Pub Date : 2024-10-11DOI: 10.1016/j.jbiotec.2024.10.005
Xinxin Chen , Bo Zhang , Xiaohan Jiang , Zhiqiang Liu , Yuguo Zheng
The biotransformation of phytosterol into high value steroid intermediates such as 9α-hydroxyandrost-4-ene-3,17-dione (9-OHAD) in Mycolicibacterium is the cornerstone of the steroid pharmaceuticals. However, the limited permeability of the dense mycobacterial cell wall severely hinders the efficient transportation of phytosterol and their bioconversion to 9-OHAD. In this study, we disrupted the genetic pathways involved in trehalose biosynthesis, trehalose recycle and by-product formation, leading to alterations in cell wall formation, cell permeability and 9-OHAD productivity. This manipulation led to an increase of 63.7% in the yield of 9-OHAD, reaching 10.8 g/L at a phytosterol concentration of 20 g/L in shake flask. The enhancement of cell permeability and 9-OHAD production were achieved through the deletion of genes TreS , TreY, OtsA, LpqY, and SugC, as well as the inactivation of regulator PadR. Notably, it was found that the increase in TMM content of cell wall components via TLC analysis directly affected the distribution of 9-OHAD within and outside the cell, ultimately leading to an increase in extracellular production of 9-OHAD from 12% to 32.1%. Therefore, this study provides with an effective strategy for enhancing 9-OHAD production by increasing cell permeability while minimizing by-product 4-AD formation.
{"title":"Improvement of 9α-hydroxyandrost-4-ene-3,17-dione production in Mycolicibacterium neoaurum by regulation of cell wall formation and transcriptional regulator PadR","authors":"Xinxin Chen , Bo Zhang , Xiaohan Jiang , Zhiqiang Liu , Yuguo Zheng","doi":"10.1016/j.jbiotec.2024.10.005","DOIUrl":"10.1016/j.jbiotec.2024.10.005","url":null,"abstract":"<div><div>The biotransformation of phytosterol into high value steroid intermediates such as 9α-hydroxyandrost-4-ene-3,17-dione (9-OHAD) in <em>Mycolicibacterium</em> is the cornerstone of the steroid pharmaceuticals. However, the limited permeability of the dense mycobacterial cell wall severely hinders the efficient transportation of phytosterol and their bioconversion to 9-OHAD. In this study, we disrupted the genetic pathways involved in trehalose biosynthesis, trehalose recycle and by-product formation, leading to alterations in cell wall formation, cell permeability and 9-OHAD productivity. This manipulation led to an increase of 63.7% in the yield of 9-OHAD, reaching 10.8 g/L at a phytosterol concentration of 20 g/L in shake flask. The enhancement of cell permeability and 9-OHAD production were achieved through the deletion of genes <em>TreS</em> <!-->, <em>TreY</em>, <em>OtsA</em>, <em>LpqY</em>, and <em>SugC</em>, as well as the inactivation of regulator PadR. Notably, it was found that the increase in TMM content of cell wall components via TLC analysis directly affected the distribution of 9-OHAD within and outside the cell, ultimately leading to an increase in extracellular production of 9-OHAD from 12% to 32.1%. Therefore, this study provides with an effective strategy for enhancing 9-OHAD production by increasing cell permeability while minimizing by-product 4-AD formation.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"396 ","pages":"Pages 10-17"},"PeriodicalIF":4.1,"publicationDate":"2024-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142446889","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-10DOI: 10.1016/j.jbiotec.2024.10.003
Thaís Fabiana Chan Salum , Daniel Day , James Sherwood , Alessandro Pellis , Thomas James Farmer
Given the urge to accelerate the substitution of petrol-derived solvents not only in more traditional fields like pharmaceuticals, personal care, or electronics but also in innovative research processes, this work focuses on the utilisation of four biobased solvents as media for the enzymatic synthesis of aliphatic-aromatic polyesters. As building blocks, the lignin-derived diethyl-2,4-pyridinedicarboxylate was selected as the potentially biobased, aromatic component while more classical diols such as 1,4-butanediol and 1,8-octanediol were used as the aliphatic portion. Results show that among the tested green solvents (cyclohexanone, phenetole, anisole and eucalyptol), the most suitable medium for lipase B from Candida antarctica-catalysed polycondensation reactions was eucalyptol that allowed reach monomer conversions >95 % and number average molecular weights up to 3500 g·mol−1. On the other hand, cyclohexanone led to the lowest monomer conversions (<80 %) and molecular weights (Mn<500 g·mol−1) confirming once again the unsuitability of ketone-containing solvents for enzymatic esterification and transesterification reactions. The lipase could be used up to three times, in eucalyptol as a solvent, without a significant decrease in monomer conversion or molecular weight.
