Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Ensemble and iterative engineering for maximal bioconversion to the blue pigment, Indigoidine from non-canonical sustainable carbon sources.

ABSTRACT: While many heterologous molecules can be produced at trace concentrations via microbial bioconversion processes, maximizing their titers, rates, and yields from lignin-derived carbon streams remains challenging. Strong growth coupling can not only increase titers and yields but also shift the production period from stationary phase to growth phase. These methods however require multi-gene edits for implementation which may be initially perceived as impractical. Here, we evaluated 4,114 potential solutions to pair growth on para-coumarate to indigoidine production and prototype two cutsets in Pseudomonas putida KT2440. The implemented design was enhanced using adaptive lab evolution and the resulting strains were characterized for emergent phenotypes. Using x-ray tomography we revealed increased cell density in this strain. Proteomics identified two upregulated peroxidases that mitigate increased reactive oxygen species formation. Nine additional stepwise modifications informed by model-guided and rational approaches realized a growth coupled strain that produced 7.3 g/L indigoidine at 77% yield in minimal salt media. These ensemble strategies provide a blueprint for producing target molecules at high product titers, rates, and yields.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Pseudomonas Putida Kt2440

SUBMITTER: Christopher Petzold

LAB HEAD: Christopher Petzold

PROVIDER: PXD040697 | Pride | 2024-02-29

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Similar Datasets

Development of Corynebacterium glutamicum as a monoterpene production platform

Project description:Monoterpenes are commonly known for their role in the flavors and fragrances industry and are also gaining attention for other uses like insect repellant and as potential renewable fuels for aviation. Corynebacterium glutamicum, a Generally Recognized as Safe microbe, has been a choice organism in industry for the annual million ton-scale bioproduction of amino acids for more than 50 years; however, efforts to produce monoterpenes in C. glutamicum have remained relatively limited. In this study, we report a further expansion of the C. glutamicum biosynthetic repertoire through the development and optimization of a mevalonate-based monoterpene platform. In the course of our initial plasmid design iterations, we demonstrated an increased flux through the mevalonate-based bypass pathway to increase isoprenol production to the highest reported titers to date at nearly 1.5 g/L. These designs also evaluated the effects of backbone, promoter, and GPP synthase homolog source on product titers. Monoterpene production was further improved by disrupting competing pathways for isoprenoid precursor supply and by implementing a biphasic production system to prevent volatilization. With this platform, we achieved 486.3 mg/L (± 54.6 mg/L) of geranoids, 557.3 mg/L of 1,8-cineole (± 12.3 mg/L), and 209.7 mg/L of linalool (±23.9 mg/L). Furthermore, we determined that C. glutamicum oxidizes geraniol through an aldehyde intermediate before asymmetrically reducing geraniol to citronellol.

2024-02-28 | PXD044193 | Pride

Proteomic analysis reveals global iron starvation response to metal mixture exposure in a Bacillus cereus strain native to a heavy metal contaminated site

Project description:Globally, multiple heavy metal contamination is an increasingly common problem. As heavy metals have the potential to disrupt microbially-mediated biogeochemical cycling, it is critical to understand their impact on microbial physiology. However, systems-level studies on the effects of a combination of heavy metals on bacteria are lacking. Here, we use a native Bacillus cereus isolate from the subsurface of the Oak Ridge Reservation (ORR; Oak Ridge, TN, USA) subsurface— representing a highly abundant species at the site— to assess the combined impact of eight metal contaminants. Using this metal mixture and individual metals, all at concentrations based on the ORR site geochemistry, we performed growth experiments and proteomic analyses of the B. cereus strain, in combination with targeted MS-based metabolomics and gene expression profiling. We found that the combination of eight metals impacts cell physiology in a manner that could not have been predicted from summing phenotypic responses to the individual metals. Specifically, exposure to the metal mixture elicited global iron starvation responses not observed in any of the individual metal treatments. As nitrate is also a significant contaminant at the ORR site and nitrate and nitrite reductases are iron-containing enzymes, we also examined the effects of the metal mixture on reduction of nitrogen oxides. We found that the metal mixture inhibits the activity of these enzymes through a combination of direct enzymatic damage and post-transcriptional and post-translational regulation. Altogether, these data suggest that metal mixture studies are critical for understanding how multiple rather than individual metals influence microbial processes in the environment.

