المؤلفون: Czajka, Jeffrey J, Nathenson, Justin A, Benites, Veronica T, Baidoo, Edward EK, Cheng, Qianshun, Wang, Yechun, Tang, Yinjie J

المصدر: Microbial Cell Factories. 17(1)

مصطلحات موضوعية: Responsible Consumption and Production, Metabolic Engineering, Norisoprenoids, Yarrowia, 13C labeling, Acetyl-CoA, Fed-batch fermentation, Machine learning, Strain stability, Terpenoid, β-carotene, Microbiology, Industrial Biotechnology, Biotechnology

الوصف: Backgroundβ-Ionone is a fragrant terpenoid that generates a pleasant floral scent and is used in diverse applications as a cosmetic and flavoring ingredient. A growing consumer desire for natural products has increased the market demand for natural β-ionone. To date, chemical extraction from plants remains the main approach for commercial natural β-ionone production. Unfortunately, changing climate and geopolitical issues can cause instability in the β-ionone supply chain. Microbial fermentation using generally recognized as safe (GRAS) yeast offers an alternative method for producing natural β-ionone. Yarrowia lipolytica is an attractive host due to its oleaginous nature, established genetic tools, and large intercellular pool size of acetyl-CoA (the terpenoid backbone precursor).ResultsA push-pull strategy via genome engineering was applied to a Y. lipolytica PO1f derived strain. Heterologous and native genes in the mevalonate pathway were overexpressed to push production to the terpenoid backbone geranylgeranyl pyrophosphate, while the carB and biofunction carRP genes from Mucor circinelloides were introduced to pull flux towards β-carotene (i.e., ionone precursor). Medium tests combined with machine learning based data analysis and 13C metabolite labeling investigated influential nutrients for the β-carotene strain that achieved > 2.5 g/L β-carotene in a rich medium. Further introduction of the carotenoid cleavage dioxygenase 1 (CCD1) from Osmanthus fragrans resulted in the β-ionone production. Utilization of in situ dodecane trapping avoided ionone loss from vaporization (with recovery efficiencies of ~ 76%) during fermentation operations, which resulted in titers of 68 mg/L β-ionone in shaking flasks and 380 mg/L in a 2 L fermenter. Both β-carotene medium tests and β-ionone fermentation outcomes indicated the last enzymatic step CCD1 (rather than acetyl-CoA supply) as the key bottleneck.ConclusionsWe engineered a GRAS Y. lipolytica platform for sustainable and economical production of the natural aroma β-ionone. Although β-carotene could be produced at high titers by Y. lipolytica, the synthesis of β-ionone was relatively poor, possibly due to low CCD1 activity and non-specific CCD1 cleavage of β-carotene. In addition, both β-carotene and β-ionone strains showed decreased performances after successive sub-cultures. For industrial application, β-ionone fermentation efforts should focus on both CCD enzyme engineering and strain stability improvement.

وصف الملف: application/pdf

الوصول الحر: https://escholarship.org/uc/item/0nq772w2Test

المؤلفون: Victor Schmidt, Hagay Enav, Timothy D. Spector, Nicholas D. Youngblut, Ruth E. Ley

المصدر: mSystems, Vol 5, Iss 5 (2020)

مصطلحات موضوعية: Bifidobacterium, lactase persistence, microbiome, strain stability, gut microbiome, human microbiome, Microbiology, QR1-502