考虑到不仅在制药、个人护理或电子产品等传统领域，而且在创新研究过程中都需要加快汽油溶剂的替代速度，这项工作重点关注利用四种生物基溶剂作为酶法合成脂肪芳香族聚酯的媒介。木质素衍生的 2,4-吡啶二羧酸二乙酯被选为潜在的生物基芳香族成分，而 1,4-丁二醇和 1,8-辛二醇等传统二元醇则被用作脂肪族部分。结果表明，在测试过的绿色溶剂（环己酮、苯乙醚、苯甲醚和桉叶油醇）中，最适合白色念珠菌脂肪酶 B 催化缩聚反应的介质是桉叶油醇，其单体转化率可达 95%，平均分子量高达 3500 g-mol-1。另一方面，环己酮的单体转化率（80%）和分子量（Mn<500 g-mol-1）最低，这再次证明含酮溶剂不适合酶促酯化和酯交换反应。以桉叶油醇为溶剂，脂肪酶最多可使用三次，而单体转化率和分子量不会显著下降。
{"title":"Enzymatic synthesis of aromatic biobased polymers in green, low-boiling solvents","authors":"Thaís Fabiana Chan Salum , Daniel Day , James Sherwood , Alessandro Pellis , Thomas James Farmer","doi":"10.1016/j.jbiotec.2024.10.003","DOIUrl":"10.1016/j.jbiotec.2024.10.003","url":null,"abstract":"<div><div>Given the urge to accelerate the substitution of petrol-derived solvents not only in more traditional fields like pharmaceuticals, personal care, or electronics but also in innovative research processes, this work focuses on the utilisation of four biobased solvents as media for the enzymatic synthesis of aliphatic-aromatic polyesters. As building blocks, the lignin-derived diethyl-2,4-pyridinedicarboxylate was selected as the potentially biobased, aromatic component while more classical diols such as 1,4-butanediol and 1,8-octanediol were used as the aliphatic portion. Results show that among the tested green solvents (cyclohexanone, phenetole, anisole and eucalyptol), the most suitable medium for lipase B from <em>Candida antarctica</em>-catalysed polycondensation reactions was eucalyptol that allowed reach monomer conversions >95 % and number average molecular weights up to 3500 g·mol<sup>−1</sup>. On the other hand, cyclohexanone led to the lowest monomer conversions (<80 %) and molecular weights (M<sub>n</sub><500 g·mol<sup>−1</sup>) confirming once again the unsuitability of ketone-containing solvents for enzymatic esterification and transesterification reactions. The lipase could be used up to three times, in eucalyptol as a solvent, without a significant decrease in monomer conversion or molecular weight.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"396 ","pages":"Pages 1-9"},"PeriodicalIF":4.1,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142446890","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-09DOI: 10.1016/j.jbiotec.2024.10.002
Bernard O. Boateng, Alan G. Ryder
Biopharmaceutical process development often involves the use of small-scale bioreactors (SSBR) for optimizing media formulations and process conditions during scale up to commercial scale production. Two key process parameters (CPP) used in SSBR studies are protein titre and viable cell density (VCD). Here, we explore the efficacy of parallel polarized total synchronous fluorescence spectroscopy (TSFS||) and Synchronous Light Scattering (SyLS||) to qualitatively monitor these CPPs and quantitatively predict titre and VCD for a large-scale cell culture media optimization SSBR study. The study involved 71 different media formulations (50+ components each), and the bioprocess was run for 13 days or more. Samples were extracted at set times (Day 0, 3, 9, and 13) and clarified by centrifugation. TSFS|| spectra showed significant emission changes along with increased light scatter over the course of the bioprocess. SyLS|| measurements strongly correlated with particle size data obtained from Dynamic Light Scattering but did not correlate well with VCD probably because of the centrifugation-based sample preparation. Statistical and principal component analysis (PCA) of the pTSFS data showed that spectral variation was greater between media formulations than due to the evolving bioprocess. This prevented the development of accurate global prediction models for media performance (e.g., predicting product titre at day 9 from media spectra measured at day 0). However, classification methods were successfully used to select media subsets with better quantitative prediction accuracy based on spectral similarities. A practical binary (high/low performance) classification model based on Support Vector Machines was generated for media formulation screening. Combining emission and scatter measurements with multivariate data analysis provides a more holistic, multi-attribute bioprocess monitoring method that minimizes the need to use different offline analytical methods. This methodology can be used to monitor process trajectories and deviations, and ultimately be used to predict bioprocess CPPs when implemented on production scale processes where there is much less compositional variation in the media. We believe this SSBR-pTSFS/SyLS approach will provide a valuable resource to develop the design/parameter space for in-process monitoring at production scale from early-stage process/media development studies.
