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Direct catalytic oxidation of rice husk lignin with hydroxide nanorod-modified copper foam and muconate production by engineered Pseudomonas sp. NGC7 利用氢氧化物纳米改性泡沫铜直接催化氧化稻壳木质素并利用工程假单胞菌 NGC7 生产粘液酸。
IF 2.3 4区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-08-26 DOI: 10.1016/j.jbiosc.2024.07.016
Akihiro Yoshida , Irwan Kurnia , Yudai Higuchi , Yuta Osaka , Chieko Yasuta , Chiho Sakamoto , Mina Tamura , Tsubasa Takamatsu , Naofumi Kamimura , Eiji Masai , Tomonori Sonoki
For the direct alkaline oxidation of rice husk lignin, we developed a copper foam-based heterogeneous catalyst that offers advantages in its recovery after the reaction mixture. The depolymerized products were utilized for muconate production by an engineered Pseudomonas sp. NGC7-based strain. A hydroxide nanorod-modified copper foam was prepared by the surface oxidation of copper foam, followed by alkaline oxidation of rice husk lignin over the catalyst. The catalyst was easily separated from the cellulosic residues in the reaction mixture, and the residues were then recovered by filtration. The resulting lignin stream was composed of a variety of aromatic monomers containing p-hydroxyphenyl, guaiacyl, and syringyl compounds. The catabolic activity of Pseudomonas sp. NGC7 was demonstrated to be more suitable for muconate production from a mixture comprising 4-hydroxybenzoate (a typical p-hydroxyphenyl compound), vanillate (a guaiacyl compound), and syringate (a syringyl compound), owing to its natural ability to grow on syringate. Thus, it was applied to produce muconate from a rice husk lignin stream prepared through hydroxide nanorod-modified copper foam-catalyzed alkaline oxidation by conferring the genes responsible for converting the acetophenone derivatives to their corresponding aromatic acids and protocatechuate decarboxylase to an NGC7-based strain deficient in protocatechuate 3,4-dioxygenase and muconate cycloisomerase. As a result, the engineered NGC7-based muconate-producing strain produced muconate selectively from the rice husk lignin stream at 93.7 mol% yield.
针对稻壳木质素的直接碱性氧化,我们开发了一种基于泡沫铜的异相催化剂,这种催化剂在反应混合物后的回收方面具有优势。解聚产物被一种基于 NGC7 的假单胞菌工程菌株用于生产粘液酸盐。通过对泡沫铜进行表面氧化,然后在催化剂上对稻壳木质素进行碱性氧化,制备出了氢氧化物纳米od 改性泡沫铜。催化剂很容易与反应混合物中的纤维素残留物分离,然后通过过滤回收残留物。生成的木质素流由多种芳香族单体组成,其中含有对羟基苯基、愈创木酰基和丁香酰基化合物。由于 NGC7 假单胞菌具有在丁香酸盐上生长的天然能力,它的分解活性被证明更适合从由 4-羟基苯甲酸酯(一种典型的对羟基苯基化合物)、香草酸酯(一种愈创木酰基化合物)和丁香酸酯(一种丁香酰基化合物)组成的混合物中生产粘液酸盐。因此,通过将负责将苯乙酮衍生物转化为相应芳香酸的基因和原儿茶酸脱羧酶赋予缺乏原儿茶酸 3,4-二氧 化酶和粘液环异构酶的 NGC7 菌株,该菌株被用于从通过氢氧化物纳米od修饰的泡沫铜催化碱性氧化制备的稻壳木质素流中生产粘液。结果,基于 NGC7 的工程化粘菌酸生产菌株从稻壳木质素流中选择性地生产出了粘菌酸,产量为 93.7 摩尔%。
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引用次数: 0
Hydrogen-producing conditions and mutation mechanisms of a highly efficient mutant strain Ethanoligenens harbinense YR-3 高效突变株 Ethanoligenens harbinense YR-3 的产氢条件和突变机制。
IF 2.3 4区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-08-22 DOI: 10.1016/j.jbiosc.2024.05.012
Guoxiang Zheng , Dongxu Tao , Nanqi Ren
In this study, the optimal hydrogen (H2) production conditions of the high-efficiency H2-producing mutant strain Ethanoligenens harbinense YR-3 (carbon-nitrogen ratio 5.5, phosphate buffer 80 mM, initial pH 6.0, biotin 1.4 mg/L) are obtained by intermittent experiments. The maximum specific H2 production rate of YR-3 (2.85 mol H2/mol glucose) was 1.4 times that of the wild strain ZGX4 (2.04 mol H2/mol glucose). The liquid-phase products are mainly ethanol and acetic acid, indicating that the metabolic pathway has not changed. Two-dimensional electrophoresis and mass spectrometry were used to compare and analyze the protein map differences between YR-3 and ZGX4. The results show that 1,6-fructose diphosphate aldolase and the flavoprotein in hydrogenase are highly expressed. This study will provide a theoretical basis for the genetic modification of high-efficiency H2-producing strains and the improvement of H2 production capacity.
