Cyclophilin B (CypB), a significant member of immunophilins family with peptidyl-prolyl cis-trans isomerase (PPIase) activity, is crucial for the growth and metabolism of prokaryotes and eukaryotes. Sporothrix globosa (S. globosa), a principal pathogen in the Sporothrix complex, causes sporotrichosis. Transcriptomic analysis identified the cypB gene as highly expressed in S. globosa. Our previous study demonstrated that the recombinant Escherichia coli strain containing SgcypB gene failed to produce sufficient product when it was induced to express the protein, implying the potential toxicity of recombinant protein to the bacterial host. Bioinformatics analysis revealed that SgCypB contains transmembrane peptides within the 52 amino acid residues at the N-terminus and 21 amino acids near the C-terminus, and 18 amino acid residues within the cytoplasm. AlphaFold2 predicted a SgCypB 3D structure in which there is an independent PPIase domain consisting of a spherical extracellular part. Hence, we chose to express the extracellular domain to yield high-level recombinant protein with PPIase activity. Finally, we successfully produced high-yield, truncated recombinant CypB protein from S. globosa (SgtrCypB) that retained characteristic PPIase activity without host bacterium toxicity. This study presents an alternative expression strategy for proteins toxic to prokaryotes, such as SgCypB.
One-sentence summary: The recombinant cyclophilin B protein of Sporothrix globosa was expressed successfully by retaining extracellular domain with peptidyl-prolyl cis-trans isomerase activity to avoid toxicity to the host bacterium.
环嗜蛋白B(CypB)是免疫嗜蛋白家族的重要成员,具有肽基-脯氨酰顺反异构酶(PPIase)活性,对原核生物和真核生物的生长和新陈代谢至关重要。球孢子虫(S.globosa)是球孢子虫复合体中的一种主要病原体,会引起孢子斑枯病。转录组分析发现,cypB 基因在球孢子虫中高度表达。我们之前的研究表明,含有 SgcypB 基因的重组大肠杆菌菌株在诱导表达该蛋白时未能产生足够的产物,这意味着重组蛋白对细菌宿主具有潜在毒性。生物信息学分析表明,SgCypB 的 N 端 52 个氨基酸残基和 C 端附近的 21 个氨基酸残基中含有跨膜肽,细胞质中含有 18 个氨基酸残基。AlphaFold2 预测了 SgCypB 的三维结构,其中有一个由球形胞外部分组成的独立 PPIase 结构域。因此,我们选择表达胞外结构域,以获得具有 PPIase 活性的高水平重组蛋白。最后,我们成功地从球囊虫中制备出了高产、截短的重组环嗜蛋白 B 蛋白(SgtrCypB),它保留了特有的 PPIase 活性,且不会对宿主菌产生毒性。这项研究为对原核生物有毒的蛋白质(如 SgCypB)提供了一种替代表达策略。
{"title":"Optimized expression of Peptidyl-prolyl cis/transisomerase cyclophilinB with prokaryotic toxicity from Sporothrix globosa.","authors":"Ling Hu, Baicheng Deng, Rong Wu, Miaorong Zhan, Xuchu Hu, Huaiqiu Huang","doi":"10.1093/jimb/kuae017","DOIUrl":"10.1093/jimb/kuae017","url":null,"abstract":"<p><p>Cyclophilin B (CypB), a significant member of immunophilins family with peptidyl-prolyl cis-trans isomerase (PPIase) activity, is crucial for the growth and metabolism of prokaryotes and eukaryotes. Sporothrix globosa (S. globosa), a principal pathogen in the Sporothrix complex, causes sporotrichosis. Transcriptomic analysis identified the cypB gene as highly expressed in S. globosa. Our previous study demonstrated that the recombinant Escherichia coli strain containing SgcypB gene failed to produce sufficient product when it was induced to express the protein, implying the potential toxicity of recombinant protein to the bacterial host. Bioinformatics analysis revealed that SgCypB contains transmembrane peptides within the 52 amino acid residues at the N-terminus and 21 amino acids near the C-terminus, and 18 amino acid residues within the cytoplasm. AlphaFold2 predicted a SgCypB 3D structure in which there is an independent PPIase domain consisting of a spherical extracellular part. Hence, we chose to express the extracellular domain to yield high-level recombinant protein with PPIase activity. Finally, we successfully produced high-yield, truncated recombinant CypB protein from S. globosa (SgtrCypB) that retained characteristic PPIase activity without host bacterium toxicity. This study presents an alternative expression strategy for proteins toxic to prokaryotes, such as SgCypB.</p><p><strong>One-sentence summary: </strong>The recombinant cyclophilin B protein of Sporothrix globosa was expressed successfully by retaining extracellular domain with peptidyl-prolyl cis-trans isomerase activity to avoid toxicity to the host bacterium.