{"title":"Monitoring small-scale bioreactor studies for media development using polarized total synchronous fluorescence spectroscopy (pTSFS) and synchronous light scattering (SyLS)","authors":"Bernard O. Boateng, Alan G. Ryder","doi":"10.1016/j.jbiotec.2024.10.002","DOIUrl":"10.1016/j.jbiotec.2024.10.002","url":null,"abstract":"<div><div>Biopharmaceutical process development often involves the use of small-scale bioreactors (SSBR) for optimizing media formulations and process conditions during scale up to commercial scale production. Two key process parameters (CPP) used in SSBR studies are protein titre and viable cell density (VCD). Here, we explore the efficacy of parallel polarized total synchronous fluorescence spectroscopy (TSFS<sub>||</sub>) and Synchronous Light Scattering (SyLS<sub>||</sub>) to qualitatively monitor these CPPs and quantitatively predict titre and VCD for a large-scale cell culture media optimization SSBR study. The study involved 71 different media formulations (50+ components each), and the bioprocess was run for 13 days or more. Samples were extracted at set times (Day 0, 3, 9, and 13) and clarified by centrifugation. TSFS<sub>||</sub> spectra showed significant emission changes along with increased light scatter over the course of the bioprocess. SyLS<sub>||</sub> measurements strongly correlated with particle size data obtained from Dynamic Light Scattering but did not correlate well with VCD probably because of the centrifugation-based sample preparation. Statistical and principal component analysis (PCA) of the pTSFS data showed that spectral variation was greater between media formulations than due to the evolving bioprocess. This prevented the development of accurate global prediction models for media performance (e.g., predicting product titre at day 9 from media spectra measured at day 0). However, classification methods were successfully used to select media subsets with better quantitative prediction accuracy based on spectral similarities. A practical binary (high/low performance) classification model based on Support Vector Machines was generated for media formulation screening. Combining emission and scatter measurements with multivariate data analysis provides a more holistic, multi-attribute bioprocess monitoring method that minimizes the need to use different offline analytical methods. This methodology can be used to monitor process trajectories and deviations, and ultimately be used to predict bioprocess CPPs when implemented on production scale processes where there is much less compositional variation in the media. We believe this SSBR-pTSFS/SyLS approach will provide a valuable resource to develop the design/parameter space for in-process monitoring at production scale from early-stage process/media development studies.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"395 ","pages":"Pages 205-215"},"PeriodicalIF":4.1,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142400370","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01DOI: 10.1016/j.jbiotec.2024.09.020
Lukas Schober , Astrid Schiefer , Margit Winkler , Florian Rudroff
Double bonds are prevalent in various substrates and renewable feedstocks, and their cleavage typically necessitates harsh reaction conditions involving high temperatures, organic solvents, and hazardous catalysts such as heavy metals or ozone. This review explores the sustainable enzymatic alternatives developed by nature for alkene cleavage. It provides a comprehensive overview of alkene-cleaving enzymes, detailing their mechanisms, substrate specificities, and applications. The enzymes discussed include those acting on aliphatic, cyclic, and activated aromatic systems. Emphasizing the significance of these biocatalysts in green chemistry and biocatalysis, this review highlights their potential to replace traditional chemical oxidants with safer, cost-effective, and environmentally friendly options. Future research directions include expanding enzyme substrate scopes, enhancing their operational stability and activity, and integrating them into scalable processes for broader application in the pharmaceutical, flavor, and fragrance industries.