本研究通过间歇实验获得了高效产氢突变株Ethanoligenens harbinense YR-3的最佳产氢条件(碳氮比5.5,磷酸盐缓冲液80 mM,初始pH 6.0,生物素1.4 mg/L)。YR-3 的最大特定 H2 产率(2.85 mol H2/mol葡萄糖)是野生菌株 ZGX4(2.04 mol H2/mol葡萄糖)的 1.4 倍。液相产物主要是乙醇和乙酸,表明代谢途径没有改变。利用二维电泳和质谱法比较分析了 YR-3 和 ZGX4 蛋白图谱的差异。结果表明,1,6-果糖二磷酸醛缩酶和氢化酶中的黄蛋白表达量较高。这项研究将为高效产氢菌株的基因改造和提高产氢能力提供理论依据。
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引用次数: 0
Removal of undesirable genes using yeast backcrossing 利用酵母回交去除不良基因。
IF 2.3 4区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-08-20 DOI: 10.1016/j.jbiosc.2024.07.015
Nobuo Fukuda, Mio Takeuchi
4-Vinylguaiacol (4-VG) is one of the causative compounds for the phenolic odor characteristics of brewed liquor. In the case of Japanese sake brewing, 4-VG is formed through the decarboxylation of ferulic acid produced by rice koji enzymes from steamed rice. PAD1 (phenylacrylic acid decarborxylase gene) and FDC1 (ferulic acid decarboxylase gene) genes are essential for decarboxylation of ferulic acid in Saccharomyces cerevisiae and the single polymorphisms of both genes show a relationship with ferulic acid decarboxylation ability. While most of the Kyokai yeasts distributed by the Brewing Society of Japan have homozygous non-function fdc1 alleles, many newly isolated natural yeasts for local specialities carry the wild-type FDC1 gene. In our previous research, we found that a crossbreed strain lost a significant amount of chromosomal DNA as it underwent meiosis-like adaptation. Here, we established a breeding approach to exclude undesirable gene alleles (such as the wild-type FDC1 genes) from yeast strains of interest by backcrossing using Kyokai yeasts. Homozygous fdc1 crossbreeds were generated through the three rounds of crossing procedures, and we confirmed the reduction of 4-VG in the culture supernatant of the homozygous fdc1 hybrid strain compared to the parental strain. Importantly, this approach does not include growth selection for the mutation of interest (the fdc1 mutant in the current case). Using various yeast strains generated throughout human history, it may become possible to design and build any yeast strains according to the purpose.