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11104532/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140908914","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Amylosucrase (EC 2.4.1.4) is a versatile enzyme with significant potential in biotechnology and food production. To facilitate its efficient preparation, a novel expression strategy was implemented in Bacillus licheniformis for the secretory expression of Neisseria polysaccharea amylosucrase (NpAS). The host strain B. licheniformis CBBD302 underwent genetic modification through the deletion of sacB, a gene responsible for encoding levansucrase that synthesizes extracellular levan from sucrose, resulting in a levan-deficient strain, B. licheniformis CBBD302B. Neisseria polysaccharea amylosucrase was successfully expressed in B. licheniformis CBBD302B using the highly efficient Sec-type signal peptide SamyL, but its extracellular translocation was unsuccessful. Consequently, the expression of NpAS via the twin-arginine translocation (TAT) pathway was investigated using the signal peptide SglmU. The study revealed that NpAS could be effectively translocated extracellularly through the TAT pathway, with the signal peptide SglmU facilitating the process. Remarkably, 62.81% of the total expressed activity was detected in the medium. This study marks the first successful secretory expression of NpAS in Bacillus species host cells, establishing a foundation for its future efficient production.
One-sentence summary: Amylosucrase was secreted in Bacillus licheniformis via the twin-arginine translocation pathway.
{"title":"Secretory expression of amylosucrase in Bacillus licheniformis through twin-arginine translocation pathway.","authors":"Caizhe Wang, Dandan Niu, Nokuthula Peace Mchunu, Meng Zhang, Suren Singh, Zhengxiang Wang","doi":"10.1093/jimb/kuae004","DOIUrl":"10.1093/jimb/kuae004","url":null,"abstract":"<p><p>Amylosucrase (EC 2.4.1.4) is a versatile enzyme with significant potential in biotechnology and food production. To facilitate its efficient preparation, a novel expression strategy was implemented in Bacillus licheniformis for the secretory expression of Neisseria polysaccharea amylosucrase (NpAS). The host strain B. licheniformis CBBD302 underwent genetic modification through the deletion of sacB, a gene responsible for encoding levansucrase that synthesizes extracellular levan from sucrose, resulting in a levan-deficient strain, B. licheniformis CBBD302B. Neisseria polysaccharea amylosucrase was successfully expressed in B. licheniformis CBBD302B using the highly efficient Sec-type signal peptide SamyL, but its extracellular translocation was unsuccessful. Consequently, the expression of NpAS via the twin-arginine translocation (TAT) pathway was investigated using the signal peptide SglmU. The study revealed that NpAS could be effectively translocated extracellularly through the TAT pathway, with the signal peptide SglmU facilitating the process. Remarkably, 62.81% of the total expressed activity was detected in the medium. This study marks the first successful secretory expression of NpAS in Bacillus species host cells, establishing a foundation for its future efficient production.</p><p><strong>One-sentence summary: </strong>Amylosucrase was secreted in Bacillus licheniformis via the twin-arginine translocation pathway.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":null,"pages":null},"PeriodicalIF":3.4,"publicationDate":"2024-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10849164/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139521156","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Bruna C Bolzico, Sofia Racca, Jorge N Khawam, Rodrigo J Leonardi, Ariel H Tomassi, Maria T Benzzo, Raul N Comelli
d-Xylose is a metabolizable carbon source for several non-Saccharomyces species, but not for native strains of S. cerevisiae. For the potential application of xylose-assimilating yeasts in biotechnological processes, a deeper understanding of pentose catabolism is needed. This work aimed to investigate the traits behind xylose utilization in diverse yeast species. The performance of 9 selected xylose-metabolizing yeast strains was evaluated and compared across 3 oxygenation conditions. Oxygenation diversely impacted growth, xylose consumption, and product accumulation. Xylose utilization by ethanol-producing species such as Spathaspora passalidarum and Scheffersomyces stipitis was less affected by oxygen restriction compared with other xylitol-accumulating species such as Meyerozyma guilliermondii, Naganishia liquefaciens, and Yamadazyma sp., for which increased aeration stimulated xylose assimilation considerably. Spathaspora passalidarum exhibited superior conversion of xylose to ethanol and showed the fastest growth and xylose consumption in all 3 conditions. By performing assays under identical conditions for all selected yeasts, we minimize bias in comparisons, providing valuable insight into xylose metabolism and facilitating the development of robust bioprocesses.
