{"title":"Biosynthesis, enzymology, and future of eunicellane diterpenoids.","authors":"Zining Li, Jeffrey D Rudolf","doi":"10.1093/jimb/kuad027","DOIUrl":"10.1093/jimb/kuad027","url":null,"abstract":"<p><p>Eunicellane diterpenoids are a remarkable family of terpene natural products and have been of high interest for over five decades. Widely distributed in soft corals and rare in plants, eunicellanes were also recently identified in actinobacteria. These terpenoids have foundational 6/10-bicyclic frameworks that are frequently oxidized into structures containing transannular ether bridges. Interest in their unique structures and promising biological activities, such as the paclitaxel-like activities of eleutherobin and the sarcodictyins, has led to advancements in natural product isolation, total synthesis, medicinal chemistry, and drug lead development. Until recently, however, there was little known about the biosynthesis and enzymology of these natural products, but several recent studies in both bacteria and coral have opened up the field. This review summarizes recent advancements in the biosynthesis and enzymology of eunicellane diterpenoids and highlights future research prospects in the field.</p><p><strong>One-sentence summary: </strong>A summary of recent advancements in the biosynthesis and enzymology of eunicellane diterpenoids, a structurally unique and biologically active family of natural products found in coral, plants, and bacteria.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":" ","pages":""},"PeriodicalIF":3.4,"publicationDate":"2023-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/f7/52/kuad027.PMC10548852.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10169404","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}
Bradley D Wahlen, Lynn M Wendt, Chelsea C St Germain, Sarah M Traynor, Caitlin Barboza, Thomas Dempster, Henri Gerken, John McGowen, Yaqi You
{"title":"Effect of nitrogen management in cultivation on the stability and microbial community of post-harvest Monoraphidium sp. algae biomass.","authors":"Bradley D Wahlen, Lynn M Wendt, Chelsea C St Germain, Sarah M Traynor, Caitlin Barboza, Thomas Dempster, Henri Gerken, John McGowen, Yaqi You","doi":"10.1093/jimb/kuad004","DOIUrl":"10.1093/jimb/kuad004","url":null,"abstract":"<p><p>Long-term storage is necessary to mitigate for seasonal variation in algae productivity, to preserve biomass quality and to guarantee a constant biomass supply to a conversion facility. While ensiling has shown promise as a solution, biomass attributes for successful storage are poorly understood. Storage studies of Monoraphidium sp. biomass indicate a strong correlation between nitrogen management in algae cultivation and stability of post-harvest algae biomass. Algae cultivated with periodic nitrogen addition were stored poorly (>20% loss, dry basis) compared to biomass from nitrogen depleted cultivation (8% loss, dry basis). A follow-up study compared the post-harvest stability of Monoraphidium biomass cultivated in nitrogen-deplete or nitrogen-replete conditions. Replete biomass experienced the largest degradation (24%, dry basis), while deplete biomass experienced the least (10%, dry basis). Dry matter loss experienced among blends of each correlated positively with nitrogen-replete biomass content. The composition of the post-storage algae microbial community was also affected by cultivation conditions, with Clostridia species being more prevalent in stored biomass obtained from nitrogen-replete cultivations. Nitrogen management has long been known to influence algae biomass productivity and biochemical composition; here, we demonstrate that it also strongly influences the stability of post-harvest algae biomass in anaerobic storage.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":" ","pages":""},"PeriodicalIF":3.4,"publicationDate":"2023-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10548854/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9492532","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":"Bacterial natural product discovery by heterologous expression.","authors":"Adjo E Kadjo, Alessandra S Eustáquio","doi":"10.1093/jimb/kuad044","DOIUrl":"10.1093/jimb/kuad044","url":null,"abstract":"<p><p>Natural products have found important applications in the pharmaceutical and agricultural sectors. In bacteria, the genes that encode the biosynthesis of natural products are often colocalized in the genome, forming biosynthetic gene clusters. It has been predicted that only 3% of natural products encoded in bacterial genomes have been discovered thus far, in part because gene clusters may be poorly expressed under laboratory conditions. Heterologous expression can help convert bioinformatics