الوصف: ABSTRACT One of the strongest associations between human genetics and the gut microbiome is a greater relative abundance of Bifidobacterium in adults with lactase gene (LCT) single nucleotide polymorphisms (SNPs) associated with lactase nonpersistence (GG genotypes), versus lactase persistence (AA/AG genotypes). To gain a finer-grained phylogenetic resolution of this association, we interrogated 1,680 16S rRNA libraries and 245 metagenomes from gut microbiomes of adults with various lactase persistence genotypes. We further employed a novel genome-capture-based enrichment of Bifidobacterium DNA from a subset of these metagenomes, including monozygotic (MZ) twin pairs, each sampled 2 or 3 times. B. adolescentis and B. longum were the most abundant Bifidobacterium species regardless of host LCT genotype. LCT genotypes could not be discriminated based on relative abundances of Bifidobacterium species or Bifidobacterium community structure. Three distinct metagenomic analysis methods of Bifidobacterium-enriched DNA revealed intraindividual temporal stability of B. longum, B. adolescentis, and B. bifidum strains against the background of a changeable microbiome. Two of our three methods also observed greater strain sharing within MZ twin pairs than within unrelated individuals for B. adolescentis, while no method revealed an effect of host LCT genotype on Bifidobacterium strain composition. Our results support a “rising tide lifts all boats” model for the dominant bifidobacteria in the adult gut: their higher abundance in lactase-nonpersistent than in lactase-persistent individuals results from an expansion at the genus level. Bifidobacterium species are known to be transmitted from mother to child and stable within individuals in infancy and childhood: our results extend this stability into adulthood. IMPORTANCE When humans domesticated animals, some adapted genetically to digest milk into adulthood (lactase persistence). The gut microbiomes of people with lactase-persistent genotypes (AA or AG) differ from those with lactase-nonpersistent genotypes (GG) by containing fewer bacteria belonging to the bifidobacteria, a group which contains beneficial species. Here, we asked if the gut microbiomes of adults with GG and AA/AG genotypes differ in the species of bifidobacteria present. In particular, we used a novel technique which allowed us to compare bifidobacteria in adults at the strain level, without the traditional need for culturing. Our results show that the GG genotype enhances the abundance of bifidobacteria regardless of species. We also noted that a person’s specific strains are recoverable several years later, and twins can share the same ones. Given that bifidobacteria are inherited from mother to child, strain stability over time in adulthood suggests long-term, multigenerational inheritance.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/2379-5077Test

الوصول الحر: https://doaj.org/article/ce9e9996ef3b4657835d64928208c8eeTest

المؤلفون: Schmidt, Victor, Enav, Hagay, Spector, Timothy D., Youngblut, Nicholas D., Ley, Ruth E.

المصدر: Schmidt , V , Enav , H , Spector , T D , Youngblut , N D & Ley , R E 2020 , ' Strain-level analysis of bifidobacterium spp. From gut microbiomes of adults with differing lactase persistence genotypes ' , mSystems , vol. 5 , no. 5 , e0091120 . https://doi.org/10.1128/mSystems.00911-20Test

مصطلحات موضوعية: Bifidobacterium, Gut microbiome, Human microbiome, Lactase persistence, Microbiome, Strain stability

الوصف: One of the strongest associations between human genetics and the gut microbiome is a greater relative abundance of Bifidobacterium in adults with lactase gene (LCT) single nucleotide polymorphisms (SNPs) associated with lactase nonpersistence (GG genotypes), versus lactase persistence (AA/AG genotypes). To gain a finer-grained phylogenetic resolution of this association, we interrogated 1,680 16S rRNA libraries and 245 metagenomes from gut microbiomes of adults with various lactase persistence genotypes. We further employed a novel genome-capture-based enrichment of Bifidobacterium DNA from a subset of these metagenomes, including monozygotic (MZ) twin pairs, each sampled 2 or 3 times. B. adolescentis and B. longum were the most abundant Bifidobacterium species regardless of host LCT genotype. LCT genotypes could not be discriminated based on relative abundances of Bifidobacterium species or Bifidobacterium community structure. Three distinct metagenomic analysis methods of Bifidobacterium-enriched DNA revealed intraindividual temporal stability of B. longum, B. adolescentis, and B. bifidum strains against the background of a changeable microbiome. Two of our three methods also observed greater strain sharing within MZ twin pairs than within unrelated individuals for B. adolescentis, while no method revealed an effect of host LCT genotype on Bifidobacterium strain composition. Our results support a “rising tide lifts all boats” model for the dominant bifidobacteria in the adult gut: their higher abundance in lactase-nonpersistent than in lactase-persistent individuals results from an expansion at the genus level. Bifidobacterium species are known to be transmitted from mother to child and stable within individuals in infancy and childhood: our results extend this stability into adulthood. IMPORTANCE When humans domesticated animals, some adapted genetically to digest milk into adulthood (lactase persistence). The gut microbiomes of people with lactase-persistent genotypes (AA or AG) differ from those with ...