4-乙烯基愈创木酚(4-VG)是造成酿造酒酚类气味特征的原因之一。在日本清酒酿造中,4-VG 是通过蒸米中的米麴酶产生的阿魏酸脱羧形成的。PAD1 基因(苯丙氨酸脱羧酶基因)和 FDC1 基因(阿魏酸脱羧酶基因)是酿酒酵母中阿魏酸脱羧的必要基因,这两个基因的单多态性与阿魏酸脱羧能力有关。日本酿造协会经销的大多数京海酵母都带有同型无功能的 Fdc1 等位基因,而许多新分离的地方特产天然酵母则带有野生型 FDC1 基因。在之前的研究中,我们发现一个杂交菌株在进行减数分裂样适应过程中丢失了大量染色体 DNA。在此,我们建立了一种育种方法,通过使用 Kyokai 酵母菌进行回交,从感兴趣的酵母菌株中排除不良基因等位基因(如野生型 FDC1 基因)。通过三轮杂交程序产生了同源 fdc1 杂交品系,我们证实同源 fdc1 杂交品系的培养上清液中 4-VG 含量比亲本品系低。重要的是,这种方法不包括对相关突变(本例中为 fdc1 突变体)的生长选择。利用人类历史上产生的各种酵母菌株,可以根据目的设计和构建任何酵母菌株。
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引用次数: 0
Biomimetic conjugation inspired from pheomelanin via thiol–quinone addition for enzymatic functionalization of fibroin 通过硫醇-醌加成从雉褐素中激发的生物模拟共轭作用,用于纤维素的酶功能化。
IF 2.3 4区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-08-20 DOI: 10.1016/j.jbiosc.2024.07.013
Xue You, Xiao Song, Yinna Wu, Mengqi Han, Wenbin Liu
Fibroin has been extensively applied in the medicine, therapy, cosmetic, and food fields. Functional modification is a common route way to expand the application potential. Tyrosinase is versatile for enzymatic functionalization of fibroin by oxidizing tyrosine residues into dopaquinone. However, grafting of functional molecules to the protein-bound dopaquinone suffers from self-crosslinking due to competitive aryl coupling or addition with other nucleophile in protein. Herein, bioinspired from pheomelanin synthesis, a new approach with superior grafting efficiency and reaction rate for enzymatic grafting of protein was developed. The high reactivity of Michael addition between thiol and dopaquinone was utilized to promote the efficiency for grafting of PEG onto fibroin. The grafting of PEG with thiol group was superior to that with amine group. It demonstrated a superior efficacy for thiol group over amino group on enzymatic functionalization. This research firstly established an effective biomimetic approach for enzymatic functionalization of protein without the unexpected self-crosslinking. It could emerged to serve as the strategy of protein functionalization.
纤维素已被广泛应用于医药、治疗、化妆品和食品领域。功能性改性是扩大应用潜力的常见途径。酪氨酸酶可将酪氨酸残基氧化为多巴醌,从而对纤维素进行酶功能化。然而,将功能分子接枝到与蛋白质结合的多巴醌上时,会因竞争性芳基偶联或与蛋白质中其他亲核物的加成而发生自交联。在此,我们从雉褐素合成中获得生物启发,开发出一种具有更高接枝效率和反应速率的酶法接枝蛋白质的新方法。利用硫醇与多巴醌之间迈克尔加成的高反应活性,提高了 PEG 与纤维蛋白的接枝效率。带有硫醇基团的 PEG 接枝效果优于带有胺基团的 PEG 接枝效果。这表明硫醇基在酶功能化方面的功效优于氨基。这项研究首次为蛋白质的酶功能化建立了一种有效的生物仿生方法,而不会出现意想不到的自交联现象。它可以作为蛋白质功能化的策略。
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引用次数: 0
Combinational manipulation of transcription factors, CreA and ClbR, is a viable strategy to improve cellulolytic enzyme production in Aspergillus aculeatus 联合操纵转录因子 CreA 和 ClbR 是提高黑曲霉纤维素分解酶产量的可行策略。
IF 2.3 4区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-08-20 DOI: 10.1016/j.jbiosc.2024.07.011
Shuji Tani , Shinya Hirose , Takashi Kawaguchi
The production of cellulolytic enzymes in response to inducible carbon sources is mainly regulated at the transcriptional level in filamentous fungi. We have identified a cellobiose-response regulator (ClbR) controlling the expression of cellulolytic enzyme-encoding genes in Aspergillus aculeatus. However, the engineering potential of combining the deletion of transcriptional repressors with the overexpression of transcriptional activators to enhance enzyme production has not been analyzed. Here, we investigated the effect of the deletion of the transcriptional repressor creA and the overexpression of the transcriptional activator clbR in enzyme production in A. aculeatus. Here, we verified that a combination of creA deletion and clbR overexpression (Δc&OE) improved cellulase, β-1,4-xylanase, and β-glucosidase production. Cellulase and β-1,4-xylanase production increased 3.4- and 8.0-fold in Δc&OE compared with the host strain (MR12) at 96-h incubation, respectively. β-Glucosidase production in ΔcreA and Δc&OE increased approximately 5.0-fold compared with that in MR12 at 240-h incubation. Transcriptional analysis revealed that the increase in enzyme production was due to increased expression of cellobiohydrolase, endo-β-1,4-glucanase, β-1,4-xylanase, and β-glucosidase 1 (bgl1). Interestingly, bgl1 expression in ΔcreA increased in a dose-dependent manner in response to glucose. Thus, combinational manipulation of transcription factors improved cellulase, xylanase, and β-glucosidase production in A. aculeatus.