predictions into products. However, challenges remain, such as gene cluster prioritization, cloning of the complete gene cluster, high level expression, product identification, and isolation of products in practical yields. Here we reviewed the literature from the past 5 years (January 2018 to June 2023) to identify studies that discovered natural products by heterologous expression. From the 50 studies identified, we present analyses of the rationale for gene cluster prioritization, cloning methods, biosynthetic class, source taxa, and host choice. Combined, the 50 studies led to the discovery of 63 new families of natural products, supporting heterologous expression as a promising way to access novel chemistry. However, the success rate of natural product detection varied from 11% to 32% based on four large-scale studies that were part of the reviewed literature. The low success rate makes it apparent that much remains to be improved. The potential reasons for failure and points to be considered to improve the chances of success are discussed.</p><p><strong>One-sentence summary: </strong>At least 63 new families of bacterial natural products were discovered using heterologous expression in the last 5 years, supporting heterologous expression as a promising way to access novel chemistry; however, the success rate is low (11-32%) making it apparent that much remains to be improved-we discuss the potential reasons for failure and points to be considered to improve the chances of success. BioRender was used to generate the graphical abstract figure.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":" ","pages":""},"PeriodicalIF":3.4,"publicationDate":"2023-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10727000/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138487743","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}
Max L Beck, Siyeon Song, Isra E Shuster, Aarzu Miharia, Allison S Walker
{"title":"Diversity and taxonomic distribution of bacterial biosynthetic gene clusters predicted to produce compounds with therapeutically relevant bioactivities.","authors":"Max L Beck, Siyeon Song, Isra E Shuster, Aarzu Miharia, Allison S Walker","doi":"10.1093/jimb/kuad024","DOIUrl":"10.1093/jimb/kuad024","url":null,"abstract":"<p><p>Bacteria have long been a source of natural products with diverse bioactivities that have been developed into therapeutics to treat human disease. Historically, researchers have focused on a few taxa of bacteria, mainly Streptomyces and other actinomycetes. This strategy was initially highly successful and resulted in the golden era of antibiotic discovery. The golden era ended when the most common antibiotics from Streptomyces had been discovered. Rediscovery of known compounds has plagued natural product discovery ever since. Recently, there has been increasing interest in identifying other taxa that produce bioactive natural products. Several bioinformatics studies have identified promising taxa with high biosynthetic capacity. However, these studies do not address the question of whether any of the products produced by these taxa are likely to have activities that will make them useful as human therapeutics. We address this gap by applying a recently developed machine learning tool that predicts natural product activity from biosynthetic gene cluster (BGC) sequences to determine which taxa are likely to produce compounds that are not only novel but also bioactive. This machine learning tool is trained on a dataset of BGC-natural product activity pairs and relies on counts of different protein domains and resistance genes in the BGC to make its predictions. We find that rare and understudied actinomycetes are the most promising sources for novel active compounds. There are also several taxa outside of actinomycetes that are likely to produce novel active compounds. We also find that most strains of Streptomyces likely produce both characterized and uncharacterized bioactive natural products. The results of this study provide guidelines to increase the efficiency of future bioprospecting efforts.</p><p><strong>One-sentence summary: </strong>This paper combines several bioinformatics workflows to identify which genera of bacteria are most likely to produce novel natural products with useful bioactivities such as antibacterial, antitumor, or antifungal activity.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":" ","pages":""},"PeriodicalIF":3.4,"publicationDate":"2023-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10548851/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10126598","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}
Liang Yang, Hao-Yi Yang, Li You, Hui Ni, Ze-Dong Jiang, Xi-Ping Du, Yan-Bing Zhu, Ming-Jing Zheng, Li-Jun Li, Rui Lin, Zhi-Peng Li, Qing-Biao Li