One-sentence summary: This work aims to expand the knowledge of xylose utilization in different yeast species, with a focus on how oxygenation impacts xylose assimilation.
{"title":"Exploring xylose metabolism in non-conventional yeasts: kinetic characterization and product accumulation under different aeration conditions.","authors":"Bruna C Bolzico, Sofia Racca, Jorge N Khawam, Rodrigo J Leonardi, Ariel H Tomassi, Maria T Benzzo, Raul N Comelli","doi":"10.1093/jimb/kuae023","DOIUrl":"10.1093/jimb/kuae023","url":null,"abstract":"<p><p>d-Xylose is a metabolizable carbon source for several non-Saccharomyces species, but not for native strains of S. cerevisiae. For the potential application of xylose-assimilating yeasts in biotechnological processes, a deeper understanding of pentose catabolism is needed. This work aimed to investigate the traits behind xylose utilization in diverse yeast species. The performance of 9 selected xylose-metabolizing yeast strains was evaluated and compared across 3 oxygenation conditions. Oxygenation diversely impacted growth, xylose consumption, and product accumulation. Xylose utilization by ethanol-producing species such as Spathaspora passalidarum and Scheffersomyces stipitis was less affected by oxygen restriction compared with other xylitol-accumulating species such as Meyerozyma guilliermondii, Naganishia liquefaciens, and Yamadazyma sp., for which increased aeration stimulated xylose assimilation considerably. Spathaspora passalidarum exhibited superior conversion of xylose to ethanol and showed the fastest growth and xylose consumption in all 3 conditions. By performing assays under identical conditions for all selected yeasts, we minimize bias in comparisons, providing valuable insight into xylose metabolism and facilitating the development of robust bioprocesses.</p><p><strong>One-sentence summary: </strong>This work aims to expand the knowledge of xylose utilization in different yeast species, with a focus on how oxygenation impacts xylose assimilation.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11247345/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141468542","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Onyinye Okonkwo, Chang Dou, Ethan Oksen, Akash Narani, Wilian Marcondes, Xiaowen Chen, Joonhoon Kim, Yuqian Gao, Meagan C Burnet, Bobbie-Jo M Webb-Robertson, Brenton C Poirier, Deepti Tanjore, Jon K Magnuson, Nathalie Munoz Munoz, James Gardner
Microbial conversion of lignocellulosic biomass represents an alternative route for production of biofuels and bioproducts. While researchers have mostly focused on engineering strains such as Rhodotorula toruloides for better bisabolene production as a sustainable aviation fuel, less is known about the impact of the feedstock heterogeneity on bisabolene production. Critical material attributes like feedstock composition, nutritional content, and inhibitory compounds can all influence bioconversion. Further, the given feedstocks can have a marked influence on selection of suitable pretreatment and hydrolysis technologies, optimizing the fermentation conditions, and possibly even modifying the microorganism's metabolic pathways, to better utilize the available feedstock. This work aimed to examine and understand how variations in corn stover batches, anatomical fractions, and storage conditions impact the efficiency of bisabolene production by R. toruloides. All of these represent different facets of feedstock heterogeneity. Deacetylation, mechanical refining, and enzymatic hydrolysis of these variable feedstocks served as the basis of this research. The resulting hydrolysates were converted to bisabolene via fermentation, a sustainable aviation fuel precursor, using an engineered R. toruloides strain. This study showed that different sources of feedstock heterogeneity can influence microbial growth and product titer in counterintuitive ways, as revealed through global analysis of protein expression. The maximum bisabolene produced by R. toruloides was on the stalk fraction of corn stover hydrolysate (8.89 ± 0.47 g/L). Further, proteomics analysis comparing the protein expression between the anatomic fractions showed that proteins relating to carbohydrate metabolism, energy production, and conversion as well as inorganic ion transport metabolism were either significantly upregulated or downregulated. Specifically, downregulation of proteins related to the iron-sulfur cluster in stalk fraction suggests a coordinated response by R. toruloides to maintain overall metabolic balance, and this was corroborated by the concentration of iron in the feedstocks.
