Enzyme and Microbial Technology最新文献

{"title":"Tailoring a cellulolytic enzyme cocktail for efficient hydrolysis of mildly pretreated lignocellulosic biomass","authors":"Eva Balaguer Moya,&nbsp;Berta Syhler,&nbsp;Giuliano Dragone,&nbsp;Solange I. Mussatto","doi":"10.1016/j.enzmictec.2024.110403","DOIUrl":"10.1016/j.enzmictec.2024.110403","url":null,"abstract":"<div><p>Commercially available cellulase cocktails frequently demonstrate high efficiency in hydrolyzing easily digestible pretreated biomass, which often lacks hemicellulose and/or lignin fractions. However, the challenge arises with enzymatic hydrolysis of mildly pretreated lignocellulosic biomasses, which contain cellulose, hemicellulose and lignin in high proportions. This study aimed to address this question by evaluating the supplementation of a commercial cellulolytic cocktail with accessory hemicellulases and two additives (H₂O₂ and Tween® 80). Statistical optimization methods were employed to enhance the release of glucose and xylose from mildly pretreated sugarcane bagasse. The optimized supplement composition resulted in the production of 304 and 124 mg g⁻¹ DM of glucose and xylose, respectively, significantly increasing glucose release by 84% and xylose release by 94% compared to using only the cellulolytic cocktail. This enhancement might be attributed to a coordinated hemicellulases action degrading hemicellulose, creating more space for cellulase activity, potentially boosted by the presence of H₂O₂ and Tween® 80. However, the addition of different concentrations of H₂O₂ in combination with hemicellulase and Tween® 80 did not result a significant difference on sugar release, which could be attributed to the limited range of concentrations studied (5 to 65 µM). The results obtained in this study using the mix of three supplements were also compared to the addition of only hemicellulase and only Tween® 80 to the cellulolytic cocktail. A significant increase in glucose release of 39% and 41%, respectively, was observed when using the optimized combination. For xylose, the increase was 38% and 41%, respectively. This study underscores the substantial potential in optimizing enzyme cocktails for the hydrolysis of mildly pretreated lignocellulosic biomass by using enzymes and additive combinations tailored to the specific biomass composition.</p></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"175 ","pages":"Article 110403"},"PeriodicalIF":3.4,"publicationDate":"2024-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0141022924000103/pdfft?md5=bec6a9262744f705eab97b18e24b8f80&pid=1-s2.0-S0141022924000103-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139558973","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}

{"title":"A novel pectate lyase with high specific activity from Bacillus sp. B58-2: Gene cloning, heterologous expression and use in ramie degumming","authors":"Sijia Liu ,&nbsp;Yan Qin ,&nbsp;Qingyan Wang ,&nbsp;Jing Zhang ,&nbsp;Jin Zhou ,&nbsp;Baoxiang He ,&nbsp;Xinquan Liang ,&nbsp;Liang Xian ,&nbsp;Junhua Wu","doi":"10.1016/j.enzmictec.2024.110395","DOIUrl":"10.1016/j.enzmictec.2024.110395","url":null,"abstract":"<div><p><span><span>Pectinase plays a crucial role in </span>ramie<span> degumming. A gene encoding a putative pectate lyase from </span></span><span><em>Bacillus</em></span> sp. strain B58–2 was cloned and heterologously expressed in <em>Escherichia coli</em>. The amplified gene <em>BvelPL1</em><span> encoded a mature protein of 400 amino acids<span>. BvelPL1 shared the highest amino acid sequence identity (78.75%) with the enzymatically characterized pectate lyase Pel from </span></span><span><em>Bacillus subtilis</em></span><span><span> strain RCK (GenBank: AFH66771.1). The purified recombinant enzyme rBvelPL1-Ec exhibited a maximum specific activity of 2433.26 U/mg at pH 8.5 and 50 °C towards polygalacturonic acid. This specific activity was higher than that of most reported pectate lyases. Remarkably, the </span>enzymatic activity<span> of rBvelPL1-Ec increased by 23.28 times in the presence of 0.4 mM calcium ion<span>. The effect of calcium ion on promoting the enzymatic activity of rBvelPL1-Ec was greater than that for all reported pectate lyases. After degumming with rBvelPL1-Ec, a weight loss of 21.27 ± 1.17% of circled ramie fibers was obtained, and the surfaces of the ramie fibers became smoother. Moreover, a weight loss of 30.47 ± 0.46% was obtained through enzymatic treated and subsequent NaOH treated circled ramie fibers. The excellent performance in degumming suggests that rBvelPL1-Ec may serve as a promising biocatalyst in the textile industry.</span></span></span></p></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"175 ","pages":"Article 110395"},"PeriodicalIF":3.4,"publicationDate":"2024-01-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139483668","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}