在丝状真菌中,针对诱导性碳源产生纤维素分解酶主要是在转录水平上进行调控的。我们已经发现了一种控制黑曲霉中纤维素分解酶编码基因表达的纤维生物糖反应调节因子(ClbR)。然而,将转录抑制因子的删除与转录激活因子的过度表达相结合以提高酶产量的工程潜力尚未得到分析。在此,我们研究了转录抑制因子 creA 的缺失和转录激活因子 clbR 的过表达对 A. aculeatus 产酶的影响。在这里,我们验证了creA缺失和clbR过表达(Δc&OE)的组合提高了纤维素酶、β-1,4-木聚糖酶和β-葡萄糖苷酶的产量。与宿主菌株(MR12)相比,培养 96 小时后,Δc&OE 的纤维素酶和 β-1,4-木聚糖酶产量分别增加了 3.4 倍和 8.0 倍。在 240 小时的培养过程中,ΔcreA 和 Δc&OE 的 β-葡萄糖苷酶产量比 MR12 增加了约 5.0 倍。转录分析表明,酶产量的增加是由于纤维生物水解酶、内-β-1,4-葡聚糖酶、β-1,4-木聚糖酶和β-葡萄糖苷酶 1(bgl1)表达的增加。有趣的是,ΔcreA 中 bgl1 的表达在葡萄糖作用下呈剂量依赖性增加。因此,对转录因子进行组合操作可提高钝顶水蚤的纤维素酶、木聚糖酶和β-葡萄糖苷酶产量。
{"title":"Combinational manipulation of transcription factors, CreA and ClbR, is a viable strategy to improve cellulolytic enzyme production in Aspergillus aculeatus","authors":"Shuji Tani ,&nbsp;Shinya Hirose ,&nbsp;Takashi Kawaguchi","doi":"10.1016/j.jbiosc.2024.07.011","DOIUrl":"10.1016/j.jbiosc.2024.07.011","url":null,"abstract":"<div><div>The production of cellulolytic enzymes in response to inducible carbon sources is mainly regulated at the transcriptional level in filamentous fungi. We have identified a cellobiose-response regulator (ClbR) controlling the expression of cellulolytic enzyme-encoding genes in <em>Aspergillus aculeatus</em>. However, the engineering potential of combining the deletion of transcriptional repressors with the overexpression of transcriptional activators to enhance enzyme production has not been analyzed. Here, we investigated the effect of the deletion of the transcriptional repressor <em>creA</em> and the overexpression of the transcriptional activator <em>clbR</em> in enzyme production in <em>A. aculeatus</em>. Here, we verified that a combination of <em>creA</em> deletion and <em>clbR</em> overexpression (Δc&amp;OE) improved cellulase, β-1,4-xylanase, and β-glucosidase production. Cellulase and β-1,4-xylanase production increased 3.4- and 8.0-fold in Δc&amp;OE compared with the host strain (MR12) at 96-h incubation, respectively. β-Glucosidase production in Δ<em>creA</em> and Δc&amp;OE increased approximately 5.0-fold compared with that in MR12 at 240-h incubation. Transcriptional analysis revealed that the increase in enzyme production was due to increased expression of cellobiohydrolase, endo-β-1,4-glucanase, β-1,4-xylanase, and β-glucosidase 1 (<em>bgl1</em>). Interestingly, <em>bgl1</em> expression in Δ<em>creA</em> increased in a dose-dependent manner in response to glucose. Thus, combinational manipulation of transcription factors improved cellulase, xylanase, and β-glucosidase production in <em>A. aculeatus</em>.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"138 5","pages":"Pages 361-368"},"PeriodicalIF":2.3,"publicationDate":"2024-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142017572","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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
Individual cell modification with cell surface specific atom transfer radical polymerization for enhanced Cr(VI) removal 利用细胞表面特异性原子转移自由基聚合对单个细胞进行改性,以提高六价铬的去除率。
IF 2.3 4区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-08-13 DOI: 10.1016/j.jbiosc.2024.07.004
Xing-Ming Zhao , Jun-Ying Liu , Heng-Chi Liu, Zhi-Zhi Yang, Han Zhao, Yang-Chun Yong
Modifying cells with polymers on the surface can enable them to gain or enhance function with various applications, wherein the atom transfer radical polymerization (ATRP) has garnered significant potential due to its biocompatibility. However, specifically initiating ATRP from the cell surface for in-situ modification remains challenging. This study established a bacterial surface-initiated ATRP method and further applied it for enhanced Cr(VI) removal. The cell surface specificity was facilely achieved by cell surface labelling with azide substrates, following alkynyl ATRP initiator specifically anchoring with azide–alkyne click chemistry. Then, the ATRP polymerization was initiated from the cell surface, and different polymers were successfully applied to in-situ modification. Further analysis revealed that the modification of Shewanella oneidensis with poly (4-vinyl pyridine) and sodium polymethacrylate improved the heavy metal tolerance and enhanced the Cr(VI) removal rate of 2.6 times from 0.088 h−1 to 0.314 h−1. This work provided a novel idea for bacterial surface modification and would extend the application of ATRP in bioremediation.