One-sentence summary: This study elucidates the effects of different sources of corn stover on bisabolene production by engineered Rhodotorula toruloides, highlighting the importance of understanding feedstock variability to enhance bioprocess efficiency and economic outcomes.
木质纤维素生物质的微生物转化是生产生物燃料和生物产品的另一条途径。研究人员主要关注如何通过工程菌株(如 Rhodotorula toruloides)来更好地生产作为可持续航空燃料(SAF)的双萘酚,但对于原料的异质性对双萘酚生产的影响却知之甚少。原料成分、营养成分和抑制性化合物等关键材料属性都会影响生物转化。此外,给定的原料会对选择合适的预处理和水解技术、优化发酵条件,甚至可能改变微生物的代谢途径产生显著影响,从而更好地利用现有原料。这项工作旨在研究和了解玉米秸秆的批次、解剖学组分和储存条件的变化如何影响 R. toruloides 生产双大麻酚烯的效率。所有这些都代表了原料异质性的不同方面。本研究以这些不同原料的脱乙酰化、机械精制和酶水解(DMR-EH)为基础。所产生的水解物通过发酵转化为双羟基甲苯,这是一种可持续的航空燃料前体,使用的是经改造的 R. toruloides 菌株。这项研究表明,不同来源的原料异质性会以反直觉的方式影响微生物的生长和产品滴度,蛋白质表达的全局分析也揭示了这一点。在玉米秸秆水解物的茎秆部分(8.89 ± 0.47 克/升),R. toruloides 产生的双酚含量最高。此外,蛋白质组学分析比较了不同解剖组分的蛋白质表达情况,结果表明,与碳水化合物代谢、能量产生和转化以及无机离子转运代谢有关的蛋白质要么明显上调,要么明显下调。具体而言,茎秆部分与铁硫簇相关的蛋白质表达下调,表明托罗拉氏菌为维持整体代谢平衡而采取了协调反应,这一点也得到了原料中铁浓度的证实。
{"title":"Corn stover variability drives differences in bisabolene production by engineered Rhodotorula toruloides.","authors":"Onyinye Okonkwo, Chang Dou, Ethan Oksen, Akash Narani, Wilian Marcondes, Xiaowen Chen, Joonhoon Kim, Yuqian Gao, Meagan C Burnet, Bobbie-Jo M Webb-Robertson, Brenton C Poirier, Deepti Tanjore, Jon K Magnuson, Nathalie Munoz Munoz, James Gardner","doi":"10.1093/jimb/kuae034","DOIUrl":"10.1093/jimb/kuae034","url":null,"abstract":"<p><p>Microbial conversion of lignocellulosic biomass represents an alternative route for production of biofuels and bioproducts. While researchers have mostly focused on engineering strains such as Rhodotorula toruloides for better bisabolene production as a sustainable aviation fuel, less is known about the impact of the feedstock heterogeneity on bisabolene production. Critical material attributes like feedstock composition, nutritional content, and inhibitory compounds can all influence bioconversion. Further, the given feedstocks can have a marked influence on selection of suitable pretreatment and hydrolysis technologies, optimizing the fermentation conditions, and possibly even modifying the microorganism's metabolic pathways, to better utilize the available feedstock. This work aimed to examine and understand how variations in corn stover batches, anatomical fractions, and storage conditions impact the efficiency of bisabolene production by R. toruloides. All of these represent different facets of feedstock heterogeneity. Deacetylation, mechanical refining, and enzymatic hydrolysis of these variable feedstocks served as the basis of this research. The resulting hydrolysates were converted to bisabolene via fermentation, a sustainable aviation fuel precursor, using an engineered R. toruloides strain. This study showed that different sources of feedstock heterogeneity can influence microbial growth and product titer in counterintuitive ways, as revealed through global analysis of protein expression. The maximum bisabolene produced by R. toruloides was on the stalk fraction of corn stover hydrolysate (8.89 ± 0.47 g/L). Further, proteomics analysis comparing the protein expression between the anatomic fractions showed that proteins relating to carbohydrate metabolism, energy production, and conversion as well as inorganic ion transport metabolism were either significantly upregulated or downregulated. Specifically, downregulation of proteins related to the iron-sulfur cluster in stalk fraction suggests a coordinated response by R. toruloides to maintain overall metabolic balance, and this was corroborated by the concentration of iron in the feedstocks.</p><p><strong>One-sentence summary: </strong>This study elucidates the effects of different sources of corn stover on bisabolene production by engineered Rhodotorula toruloides, highlighting the importance of understanding feedstock variability to enhance bioprocess efficiency and economic outcomes.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11467694/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142348233","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Benjamin Ingham, Katherine Hollywood, Phavit Wongsirichot, Alistair Veitch, James Winterburn
The application of liquid chromatography and mass spectrometry (MS) is a challenging area of research for structural identification of sophorolipids, owing to the large number of possible variations in structure and limited knowledge on the separation and fragmentation characteristics of the variants. The aims of this work was to provide a comprehensive analysis of the expected characteristics and fragmentation patterns of a wide range of sophorolipid biosurfactant congeners, providing a methodology and process alongside freely available data to inform and enable future research of commercial or novel sophorolipids. Samples of acidic and lactonic sophorolipid standards were tested using reverse-phase ultra-high performance liquid chromatography and identified using electrospray ionization MS. 37 sophorolipid variants were identified and compared for their elution order and fragmentation pattern under MS/MS. The retention time of sophorolipids was increased by the presence of lactonization, unsaturation, chain length, and acetylation as hydrophobic interactions with the C18 stationary phase increased. A key finding that acidic forms can elute later than lactonic variants was obtained when the fatty acid length and unsaturation and acetylation are altered, in contradiction to previous literature statements. Fragmentation pathways were determined for lactonic and acidic variants under negative [M-H]- and positive [M+NH4]+ ionization, and unique patterns/pathways were identified to help determine the structural components present. The first publicly available database of chromatograms and MS2 spectra has been made available to aid in the identification of sophorolipid components and provide a reliable dataset to accelerate future research into novel sophorolipids and shorten the time to innovation.