{"title":"Enhanced theanine production with reduced ATP supply by alginate entrapped Escherichia coli co-expressing γ-glutamylmethylamide synthetase and polyphosphate kinase","authors":"Do Hyun Cho ,&nbsp;Suwon Kim ,&nbsp;Yeda Lee ,&nbsp;Yuni Shin ,&nbsp;Suhye Choi ,&nbsp;Jinok Oh ,&nbsp;Hee Taek Kim ,&nbsp;See-Hyoung Park ,&nbsp;Kyungmoon Park ,&nbsp;Shashi Kant Bhatia ,&nbsp;Yung-Hun Yang","doi":"10.1016/j.enzmictec.2024.110394","DOIUrl":"10.1016/j.enzmictec.2024.110394","url":null,"abstract":"<div><p><span><span><span>L-theanine is an amino acid with a unique flavor and many therapeutic effects. Its enzymatic synthesis has been actively studied and γ-Glutamylmethylamide synthetase (GMAS) is one of the promising </span>enzymes<span> in the biological synthesis of theanine. However, the theanine biosynthetic pathway with GMAS is highly ATP-dependent and the supply of external ATP was needed to achieve high concentration of theanine production. As a result, this study aimed to investigate polyphosphate kinase 2 (PPK2) as ATP regeneration system with hexametaphosphate. Furthermore, the </span></span>alginate entrapment method was employed to immobilize whole cells containing both </span><em>gmas</em> and <em>ppk2</em> together resulting in enhanced reusability of the theanine production system with reduced supply of ATP. After immobilization, theanine production was increased to 239 mM (41.6 g/L) with a conversion rate of 79.7% using 15 mM ATP and the reusability was enhanced, maintaining a 100% conversion rate up to the fifth cycles and 60% of conversion up to eighth cycles. It could increase long-term storage property for future uses up to 35 days with 75% activity of initial activity. Overall, immobilization of both production and cofactor regeneration system could increase the stability and reusability of theanine production system.</p></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"175 ","pages":"Article 110394"},"PeriodicalIF":3.4,"publicationDate":"2024-01-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139469429","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}

{"title":"A concerted enzymatic de-structuring of lignocellulosic materials using a compost-derived microbial consortia favoring the consolidated pretreatment and bio-saccharification","authors":"Gunasekaran Rajeswari ,&nbsp;Vinod Kumar ,&nbsp;Samuel Jacob","doi":"10.1016/j.enzmictec.2023.110393","DOIUrl":"10.1016/j.enzmictec.2023.110393","url":null,"abstract":"<div><p><span>The robustness of microbial consortia isolated from compost habitat encompasses the complementary metabolism that aids in consolidated bioprocessing (CBP) of lignocellulosic biomass (LCB) by division of labor across the </span>symbionts<span>. Composting of organic waste is deemed to be an efficient way of carbon recycling, where the syntrophic microbial population exerts a concerted action of lignin and polysaccharide<span> (hemicellulose and cellulose) component of plant biomass. The potential of this interrelated microorganism could be enhanced through adaptive laboratory evolution (ALE) with LCB for its desired functional capabilities. Therefore, in this study, microbial symbionts derived from organic compost was enriched on saw dust (SD) (woody biomass), aloe vera leaf rind (AVLR) (agro-industrial waste) and commercial filter paper (FP) (pure cellulose) through ALE under different conditions. Later, the efficacy of enriched consortium (EC) on consolidated pretreatment and bio-saccharification was determined based on substrate degradation, endo-enzymes profiling and fermentable sugar yield. Among the treatment sets, AVLR biomass treated with EC-5 has resulted in the higher degradation rate of lignin (47.01 ± 0.66%, w/w) and polysaccharides (45.87 ± 1.82%, w/w) with a total sugar yield of about 60.01 ± 4.24 mg/g. In addition, the extent of structural disintegration of substrate after EC-treatment was clearly deciphered by FTIR and XRD analysis. And the factors of Pearson correlation matrix reinforces the potency of EC-5 by exhibiting a strong positive correlation between AVLR degradation and the sugar release. Thus, a consortium based CBP could promote the feasibility of establishing a sustainable second generation biorefinery framework.</span></span></p></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"174 ","pages":"Article 110393"},"PeriodicalIF":3.4,"publicationDate":"2024-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139083818","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}