الإتاحة: https://doi.org/10.1128/mSystems.00911-20Test
https://kclpure.kcl.ac.uk/portal/en/publications/1ce9d697-d959-490c-8568-5b62f171e5d0Test
http://www.scopus.com/inward/record.url?scp=85092320422&partnerID=8YFLogxKTest

المؤلفون: Wei Du, Joeri A. Jongbloets, Coco van Boxtel, Hugo Pineda Hernández, David Lips, Brett G. Oliver, Klaas J. Hellingwerf, Filipe Branco dos Santos

المصدر: Biotechnology for Biofuels, Vol 11, Iss 1, Pp 1-13 (2018)

مصطلحات موضوعية: Strain stability, Growth coupled, Metabolic modeling, Cyanobacteria, Acetate production, Fuel, TP315-360, Biotechnology, TP248.13-248.65

الوصف: Abstract Background Microbial bioengineering has the potential to become a key contributor to the future development of human society by providing sustainable, novel, and cost-effective production pipelines. However, the sustained productivity of genetically engineered strains is often a challenge, as spontaneous non-producing mutants tend to grow faster and take over the population. Novel strategies to prevent this issue of strain instability are urgently needed. Results In this study, we propose a novel strategy applicable to all microbial production systems for which a genome-scale metabolic model is available that aligns the production of native metabolites to the formation of biomass. Based on well-established constraint-based analysis techniques such as OptKnock and FVA, we developed an in silico pipeline—FRUITS—that specifically ‘Finds Reactions Usable in Tapping Side-products’. It analyses a metabolic network to identify compounds produced in anabolism that are suitable to be coupled to growth by deletion of their re-utilization pathway(s), and computes their respective biomass and product formation rates. When applied to Synechocystis sp. PCC6803, a model cyanobacterium explored for sustainable bioproduction, a total of nine target metabolites were identified. We tested our approach for one of these compounds, acetate, which is used in a wide range of industrial applications. The model-guided engineered strain shows an obligatory coupling between acetate production and photoautotrophic growth as predicted. Furthermore, the stability of acetate productivity in this strain was confirmed by performing prolonged turbidostat cultivations. Conclusions This work demonstrates a novel approach to stabilize the production of target compounds in cyanobacteria that culminated in the first report of a photoautotrophic growth-coupled cell factory. The method developed is generic and can easily be extended to any other modeled microbial production system.

وصف الملف: electronic resource

العلاقة: http://link.springer.com/article/10.1186/s13068-018-1037-8Test; https://doaj.org/toc/1754-6834Test

الوصول الحر: https://doaj.org/article/f621be8101a1493bb80e286580e182c4Test

المؤلفون: Kurt Fenster, Barbara Freeburg, Chris Hollard, Connie Wong, Rune Rønhave Laursen, Arthur C. Ouwehand

المصدر: Microorganisms; Volume 7; Issue 3; Pages: 83

مصطلحات موضوعية: probiotics, Lactobacillus, Bifidobacterium, manufacturing, strain stability

جغرافية الموضوع: agris

الوصف: To successfully deliver probiotic benefits to the consumer, several criteria must be met. Here, we discuss the often-forgotten challenges in manufacturing the strains and incorporating them in consumer products that provide the required dose at the end of shelf life. For manufacturing, an intricate production process is required that ensures both high yield and stability and must also be able to meet requirements such as the absence of specific allergens, which precludes some obvious culture media ingredients. Reproducibility is important to ensure constant high performance and quality. To ensure this, quality control throughout the whole production process, from raw materials to the final product, is essential, as is the documentation of this quality control. Consumer product formulation requires extensive skill and experience. Traditionally, probiotic lactic acid bacteria and bifidobacteria have been incorporated in fermented dairy products, with limited shelf life and refrigerated storage. Currently, probiotics may be incorporated in dietary supplements and other “dry” food matrices which are expected to have up to 24 months of stability at ambient temperature and humidity. With the right choice of production process, product formulation, and strains, high-quality probiotics can be successfully included in a wide variety of delivery formats to suit consumer requirements.