One-sentence summary: This article describes the process and challenges in identifying different structures of eco-friendly biosurfactants, providing a novel database to compare results.
{"title":"Uncovering the fragmentation and separation characteristics of sophorolipid biosurfactants with LC-MS-ESI.","authors":"Benjamin Ingham, Katherine Hollywood, Phavit Wongsirichot, Alistair Veitch, James Winterburn","doi":"10.1093/jimb/kuae035","DOIUrl":"10.1093/jimb/kuae035","url":null,"abstract":"<p><p>The application of liquid chromatography and mass spectrometry (MS) is a challenging area of research for structural identification of sophorolipids, owing to the large number of possible variations in structure and limited knowledge on the separation and fragmentation characteristics of the variants. The aims of this work was to provide a comprehensive analysis of the expected characteristics and fragmentation patterns of a wide range of sophorolipid biosurfactant congeners, providing a methodology and process alongside freely available data to inform and enable future research of commercial or novel sophorolipids. Samples of acidic and lactonic sophorolipid standards were tested using reverse-phase ultra-high performance liquid chromatography and identified using electrospray ionization MS. 37 sophorolipid variants were identified and compared for their elution order and fragmentation pattern under MS/MS. The retention time of sophorolipids was increased by the presence of lactonization, unsaturation, chain length, and acetylation as hydrophobic interactions with the C18 stationary phase increased. A key finding that acidic forms can elute later than lactonic variants was obtained when the fatty acid length and unsaturation and acetylation are altered, in contradiction to previous literature statements. Fragmentation pathways were determined for lactonic and acidic variants under negative [M-H]- and positive [M+NH4]+ ionization, and unique patterns/pathways were identified to help determine the structural components present. The first publicly available database of chromatograms and MS2 spectra has been made available to aid in the identification of sophorolipid components and provide a reliable dataset to accelerate future research into novel sophorolipids and shorten the time to innovation.</p><p><strong>One-sentence summary: </strong>This article describes the process and challenges in identifying different structures of eco-friendly biosurfactants, providing a novel database to compare results.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11484030/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142348235","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Moriel J Dror, Joshua Misa, Danielle A Yee, Angela M Chu, Rachel K Yu, Bradley B Chan, Lauren S Aoyama, Anjali P Chaparala, Sarah E O'Connor, Yi Tang
Monoterpene indole alkaloids (MIAs) are a class of natural products comprised of thousands of structurally unique bioactive compounds with significant therapeutic values. Due to difficulties associated with isolation from native plant species and organic synthesis of these structurally complex molecules, microbial production of MIAs using engineered hosts are highly desired. In this work, we report the engineering of fully integrated Saccharomyces cerevisiae strains that allow de novo access to strictosidine, the universal precursor to thousands of MIAs at 30-40 mg/L. The optimization efforts were based on a previously reported yeast strain that is engineered to produce high titers of the monoterpene precursor geraniol through compartmentalization of mevalonate pathway in the mitochondria. Our approaches here included the use of CRISPR-dCas9 interference to identify mitochondria diphosphate transporters that negatively impact the titer of the monoterpene, followed by genetic inactivation; the overexpression of transcriptional regulators that increase cellular respiration and mitochondria biogenesis. Strain construction included the strategic integration of genes encoding both MIA biosynthetic and accessory enzymes into the genome under a variety of constitutive and inducible promoters. Following successful de novo production of strictosidine, complex alkaloids belonging to heteroyohimbine and corynantheine families were reconstituted in the host with introduction of additional downstream enzymes. We demonstrate that the serpentine/alstonine pair can be produced at ∼5 mg/L titer, while corynantheidine, the precursor to mitragynine can be produced at ∼1 mg/L titer. Feeding of halogenated tryptamine led to the biosynthesis of analogs of alkaloids in both families. Collectively, our yeast strain represents an excellent starting point to further engineer biosynthetic bottlenecks in this pathway and to access additional MIAs and analogs through microbial fermentation.