{"title":"A green pathway for lignin valorization: Enzymatic lignin depolymerization in biocompatible ionic liquids and deep eutectic solvents","authors":"Enshi Liu ,&nbsp;Martha Inés Vélez Mercado ,&nbsp;Fernando Segato ,&nbsp;Mark R. Wilkins","doi":"10.1016/j.enzmictec.2023.110392","DOIUrl":"10.1016/j.enzmictec.2023.110392","url":null,"abstract":"<div><p><span><span>Lignin depolymerization<span>, which enables the breakdown of a complex and heterogeneous aromatic polymer into relatively uniform derivatives, serves as a critical process in valorization of lignin. Enzymatic lignin depolymerization has become a promising biological strategy to overcome the heterogeneity of lignin, due to its mild reaction conditions and high specificity. However, the low solubility of lignin compounds in aqueous environments prevents efficient lignin depolymerization by lignin-degrading </span></span>enzymes. The employment of biocompatible </span>ionic liquids<span> (ILs) and deep eutectic solvents (DESs) in lignin fractionation has created a promising pathway to enzymatically depolymerize lignin within these green solvents to increase lignin solubility. In this review, recent research progress on enzymatic lignin depolymerization, particularly in a consolidated process involving ILs/DESs is summarized. In addition, the interactions between lignin-degrading enzymes and solvent systems are explored, and potential protein engineering methodology to improve the performance of lignin-degrading enzymes is discussed. Consolidation of enzymatic lignin depolymerization and biocompatible ILs/DESs paves a sustainable, efficient, and synergistic way to convert lignin into value-added products.</span></p></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"174 ","pages":"Article 110392"},"PeriodicalIF":3.4,"publicationDate":"2023-12-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139052709","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}

{"title":"Evaluation of alternate hosts for recombinant expression of a reductive dehalogenase","authors":"Rabeya Rahmatullah, Christopher Marquis","doi":"10.1016/j.enzmictec.2023.110390","DOIUrl":"https://doi.org/10.1016/j.enzmictec.2023.110390","url":null,"abstract":"<p>Organohalides are recalcitrant, toxic environmental pollutants. Reductive dehalogenase enzymes (RDases) found in organohalide respiring bacteria (OHRB) utilise organohalides as electron acceptors for cellular energy and growth, producing lesser-halogenated compounds. Consequently, microbial reductive dehalogenation via organohalide respiration represents a promising solution for clean-up of organohalide pollutants. <em>Dehalobacter</em> sp. UNSWDHB is an OHRB capable of respiring highly toxic chloroform (CF) and converting it to dichloromethane (DCM). TmrA has been identified as an RDase responsible for this conversion and different strategies for generation of functional recombinant TmrA is the focus of this article. In this study, TmrA was recovered from inclusion bodies expressed in <em>E. coli</em> and refolded in the presence of FeCl3, Na2S and cobalamin to yield functional enzyme. TmrA has been previously expressed in a soluble and functional form in the corrinoid-producing <em>Bacillus megaterium</em>. Using a fractional experimental design for cultivation and induction combined with purification under anaerobic conditions resulted in substantially higher activity of recombinant and native TmrA than previously reported. TmrA was then expressed in a soluble and active form in <em>Shimwellia blattae.</em> Co-expression with two different putative chaperone proteins from the original host did not increase the level of soluble expression in <em>S. blattae</em>, however activity assays showed that removing the TAT signal from TmrA increases the dechlorination activity compared to when the TAT signal is present. Finally, TmrA was successfully expressed in a soluble and active form in the H2-oxidizing <em>C. necator</em> H16, a novel host for the expression of RDases.</p>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"2 1","pages":""},"PeriodicalIF":3.4,"publicationDate":"2023-12-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139029352","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}