在细胞表面使用聚合物对细胞进行改性,可使细胞获得或增强多种应用功能,其中原子转移自由基聚合(ATRP)因其生物相容性而具有巨大潜力。然而,从细胞表面特异性地引发原子转移自由基聚合以进行原位改性仍具有挑战性。本研究建立了一种细菌表面引发的 ATRP 方法,并将其进一步应用于增强六价铬的去除。细胞表面特异性是通过叠氮基质标记细胞表面,然后炔基 ATRP 引发剂通过叠氮-炔烃点击化学特异性锚定后轻松实现的。然后,从细胞表面开始 ATRP 聚合,成功地将不同的聚合物用于原位修饰。进一步的分析表明,用聚(4-乙烯基吡啶)和聚甲基丙烯酸钠修饰 Shewanella oneidensis 提高了其对重金属的耐受性,并将 Cr(VI) 的去除率从 0.088 h-1 提高到 0.314 h-1 的 2.6 倍。这项工作为细菌表面改性提供了一种新的思路,并将扩大 ATRP 在生物修复中的应用。
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引用次数: 0
Structural and functional analysis of l-methionine oxidase identified through sequence data mining 通过序列数据挖掘确定的蛋氨酸氧化酶的结构和功能分析。
IF 2.3 4区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-08-13 DOI: 10.1016/j.jbiosc.2024.07.014
Yui Kawamura, Sayaka Sugiura, Hayato Araseki, Taichi Chisuga, Shogo Nakano
l-Amino acid oxidase (LAAO), an FAD-dependent enzyme, catalyzes the oxidation of l-amino acids (l-AAs) to their corresponding imino acids. While LAAOs, which can oxidize charged or aromatic l-AAs specifically, have been extensively characterized across various species, LAAOs that have high specificity toward alkyl-chain l-AAs, such as l-Met, are hardly characterized for now. In this study, we screened a highly specific l-Met oxidizing LAAOs from Burkholderiales bacterium (BbMetOx) and Undibacterium sp. KW1 (UndMetOx) using sequence similarity network (SSN) analysis. These enzymes displayed an order of magnitude higher specific activity towards l-Met compared to other l-AAs. Enzyme activity assays showed that these LAAOs operate optimally at moderate condition because the optimal pH and Tm values were pH 7.0 and 58–60°C. We determined the crystal structures of wild-type BbMetOx (BbMetOx(WT)) and an inactivated mutant, BbMetOx (K304A), at 2.7 Å and 2.2 Å resolution, respectively. The overall structure of BbMetOx is closely similar to other known LAAOs of which structures were determined. Comparative analysis of the BbMetOx structures revealed significant conformational changes in the catalytic domain, particularly a movement of approximately 8 Å in the Cα atom of residue Y180. Further analysis highlighted four residues, i.e., Y180, M182, F300, and M302, as critical for l-Met recognition, with alanine substitution at these positions resulting in loss of activity. This study not only underscores the utility of SSN for discovering novel LAAOs but also advances our understanding of substrate specificity in this enzyme family.