2022-09-19 | PXD035730 | Pride

Complete integration of carbene-transfer chemistry into biosynthesis

Project description:Biosynthesis is an environmentally friendly and renewable way to synthesize a broad range of natural and some new-to-nature products; however, it lacks many of the reactions and flexibility of synthetic chemistry, an example being carbene mediated reactions. While it was recently shown that these reactions can be imported into the cell, carbene donors and unnatural cofactors needed to be added exogenously to the cells, precluding cost-effective scale-up of the reaction. Here we identified a biosynthetic gene cluster that when expressed in Streptomyces albus will produce the diazo compound azaserine, which we also demonstrated can serve as a carbene donor across a carbon-carbon double bond in another intracellularly produced molecule, styrene, thereby producing a cyclopropanated product. The reaction was catalyzed with P450 mutants harboring a native cofactor with excellent diastereoselectivity and moderate yield. This work establishes a platform for introducing carbene mediated reactions into microbes for bio-manufacture and contributes to sustainable green chemistry.

2023-05-03 | PXD037509 | Pride

Edible mycelium bioengineered for enhanced nutritional value and sensory appeal using a new synthetic biology toolkit

Project description:Genetic engineering of filamentous fungi has promise for accelerating the transition to a more sustainable food system and enhancing the nutritional value, sensory appeal, and scalability of microbial foods. However, genetic tools and demonstrated use cases for bioengineered food production by edible strains are lacking. Here, we developed a synthetic biology toolkit for Aspergillus oryzae, an edible fungus traditionally used in fermented foods and currently used in protein production and meat alternatives. Our toolkit includes a CRISPR-Cas9 method for genome integration, neutral loci, and new promoters. We use these tools to enhance the elevate levels of the nutraceutical ergothioneine and intracellular heme in the edible biomass. The biomass overproducing heme is red in color and is readily formulated into imitation meat patties with minimal processing. These findings highlight the promise of genetic approaches to enhance fungal meat alternatives and provide useful engineering tools for diverse applications in fungal food production and beyond.

2024-03-22 | PXD043152 | Pride

Maximizing Heterologous Expression of Engineered Type I Polyketide Synthases: Investigating Codon Optimization Strategies

Project description:Type I polyketide synthases (T1PKSs) hold an enormous potential as a rational production platform for the biosynthesis of speciality chemicals. However, despite the great progress in this field, the heterologous expression of PKSs remains a major challenge. One of the first measures to improve heterologous gene expression can be codon optimization. Although controversial, choosing the wrong codon optimization strategy can have detrimental effects on protein and product levels. In this study, we analyzed 11 different codon variants of an engineered T1PKS and investigated in a systematic approach their influence on heterologous expression in Corynebacterium glutamicum, Escherichia coli, and Pseudomonas putida. Our best performing codon variants exhibited a minimum 50-fold increase in PKS protein levels, which also enables the production of an unnatural polyketide in each of the hosts. Furthermore, we developed a free online tool (https://basebuddy.lbl.gov) that offers transparent and highly customizable codon optimization with up-to-date codon usage tables. Here, we not only highlight the significance of codon optimization but also establish the groundwork for high-throughput assembly and characterization of PKS pathways in alternative hosts.

2023-09-01 | PXD042749 | Pride

Alkaline-SDS cell lysis with acetone protein precipitation for proteomic sample preparation of microbes in 96-well plate format

Project description:Automated proteomic sample preparation offers a solution to the large sample throughput demands in the biotechnology field where hundreds or thousands of engineered microbes are constructed for testing is routine. Meanwhile, sample preparation methods that work efficiently on broader microbial groups are desirable for new applications of proteomics in other fields, such as microbial communities. Here, we detail a step-by-step protocol that consists of cell lysis in alkaline chemical buffer (NaOH/SDS) followed by protein precipitation with high-ionic strength acetone in 96-well format. The protocol works for a broad range of microbes (e.g Gram-negative bacteria, Gram-positive bacteria, non-filamentous fungi) and the resulting proteins are ready for tryptic digestion for bottom-up quantitative proteomic analysis without the need for desalting column cleanup.