{"title":"Evaluation of alternate hosts for recombinant expression of a reductive dehalogenase","authors":"Rabeya Rahmatullah,&nbsp;Christopher P. Marquis","doi":"10.1016/j.enzmictec.2023.110390","DOIUrl":"10.1016/j.enzmictec.2023.110390","url":null,"abstract":"<div><p>Organohalides are recalcitrant, toxic environmental pollutants. Reductive dehalogenase enzymes (RDases) found in organohalide respiring bacteria (OHRB) utilise organohalides as electron acceptors for cellular energy and growth, producing lesser-halogenated compounds. Consequently, microbial reductive dehalogenation via organohalide respiration represents a promising solution for clean-up of organohalide pollutants. <em>Dehalobacter</em> sp. UNSWDHB is an OHRB capable of respiring highly toxic chloroform (CF) and converting it to dichloromethane (DCM). TmrA has been identified as an RDase responsible for this conversion and different strategies for generation of functional recombinant TmrA is the focus of this article. In this study, TmrA was recovered from inclusion bodies expressed in <em>E. coli</em> and refolded in the presence of FeCl₃, Na₂S and cobalamin to yield functional enzyme. TmrA has been previously expressed in a soluble and functional form in the corrinoid-producing <em>Bacillus megaterium</em>. Using a fractional experimental design for cultivation and induction combined with purification under anaerobic conditions resulted in substantially higher activity of recombinant and native TmrA than previously reported. TmrA was then expressed in a soluble and active form in <em>Shimwellia blattae.</em> Co-expression with two different putative chaperone proteins from the original host did not increase the level of soluble expression in <em>S. blattae</em>, however activity assays showed that removing the TAT signal from TmrA increases the dechlorination activity compared to when the TAT signal is present. Finally, TmrA was successfully expressed in a soluble and active form in the H₂-oxidizing <em>C. necator</em> H16, a novel host for the expression of RDases.</p></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"174 ","pages":"Article 110390"},"PeriodicalIF":3.4,"publicationDate":"2023-12-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0141022923001989/pdfft?md5=906170bb18c03e411260ac2a88f5f4d7&pid=1-s2.0-S0141022923001989-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139024610","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}

{"title":"Split-GFP complementation at the bacterial cell surface for antibody-free labeling and quantification of heterologous protein display","authors":"David Gercke,&nbsp;Florian Lenz,&nbsp;Joachim Jose","doi":"10.1016/j.enzmictec.2023.110391","DOIUrl":"10.1016/j.enzmictec.2023.110391","url":null,"abstract":"<div><p>The split-GFP system is a versatile tool with numerous applications, but it has been underutilized for the labeling of heterologous surface-displayed proteins. By inserting the 16 amino acid sequence of the GFP11-tag between a protein of interest and an autotransporter protein, it is possible to present a protein at the outer membrane of gram-negative bacteria and to fluorescently label it by complementation with externally added GFP1–10. The labeled cells could be clearly discerned from cells without the protein of interest using flow cytometry and the insertion of the GFP11-tag caused no significant alteration of the catalytic activity for the tested model enzyme CsBglA. Furthermore, the amount of the protein of interest on the cells could be quantified by comparing the green fluorescence resulting from the complementation to that of standards with known concentrations. This allows a precise characterization of whole-cell biocatalysts, which is difficult with existing methods. The split-GFP complementation approach was shown to be specific, in a similar manner as commercial antibodies. It is cost-efficient, minimizes the possibility of adverse effects on protein expression or solubility, and can be performed at high throughput.</p></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"174 ","pages":"Article 110391"},"PeriodicalIF":3.4,"publicationDate":"2023-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0141022923001990/pdfft?md5=2ce92ee2b31db9ab8cd904db7f8b908d&pid=1-s2.0-S0141022923001990-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138820224","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}