One sentence summary: An Saccharomyces cerevisiae-based microbial platform was developed for the biosynthesis of monoterpene indole alkaloids, including the universal precursor strictosidine and further modified heteroyohimbine and corynantheidine alkaloids.
{"title":"Engineered biosynthesis of plant heteroyohimbine and corynantheine alkaloids in Saccharomyces cerevisiae.","authors":"Moriel J Dror, Joshua Misa, Danielle A Yee, Angela M Chu, Rachel K Yu, Bradley B Chan, Lauren S Aoyama, Anjali P Chaparala, Sarah E O'Connor, Yi Tang","doi":"10.1093/jimb/kuad047","DOIUrl":"10.1093/jimb/kuad047","url":null,"abstract":"<p><p>Monoterpene indole alkaloids (MIAs) are a class of natural products comprised of thousands of structurally unique bioactive compounds with significant therapeutic values. Due to difficulties associated with isolation from native plant species and organic synthesis of these structurally complex molecules, microbial production of MIAs using engineered hosts are highly desired. In this work, we report the engineering of fully integrated Saccharomyces cerevisiae strains that allow de novo access to strictosidine, the universal precursor to thousands of MIAs at 30-40 mg/L. The optimization efforts were based on a previously reported yeast strain that is engineered to produce high titers of the monoterpene precursor geraniol through compartmentalization of mevalonate pathway in the mitochondria. Our approaches here included the use of CRISPR-dCas9 interference to identify mitochondria diphosphate transporters that negatively impact the titer of the monoterpene, followed by genetic inactivation; the overexpression of transcriptional regulators that increase cellular respiration and mitochondria biogenesis. Strain construction included the strategic integration of genes encoding both MIA biosynthetic and accessory enzymes into the genome under a variety of constitutive and inducible promoters. Following successful de novo production of strictosidine, complex alkaloids belonging to heteroyohimbine and corynantheine families were reconstituted in the host with introduction of additional downstream enzymes. We demonstrate that the serpentine/alstonine pair can be produced at ∼5 mg/L titer, while corynantheidine, the precursor to mitragynine can be produced at ∼1 mg/L titer. Feeding of halogenated tryptamine led to the biosynthesis of analogs of alkaloids in both families. Collectively, our yeast strain represents an excellent starting point to further engineer biosynthetic bottlenecks in this pathway and to access additional MIAs and analogs through microbial fermentation.</p><p><strong>One sentence summary: </strong>An Saccharomyces cerevisiae-based microbial platform was developed for the biosynthesis of monoterpene indole alkaloids, including the universal precursor strictosidine and further modified heteroyohimbine and corynantheidine alkaloids.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":null,"pages":null},"PeriodicalIF":3.4,"publicationDate":"2024-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10995622/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138885046","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Łukasz Wysocki, Patrycja Adamczuk, Paula Bardadyn, Anna Gabor, Karolina Jelonek, Monika Kudelska, Maksymilian Kukuć, Adrianna Piasek, Marta Pietras, Monika Słomka, Zoja Trojan, Wiktoria Tybulczuk, Anna Sobiepanek, Joanna Żylińska-Urban, Joanna Cieśla
Spent coffee grounds (SCG) are commercial waste that are still rich in numerous valuable ingredients and can be further processed into useful products such as coffee oil, antioxidant extract, lactic acid, and lignin. The challenge and innovation is to develop the SCG processing technology, maximizing the use of raw material and minimizing the use of other resources within the sequential process. The presented research is focused on the aspect of biotechnological production of lactic acid from SCG by using the Lacticaseibacillus rhamnosus strain isolated from the environment. Thanks to the optimization of the processes of acid hydrolysis, neutralization, enzymatic hydrolysis of SCG, and fermentation, the obtained concentration of lactic acid was increased after 72 hr of culture from the initial 4.60 g/l to 48.6 g/l. In addition, the whole process has been improved, taking into account the dependence on other processes within the complete SCG biorefinery, economy, energy, and waste aspects. Costly enzymatic hydrolysis was completely eliminated, and it was proven that supplementation of SCG hydrolysate with expensive yeast extract can be replaced by cheap waste from the agri-food industry.
