Journal of Industrial Microbiology & Biotechnology最新文献

{"title":"Development of Modular Expression Across Phylogenetically Distinct Diazotrophs","authors":"Shawn Kulakowski, Alex Rivier, Rita Kuo, Sonya Mengel, Thomas Eng","doi":"10.1093/jimb/kuae033","DOIUrl":"https://doi.org/10.1093/jimb/kuae033","url":null,"abstract":"Diazotrophic bacteria can reduce atmospheric nitrogen into ammonia enabling bioavailability of the essential element. Many diazotrophs closely associate with plant roots increasing nitrogen availability, acting as plant growth promoters. These associations have the potential to reduce the need for costly synthetic fertilizers if they could be engineered for agricultural applications. However, despite the importance of diazotrophic bacteria, genetic tools are poorly developed in a limited number of species, in turn narrowing the crops and root microbiomes that can be targeted. Here we report optimized protocols and plasmids to manipulate phylogenetically diverse diazotrophs with the goal of enabling synthetic biology and genetic engineering. Three broad-host-range plasmids can be used across multiple diazotrophs, with the identification of one specific plasmid (containing origin of replication RK2 and a kanamycin resistance marker) showing the highest degree of compatibility across bacteria tested. We then demonstrated modular expression by testing seven promoters and eleven ribosomal binding sites using proxy fluorescent proteins. Finally, we tested four small molecule inducible systems to report expression in three diazotrophs and demonstrated genome editing in Klebsiella michiganensis M5al.","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":"4 1","pages":""},"PeriodicalIF":3.4,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142205949","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}

{"title":"Simultaneous enumeration of yeast and bacterial cells in the context of industrial bioprocesses","authors":"Carolina Teixeira Martins, Ana Paula Jacobus, Renilson Conceição, Douglas Fernandes Barbin, Helena Bolini, Andreas Karoly Gombert","doi":"10.1093/jimb/kuae029","DOIUrl":"https://doi.org/10.1093/jimb/kuae029","url":null,"abstract":"In scenarios where yeast and bacterial cells coexist, it is of interest to simultaneously quantify the concentrations of both cell types, since traditional methods used to determine these concentrations individually take more time and resources. Here, we compared different methods for quantifying the fuel ethanol Saccharomyces cerevisiae PE-2 yeast strain and cells from the probiotic Lactiplantibacillus plantarum strain in microbial suspensions. Individual suspensions were prepared, mixed in 1:1 or 100:1 yeast-to-bacteria ratios, covering the range typically encountered in sugarcane biorefineries, and analyzed using bright field microscopy, manual and automatic Spread-plate and Drop-plate counting, flow cytometry (at 1:1 and 100:1 ratios), and a Coulter Counter (at 1:1 and 100:1 ratios). We observed that for yeast cell counts in the mixture (1:1 and 100:1 ratios), flow cytometry, the Coulter Counter, and both Spread-plate options (manual and automatic CFU counting) yielded statistically similar results, while the Drop-plate and microscopy-based methods gave statistically different results. For bacterial cell quantification, the microscopy-based method, Drop-plate, and both Spread-plate plating options and flow cytometry (1:1 ratio) produced no significantly different results (p > .05). In contrast, the Coulter Counter (1:1 ratio) and flow cytometry (100:1 ratio) presented results statistically different (p < .05). Additionally, quantifying bacterial cells in a mixed suspension at a 100:1 ratio wasn't possible due to an overlap between yeast cell debris and bacterial cells. We conclude that each method has limitations, advantages, and disadvantages. One-Sentence Summary This study compares methods for simultaneously quantifying yeast and bacterial cells in a mixed sample, highlighting that in different cell proportions, some methods cannot quantify both cell types and present distinct advantages and limitations regarding time, cost, and precision.","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":"57 1","pages":""},"PeriodicalIF":3.4,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142205852","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}