{"title":"Cloning and heterologous expression of Fusarium oxysporum nitrilase gene in Escherichia coli and evaluation in cyanide degradation","authors":"Azamsadat Moosavizadeh ,&nbsp;Mostafa Motallebi ,&nbsp;Zahra Moghaddassi Jahromi ,&nbsp;Lukhanyo Mekuto","doi":"10.1016/j.enzmictec.2023.110389","DOIUrl":"10.1016/j.enzmictec.2023.110389","url":null,"abstract":"<div><p>Cyanide is widely utilized in the extraction of precious metal extraction even though it has been deemed as the most toxic compound. <em>Fusarium oxysporum</em> has been shown to degrade cyanide through the activity of the Nitrilase enzyme. In this study, the coding sequence of <em>nitrilase</em> gene from <em>F. oxysporum</em> genomic DNA was optimized for cloning and expression in <em>E. coli</em>. The pUC57 containing synthetic optimized <em>nitrilase</em> gene was transferred into <em>E. coli</em> DH5α strain. This <em>nitrilase</em> gene was sub-cloned into pET26b (+) expression vector containing an in-built His-tag at the C-terminal end to facilitate its purification. The recombinant plasmid, pETAM1, was confirmed by PCR, digestion pattern, and sequencing. The recombinant protein was overproduced in <em>E. coli</em> BL21 (DE3). The results of the SDS-PAGE pattern and Western blot analysis confirmed the expression of the expected recombinant protein. For expression optimization of Nitrilase protein, M16 orthogonal experimental design of the Taguchi method was used. The effect of induction time, temperature and IPTG concentration were examined using four levels for each factors. Estimation of the amount of the expressed protein was calculated via densitometry on SDS-PAGE. The enzyme activity and expression in <em>E. coli</em> proved to be successful since there was ammonia production when potassium cyanide and acrylonitrile were used as substrates while the highest enzyme activity of 88% was expressed at 30 °C. The K_m and V_m values of the expressed Nitrilase enzyme were determined to be 0.68 mM and 0.48 mM/min respectively.</p></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"174 ","pages":"Article 110389"},"PeriodicalIF":3.4,"publicationDate":"2023-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0141022923001977/pdfft?md5=a2b981cfcf24ef4d1b64962f9f0c8fef&pid=1-s2.0-S0141022923001977-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138820165","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}

{"title":"Expression of the two-component regulator StyS/StyR enhanced transcription of the styrene monooxygenase gene styAB and indigo biosynthesis in Escherichia coli","authors":"Sheng Yin ,&nbsp;Yujie Li ,&nbsp;Jialing Hou","doi":"10.1016/j.enzmictec.2023.110381","DOIUrl":"10.1016/j.enzmictec.2023.110381","url":null,"abstract":"<div><p><span>Indigo, an economically important dye, could be biosynthesized from indole<span><span> by catalysis of the styrene<span> monooxygenase StyAB. To enhance indigo </span></span>biosynthesis, the </span></span><em>styAB</em><span> gene and its transcription regulator gene </span><em>styS</em>/<em>styR</em> in styrene catabolism were cloned from <span><em>Pseudomonas putida</em></span> and coexpressed in <em>Escherichia coli</em>. The presence of the intact regulator gene <em>styS/styR</em> dramatically increased the transcriptional levels of <em>styA</em> and <em>styB</em> by approximately 120-fold in the recombinant strain SRAB2 with coexpression of <em>styS/styR</em> and <em>styAB</em> compared to the control strain ABST with solo expression of <em>styAB</em>. A yield of 67.6 mg/L indigo was detected in strain SRAB2 after 24 h of fermentation with 120 μg/mL indole, which was approximately 14-fold higher than that in the control strain ABST. The maximum yield of indigo was produced from 160 μg/mL indole in fermentation of strain SRAB2. However, the addition of styrene to the media significantly inhibited the transcription of <em>styA</em> and <em>styB</em> and consequent indigo biosynthesis in recombinant <em>E. coli</em><span> strains. Furthermore, the substitution of indole with tryptophan as the fermentation substrate remarkably boosted indigo production, and the maximal yield of 565.6 mg/L was detected in strain SRAB2 in fermentation with 1.2 mg/mL tryptophan. The results revealed that the regulation of </span><em>styAB</em> transcription by the two-component regulator StyS/StyR in styrene catabolism in <em>P. putida</em> was effective in <em>E. coli</em>, which provided a new strategy for the development of engineered <em>E. coli</em> strains with the capacity for highly efficient indigo production.</p></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"174 ","pages":"Article 110381"},"PeriodicalIF":3.4,"publicationDate":"2023-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138686419","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}