{"title":"Harnessing nature's catalysts: Advances in enzymatic alkene cleavage","authors":"Lukas Schober , Astrid Schiefer , Margit Winkler , Florian Rudroff","doi":"10.1016/j.jbiotec.2024.09.020","DOIUrl":"10.1016/j.jbiotec.2024.09.020","url":null,"abstract":"<div><div>Double bonds are prevalent in various substrates and renewable feedstocks, and their cleavage typically necessitates harsh reaction conditions involving high temperatures, organic solvents, and hazardous catalysts such as heavy metals or ozone. This review explores the sustainable enzymatic alternatives developed by nature for alkene cleavage. It provides a comprehensive overview of alkene-cleaving enzymes, detailing their mechanisms, substrate specificities, and applications. The enzymes discussed include those acting on aliphatic, cyclic, and activated aromatic systems. Emphasizing the significance of these biocatalysts in green chemistry and biocatalysis, this review highlights their potential to replace traditional chemical oxidants with safer, cost-effective, and environmentally friendly options. Future research directions include expanding enzyme substrate scopes, enhancing their operational stability and activity, and integrating them into scalable processes for broader application in the pharmaceutical, flavor, and fragrance industries.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"395 ","pages":"Pages 189-204"},"PeriodicalIF":4.1,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142371877","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-30DOI: 10.1016/j.jbiotec.2024.09.011
Chuan Wang , Xiaolei Guo , Mingtao Fan , Long Yue , Hang Wang , Jiadao Wang , Zhengqi Zha , Hongping Yin
Collagen is the most abundant protein in human and mammalian structures and is a component of the mammalian extracellular matrix (ECM). Recombinant collagen is a suitable alternative to native collagen extracted from animal tissue for various biomaterials. However, due to the limitations of the expression system, most recombinant collagens are collagen fragments and lack triple helix structures. In this study, Chinese hamster ovary (CHO) cells were used to express the full-length human type I collagen α1 chain (rhCol1α1). Moreover, Endo180 affinity chromatography and pepsin were used to purify pepsin-soluble rhCol1α1 (PSC1). The amino acid composition of PSC1 was closer to that of native human type I collagen, and PSC1 contained 9.1 % hydroxyproline. Analysis of the CD spectra and molecular weight distribution results revealed that PSC1 forms a stable triple helix structure that is resistant to pepsin hydrolysis and has some tolerance to MMP1, MMP2 and MMP8 hydrolysis. Atomic force microscopy (AFM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) revealed that PSC1 can self-assemble into fibers at a concentration of 1 mg/ml; moreover, PSC1 can promote the proliferation and migration of NIH 3T3 cells. In conclusion, our data suggest that PSC1 is a highly similar type of recombinant collagen that may have applications in biomaterials and other medical fields.
胶原蛋白是人体和哺乳动物结构中含量最高的蛋白质，也是哺乳动物细胞外基质（ECM）的组成部分。重组胶原蛋白可以替代从动物组织中提取的原生胶原蛋白，用于制造各种生物材料。然而，由于表达系统的限制，大多数重组胶原都是胶原片段，缺乏三螺旋结构。本研究利用中国仓鼠卵巢（CHO）细胞表达了全长的人 I 型胶原蛋白 α1 链（rhCol1α1）。此外，还利用 Endo180 亲和层析和胃蛋白酶纯化了胃蛋白酶可溶性 rhCol1α1 （PSC1）。PSC1 的氨基酸组成更接近于原生人类 I 型胶原蛋白，PSC1 含有 9.1% 的羟脯氨酸。对 CD 光谱和分子量分布结果的分析表明，PSC1 形成了稳定的三重螺旋结构，可抗胃蛋白酶水解，对 MMP1、MMP2 和 MMP8 的水解也有一定的耐受性。原子力显微镜（AFM）、透射电子显微镜（TEM）和扫描电子显微镜（SEM）显示，PSC1 在浓度为 1 毫克/毫升时能自组装成纤维；此外，PSC1 还能促进 NIH 3T3 细胞的增殖和迁移。总之，我们的数据表明，PSC1 是一种高度相似的重组胶原蛋白，可应用于生物材料和其他医学领域。
{"title":"Production of recombinant human type I collagen homotrimers in CHO cells and their physicochemical and functional properties","authors":"Chuan Wang , Xiaolei Guo , Mingtao Fan , Long Yue , Hang Wang , Jiadao Wang , Zhengqi Zha , Hongping Yin","doi":"10.1016/j.jbiotec.2024.09.011","DOIUrl":"10.1016/j.jbiotec.2024.09.011","url":null,"abstract":"<div><div>Collagen is the most abundant protein in human and mammalian structures and is a component of the mammalian extracellular matrix (ECM). Recombinant collagen is a suitable alternative to native collagen extracted from animal tissue for various biomaterials. However, due to the limitations of the expression system, most recombinant collagens are collagen fragments and lack triple helix structures. In this study, Chinese hamster ovary (CHO) cells were used to express the full-length human type I collagen α1 chain (rhCol1α1). Moreover, Endo180 affinity chromatography and pepsin were used to purify pepsin-soluble rhCol1α1 (PSC1). The amino acid composition of PSC1 was closer to that of native human type I collagen, and PSC1 contained 9.1 % hydroxyproline. Analysis of the CD spectra and molecular weight distribution results revealed that PSC1 forms a stable triple helix structure that is resistant to pepsin hydrolysis and has some tolerance to MMP1, MMP2 and MMP8 hydrolysis. Atomic force microscopy (AFM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) revealed that PSC1 can self-assemble into fibers at a concentration of 1 mg/ml; moreover, PSC1 can promote the proliferation and migration of NIH 3T3 cells. In conclusion, our data suggest that PSC1 is a highly similar type of recombinant collagen that may have applications in biomaterials and other medical fields.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"395 ","pages":"Pages 149-160"},"PeriodicalIF":4.1,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142365347","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-29DOI: 10.1016/j.jbiotec.2024.09.012