{"title":"Multi-modular metabolic engineering and efflux engineering for enhanced lycopene production in recombinant Saccharomyces cerevisiae","authors":"Guangxi Huang, Jiarong Li, Jingyuan Lin, Changqing Duan, Guoliang Yan","doi":"10.1093/jimb/kuae015","DOIUrl":"https://doi.org/10.1093/jimb/kuae015","url":null,"abstract":"Lycopene has been widely used in the food industry and medical field due to its antioxidant, anti-cancer, and anti-inflammatory properties. However, achieving efficient manufacture of lycopene using chassis cells on an industrial scale remains a major challenge. Herein, we attempted to integrate multiple metabolic engineering strategies to establish an efficient and balanced lycopene biosynthetic system in Saccharomyces cerevisiae. First, the lycopene synthesis pathway was modularized to sequentially enhance the metabolic flux of the Mevalonate pathway, the acetyl-CoA supply module, and lycopene exogenous enzymatic module. The modular operation enabled the efficient conversion of acetyl-CoA to downstream pathway of lycopene synthesis, resulting in a 3.1-fold increase of lycopene yield. Second, we introduced acetate as an exogenous carbon source and utilized an acetate-repressible promoter to replace the natural ERG9 promoter. This approach not only enhanced the supply of acetyl-CoA but also concurrently diminished the flux towards the competitive ergosterol pathway. As a result, a further 42.3% increase in lycopene production was observed. Third, we optimized NADPH supply and mitigated cytotoxicity by overexpressing ABC transporters to promote lycopene efflux. The obtained strain YLY-PDR11 showed a 12.7-fold increase in extracellular lycopene level compared to the control strain. Finally, the total lycopene yield reached 343.7mg/L, which was 4.3 times higher than that of the initial strain YLY-04. Our results demonstrate that combining multi-modular metabolic engineering with efflux engineering is an effective approach to improve the production of lycopene. This strategy can also be applied to the overproduction of other desirable isoprenoid compounds with similar synthesis and storage patterns in S. cerevisiae.","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":"414 1","pages":""},"PeriodicalIF":3.4,"publicationDate":"2024-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140584408","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}

{"title":"Application of the surface engineered recombinant Escherichia coli to the industrial battery waste solution for lithium recovery","authors":"Jaehoon Jeong, Vidhya Selvamani, Murali kannan Maruthamuthu, Kulandaisamy Arulsamy, Soon Ho Hong","doi":"10.1093/jimb/kuae012","DOIUrl":"https://doi.org/10.1093/jimb/kuae012","url":null,"abstract":"Escherichia coli were engineered to selectively adsorb and recover lithium from the environment by employing bacterial cell surface display strategy. Lithium binding peptide LBP1 was integrated to the Escherichia coli membrane protein OmpC. The effect of environmental conditions on the adsorption of lithium by recombinant strain was evaluated, and lithium particles on cellular surface was analysed by FE-SEM and XRD. To elevate the lithium adsorption, dimeric, trimeric and tetrameric repeats of the LBP1 peptide was constructed and displayed on the surface of E. coli. The constructed recombinant E. coli displaying LBP1 trimer was applied to real industrial lithium battery wastewater to recover lithium.","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":"35 1","pages":""},"PeriodicalIF":3.4,"publicationDate":"2024-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140584320","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}

{"title":"Harnessing regulatory networks in Actinobacteria for natural product discovery","authors":"Hannah E Augustijn, Anna M Roseboom, Marnix H Medema, Gilles P van Wezel","doi":"10.1093/jimb/kuae011","DOIUrl":"https://doi.org/10.1093/jimb/kuae011","url":null,"abstract":"Microbes typically live in complex habitats where they need to rapidly adapt to continuously changing growth conditions. To do so, they produce an astonishing array of natural products with diverse structures and functions. Actinobacteria stand out for their prolific production of bioactive molecules, including antibiotics, anticancer agents, antifungals, and immunosuppressants. Attention has been directed especially towards the identification of the compounds they produce and the mining of the large diversity of biosynthetic gene clusters (BGCs) in their genomes. However, the current return on investment in random screening for bioactive compounds is low, while it is hard to predict which of the millions of BGCs should be prioritized. Moreover, many of the BGCs for yet undiscovered natural products are silent or cryptic under laboratory growth conditions. To identify ways to prioritize and activate these BGCs, knowledge regarding the way their expression is controlled is crucial. Intricate regulatory networks control global gene expression in Actinobacteria, governed by a staggering number of up to 1000 transcription factors per strain. This review highlights recent advances in experimental and computational methods for characterizing and predicting transcription factor binding sites and their applications to guide natural product discovery. We propose that regulation-guided genome mining approaches will open new avenues toward eliciting the expression of BGCs, as well as prioritizing subsets of BGCs for expression using synthetic biology approaches. One-Sentence Summary This review provides insights into advances in experimental and computational methods aimed at predicting transcription factor binding sites and their applications to guide natural product discovery.","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":"22 1","pages":""},"PeriodicalIF":3.4,"publicationDate":"2024-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140584317","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}