l-氨基酸氧化酶(LAAO)是一种依赖于 FAD 的酶,可催化 l-氨基酸(l-As)氧化为相应的亚胺酸。虽然能特异性氧化带电或芳香族 l-AAs 的 LAAO 已在不同物种中得到广泛表征,但对烷基链 l-AAs(如 l-Met)具有高度特异性的 LAAO 目前几乎没有表征。在这项研究中,我们利用序列相似性网络(SSN)分析法筛选了来自伯克霍尔德氏菌(BbMetOx)和KW1 Undibacterium sp.(UndMetOx)的高特异性l-Met氧化LAAOs。与其他 l-AAs 相比,这些酶对 l-Met 的特异性活性高出一个数量级。酶活性测定显示,这些LAAOs在中等条件下运行最佳,因为最佳pH值和Tm值分别为pH7.0和58-60°C。我们分别以 2.7 Å 和 2.2 Å 的分辨率测定了野生型 BbMetOx(BbMetOx(WT))和失活突变体 BbMetOx(K304A)的晶体结构。BbMetOx 的整体结构与已确定结构的其他已知 LAAOs 非常相似。对 BbMetOx 结构的比较分析表明,催化结构域的构象发生了显著变化,尤其是残基 Y180 的 Cα 原子发生了约 8 Å 的移动。进一步的分析突出表明,Y180、M182、F300 和 M302 这四个残基对 l-Met 的识别至关重要,这些位置上的丙氨酸取代会导致活性丧失。这项研究不仅强调了 SSN 在发现新型 LAAOs 方面的作用,而且还加深了我们对该酶家族底物特异性的了解。
{"title":"Structural and functional analysis of l-methionine oxidase identified through sequence data mining","authors":"Yui Kawamura,&nbsp;Sayaka Sugiura,&nbsp;Hayato Araseki,&nbsp;Taichi Chisuga,&nbsp;Shogo Nakano","doi":"10.1016/j.jbiosc.2024.07.014","DOIUrl":"10.1016/j.jbiosc.2024.07.014","url":null,"abstract":"<div><div><span>l</span>-Amino acid oxidase (LAAO), an FAD-dependent enzyme, catalyzes the oxidation of <span>l</span>-amino acids (<span>l</span>-AAs) to their corresponding imino acids. While LAAOs, which can oxidize charged or aromatic <span>l</span>-AAs specifically, have been extensively characterized across various species, LAAOs that have high specificity toward alkyl-chain <span>l</span>-AAs, such as <span>l</span>-Met, are hardly characterized for now. In this study, we screened a highly specific <span>l</span>-Met oxidizing LAAOs from <em>Burkholderiales</em> bacterium (BbMetOx) and <em>Undibacterium</em> sp. KW1 (UndMetOx) using sequence similarity network (SSN) analysis. These enzymes displayed an order of magnitude higher specific activity towards <span>l</span>-Met compared to other <span>l</span>-AAs. Enzyme activity assays showed that these LAAOs operate optimally at moderate condition because the optimal pH and <em>T</em><sub>m</sub> values were pH 7.0 and 58–60°C. We determined the crystal structures of wild-type BbMetOx (BbMetOx(WT)) and an inactivated mutant, BbMetOx (K304A), at 2.7 Å and 2.2 Å resolution, respectively. The overall structure of BbMetOx is closely similar to other known LAAOs of which structures were determined. Comparative analysis of the BbMetOx structures revealed significant conformational changes in the catalytic domain, particularly a movement of approximately 8 Å in the C<sub>α</sub> atom of residue Y180. Further analysis highlighted four residues, i.e., Y180, M182, F300, and M302, as critical for <span>l</span>-Met recognition, with alanine substitution at these positions resulting in loss of activity. This study not only underscores the utility of SSN for discovering novel LAAOs but also advances our understanding of substrate specificity in this enzyme family.</div></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":"138 5","pages":"Pages 391-398"},"PeriodicalIF":2.3,"publicationDate":"2024-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141982307","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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
Screening of protein-based inhibitors for the intracellular domain of epidermal growth factor receptor by directed evolution using the yeast Gγ recruitment system 利用酵母 Gγ 招募系统,通过定向进化筛选表皮生长因子受体胞内结构域的蛋白抑制剂。
IF 2.3 4区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-08-09 DOI: 10.1016/j.jbiosc.2024.07.007
Ririka Asama , Masahiro Tominaga , Sayaka Ito , Yoichiro Ito , Kazuhiro Takemura , Shun Sakuraba , Kohei Katsurada , Nobuo Fukuda , Akihiko Kondo , Jun Ishii
Protein-based therapeutics, including antibodies and antibody-like-proteins, have increasingly attracted attention due to their high specificity compared to small-molecular drugs. The Gγ recruitment system, one of the in vivo yeast two-hybrid systems for detecting protein–protein interactions, has been previously developed using yeast signal transduction machinery. In this study, we modified the Gγ recruitment system to screen the protein mutants that efficiently bind to the intracellular domain of the epidermal growth factor receptor L858R mutant (cytoEGFRL858R). Using the modified platform, we performed in vivo directed evolution for growth factor receptor-bound protein 2 (Grb2) and its truncated variant containing only the Src-homology 2 (SH2) domain, successfully identifying several mutants that more strongly bound to cytoEGFRL858R than their parental proteins. Some of them contained novel beneficial mutations (F108Y and Q144H) and specifically bound to the recombinant cytosolic phosphorylated EGFR in vitro, highlighting the utility of the evolutionary platform.
