Metabolic engineering of Saccharomyces cerevisiae for isoprenoids production ...rutayisirer
Metabolic engineering of Saccharomyces cerevisiae for isoprenoids production : S. cerevisae naturally uses MVA pathway to produce isoprenoids, which constitute one of the most used natural products in pharmaceuticals (anticancer: Paclitaxel-Taxol, antimalaria: Artemisin, nutraceuticals, food additives, fragrances, etc. And this microorganism can be engineered to overproduce these chemicals, and other metabolic pathways can be incorporated for this effect.
Isoprenoids are the most abundant and highly diverse group of natural and chemical products with a variety of applications in pharmaceuticals, nutraceuticals, flavors, cosmetics, food additives and biofuels. The plant extraction, chemical synthesis and in vitro enzymatic production of these compounds have been the traditional options for large-scale production and these methods have shown drawbacks and they are impractical. In response to this problem and a poor production efficiency with an increase demand in isoprenoids, the focus has been set to the microbial production, which is an excellent alternative for overcoming these limitations. Microbes require little and naturally produce the building blocks of all isoprenoids: isopentenyl diphosphate (IPP) and its isomer dimethyl allyl diphosphate (DMAPP). IPP and DMAPP can be produced by two metabolic pathways, the mevalonate pathway (MVA or MEV) and the methyl-erythritol phosphate or deoxy xylulose phosphate pathway (MEP or DXP). S. cerevisae naturally uses MVA pathway to produce isoprenoids. The availability of its entire genome sequence allows the application of metabolic engineering and synthetic biology approaches for improving its enormous biosynthetic potential.
Bioconversion of Penicillin to CephalosporinIOSR Journals
Cephalosporins are known as 3rd generation broad spectrum Beta lactam antibiotics, which can also be produced synthetically. Commonly, chemical ring expansion followed by an enzymatic removal of the phenylacetyl side chain is commonly employed to convert penicillin G into 7-aminodeacetoxycephalosporanic acid, the precursor for the manufacture of semisynthetic cephalosporins. This process requires several steps, is expensive and highly polluting. Thus there is a need to device a simple biological route to replace the chemical process. A mutant of Streptomyces clavuligerus NP1 was reported to converts Penicillin G to Deacetoxycephalosporin G (DAOG;phenylacetyl-7-aminodeacetoxycephalosporanic acid) enzymatically[5,8] . This enzyme, deacetoxycephalosporin synthase has the potential for the large scale transformation of Penicillin G to deacetoxycephalosporin. The present work studies the conditions required for efficient transformation of Penicillin G to Deacetoxycephalosporin using the wild type strain Streptomyces clavuligerus . Detection of cephalosporin was carried out using various methods. Additionally succinic acid formation was also studied as it could be used as a commercially important by product of the transformation. Deacetoxycephalosporin synthase also extracted and partially purified and characterised.
Determination of Immobilization Process Parameters of Corynebacterium glutami...IJERA Editor
The parameters of the immobilized process of Corynebacterium glutamicum on kappa carrageenan were identified by
Plackett-Burman matrix, and the experiments were designed by response surface methodology having the central
composite designs (RSM-CCD). The maximum yield of cell immobilization on kappa carrageenan carrier reached at
78% ± 2%. Optimal parameters were 3 grams kappa carrageenan per 100 militters sterile water and 58.58 million
cfu/mL, forming gels at 100C for 25 minutes and the speed when soaking particles of 150 rpm for 120 minutes in 0.58
M potassium chlorua solvent. The immobile finished products are applied in L-lysine production, their reusing ability
is 3 times and the total yield of L-lysine was accumulated 93 g/L in medium during 96 fermented hours. The L-lysine
productivity of the batch fermentation was 0.969 g.L-1
.h-1
. And the set-up storage conditions are the mixed solvent of
CaCl2 0.5% (w/v) and KCl 0.5% (w/v); pH is 7.0 in 40C. After 60 storage days, the survival cell rate was remained
51%.
Emile Van Schaftingen-Homenaje al bioquímico español Alberto Sols_20_21_02201...Fundación Ramón Areces
La figura de Alberto Sols fue durante décadas una referencia para los bioquímicos españoles. Los días 20 y 21 de febrero de 2017, la Fundación Ramón Areces dedicó un simposio internacional a su memoria, rindiendo homenaje a sus principales logros: las levaduras como modelo experimental, la enzimología y regulación metabólica y la patología molecular. El encuentro científico, que estuvo coordinado por Carlos Gancedo y Joan Guinovart, reunió a expertos internacionales para debatir sobre el legado de este científico español.