وصف الملف: application/pdf

العلاقة: https://dx.doi.org/10.3390/microorganisms7030083Test

الإتاحة: https://doi.org/10.3390/microorganisms7030083Test

المؤلفون: Bryan Charleston, Ginette Wilsden, Julian Seago, Ben Jackson, Yongjie Harvey, Eva Pérez-Martín, Nicholas Juleff

المصدر: Vaccine

مصطلحات موضوعية: Serotype, animal diseases, viruses, Serogroup, Article, Virus, Serology, Vaccine strain, medicine, Animals, Strain stability, General Veterinary, General Immunology and Microbiology, biology, Foot-and-mouth disease, Foot-and-mouth disease virus, Public Health, Environmental and Occupational Health, virus diseases, Viral Vaccines, biochemical phenomena, metabolism, and nutrition, biology.organism_classification, medicine.disease, Virology, Vaccination, Infectious Diseases, Capsid, Virus neutralisation test, Foot-and-Mouth Disease, Molecular Medicine, Capsid Proteins, FMD vaccine, human activities

الوصف: Highlights • Development of improved FMD vaccines for East Africa. • Selection of candidate FMDV vaccine strains based on stability and serology. • Stability diversity between and within FMDV serotypes.
Foot-and-mouth disease (FMD) is a global burden on the livestock industry. The causative agent, FMD virus (FMDV), is highly infectious and exists in seven distinct serotypes. Vaccination remains the most effective control strategy in endemic regions and current FMD vaccines are made from inactivated preparations of whole virus. The inherent instability of FMDV and the emergence of new strains presents challenges to efficacious vaccine development. Currently, vaccines available in East Africa are comprised of relatively historic strains with unreported stabilities. As an initial step to produce an improved multivalent FMD vaccine we have identified naturally stable East African FMDV strains for each of the A, O, SAT1 and SAT2 serotypes and investigated their potential for protecting ruminants against strains that have recently circulated in East Africa. Interestingly, high diversity in stability between and within serotypes was observed, and in comparison to non-African A serotype viruses reported to date, the East African strains tested in this study are less stable. Candidate vaccine strains were adapted to propagation in BHK-21 cells with minimal capsid changes and used to generate vaccinate sera that effectively neutralised a panel of FMDV strains selected to improve FMD vaccines used in East Africa. This work highlights the importance of combining tools to predict and assess FMDV vaccine stability, with cell culture adaptation and serological tests in the development of FMD vaccines.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::170dea3f81dce73d81722d238d1f0c1eTest
https://doi.org/10.1016/j.vaccine.2021.07.001Test

المؤلفون: Du, Wei, Jongbloets, Joeri A., van Boxtel, C., Hernández, Hugo Pineda, Lips, David, Olivier, B.G., Hellingwerf, Klaas J., dos Santos, Filipe Branco

المصدر: Du , W , Jongbloets , J A , van Boxtel , C , Hernández , H P , Lips , D , Olivier , B G , Hellingwerf , K J & dos Santos , F B 2018 , ' Alignment of microbial fitness with engineered product formation : Obligatory coupling between acetate production and photoautotrophic growth ' , Biotechnology for Biofuels , vol. 11 , no. 38 , 38 , pp. 1-13 . https://doi.org/10.1186/s13068-018-1037-8Test

مصطلحات موضوعية: Acetate production, Cyanobacteria, Growth coupled, Metabolic modeling, Strain stability, /dk/atira/pure/sustainabledevelopmentgoals/industry_innovation_and_infrastructure, name=SDG 9 - Industry, Innovation, and Infrastructure