Klaudia Žigová , Zuzana Marčeková , Tatiana Petrovičová , Katarína Lorková , František Čacho , Vladimír Krasňan , Martin Rebroš
Alcohol dehydrogenase I from Zymomonas mobilis (zmADH1) is a zinc-dependent oxidoreductase that catalyses the oxidation of primary or secondary alcohols to the corresponding aldehydes or ketones using NAD+/NADH as a cofactor. Efforts to express zmADH1 in Escherichia coli in a soluble form have been laden with solubility difficulties. A soluble form of recombinant zmADH1 was achieved by the addition of 1 mM zinc into media. Zinc addition facilitates the proper folding of recombinant zmADH1 and significantly reduces the formation of inclusion bodies. The yield of recombinant zmADH1 represents approximately 30 mg/1 L Luria-Bertani media. Intensified production in fermenters showed a striking difference between the specific and total activities of zmADH1 produced at different zinc concentrations. The zmADH1 showed an affinity to medium-chain alcohols, especially 1-pentanol, which could be used in new greener routes for preparation of aldehydes and alcohols.
{"title":"Intensified functional expression of recombinant Zymomonas mobilis zinc-dependent alcohol dehydrogenase I","authors":"Klaudia Žigová , Zuzana Marčeková , Tatiana Petrovičová , Katarína Lorková , František Čacho , Vladimír Krasňan , Martin Rebroš","doi":"10.1016/j.jbiotec.2024.09.012","DOIUrl":"10.1016/j.jbiotec.2024.09.012","url":null,"abstract":"<div><div>Alcohol dehydrogenase I from <em>Zymomonas mobilis</em> (zmADH1) is a zinc-dependent oxidoreductase that catalyses the oxidation of primary or secondary alcohols to the corresponding aldehydes or ketones using NAD<sup>+</sup>/NADH as a cofactor. Efforts to express zmADH1 in <em>Escherichia coli</em> in a soluble form have been laden with solubility difficulties. A soluble form of recombinant zmADH1 was achieved by the addition of 1 mM zinc into media. Zinc addition facilitates the proper folding of recombinant zmADH1 and significantly reduces the formation of inclusion bodies. The yield of recombinant zmADH1 represents approximately 30 mg/1 L Luria-Bertani media. Intensified production in fermenters showed a striking difference between the specific and total activities of zmADH1 produced at different zinc concentrations. The zmADH1 showed an affinity to medium-chain alcohols, especially 1-pentanol, which could be used in new greener routes for preparation of aldehydes and alcohols.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"395 ","pages":"Pages 141-148"},"PeriodicalIF":4.1,"publicationDate":"2024-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142347202","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-28DOI: 10.1016/j.jbiotec.2024.09.015
Hyun Jin Kim , Byung Chan Kim , Hanna Park , Geunsang Cho , Taekyu Lee , Hee Taek Kim , Shashi Kant Bhatia , Yung-Hun Yang
Levulinic acid(LA) is produced through acid-catalyzed hydrolysis and dehydration of lignocellulosic biomass. It is a key platform chemical used as an intermediate in various industries including biofuels, cosmetics, pharmaceuticals, and polymers. Traditional LA production uses chemical conversion, which requires high temperatures and pressures, strong acids, and produces undesirable side reactions, repolymerization products, and waste problems Therefore, we designed an integrated process to produce LA from glucose through metabolic engineering of Pseudomonas putida KT2440. As a metabolic engineering strategy, codon optimized phospho-2-dehydro-3-deoxyheptonate aldolase (AroG), 3-dehydroshikimate dehydratase (AsbF), and acetoacetate decarboxylase (Adc) were introduced to express genes of the shikimate and β-ketoadipic acid pathways, and the 3-oxoadipate CoA-transferase (pcaIJ) gene was deleted to prevent loss of biosynthetic intermediates. To increase the accumulation of the produced LA, the lva operon encoding levulinyl-CoA synthetase (LvaE) was deleted resulting in the high LA-producing strain P. putida HP203. Culture conditions such as medium, temperature, glucose concentration, and nitrogen source were optimized, and under optimal conditions, P. putida HP203 strain biosynthesized 36.3 mM (4.2 g/L) LA from glucose in a fed-batch fermentation system. When lignocellulosic biomass hydrolysate was used as the substrate, this strain produced 7.31 mM of LA. This is the first report of microbial production of LA from glucose by P. putida. This study suggests the possibility of manipulating biosynthetic pathway to produce biological products from glucose for various applications.