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
The increased availability of biomedical data, particularly in the public domain, offers the opportunity to better understand human health and to develop effective therapeutics for a wide range of unmet medical needs. However, data scientists remain stymied by the fact that data remain hard to find and to productively reuse because data and their metadata i) are wholly inaccessible, ii) are in non-standard or incompatible representations, iii) do not conform to community standards, and iv) have unclear or highly restricted terms and conditions that preclude legitimate reuse. These limitations require a rethink on data can be made machine and AI-ready - the key motivation behind the FAIR Guiding Principles. Concurrently, while recent efforts have explored the use of deep learning to fuse disparate data into predictive models for a wide range of biomedical applications, these models often fail even when the correct answer is already known, and fail to explain individual predictions in terms that data scientists can appreciate. These limitations suggest that new methods to produce practical artificial intelligence are still needed.
In this talk, I will discuss our work in (1) building an integrative knowledge infrastructure to prepare FAIR and "AI-ready" data and services along with (2) neurosymbolic AI methods to improve the quality of predictions and to generate plausible explanations. Attention is given to standards, platforms, and methods to wrangle knowledge into simple, but effective semantic and latent representations, and to make these available into standards-compliant and discoverable interfaces that can be used in model building, validation, and explanation. Our work, and those of others in the field, creates a baseline for building trustworthy and easy to deploy AI models in biomedicine.
Bio
Dr. Michel Dumontier is the Distinguished Professor of Data Science at Maastricht University, founder and executive director of the Institute of Data Science, and co-founder of the FAIR (Findable, Accessible, Interoperable and Reusable) data principles. His research explores socio-technological approaches for responsible discovery science, which includes collaborative multi-modal knowledge graphs, privacy-preserving distributed data mining, and AI methods for drug discovery and personalized medicine. His work is supported through the Dutch National Research Agenda, the Netherlands Organisation for Scientific Research, Horizon Europe, the European Open Science Cloud, the US National Institutes of Health, and a Marie-Curie Innovative Training Network. He is the editor-in-chief for the journal Data Science and is internationally recognized for his contributions in bioinformatics, biomedical informatics, and semantic technologies including ontologies and linked data.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
PRESENTATION ABOUT PRINCIPLE OF COSMATIC EVALUATION
JBEI Highlights - October 2014
1. Yeast tolerance to the ionic liquid 1-ethyl-
3-methylimidazolium acetate
Outcomes
• 13 yeast strains tolerant to 5% [C2Ciim][OAc] were identified.
• Galactomyces geotrichum was the most tolerant yeast. This yeast actually exhibited enhanced growth in IL medium.1
Several yeasts capable of rapid growth and high cell density in 5% IL were identified.
1Sitepu, et al., “Yeast tolerance to the ionic liquid 1-ethyl-3-methylimidazolium acetate.” FEMS Yeast Research
(in press)
Background
• Pretreatment of biomass with
ionic liquids (ILs) can
improve hydrolysis. However,
residual IL on pretreated
biomass can inhibit many
fermentative
microorganisms.
• There is a need for yeasts
that can tolerate up to 4% of
the IL 1-ethyl-3-
methylimidazolium acetate
([C2Ciim][OAc]), a promsing
IL for pretreatment.
Significance
• These [C2Ciim][OAc]-tolerant yeasts could be used for fermentation of hydrolysates
from IL-pretreated biomass or serve as a source of yeast IL-tolerance pathways.
Finalcultureabsorbanceat600nm
Approach
• 168 strains of yeasts spanning multiple phyla from the Phaff Yeast Culture Collection at UC Davis were screened by
culturing in media containing varying levels of [C2Ciim][OAc].
2. Expression of a bacterial 3-dehydroshikimate dehydratase
(QsuB) reduces lignin content and improves biomass
saccharification efficiency.
Outcomes
• Arabidopsis plants expressing QsuB show drastic lignin reductions (~50%), enrichment of H-units, and reduced lignin DP.
• The biomass from the engineered Arabidopsis lines display a two-fold increase of saccharification efficiency.
1 Eudes, A., Noppadon, S., Baidoo, E., George, A., Liang, Y., Yang, F., Singh, S., Keasling, J., Simmons, B., Loque, D. (2014).
Expression of a bacterial 3‐dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification
efficiency. Plant Biotechnology Journal.
Background
• Lignin is a polymer that
confers recalcitrance to plant
biomass.
• Novel approaches to reduce
lignin in bioenergy crops
without affecting biomass
yield are desired for
economical production of
second-generation biofuels
Approach
• Identify enzymes whose
expression in Arabidopsis
leads to lignin reduction1.
Such enzymes will be
expressed in a tissue-specific
manner in bioenergy crops to
avoid the adverse effects of
lignin reduction.
Significance
• Expression of 3-dehydroshikimate dehydratase is a novel strategy to reduce lignin.
• Crops expressing QsuB under the control of tissue-specific promoters are being developed.