الوصف: Background: Microbial bioengineering has the potential to become a key contributor to the future development of human society by providing sustainable, novel, and cost-effective production pipelines. However, the sustained productivity of genetically engineered strains is often a challenge, as spontaneous non-producing mutants tend to grow faster and take over the population. Novel strategies to prevent this issue of strain instability are urgently needed. Results: In this study, we propose a novel strategy applicable to all microbial production systems for which a genome-scale metabolic model is available that aligns the production of native metabolites to the formation of biomass. Based on well-established constraint-based analysis techniques such as OptKnock and FVA, we developed an in silico pipeline - FRUITS - that specifically 'Finds Reactions Usable in Tapping Side-products'. It analyses a metabolic network to identify compounds produced in anabolism that are suitable to be coupled to growth by deletion of their re-utilization pathway(s), and computes their respective biomass and product formation rates. When applied to Synechocystis sp. PCC6803, a model cyanobacterium explored for sustainable bioproduction, a total of nine target metabolites were identified. We tested our approach for one of these compounds, acetate, which is used in a wide range of industrial applications. The model-guided engineered strain shows an obligatory coupling between acetate production and photoautotrophic growth as predicted. Furthermore, the stability of acetate productivity in this strain was confirmed by performing prolonged turbidostat cultivations. Conclusions: This work demonstrates a novel approach to stabilize the production of target compounds in cyanobacteria that culminated in the first report of a photoautotrophic growth-coupled cell factory. The method developed is generic and can easily be extended to any other modeled microbial production system.

العلاقة: https://research.vu.nl/en/publications/ee5f0d1e-a100-479b-9c68-1a7ec05df047Test

الإتاحة: https://doi.org/10.1186/s13068-018-1037-8Test
https://research.vu.nl/en/publications/ee5f0d1e-a100-479b-9c68-1a7ec05df047Test
https://hdl.handle.net/1871.1/ee5f0d1e-a100-479b-9c68-1a7ec05df047Test
http://www.scopus.com/inward/record.url?scp=85041891976&partnerID=8YFLogxKTest
http://www.scopus.com/inward/citedby.url?scp=85041891976&partnerID=8YFLogxKTest

المؤلفون: Jingwen Zhou, Yang Gu, Yongkun Lv, Jingliang Xu, Peng Xu

المصدر: Metabolic Engineering

مصطلحات موضوعية: 0106 biological sciences, Naringenin, media_common.quotation_subject, Auxotrophy, Cell, Yarrowia, Bioengineering, Biology, 01 natural sciences, Applied Microbiology and Biotechnology, Metabolic engineering, 03 medical and health sciences, chemistry.chemical_compound, 010608 biotechnology, medicine, Homeostasis, Strain stability, Cell fitness, Autoregulation, Original Research Article, Metabolic addiction, Synthetic biology, media_common, 030304 developmental biology, 2. Zero hunger, 0303 health sciences, Addiction, food and beverages, Phenotype, Bioproduction, Yeast, Cell biology, Malonyl Coenzyme A, medicine.anatomical_structure, Metabolic Engineering, chemistry, Cell culture, Metabolic heterogeneity, Leucine, Negative autoregulation, Biotechnology

الوصف: Metabolic addiction, an organism that is metabolically addicted with a compound to maintain its growth fitness, is an underexplored area in metabolic engineering. Microbes with heavily engineered pathways or genetic circuits tend to experience metabolic burden leading to degenerated or abortive production phenotype during long-term cultivation or scale-up. A promising solution to combat metabolic instability is to tie up the end-product with an intermediary metabolite that is essential to the growth of the producing host. Here we present a simple strategy to improve both metabolic stability and pathway yield by coupling chemical addiction with negative autoregulatory genetic circuits. Naringenin and lipids compete for the same precursor malonyl-CoA with inversed pathway yield in oleaginous yeast. Negative autoregulation of the lipogenic pathways, enabled by CRISPRi and fatty acid-inducible promoters, repartitions malonyl-CoA to favor flavonoid synthesis and increased naringenin production by 74.8%. With flavonoid-sensing transcriptional activator FdeR and yeast hybrid promoters to control leucine synthesis and cell grwoth fitness, this amino acid feedforward metabolic circuit confers a flavonoid addiction phenotype that selectively enrich the naringenin-producing pupulation in the leucine auxotrophic yeast. The engineered yeast persisted 90.9% of naringenin titer up to 324 generations. Cells without flavonoid addiction regained growth fitness but lost 94.5% of the naringenin titer after cell passage beyond 300 generations. Metabolic addiction and negative autoregulation may be generalized as basic tools to eliminate metabolic heterogeneity, improve strain stability and pathway yield in long-term and large-scale bioproduction.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::e4db42a4db7445e7dd4e13610ada9253Test
https://doi.org/10.1016/j.ymben.2020.05.005Test