2023-06-23 | PXD039268 | Pride

Arabidopsis thaliana mitochondrial proteome in mtran mutants

Project description:Mitochondrial proteomes from mtran double mutants were compared to wild type mitochondrial proteomes

2023-09-01 | PXD043729 | Pride

Rationally designed chromosome fusion is not a limiting factor for rapid growth of Vibrio natriegens

Project description:DNA replication prior to cell division is essential for the proliferation of all cells. Bacterial chromosomes are replicated bidirectionally from a single origin of replication, with replication proceeding at about 1000 bp per second. For the best-studied model organism, Escherichia coli, this translates into a replication time of about 40 min for its 4.6 Mb chromosome. Nevertheless, E. coli can propagate by overlapping replication cycles with a maximum short doubling time of 20 min. The fastest growing bacterium known today, Vibrio natriegens, is able to replicate with a generation time of less than 10 min. It has a bipartite genome with chromosome sizes of 3.2 and 1.9 Mb. Is simultaneous replication from two origins a prerequisite for its rapid growth? We fused the two chromosomes of V. natriegens to create a strain carrying a 5.2 Mb chromosome with a single origin of replication. Compared to the wild-type, this strain showed little deviation in growth rate. This suggests that the split genome is not a prerequisite for rapid growth, and that DNA replication is not an important growth rate-limiting factor.

2024-04-02 | PXD049476 | Pride

Metabolome and proteome analyses of glycerol overproducing E. coli unravel transcriptional misregulation of glycolysis and guide optimization strategies

Project description:Feedback-regulation of gene expression in synthetic metabolic pathways can improve production titers and yields. However, the dynamic nature of metabolism makes it difficult to engineer such regulation on a rational basis. Here, we expressed enzymes in a synthetic glycerol production pathway with static or feedback-regulated promoters, and explored the consequences for E. coli metabolism. Expressing the synthetic glycerol pathway with static promoters caused a strong growth burden, resulting in low biomass-levels and low glycerol titers. We show that the growth burden is due to a regulatory interaction between fructose-1,6-bisphoshate (FBP) and the transcription factor Cra, which switches between glycolysis and gluconeogenesis in E. coli. This regulation activated gluconeogenesis at higher induction of the glycerol pathway, because FBP levels were low. Feedback-regulated promoters, in contrast, stabilized FBP levels and enabled robust expression of the synthetic glycerol pathway. We show the broad utility of this approach by engineering different feedback-regulated promoters (pBAD, pTet, pConstitutive) and by applying one of them to carotenoid production in E. coli.

2021-07-21 | PXD022965 | Pride

Deletion of genes linked to the C1-fixing gene cluster affects growth, by-products, and proteome of Clostridium autoethanogenum

Project description:Gas fermentation has emerged as a sustainable route to produce fuels and chemicals by recycling inexpensive one-carbon (C1) feedstocks from gaseous and solid waste using gas-fermenting microbes. Currently, acetogens that utilise the Wood-Ljungdahl pathway to convert carbon oxides (CO and CO2) into valuable products are the most advanced biocatalysts for gas fermentation. However, our understanding of the functionalities of the genes involved in the C1-fixing gene cluster and its closely-linked genes is incomplete. Here, we investigate the role of two genes with unclear functions – hypothetical protein (hp; LABRINI_07945) and CooT nickel binding protein (nbp; LABRINI_07950) – directly adjacent and expressed at similar levels to the C1-fixing gene cluster in the gas-fermenting model-acetogen Clostridium autoethanogenum. Targeted deletion of either the hp or nbp gene using CRISPR/nCas9, and phenotypic characterisation in heterotrophic and autotrophic batch and autotrophic bioreactor continuous cultures revealed significant growth defects and altered by-product profiles for both ∆hp and ∆nbp strains. Variable effects of gene deletion on autotrophic batch growth on rich or minimal media suggest that both genes affect the utilisation of complex nutrients. Autotrophic chemostat cultures showed lower acetate and ethanol production rates and higher carbon flux to CO2 and biomass for both deletion strains. Additionally, proteome analysis revealed that disruption of either gene affects the expression of proteins of the C1-fixing gene cluster and ethanol synthesis pathways. Our work contributes to a better understanding of genotype-phenotype relationships in acetogens and offers engineering targets to improve carbon fixation efficiency in gas fermentation.

2023-05-10 | PXD040151 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data