乙酰丙酸（LA）是通过酸催化水解和脱水木质纤维素生物质产生的。它是一种重要的平台化学品，在生物燃料、化妆品、医药和聚合物等多个行业中用作中间体。传统的 LA 生产采用化学转化法，需要高温高压和强酸，并会产生不良副反应、再聚合产物和废弃物等问题。作为一种代谢工程策略，我们引入了经过密码子优化的磷酸-2-脱氢-3-脱氧庚二酸醛缩酶（AroG）、3-脱氢莽草酸脱水酶（AsbF）和乙酰乙酸脱羧酶（Adc）来表达莽草酸和β-酮基二酸途径的基因，并删除了3-氧代二酸 CoA-转移酶（pcaIJ）基因以防止生物合成中间产物的损失。为了增加所产生的 LA 的积累，删除了编码左旋丙烯酰-CoA 合成酶（LvaE）的 lva 操作子，从而产生了高产 LA 菌株 P. putida HP203。对培养基、温度、葡萄糖浓度和氮源等培养条件进行了优化，在最佳条件下，P. putida HP203菌株在饲料批量发酵系统中从葡萄糖中生物合成了36.3mM（4.2g/L）的LA。当使用木质纤维素生物质水解物作为底物时，该菌株产生了 7.31mM 的 LA。这是首次报道 P. putida 微生物利用葡萄糖生产 LA。这项研究表明，有可能通过操纵生物合成途径，从葡萄糖中生产出生物产品，用于各种用途。
{"title":"Microbial production of levulinic acid from glucose by engineered Pseudomonas putida KT2440","authors":"Hyun Jin Kim , Byung Chan Kim , Hanna Park , Geunsang Cho , Taekyu Lee , Hee Taek Kim , Shashi Kant Bhatia , Yung-Hun Yang","doi":"10.1016/j.jbiotec.2024.09.015","DOIUrl":"10.1016/j.jbiotec.2024.09.015","url":null,"abstract":"<div><div>Levulinic acid(LA) is produced through acid-catalyzed hydrolysis and dehydration of lignocellulosic biomass. It is a key platform chemical used as an intermediate in various industries including biofuels, cosmetics, pharmaceuticals, and polymers. Traditional LA production uses chemical conversion, which requires high temperatures and pressures, strong acids, and produces undesirable side reactions, repolymerization products, and waste problems Therefore, we designed an integrated process to produce LA from glucose through metabolic engineering of <em>Pseudomonas putida</em> KT2440. As a metabolic engineering strategy, codon optimized phospho-2-dehydro-3-deoxyheptonate aldolase (AroG), 3-dehydroshikimate dehydratase (AsbF), and acetoacetate decarboxylase (Adc) were introduced to express genes of the shikimate and β-ketoadipic acid pathways, and the 3-oxoadipate CoA-transferase (<em>pcaIJ)</em> gene was deleted to prevent loss of biosynthetic intermediates. To increase the accumulation of the produced LA, the <em>lva</em> operon encoding levulinyl-CoA synthetase (LvaE) was deleted resulting in the high LA-producing strain <em>P. putida</em> HP203. Culture conditions such as medium, temperature, glucose concentration, and nitrogen source were optimized, and under optimal conditions, <em>P. putida</em> HP203 strain biosynthesized 36.3 mM (4.2 g/L) LA from glucose in a fed-batch fermentation system. When lignocellulosic biomass hydrolysate was used as the substrate, this strain produced 7.31 mM of LA. This is the first report of microbial production of LA from glucose by <em>P. putida</em>. This study suggests the possibility of manipulating biosynthetic pathway to produce biological products from glucose for various applications.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"395 ","pages":"Pages 161-169"},"PeriodicalIF":4.1,"publicationDate":"2024-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142347213","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-28DOI: 10.1016/j.jbiotec.2024.09.017
Paweł Jamróz , Andrzej Świeży , Małgorzata Noworyta , Katarzyna Starzak , Patrycja Środa , Weronika Wielgus , Patryk Szymaszek , Małgorzata Tyszka-Czochara , Joanna Ortyl
In the present study, we focused on the development and characterization of formulations that function as biological inks. These inks were doped with coumarin derivatives to act as molecular luminescent sensors that allow the monitoring of the kinetics of in situ photopolymerization in 3D (DLP) printing and bioprinting using pneumatic extrusion techniques, making it possible to study the changes in the system in real time. The efficiency of the systems was tested on compositions containing monomers: poly(ethylene glycol) diacrylates and photoinitiators: 2,4,6-trimethylbenzoyldi-phenylphosphinate and lithium phenyl-2,4,6-trimethylbenzoylphosphinate. The selected formulations were spectroscopically characterized and examined for their photopolymerization kinetics and rheological properties. This is important because of the fact that spectroscopic characterization, examination of photopolymerization kinetics, and rheological properties provide valuable insights into the behaviour of photocurable resin dedicated for 3D printing processes. The next step involved printing tests on commercially available 3D printers. In turn, printing carried out as part of the work on commercially available 3D printers further verified the effectiveness of the formulations. Moreover the formulation components and the resulting 3D objects were tested for their antiproliferative effects on the selected Chinese hamster ovary cell line, CHO-K1.