{"title":"Transcriptomics analysis and fed-batch regulation of high astaxanthin-producing Phaffia rhodozyma/Xanthophyllomyces dendrorhous obtained through adaptive laboratory evolution.","authors":"Liang Yang, Hao-Yi Yang, Li You, Hui Ni, Ze-Dong Jiang, Xi-Ping Du, Yan-Bing Zhu, Ming-Jing Zheng, Li-Jun Li, Rui Lin, Zhi-Peng Li, Qing-Biao Li","doi":"10.1093/jimb/kuad015","DOIUrl":"10.1093/jimb/kuad015","url":null,"abstract":"<p><p>Astaxanthin has high utilization value in functional food because of its strong antioxidant capacity. However, the astaxanthin content of Phaffia rhodozyma is relatively low. Adaptive laboratory evolution is an excellent method to obtain high-yield strains. TiO2 is a good inducer of oxidative stress. In this study, different concentrations of TiO2 were used to domesticate P. rhodozyma, and at a concentration of 1000 mg/L of TiO2 for 105 days, the optimal strain JMU-ALE105 for astaxanthin production was obtained. After fermentation, the astaxanthin content reached 6.50 mg/g, which was 41.61% higher than that of the original strain. The ALE105 strain was fermented by batch and fed-batch, and the astaxanthin content reached 6.81 mg/g. Transcriptomics analysis showed that the astaxanthin synthesis pathway, and fatty acid, pyruvate, and nitrogen metabolism pathway of the ALE105 strain were significantly upregulated. Based on the nitrogen metabolism pathway, the nitrogen source was adjusted by ammonium sulphate fed-batch fermentation, which increased the astaxanthin content, reaching 8.36 mg/g. This study provides a technical basis and theoretical research for promoting industrialization of astaxanthin production of P. rhodozyma.</p><p><strong>One-sentence summary: </strong>A high-yield astaxanthin strain (ALE105) was obtained through TiO2 domestication, and its metabolic mechanism was analysed by transcriptomics, which combined with nitrogen source regulation to further improve astaxanthin yield.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":"50 1","pages":""},"PeriodicalIF":3.4,"publicationDate":"2023-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/1f/e1/kuad015.PMC10448994.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10426361","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}
YuXuan Yao, ShengLan Zheng, ShiLin Chi, Feng Chen, Ning Cai, ZhenZhen Cai, Zhipeng Li, Hui Ni
{"title":"Characterization of the off-flavor from Pichia pastoris GS115 during the overexpression of an α-l-rhamnosidase.","authors":"YuXuan Yao, ShengLan Zheng, ShiLin Chi, Feng Chen, Ning Cai, ZhenZhen Cai, Zhipeng Li, Hui Ni","doi":"10.1093/jimb/kuad035","DOIUrl":"10.1093/jimb/kuad035","url":null,"abstract":"<p><p>The off-flavor of Pichia pastoris strains is a negative characteristic of proteins overexpressed with this yeast. In the present study, P. pastoris GS115 overexpressing an α-l-rhamnosidase was taken as the example to characterize the off-flavor via sensory evaluation, gas chromatography-mass spectrometer, gas chromatography-olfaction, and omission test. The result showed that the off-flavor was due to the strong sweaty note, and moderate metallic and plastic notes. Four volatile compounds, that is, tetramethylpyrazine, 2,4-di-tert-butylphenol, isovaleric acid, and 2-methylbutyric acid, were identified to be major contributors to the sweaty note. Dodecanol and 2-acetylbutyrolactone were identified to be contributors to the metallic and plastic notes, respectively. It is the first study on the off-flavor of P. pastoris strains, helping understand metabolites with off-flavor of this yeast. Interestingly, it is the first study illustrating 2-acetylbutyrolactone and dodecanol with plastic and metallic notes, providing new information about the aromatic contributors of biological products.</p><p><strong>Importance: </strong>The methylotrophic yeast Pichia pastoris is an important host for the industrial expression of functional proteins. In our previous studies, P. pastoris strains have been sniffed with a strong off-flavor during the overexpression of various functional proteins, limiting the application of these proteins. Although many yeast strains have been reported with off-flavor, no attention has been paid to characterize the off-flavor in P. pastoris so far. Considering that P. pastoris has advantages over other established expression systems of functional proteins, it is of interest to identify the compounds with off-flavor synthesized in the overexpression of functional proteins with P. pastoris strains. In this study, the off-flavor synthesized from P. pastoris GS115 was characterized during the overexpression of an α-l-rhamnosidase, which helps understand the aromatic metabolites with off-flavor of P. pastoris strains. In addition, 2-acetylbutyrolactone and dodecanol were newly revealed with plastic and metallic notes, enriching the aromatic contributors of biological products. Thus, this study is important for understanding the metabolites with off-flavor of P. pastoris strains and other organisms, providing important knowledge to improve the flavor of products yielding with P. pastoris strains and other organisms.