One-sentence summary: A process for efficient lactic acid production from spent coffee grounds using the Lacticaseibacillus rhamnosus strain was developed and optimized, including nutrient solution preparation, supplementation and fermentation.
{"title":"Development of lactic acid production from coffee grounds hydrolysate by fermentation with Lacticaseibacillus rhamnosus.","authors":"Łukasz Wysocki, Patrycja Adamczuk, Paula Bardadyn, Anna Gabor, Karolina Jelonek, Monika Kudelska, Maksymilian Kukuć, Adrianna Piasek, Marta Pietras, Monika Słomka, Zoja Trojan, Wiktoria Tybulczuk, Anna Sobiepanek, Joanna Żylińska-Urban, Joanna Cieśla","doi":"10.1093/jimb/kuae032","DOIUrl":"10.1093/jimb/kuae032","url":null,"abstract":"<p><p>Spent coffee grounds (SCG) are commercial waste that are still rich in numerous valuable ingredients and can be further processed into useful products such as coffee oil, antioxidant extract, lactic acid, and lignin. The challenge and innovation is to develop the SCG processing technology, maximizing the use of raw material and minimizing the use of other resources within the sequential process. The presented research is focused on the aspect of biotechnological production of lactic acid from SCG by using the Lacticaseibacillus rhamnosus strain isolated from the environment. Thanks to the optimization of the processes of acid hydrolysis, neutralization, enzymatic hydrolysis of SCG, and fermentation, the obtained concentration of lactic acid was increased after 72 hr of culture from the initial 4.60 g/l to 48.6 g/l. In addition, the whole process has been improved, taking into account the dependence on other processes within the complete SCG biorefinery, economy, energy, and waste aspects. Costly enzymatic hydrolysis was completely eliminated, and it was proven that supplementation of SCG hydrolysate with expensive yeast extract can be replaced by cheap waste from the agri-food industry.</p><p><strong>One-sentence summary: </strong>A process for efficient lactic acid production from spent coffee grounds using the Lacticaseibacillus rhamnosus strain was developed and optimized, including nutrient solution preparation, supplementation and fermentation.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11399779/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142125953","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ihtisham Ul Haq, Sajida Maryam, Divine Y Shyntum, Taj A Khan, Fan Li
The growing prevalence of fungal infections alongside rising resistance to antifungal drugs poses a significant challenge to public health safety. At the close of the 2000s, major pharmaceutical firms began to scale back on antimicrobial research due to repeated setbacks and diminished economic gains, leaving only smaller companies and research labs to pursue new antifungal solutions. Among various natural sources explored for novel antifungal compounds, antifungal peptides (AFPs) emerge as particularly promising. Despite their potential, AFPs receive less focus than their antibacterial counterparts. These peptides have been sourced extensively from nature, including plants, animals, insects, and especially bacteria and fungi. Furthermore, with advancements in recombinant biotechnology and computational biology, AFPs can also be synthesized in lab settings, facilitating peptide production. AFPs are noted for their wide-ranging efficacy, in vitro and in vivo safety, and ability to combat biofilms. They are distinguished by their high specificity, minimal toxicity to cells, and reduced likelihood of resistance development. This review aims to comprehensively cover AFPs, including their sources-both natural and synthetic-their antifungal and biofilm-fighting capabilities in laboratory and real-world settings, their action mechanisms, and the current status of AFP research.