{"title":"Photocurable biomaterials labeled with luminescent sensors dedicated to bioprinting","authors":"Paweł Jamróz , Andrzej Świeży , Małgorzata Noworyta , Katarzyna Starzak , Patrycja Środa , Weronika Wielgus , Patryk Szymaszek , Małgorzata Tyszka-Czochara , Joanna Ortyl","doi":"10.1016/j.jbiotec.2024.09.017","DOIUrl":"10.1016/j.jbiotec.2024.09.017","url":null,"abstract":"<div><div>In the present study, we focused on the development and characterization of formulations that function as biological inks. These inks were doped with coumarin derivatives to act as molecular luminescent sensors that allow the monitoring of the kinetics of in situ photopolymerization in 3D (DLP) printing and bioprinting using pneumatic extrusion techniques, making it possible to study the changes in the system in real time. The efficiency of the systems was tested on compositions containing monomers: poly(ethylene glycol) diacrylates and photoinitiators: 2,4,6-trimethylbenzoyldi-phenylphosphinate and lithium phenyl-2,4,6-trimethylbenzoylphosphinate. The selected formulations were spectroscopically characterized and examined for their photopolymerization kinetics and rheological properties. This is important because of the fact that spectroscopic characterization, examination of photopolymerization kinetics, and rheological properties provide valuable insights into the behaviour of photocurable resin dedicated for 3D printing processes. The next step involved printing tests on commercially available 3D printers. In turn, printing carried out as part of the work on commercially available 3D printers further verified the effectiveness of the formulations. Moreover the formulation components and the resulting 3D objects were tested for their antiproliferative effects on the selected Chinese hamster ovary cell line, CHO-K1.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"395 ","pages":"Pages 122-140"},"PeriodicalIF":4.1,"publicationDate":"2024-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142347215","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-28DOI: 10.1016/j.jbiotec.2024.09.019
Pooja R. Aggarwal , Mehanathan Muthamilarasan , Pooja Choudhary
The rapid depletion of conventional fuel resources and rising energy demand has accelerated the search for alternative energy sources. Further, the expanding need to use bioenergy crops for sustainable fuel production has enhanced the competition for agricultural land, raising the “food vs. fuel” competition. Considering this, producing bioenergy crops on marginal land has a great perspective for achieving sustainable bioenergy production and mitigating the negative impacts of climate change. C4 crops are dual-purpose crops with better efficiency to fix atmospheric CO2 and convert solar energy into lignocellulosic biomass. Of these, millets have gained worldwide attention due to their climate resilience and nutraceutical properties. Due to close synteny with contemporary C4 bioenergy crops, millets are being considered a model crop for studying diverse agronomically important traits associated with biomass production. Millets can be cultivated on marginal land with minimum fertilizer inputs and maximum biomass production. In this regard, advanced molecular approaches, including marker-assisted breeding, multi-omics approaches, and gene-editing technologies, can be employed to genetically engineer these crops for enhanced biofuel production efficiency. The current study aims to provide an overview of millets as a sustainable bioenergy source and underlines the significance of millets as a C4 model to elucidate the genes and pathways involved in lignocellulosic biomass production using advanced molecular biology approaches.