</p><p><strong>One-sentence summary: </strong>Characterize the sensory and chemical profile of the off-flavor produced by one strain of P. pastoris in vitro.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":" ","pages":""},"PeriodicalIF":3.4,"publicationDate":"2023-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10696632/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"71521675","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":"Enhancing the production of a heterologous Trametes laccase (LacA) by replacement of the major cellulase CBH1 in Trichoderma reesei.","authors":"Jiaxin Zhang, Yu Hong, Kehang Li, Yu Sun, Cheng Yao, Jianya Ling, Yaohua Zhong","doi":"10.1093/jimb/kuad002","DOIUrl":"10.1093/jimb/kuad002","url":null,"abstract":"<p><p>The laccases from white-rot fungi exhibit high redox potential in treating phenolic compounds. However, their application in commercial purposes has been limited because of the relatively low productivity of the native hosts. Here, the laccase A-encoding gene lacA of Trametes sp. AH28-2 was overexpressed under the control of the strong promoter of cbh1 (Pcbh1), the gene encoding the endogenous cellobiohydrolase 1 (CBH1), in the industrial workhorse fungus Trichoderma reesei. Firstly, the lacA expression cassette was randomly integrated into the T. reesei chromosome by genetic transformation. The lacA gene was successfully transcribed, but the laccase couldn't be detected in the liquid fermentation condition. Meanwhile, it was found that the endoplasmic reticulum-associated degradation (ERAD) was strongly activated, indicating that the expression of LacA probably triggered intense endoplasmic reticulum (ER) stress. Subsequently, the lacA expression cassette was added with the downstream region of cbh1 (Tcbh1) to construct the new expression cassette lacA::Δcbh1, which could replace the cbh1 locus in the genome via homologous recombination. After genetic transformation, the lacA gene was integrated into the cbh1 locus and transcribed. And the unfolded protein response (UPR) and ERAD were only slightly induced, for which the loss of endogenous cellulase CBH1 released the pressure of secretion. Finally, the maximum laccase activity of 168.3 U/l was obtained in the fermentation broth. These results demonstrated that the reduction of secretion pressure by deletion of endogenous protein-encoding genes would be an efficient strategy for the secretion of heterologous target proteins in industrial fungi.</p><p><strong>One-sentence summary: </strong>The reduction of the secretion pressure by deletion of the endogenous cbh1 gene can contribute to heterologous expression of the laccase (LacA) from Trametes sp. AH28-2 in Trichoderma reesei.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":"50 1","pages":""},"PeriodicalIF":3.2,"publicationDate":"2023-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/d4/e5/kuad002.PMC10124127.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9716403","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":"Diverse enzymatic chemistry for propionate side chain cleavages in tetrapyrrole biosynthesis.","authors":"Richiro Ushimaru, Jiaqi Lyu, Ikuro Abe","doi":"10.1093/jimb/kuad016","DOIUrl":"10.1093/jimb/kuad016","url":null,"abstract":"<p><p>Tetrapyrroles represent a unique class of natural products that possess diverse chemical architectures and exhibit a broad range of biological functions. Accordingly, they attract keen attention from the natural product community. Many metal-chelating tetrapyrroles serve as enzyme cofactors essential for life, while certain organisms produce metal-free porphyrin metabolites with biological activities potentially beneficial for the producing organisms and for human use. The unique properties of tetrapyrrole natural products derive from their extensively modified and highly conjugated macrocyclic core structures. Most of these various tetrapyrrole natural products biosynthetically originate from a branching point precursor, uroporphyrinogen III, which contains propionate and acetate side chains on its macrocycle. Over the past few decades, many modification enzymes with unique catalytic activities, and the diverse enzymatic chemistries employed to cleave the propionate side chains from the macrocycles, have been identified. In this review, we highlight the tetrapyrrole biosynthetic enzymes required for the propionate side chain removal processes and discuss their various chemical mechanisms.