{"title":"One-pot chemoenzymatic syntheses of non-canonical amino acids.","authors":"Tsung-Han Chao, Xiangyu Wu, Hans Renata","doi":"10.1093/jimb/kuae005","DOIUrl":"10.1093/jimb/kuae005","url":null,"abstract":"<p><p>Despite their prevalent use in drug discovery and protein biochemistry, non-canonical amino acids are still challenging to synthesize through purely chemical means. In recent years, biocatalysis has emerged as a transformative paradigm for small-molecule synthesis. One strategy to further empower biocatalysis is to use it in combination with modern chemical reactions and take advantage of the strengths of each method to enable access to challenging structural motifs that were previously unattainable using each method alone. In this Mini-Review, we highlight several recent case studies that feature the synergistic use of chemical and enzymatic transformations in one pot to synthesize novel non-canonical amino acids.</p><p><strong>One-sentence summary: </strong>This Mini-Review highlights several recent case studies that feature the synergistic use of chemical and enzymatic transformations in one pot to synthesize novel non-canonical amino acids.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":" ","pages":""},"PeriodicalIF":3.4,"publicationDate":"2024-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10853765/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139564289","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":"Performance evaluation of a low-throughput qPCR-based Legionella assay for utility as an onsite industrial water system monitoring method.","authors":"Alexsandra Corrigan, Benjamin Niemaseck, Mackenzie Moore, Douglas McIlwaine, Jeremy Duguay","doi":"10.1093/jimb/kuae030","DOIUrl":"10.1093/jimb/kuae030","url":null,"abstract":"<p><p>Legionella is a bacterial genus found in natural aquatic environments, as well as domestic and industrial water systems. Legionella presents potential human health risks when aerosolized and inhaled by at-risk individuals and is commonly monitored at locations with likelihood of proliferation and human exposure. Legionella monitoring is widely performed using culture-based testing, which faces limitations including turnaround time and interferences. Molecular biology methodologies, including quantitative polymerase chain reaction (qPCR), are being explored to supplement or replace culture-based testing because of faster turnaround and lower detection limits, allowing for more rapid water remediation measures. In this study, three methods were compared by testing industrial water samples: culture-based testing by a certified lab, high throughput qPCR testing (HT qPCR), and field deployable low throughput qPCR testing (LT qPCR). The qPCR test methods reported more positive results than culture testing, indicating improved sensitivity and specificity. The LT qPCR test is portable with quick turnaround times, and can be leveraged for environmental surveillance, process optimization, monitoring, and onsite case investigations. The LT qPCR test had high negative predictive value and would be a useful tool for negative screening of Legionella samples from high-risk environments and/or outbreak investigations to streamline samples for culture testing.</p><p><strong>One-sentence summary: </strong>This study compared three test methods for Legionella to evaluate performance of a low throughput quantitative polymerase chain reaction (LT qPCR) test for Legionella that can be used onsite; the study found that the high throughput (HT) and LT qPCR tests used in this study gave more positive results than culture testing, and the results indicated a similar negative predictive value for the HT and LT qPCR tests, supporting that the LT qPCR method could be useful for negative screening of Legionella samples in industrial water systems onsite.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11388924/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142008897","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":"Automated yeast cultivation control using a biosensor and flow cytometry.","authors":"Raquel Perruca Foncillas, Sara Magnusson, Basel Al-Rudainy, Ola Wallberg, Marie F Gorwa-Grauslund, Magnus Carlquist","doi":"10.1093/jimb/kuae039","DOIUrl":"10.1093/jimb/kuae039","url":null,"abstract":"<p><p>Effective microbial bioprocessing relies on maintaining ideal cultivation conditions, highlighting the necessity for tools that monitor and regulate cellular performance and robustness. This study evaluates a fed-batch cultivation control system based on at-line flow cytometry monitoring