One-sentence summary: This comprehensive review of AFPs will be helpful for further research in antifungal research.
{"title":"Exploring the frontiers of therapeutic breadth of antifungal peptides: A new avenue in antifungal drugs.","authors":"Ihtisham Ul Haq, Sajida Maryam, Divine Y Shyntum, Taj A Khan, Fan Li","doi":"10.1093/jimb/kuae018","DOIUrl":"10.1093/jimb/kuae018","url":null,"abstract":"<p><p>The growing prevalence of fungal infections alongside rising resistance to antifungal drugs poses a significant challenge to public health safety. At the close of the 2000s, major pharmaceutical firms began to scale back on antimicrobial research due to repeated setbacks and diminished economic gains, leaving only smaller companies and research labs to pursue new antifungal solutions. Among various natural sources explored for novel antifungal compounds, antifungal peptides (AFPs) emerge as particularly promising. Despite their potential, AFPs receive less focus than their antibacterial counterparts. These peptides have been sourced extensively from nature, including plants, animals, insects, and especially bacteria and fungi. Furthermore, with advancements in recombinant biotechnology and computational biology, AFPs can also be synthesized in lab settings, facilitating peptide production. AFPs are noted for their wide-ranging efficacy, in vitro and in vivo safety, and ability to combat biofilms. They are distinguished by their high specificity, minimal toxicity to cells, and reduced likelihood of resistance development. This review aims to comprehensively cover AFPs, including their sources-both natural and synthetic-their antifungal and biofilm-fighting capabilities in laboratory and real-world settings, their action mechanisms, and the current status of AFP research.</p><p><strong>One-sentence summary: </strong>This comprehensive review of AFPs will be helpful for further research in antifungal research.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11119867/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140861537","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Correction to: Characterization and catalytic investigation of fungal single-module nonribosomal peptide synthetase in terpene-amino acid meroterpenoid biosynthesis.","authors":"","doi":"10.1093/jimb/kuae002","DOIUrl":"10.1093/jimb/kuae002","url":null,"abstract":"","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":null,"pages":null},"PeriodicalIF":3.4,"publicationDate":"2024-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10845890/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139485729","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The increasing global population and climate change pose significant challenges to agriculture, particularly in managing plant diseases caused by phytopathogens. Traditional methods, including chemical pesticides and antibiotics, have become less effective due to pathogen resistance and environmental concerns. Phage therapy emerges as a promising alternative, offering a sustainable and precise approach to controlling plant bacterial diseases without harming beneficial soil microorganisms. This review explores the potential of bacteriophages as biocontrol agents, highlighting their specificity, rapid multiplication, and minimal environmental impact. We discuss the historical context, current applications, and prospects of phage therapy in agriculture, emphasizing its role in enhancing crop yield and quality. Additionally, the paper examines the integration of phage therapy with modern agricultural practices and the development phage cocktails and genetically engineered phages to combat resistant pathogens. The findings suggest that phage therapy could revolutionize phytopathological management, contributing to global food security and sustainable agricultural practices.
One-sentence summary: The burden of plant diseases and phage-based phytopathological treatment.
{"title":"Phytopathological management through bacteriophages: enhancing food security amidst climate change.","authors":"Ihtisham Ul Haq, Mehtab Khan, Imran Khan","doi":"10.1093/jimb/kuae031","DOIUrl":"10.1093/jimb/kuae031","url":null,"abstract":"<p><p>The increasing global population and climate change pose significant challenges to agriculture, particularly in managing plant diseases caused by phytopathogens. Traditional methods, including chemical pesticides and antibiotics, have become less effective due to pathogen resistance and environmental concerns. Phage therapy emerges as a promising alternative, offering a sustainable and precise approach to controlling plant bacterial diseases without harming beneficial soil microorganisms. This review explores the potential of bacteriophages as biocontrol agents, highlighting their specificity, rapid multiplication, and minimal environmental impact. We discuss the historical context, current applications, and prospects of phage therapy in agriculture, emphasizing its role in enhancing crop yield and quality. Additionally, the paper examines the integration of phage therapy with modern agricultural practices and the development phage cocktails and genetically engineered phages to combat resistant pathogens. The findings suggest that phage therapy could revolutionize phytopathological management, contributing to global food security and sustainable agricultural practices.</p><p><strong>One-sentence summary: </strong>The burden of plant diseases and phage-based phytopathological treatment.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11388930/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142108120","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}