</p><p><strong>One-sentence summary: </strong>This mini-review describes various enzymes involved in the propionate side chain cleavages during the biosynthesis of tetrapyrrole cofactors and secondary metabolites.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":" ","pages":""},"PeriodicalIF":3.4,"publicationDate":"2023-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10548856/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9758848","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":"Biosensor development for single-cell detection of glucuronate.","authors":"Jennifer Kaczmarek Nash, Kristala L J Prather","doi":"10.1093/jimb/kuad013","DOIUrl":"https://doi.org/10.1093/jimb/kuad013","url":null,"abstract":"<p><p>Recent work in biosensors has shown promise to enable high throughput searches through large genetic libraries. However, just as physiological limitations and lack of in-depth mechanistic knowledge can prevent us from achieving high titers in microbial systems; similar roadblocks can appear in the application of biosensors. Here, we characterized a previously developed transcription-factor (ExuR) based galacturonate biosensor for its other cognate ligand, glucuronate. Though we saw an ideal response to glucuronate from the biosensor in controlled and ideal experimental circumstances, these results began to deviate from a well-behaved system when we explored the application of the sensor to different MIOX homologs. Through modifications to circuit architecture and culture conditions, we were able to decrease this variation and use these more optimal conditions to apply the biosensor for the separation of two closely related MIOX homologs.</p><p><strong>One-sentence summary: </strong>In this work, a transcription-factor biosensor was investigated for its potential to screen a library of myo -inositol oxygenase variants while seeking to mitigate the impact the production pathway appeared to have on the biosensor.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":"50 1","pages":""},"PeriodicalIF":3.4,"publicationDate":"2023-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/e7/ab/kuad013.PMC10294642.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9766055","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}
Adrian Sven Geissler, Annaleigh Ohrt Fehler, Line Dahl Poulsen, Enrique González-Tortuero, Thomas Beuchert Kallehauge, Ferhat Alkan, Christian Anthon, Stefan Ernst Seemann, Michael Dolberg Rasmussen, Anne Breüner, Carsten Hjort, Jeppe Vinther, Jan Gorodkin
{"title":"CRISPRi screen for enhancing heterologous α-amylase yield in Bacillus subtilis.","authors":"Adrian Sven Geissler, Annaleigh Ohrt Fehler, Line Dahl Poulsen, Enrique González-Tortuero, Thomas Beuchert Kallehauge, Ferhat Alkan, Christian Anthon, Stefan Ernst Seemann, Michael Dolberg Rasmussen, Anne Breüner, Carsten Hjort, Jeppe Vinther, Jan Gorodkin","doi":"10.1093/jimb/kuac028","DOIUrl":"10.1093/jimb/kuac028","url":null,"abstract":"<p><p>Yield improvements in cell factories can potentially be obtained by fine-tuning the regulatory mechanisms for gene candidates. In pursuit of such candidates, we performed RNA-sequencing of two α-amylase producing Bacillus strains and predict hundreds of putative novel non-coding transcribed regions. Surprisingly, we found among hundreds of non-coding and structured RNA candidates that non-coding genomic regions are proportionally undergoing the highest changes in expression during fermentation. Since these classes of RNA are also understudied, we targeted the corresponding genomic regions with CRIPSRi knockdown to test for any potential impact on the yield. From differentially expression analysis, we selected 53 non-coding candidates. Although CRISPRi knockdowns target both the sense and the antisense strand, the CRISPRi experiment cannot link causes for yield changes to the sense or antisense disruption. Nevertheless, we observed on several instances with strong changes in enzyme yield. The knockdown targeting the genomic region for a putative antisense RNA of the 3' UTR of the skfA-skfH operon led to a 21% increase in yield. In contrast, the knockdown targeting the genomic regions of putative antisense RNAs of the cytochrome c oxidase subunit 1 (ctaD), the sigma factor sigH, and the uncharacterized gene yhfT decreased yields by 31 to 43%.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":"50 1","pages":""},"PeriodicalIF":3.2,"publicationDate":"2023-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9936203/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10528673","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}