新文献_第3页

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

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

{"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

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

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

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

{"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

Identification and characterization of endo-xylanases from families GH10 and GH11 sourced from marine thermal environments

IF 3.4 3区生物学

Enzyme and Microbial Technology Pub Date : 2025-01-31 DOI: 10.1016/j.enzmictec.2025.110592

Lara Chrystina Malta Neri , Hörður Guðmundsson , Gaëlle Meurrens , Amélie Robert , Olafur H. Fridjonsson , Gudmundur Oli Hreggvidsson , Bjorn Thor Adalsteinsson

{"title":"Identification and characterization of endo-xylanases from families GH10 and GH11 sourced from marine thermal environments","authors":"Lara Chrystina Malta Neri , Hörður Guðmundsson , Gaëlle Meurrens , Amélie Robert , Olafur H. Fridjonsson , Gudmundur Oli Hreggvidsson , Bjorn Thor Adalsteinsson","doi":"10.1016/j.enzmictec.2025.110592","DOIUrl":"10.1016/j.enzmictec.2025.110592","url":null,"abstract":"<div><div>Seaweed biomass is an underutilized resource that is rich in polysaccharides, including xylan. Seaweed polysaccharides could be used as a feedstock in industrial microbiology and and for production of prebiotic oligosaccharides and rare monosaccharides - processes that would benefit from the availability of robust enzymes that break down the seaweed polysaccharides. The present study aimed to identify genes encoding endo-xylanases in bacterial genomes and metagenomes sourced from marine thermal environments, and to characterize the respective enzymes. Twelve endo-xylanases were studied which displayed 59 % median maximal sequence similarity to characterized GH10 or GH11 enzymes. Overall, most of the enzymes functioned optimally at high temperatures, in the presence of salt, and at circumneutral pH. Eight enzymes functioned optimally at temperatures of 50°C or higher, and in the most extreme cases at 85°C to 95°C. Six enzymes retained activity after three-hour incubation at 60°C or higher. Ten enzymes displayed improved catalytic function in the presence of salt, and several retained high catalytic function at 10 % NaCl concentration. All the enzymes hydrolyzed xylan from diverse sources, including crude biomass. The study contributes to an increased understanding of the structural diversity of xylanases; it expands the availability of thermostable xylanases of marine origin; and contributes to increased valorization of seaweed biomass.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"187 ","pages":"Article 110592"},"PeriodicalIF":3.4,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143576749","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

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

{"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

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

{"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

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

{"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

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

{"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

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

{"title":"Development of translationally active cell lysates from different filamentous fungi for application in cell-free protein synthesis","authors":"Stephanie Friedrich , Marina Schramm , Jan Kiebist , Kai-Uwe Schmidtke , Katrin Scheibner","doi":"10.1016/j.enzmictec.2025.110588","DOIUrl":"10.1016/j.enzmictec.2025.110588","url":null,"abstract":"<div><div>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.</div><div>In 12 of the 22 different filamentous fungi examined, <em>in vitro</em> 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 <em>Cyclocybe</em> (<em>Agrocybe) aegerita.</em></div><div>For the most promising candidate <em>Neurospora crassa</em>, the influence of different conditions of cultivation and lysate preparation on <em>in vitro</em> 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 <em>N. crassa</em> 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 <em>Aspergillus niger</em> were cell-free expressed using the different lysates from <em>N. crassa</em>.</div><div>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.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"185 ","pages":"Article 110588"},"PeriodicalIF":3.4,"publicationDate":"2025-01-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143051971","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

Pylb-based overexpression of cytochrome P450 in Bacillus subtilis 168

IF 3.4 3区生物学

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

{"title":"Pylb-based overexpression of cytochrome P450 in Bacillus subtilis 168","authors":"Thanaporn Wichai , Sarintip Sooksai , Sajee Noitang , Alisa S. Vangnai , Panaya Kotchaplai","doi":"10.1016/j.enzmictec.2025.110587","DOIUrl":"10.1016/j.enzmictec.2025.110587","url":null,"abstract":"<div><div>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 <em>Bacillus subtilis</em> 168, to develop an inducer-free expression system, especially cytochrome P450 expression, in <em>B. subtilis</em>, 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 <em>Escherichia coli</em> and <em>B. subtilis</em>. Recombinant <em>B. subtilis</em> 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 <em>B. subtilis</em> 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 <em>B. subtilis</em> 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.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"185 ","pages":"Article 110587"},"PeriodicalIF":3.4,"publicationDate":"2025-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143046032","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