{"title":"Millet as a promising C4 model crop for sustainable biofuel production","authors":"Pooja R. Aggarwal , Mehanathan Muthamilarasan , Pooja Choudhary","doi":"10.1016/j.jbiotec.2024.09.019","DOIUrl":"10.1016/j.jbiotec.2024.09.019","url":null,"abstract":"<div><div>The rapid depletion of conventional fuel resources and rising energy demand has accelerated the search for alternative energy sources. Further, the expanding need to use bioenergy crops for sustainable fuel production has enhanced the competition for agricultural land, raising the “food vs. fuel” competition. Considering this, producing bioenergy crops on marginal land has a great perspective for achieving sustainable bioenergy production and mitigating the negative impacts of climate change. C4 crops are dual-purpose crops with better efficiency to fix atmospheric CO<sub>2</sub> and convert solar energy into lignocellulosic biomass. Of these, millets have gained worldwide attention due to their climate resilience and nutraceutical properties. Due to close synteny with contemporary C4 bioenergy crops, millets are being considered a model crop for studying diverse agronomically important traits associated with biomass production. Millets can be cultivated on marginal land with minimum fertilizer inputs and maximum biomass production. In this regard, advanced molecular approaches, including marker-assisted breeding, multi-omics approaches, and gene-editing technologies, can be employed to genetically engineer these crops for enhanced biofuel production efficiency. The current study aims to provide an overview of millets as a sustainable bioenergy source and underlines the significance of millets as a C4 model to elucidate the genes and pathways involved in lignocellulosic biomass production using advanced molecular biology approaches.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"395 ","pages":"Pages 110-121"},"PeriodicalIF":4.1,"publicationDate":"2024-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142347214","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-26DOI: 10.1016/j.jbiotec.2024.09.016
Eunhui Yoo, Hee Jung Choi, Jae Yong Han
Adiponectin (ADPN) exerts various cellular and metabolic functions by activating signaling pathways, including extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) pathways, the protein kinase B (Akt) pathway, and the p38 mitogen-activated protein kinase (MAPK) pathway. However, generating functional recombinant human adiponectin (hADPN) in bacterial or mammalian cells is challenging. Although ADPN agonist peptides have been developed, problems like stability, solubility, and affinity for receptors remain. Recently, a genome-edited chicken bioreactor system was established, ensuring efficient ADPN production with optimal post-transcriptional modifications. We assessed the ability of egg white (EW)-derived hADPN, commercial hADPN, various ADPN agonist peptides, and globular ADPN on activation of the ERK1/2, Akt, and p38 MAPK pathways. EW-derived hADPN, abundant in hexamers and high molecular weight multimers, significantly phosphorylated ERK1/2 in serum-starved HEK293 cells after 15 min of treatment. Comparative analysis revealed that EW-derived hADPN and commercial hADPN induced greater phosphorylation of ERK1/2, Akt, and p38 MAPK than ADPN agonist peptides and globular ADPN, with EW-derived hADPN showing the highest activation. In summary, the finding that EW-derived hADPN strongly activates the ERK1/2, Akt, p38 MAPK signaling pathways highlights that an ADPN production system based on genome-edited chickens is an advantageous alternative to existing methods.
{"title":"Enhanced activation of signaling pathway by recombinant human adiponectin from genome-edited chickens","authors":"Eunhui Yoo, Hee Jung Choi, Jae Yong Han","doi":"10.1016/j.jbiotec.2024.09.016","DOIUrl":"10.1016/j.jbiotec.2024.09.016","url":null,"abstract":"<div><div>Adiponectin (ADPN) exerts various cellular and metabolic functions by activating signaling pathways, including extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) pathways, the protein kinase B (Akt) pathway, and the p38 mitogen-activated protein kinase (MAPK) pathway. However, generating functional recombinant human adiponectin (hADPN) in bacterial or mammalian cells is challenging. Although ADPN agonist peptides have been developed, problems like stability, solubility, and affinity for receptors remain. Recently, a genome-edited chicken bioreactor system was established, ensuring efficient ADPN production with optimal post-transcriptional modifications. We assessed the ability of egg white (EW)-derived hADPN, commercial hADPN, various ADPN agonist peptides, and globular ADPN on activation of the ERK1/2, Akt, and p38 MAPK pathways. EW-derived hADPN, abundant in hexamers and high molecular weight multimers, significantly phosphorylated ERK1/2 in serum-starved HEK293 cells after 15 min of treatment. Comparative analysis revealed that EW-derived hADPN and commercial hADPN induced greater phosphorylation of ERK1/2, Akt, and p38 MAPK than ADPN agonist peptides and globular ADPN, with EW-derived hADPN showing the highest activation. In summary, the finding that EW-derived hADPN strongly activates the ERK1/2, Akt, p38 MAPK signaling pathways highlights that an ADPN production system based on genome-edited chickens is an advantageous alternative to existing methods.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"395 ","pages":"Pages 95-99"},"PeriodicalIF":4.1,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142327763","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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