المؤلفون: Beatrice, Battaglino, Wei, Du, Cristina, Pagliano, Joeri A, Jongbloets, Angela, Re, Guido, Saracco, Filipe, Branco Dos Santos

المصدر: ACS Synthetic Biology

مصطلحات موضوعية: thermodynamically favored reactions, growth-coupled production, Malates, Synechocystis, malate production, cyanobacteria, Metabolic Networks and Pathways, strain stability, Research Article

الوصف: Powered by (sun)light to oxidize water, cyanobacteria can directly convert atmospheric CO2 into valuable carbon-based compounds and meanwhile release O2 to the atmosphere. As such, cyanobacteria are promising candidates to be developed as microbial cell factories for the production of chemicals. Nevertheless, similar to other microbial cell factories, engineered cyanobacteria may suffer from production instability. The alignment of product formation with microbial fitness is a valid strategy to tackle this issue. We have described previously the “FRUITS” algorithm for the identification of metabolites suitable to be coupled to growth (i.e., side products in anabolic reactions) in the model cyanobacterium Synechocystis. sp PCC6803. However, the list of candidate metabolites identified using this algorithm can be somewhat limiting, due to the inherent structure of metabolic networks. Here, we aim at broadening the spectrum of candidate compounds beyond the ones predicted by FRUITS, through the conversion of a growth-coupled metabolite to downstream metabolites via thermodynamically favored conversions. We showcase the feasibility of this approach for malate production using fumarate as the growth-coupled substrate in Synechocystis mutants. A final titer of ∼1.2 mM was achieved for malate during photoautotrophic batch cultivations. Under prolonged continuous cultivation, the most efficient malate-producing strain can maintain its productivity for at least 45 generations, sharply contrasting with other producing Synechocystis strains engineered with classical approaches. Our study also opens a new possibility for extending the stable production concept to derivatives of growth-coupled metabolites, increasing the list of suitable target compounds.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=pmid________::67ee5d551b617f6547e388797531c461Test
https://pubmed.ncbi.nlm.nih.gov/34808039Test

المؤلفون: Chen Ming-Tang, Lin Song, Shandil Ishaan, Andrews Dewan, Stadheim Terrance A, Choi Byung-Kwon

المصدر: Microbial Cell Factories, Vol 11, Iss 1, p 91 (2012)

مصطلحات موضوعية: Mating, Diploid, Pichia pastoris, Strain stability, Fermentation, Recombinant protein expression, Microbiology, QR1-502

الوصف: Background Yeast mating provides an efficient means for strain and library construction. However, biotechnological applications of mating in the methylotrophic yeast Pichia pastoris have been hampered because of concerns about strain stability of P. pastoris diploids. The aim of the study reported here is to investigate heterologous protein expression in diploid P. pastoris strains and to evaluate diploid strain stability using high cell density fermentation processes. Results By using a monoclonal antibody as a target protein, we demonstrate that recombinant protein production in both wild-type and glycoengineered P. pastoris diploids is stable and efficient during a nutrient rich shake flask cultivation. When diploid strains were cultivated under bioreactor conditions, sporulation was observed. Nevertheless, both wild-type and glycoengineered P. pastoris diploids showed robust productivity and secreted recombinant antibody of high quality. Specifically, the yeast culture maintained a diploid state for 240 h post-induction phase while protein titer and N-linked glycosylation profiles were comparable to that of a haploid strain expressing the same antibody. As an application of mating, we also constructed an antibody display library and used mating to generate novel full-length antibody sequences . Conclusions To the best of our knowledge, this study reports for the first time a comprehensive characterization of recombinant protein expression and fermentation using diploid P. pastoris strains. Data presented here support the use of mating for various applications including strain consolidation, variable-region glycosylation antibody display library, and process optimization.

العلاقة: http://www.microbialcellfactories.com/content/11/1/91Test; https://doaj.org/toc/1475-2859Test; https://doaj.org/article/7f52e55720d44006b570d7bae1384242Test

الإتاحة: https://doi.org/10.1186/1475-2859-11-91Test
https://doaj.org/article/7f52e55720d44006b570d7bae1384242Test