of intact yeast cells having a fluorescent transcription factor-based redox biosensor. Specifically, the biosensor assesses the response of an industrial xylose-fermenting Saccharomyces cerevisiae strain carrying the TRX2p-yEGFP biosensor for NADPH/NADP+ ratio imbalance when exposed to furfural. The developed control system successfully detected biosensor output and automatically adjusted furfural feed rate, ensuring physiological fitness at high furfural levels. Moreover, the single-cell measurements enabled the monitoring of subpopulation dynamics, enhancing control precision over traditional methods. The presented automated control system highlights the potential of combining biosensors and flow cytometry for robust microbial cultivations by leveraging intracellular properties as control inputs.</p><p><strong>One-sentence summary: </strong>An automated control system using flow cytometry and biosensors enhances microbial bioprocessing by regulating cellular performance in response to the environmental stressor furfural.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11561399/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142467551","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":"Sustainable production of 2,3,5,6-Tetramethylpyrazine at high titer in engineered Corynebacterium glutamicum.","authors":"Aparajitha Srinivasan, Kevin Chen-Xiao, Deepanwita Banerjee, Asun Oka, Venkataramana R Pidatala, Aymerick Eudes, Blake A Simmons, Thomas Eng, Aindrila Mukhopadhyay","doi":"10.1093/jimb/kuae026","DOIUrl":"10.1093/jimb/kuae026","url":null,"abstract":"<p><p>The industrial amino acid production workhorse, Corynebacterium glutamicum naturally produces low levels of 2,3,5,6-tetramethylpyrazine (TMP), a valuable flavor, fragrance, and commodity chemical. Here, we demonstrate TMP production (∼0.8 g L-1) in C. glutamicum type strain ATCC13032 via overexpression of acetolactate synthase and/or α-acetolactate decarboxylase from Lactococcus lactis in CGXII minimal medium supplemented with 40 g L-1 glucose. This engineered strain also demonstrated growth and TMP production when the minimal medium was supplemented with up to 40% (v v-1) hydrolysates derived from ionic liquid-pretreated sorghum biomass. A key objective was to take the fully engineered strain developed in this study and interrogate medium parameters that influence the production of TMP, a critical post-strain engineering optimization. Design of experiments in a high-throughput plate format identified glucose, urea, and their ratio as significant components affecting TMP production. These two components were further optimized using response surface methodology. In the optimized CGXII medium, the engineered strain could produce up to 3.56 g L-1 TMP (4-fold enhancement in titers and 2-fold enhancement in yield, mol mol-1) from 80 g L-1 glucose and 11.9 g L-1 urea in shake flask batch cultivation.</p><p><strong>One-sentence summary: </strong>Corynebacterium glutamicum was metabolically engineered to produce 2,3,5,6-tetramethylpyrazine followed by a design of experiments approach to optimize medium components for high-titer production.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11302136/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141626920","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":"Expanding the synthetic biology toolbox of Cupriavidus necator for establishing fatty acid production.","authors":"Shivangi Mishra, Paul M Perkovich, Wayne P Mitchell, Maya Venkataraman, Brian F Pfleger","doi":"10.1093/jimb/kuae008","DOIUrl":"10.1093/jimb/kuae008","url":null,"abstract":"<p><p>The Gram-negative betaproteobacterium Cupriavidus necator is a chemolithotroph that can convert carbon dioxide into biomass. Cupriavidus necator has been engineered to produce a variety of high-value chemicals in the past. However, there is still a lack of a well-characterized toolbox for gene expression and genome engineering. Development and optimization of biosynthetic pathways in metabolically engineered microorganisms necessitates control of gene expression via functional genetic elements such as promoters, ribosome binding sites (RBSs), and codon optimization. In this work, a set of inducible and constitutive promoters were validated and characterized in C. necator, and a library of RBSs was designed and tested to show a 50-fold range of expression for green fluorescent protein (gfp). The effect of codon optimization on gene expression in C. necator was studied by expressing gfp and mCherry genes with varied codon-adaptation indices and was validated by expressing codon-optimized variants of a C12-specific fatty acid thioesterase to produce dodecanoic acid. We discuss further hurdles that will need to be overcome for C. necator to be widely used for biosynthetic processes.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10926325/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139898044","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}