3-dehydroshikimate
shikimate
phenylalanine
p-coumaroyl-CoA
p-coumaroyl-shikimate
feruloyl-CoA
p-coumaryl alcohol
coniferyl alcohol
sinapyl alcohol
H-unit
G-unit
HCT shikimate
PCA
QsuB
phenylalanine
coniferaldehyde
sinapaldehydeS-unit
phosphoenol pyruvate
+
p-coumaraldehyde
erythrose-4-phosphate
1) The lignin pathway and expression of QsuB
PCA, protocatechuate
0
2
4
6
8
10
12
14
16
18
WT
qsuB‐1
qsuB‐3
qsuB‐6
qsuB‐7
3) Lignin content, composition (2D‐NMR), and structure (SEC) in qsuB transgenic lines
qsuB-1 WT qsuB-3 qsuB-6 qsuB-7
2) QsuB plants do not show drastic biomass reduction
Lignin (% Cell wall)
H: 3.8%
S: 20.0%
G: 76.2%
H: 27.2%
S: 30.2%
G: 42.6%
0
50
100
150
200
250
300
350
400
450 WT qsuB‐1
qsuB‐3 qsuB‐6
qsuB‐7
4) Biomass saccharification
Sugars (µg mg‐1biomass)
0.00
0.05
0.10
0.15
0.20
0.25
5 10 15 20 25
Elution time (min)
Normalized intensity
m > 22 kDa 22 kDa > m > 0.74 kDa
WT
qsuB-1
m < 0.74 kDa
3. Background
• Protein-protein interactions are important for
enzyme function, including cell wall
biosynthesis
• Current methods to assess interactions have
limitations
Approach
• Develop and validate split luciferase as
method for determining interactions between
plant membrane proteins
• Compare with other methods (BiFC, Y2H)
Outcomes
• Split luciferase was demonstrated to be a
robust method for assessing protein
interactions
A reversible Renilla luciferase protein
complementation assay for rapid identification of
protein-protein interactions reveals the existence of
an interaction network involved in xyloglucan
biosynthesis in the plant Golgi apparatus
Lund, C. H., Bromley, J. R., Stenbaek, A., Rasmussen, R. E., Scheller, H. V., & Sakuragi, Y. (2014). "A
reversible Renilla luciferase protein complementation assay for rapid identification of protein-protein
interactions reveals the existence of an interaction network involved in xyloglucan biosynthesis in the plant
Golgi apparatus". J Exp Bot, 18. doi, 10.1093/jxb/eru401
Significance
• The method is an important tool for understanding
cell wall biosynthesis and engineer improved
biofuel crops
FUT1*
XXT2*
XXT5XXT1
CSLC4
*
MUR3
*
Golgi apparatus
When protein fusions
with the N- and C-
termini of luciferase
interact, a functional
luciferase is generated
The method can be used with proteins
transiently expressed in tobacco
The method was used to investigate
interactions between
glycosyltransferases involved in
xyloglucan biosynthesis
4. OH
O
P
O
O
OH
OH
D-glyceraldehyde 3-phosphate
O
O
O-
pyruvate
HO
OH
OHO
P
O
O
OH
OH
D-ribulose 5-phosphate
OH
OHO
P
O
O OH
OH
1-deoxy-D-xylulose 5-phosphate (DXP)
CO2
2C-methyl-D-erythritol 4-phosphate (MEP)
4-diphosphocytidyl-2C-methyl D-erythritol (CDP-ME)
Dxr (ispC)
Dxs
nDXP
(ribB mutants
or yajO)
IspD
IspE
4-diphosphocytidyl-2C-methyl D-erythritol 2-phosphate (CDP-MEP)
IspF
2C-methyl-D-erythritol 2,4-cyclodiphosphate (MEcPP)
IspG
IspH
(E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP)
IPP DMAPP
Idi
Enhancing terpene yield from sugars via
novel routes to DXP (1-deoxy-D-xylulose 5-phosphate)
1Linshiz, et al., “PaR-PaR: Laboratory Automation System.” ACS Synth. Biol. 2:216-222 (2013).
2Linshiz, et al., “PR-PR: Cross-Platform Laboratory System.” ACS Synth. Biol. Article ASAP (2014).
Background
• Terpene production via the DXP pathway entails condensation
of pyruvate and G3P by DXP synthase (Dxs). Carbon is lost
as CO2 in the process.
• To avoid this carbon-loss and circumvent allosteric regulation
of DXS we searched for alternative routes to DXP from a
pentose phosphate.
Approach
• The dxs gene was knocked out in E. coli and we used both
directed evolution and rational gene selection approaches to
discover alternative routes to DXP.
Significance
• The theoretical pathway yield to terpene biofuels has been increased significantly, almost reaching
the maximum biochemical yield. nDXP provides a direct route from C5 sugars to terpenes.