与小分子药物相比,以蛋白质为基础的疗法(包括抗体和抗体样蛋白)具有高度特异性,因此越来越受到关注。Gγ 招募系统是体内酵母双杂交系统之一,用于检测蛋白质与蛋白质之间的相互作用。在本研究中,我们对 Gγ 招募系统进行了改进,以筛选能与表皮生长因子受体 L858R 突变体(cytoEGFRL858R)胞内结构域有效结合的蛋白质突变体。利用改进后的平台,我们对生长因子受体结合蛋白2(Grb2)及其仅含有Src-homology 2(SH2)结构域的截短变体进行了体内定向进化,成功鉴定出几种突变体,它们与cytoEGFRL858R的结合比亲代蛋白更强。其中一些突变体含有新的有益突变(F108Y和Q144H),并在体外与重组的细胞磷酸化表皮生长因子受体特异性结合,凸显了进化平台的实用性。
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引用次数: 0
Transcriptomic analysis reveals insights into the responses of Synechocystis sp. PCC 6803 to acidification during cultivation with ammonium salts as a nitrogen source 转录组分析揭示了 Synechocystis sp.
IF 2.3 4区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-08-06 DOI: 10.1016/j.jbiosc.2024.07.005
Kotaro Kobayashi , Kohei Yoneda , Yoshiaki Maeda , Iwane Suzuki

Utilizing ammonium in wastewater is a prospective way to reduce costs for bioproduction by photosynthetic organisms. A model cyanobacterium Synechocystis sp. PCC 6803 takes advantage of tolerance to ammonium compared to other microalgae. However, in this study, we report that Synechocystis growth was inhibited when cultured in a medium containing ammonium. This may be due to the pH decreasing below 6 caused by consuming ammonium. Transcriptomic analysis by RNA-seq revealed that the expression of the genes for proteases, chaperones, and antioxidant-scavenging enzymes was induced, but photosynthetic components were repressed. Although these regulations are similar to the previous studies on acidic stress in nitrate-containing culture, the expression of genes such as sigD, slr0042, slr0373, slr0374, and slr1501 was different, indicating that these phenomena are not simply identical to the known responses to acidic stress. The expression of the genes for photosynthesis, gluconeogenesis, and nitrogen assimilation was repressed, and glycolysis and the tricarboxylic acid cycle were induced. Despite the up-regulation of the carbon catabolism and down-regulation of nitrogen assimilation, the 2-oxoglutarate content in the ammonium-grown cells was lower than that in the nitrate-grown cells, and the contents of the major amino acids, such as Glu, Ala, Asp, and Gly were decreased, while the minor amino acids were the same or increased, especially Arg, Lys, Val, and Ile. These results demonstrated that the acidic stress induced by the consumption of ammonium ions differs from the sudden pH drop, and the Synechocystis cell manages amino acid levels to endure carbon limitation under the stress.