Metabolic route γP Bisabolene (g/g)
Maximum Biochemical Yield ‐ 0.324
Glu, mevalonate 77.8 0.252
Glu, DXP via Dxs 86.2 0.279
Glu, DXP via nDXP 95.4 0.309
Xyl, DXP via Dxs 73.3 0.237
Xyl, DXP via nDXP 79.6 0.258
Kirby et al., “Enhancing Terpene Yield from Sugars via Novel Routes to 1‐Deoxy‐D‐Xylulose 5‐Phosphate”
Appl. Environ. Microbiol. 81: AEM.02920‐14 (2014).
Genome sequencing at the
JGI identified ribB mutants
that complemented a dxs
knockout in E. coli
Outcomes
• Two novel routes to DXP
(nDXP) were discovered
and used to increase
terpene titers in E. coli
grown on either glucose or
xylose.
5. Understanding the Role of Histidine in the GHSxG
Acyltransferase Active Site Motif: Evidence for Histidine
Stabilization of the Malonyl-Enzyme Intermediate
Outcomes
• A histidine to alanine mutation (H640A) in the GHSxG motif yersiniabactin acyltransferase results in an approximately
seven-fold higher hydrolysis rate over the wildtype enzyme, while retaining transacylation activity.
Poust et al., “Understanding the Role of Histidine in the GHSxG Acyltransferase Active Site Motif: Evidence for
Histidine Stabilization of the Malonyl-Enzyme Intermediate.” PloS One. 9: e109421 (2014).
Background
• The role of the conserved
histidine in the active site
motif of acyltransferase
active sites is unclear,
contradictory findings exist in
the literature
Approach
• Utilize high resolution mass
spectrometry and
colorimetric kinetic assays to
determine the acyltransfer
activity and hydrolysis rates
of active site knockouts of
the yersiniabactin
acyltransferase
Significance
• Suggests that acyltransferases have evolved to protect acyl intermediates, functionally diverging from their α/β
hydrolase relatives
6. A droplet-to-digital (D2D) microfluidics
platform for single cell assays1
Outcomes
• A microfluidic device was successfully used to screen the effects of ionic liquids (16 conditions) on cell growth and biofuel
production (comparable to well-plate methods).
• This is a first step towards a higher-throughput microfluidic method which will screen over 400 ionic liquid conditions on
enzymes and biofuel producing microorganisms.
1Shih et al., “A droplet-to-digital (D2D) microfluidic device for single cell assays.” Lab on a Chip. DOI:
10.1039/c4lc00794h
Background
• Ionic liquid (IL) pretreatment
has been shown to efficiently
dissolve biomass and
produce higher lignin-free
cellulose.
• Some IL are toxic to most
enzymes and organisms
used for sacchrification and
fermentation.
Approach
• Develop a microfluidic device
to isolate and to analyze
single cells for toxicity
towards four types of ILs in
various concentrations.
Significance
• This is automated microfluidic method which allows fast determination of toxic IL
with 600-fold reduction in volumes (compared to well-plate methods) and with only
6 pipetting steps.
Microfluidic device (D2D)
Microfluidic automation system
Single yeast cell grown on a
microfluidic device
Growth curves / biofuel production
![Yeast tolerance to the ionic liquid 1-ethyl-
3-methylimidazolium acetate
Outcomes
• 13 yeast strains tolerant to 5% [C2Ciim][OAc] were identified.
• Galactomyces geotrichum was the most tolerant yeast. This yeast actually exhibited enhanced growth in IL medium.1
Several yeasts capable of rapid growth and high cell density in 5% IL were identified.
1Sitepu, et al., “Yeast tolerance to the ionic liquid 1-ethyl-3-methylimidazolium acetate.” FEMS Yeast Research
(in press)
Background
• Pretreatment of biomass with
ionic liquids (ILs) can
improve hydrolysis. However,
residual IL on pretreated
biomass can inhibit many
fermentative
microorganisms.
• There is a need for yeasts
that can tolerate up to 4% of
the IL 1-ethyl-3-
methylimidazolium acetate
([C2Ciim][OAc]), a promsing
IL for pretreatment.
Significance
• These [C2Ciim][OAc]-tolerant yeasts could be used for fermentation of hydrolysates
from IL-pretreated biomass or serve as a source of yeast IL-tolerance pathways.
Finalcultureabsorbanceat600nm
Approach
• 168 strains of yeasts spanning multiple phyla from the Phaff Yeast Culture Collection at UC Davis were screened by
culturing in media containing varying levels of [C2Ciim][OAc].](https://image.slidesharecdn.com/jbeihighlightsoctober2014-161017203800/85/JBEI-Highlights-October-2014-1-320.jpg)