利用废水中的铵是光合生物降低生物生产成本的一种可行方法。模式蓝藻 Synechocystis sp. PCC 6803 与其他微藻相比,具有耐氨的优势。然而,在本研究中,我们发现 Synechocystis 在含铵的培养基中生长受到抑制。这可能是由于消耗铵导致 pH 值下降到 6 以下所致。通过 RNA-seq 进行转录组分析发现,蛋白酶、伴侣蛋白和抗氧化清除酶基因的表达受到诱导,但光合成分的表达受到抑制。虽然这些调控与之前含硝酸盐培养物中酸性胁迫的研究相似,但 sigD、slr0042、slr0373、slr0374 和 slr1501 等基因的表达却有所不同,表明这些现象与已知的酸性胁迫反应并不简单相同。光合作用、葡萄糖生成和氮同化基因的表达受到抑制,而糖酵解和三羧酸循环基因则被诱导。尽管碳分解代谢基因上调,氮同化作用基因下调,但铵盐生长细胞中 2-氧代戊二酸含量低于硝酸盐生长细胞,主要氨基酸如 Glu、Ala、Asp 和 Gly 的含量降低,而次要氨基酸含量不变或增加,尤其是 Arg、Lys、Val 和 Ile。这些结果表明,铵离子消耗引起的酸性胁迫不同于 pH 值的骤降,Synechocystis 细胞在胁迫下通过管理氨基酸水平来承受碳限制。
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引用次数: 0
Novel multifunctional plant growth-promoting bacteria isolated from the oil palm rhizosphere under long-term organic matter application 在长期施用有机物的条件下,从油棕根瘤菌中分离出的新型多功能植物生长促进菌。
IF 2.3 4区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-08-06 DOI: 10.1016/j.jbiosc.2024.07.008
Fandi Hidayat , Rizki Desika Putri Pane , Fadilla Sapalina , Eka Listia , Winarna , Muhammad Edwin Syahputra Lubis , Mugihito Oshiro , Kenji Sakai , Yukihiro Tashiro
Most agricultural products are presently cultivated on marginal lands with poor soil properties and unfavorable environmental conditions (diseases and abiotic stresses), which can threaten plant growth and yield. Plant growth-promoting bacteria (PGPB) are beneficial bacteria that promote plant growth and biomass and act as biocontrols against diseases and stress. However, most isolated PGPBs have a single function and low survival rates owing to their limited growth behaviors. In this study, we isolated multifunctional PGPB from oil palm rhizosphere, quantitatively measured their activities, and evaluated their effectiveness in Brassica rapa (Komatsuna) cultivation. This is the first study to report the isolation of three multifunctional PGPB strains with ammonium production, phosphate-potassium-silicate solubilization, and indole-3-acetic acid (IAA) production from the oil palm rhizosphere, namely Kosakonia oryzendophytica AJLB38, Enterobacter quasimori AJTS77, and Lelliottia jeotgali AJTS83. Additionally, these strains showed antifungal activity against the oil palm pathogen Ganoderma boninense. These strains grow under high temperature, acidic and alkaline pH, and high salt concentration, which would result in their proliferation in various environmental conditions. The cultivation experiments revealed these strains improved the growth and biomass with half the dosage of chemical fertilizer application, which was not significantly different to the full dosage. Furthermore, the overall plant growth-promoting activities in quantitative assays and overall B. rapa growth in cultivation experiments were statistically correlated, which could contribute to the prediction of plant growth promotion without plant cultivation experiments. Thus, the selected PGPB could be valuable as a biofertilizer to improve soil health and quality and promote agricultural sustainability.
目前,大多数农产品都是在土壤贫瘠、环境条件不利(病害和非生物胁迫)的贫瘠土地上种植的,这可能会威胁到植物的生长和产量。植物生长促进菌(PGPB)是促进植物生长和生物量的有益细菌,也是抵御病害和胁迫的生物控制菌。然而,由于其生长行为有限,大多数分离出的 PGPB 功能单一且存活率低。在这项研究中,我们从油棕根瘤菌中分离出了多功能 PGPB,定量测定了它们的活性,并评估了它们在甘蓝型油菜(小松菜)栽培中的有效性。这是首次报道从油棕根瘤菌中分离出三种多功能 PGPB 菌株,即 Kosakonia oryzendophytica AJLB38、Enterobacter quasimori AJTS77 和 Lelliottia jeotgali AJTS83,它们具有产铵、磷酸-钾-硅酸盐增溶和产生吲哚-3-乙酸(IAA)的功能。此外,这些菌株对油棕病原体 Ganoderma boninense 具有抗真菌活性。这些菌株可在高温、酸碱 pH 值和高盐浓度条件下生长,因此可在各种环境条件下增殖。栽培实验表明,这些菌株在化肥施用量减半的情况下就能改善生长和生物量,与施用全量化肥相比没有显著差异。此外,定量检测中的总体植物生长促进活性与栽培实验中的蚕豆总体生长情况在统计学上存在相关性,这有助于在不进行植物栽培实验的情况下预测植物的生长促进情况。因此,所选的 PGPB 可作为生物肥料改善土壤健康和质量,促进农业可持续发展。
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引用次数: 0
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Journal of bioscience and bioengineering
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