e-ISSN: 2807-2820 Natural Sciences Engineering Technology Journal [NASET Journa.
https://nasetjournal.
Targeted Metabolic Engineering of Saccharomyces cerevisiae for High-Efficiency Valorization of Lignocellulosic Biomass into Superior-Quality Bioplastics Nur Diana1*.
Zaki Ahmad2.
Selma Fajic3 Department of Molecular Biology.
CMHC Research Center.
Palembang.
Indonesia Department of Physics Education.
Enigma Institute.
Palembang.
Indonesia Division of Natural Resources Economy.
Banja Luka College.
Banja Luka.
Bosnia and Herzegovina
ARTIC LE
INFO
Keywords:
Saccharomyces cerevisiae Metabolic Engineering Synthetic Biology Lignocellulosic Waste Circular Bioeconomy *Corresp ondi ng author:
Nur Diana E-mail address:
nurdiananurdiana353@gmail.
All authors have reviewed and approved th e Anal version of the manuscript.
https://doi.
org/10.
37275/nasetjournal.
A B S T R A C T
The global transition towards a sustainable circular bioeconomy urgently requires innovative platforms for converting renewable waste streams into value-added products.
Lignocellulosic biomass, particularly agricultural residue like rice straw, stands as a vast, underutilized carbon source.
This study details the systematic metabolic engineering of Saccharomyces cerevisiae for the high-efficiency production of poly.
-hydroxybutyrat.
(PHB), a biodegradable bioplastic, from rice straw hydrolysate.
A multi faceted synthetic biology approach was implemented in S.
CEN.
PK2-1C.
A robust xylose co-utilization pathway was integrated using codon-optimized genes from Scheffersomyces stipitis.
The PHB biosynthesis pathway from Cupriavidus necator was introduced using a cassette of strong, constitutive yeast promoters .
TDH3, pTEF1, pPGK.
To maximize carbon flux towards PHB, key competing pathways were eliminated via CRISPR Cas9-mediated gene knockouts of the primary alcohol dehydrogenase (ADH.
and glycerol-3-phosphate dehydrogenase (GPD.
The performance of the final engineered strain was evaluated in high -cell-density fed-batch fermentation using detoxified rice straw hydrolysate sourced from Palembang.
Indonesia.
The final engineered strain.
YL-PHB-05 .
adh1 igpd.
, demonstrated superior performance.
In fed-batch bioreactor cultivation, it achieved a final cell dry weight of 33.
8 A 1.
5 g/L and a PHB titer of 15.
2 A 0.
7 g/L, with an intracellular PHB accumulation of 45.
0 A 1.
of cell dry weight.
This corresponds to a high yield of 0.
28 g PHB per gram of consumed sugars.
Crucially, the produced PHB exhibited a superior weight-average molecular weight (M.
2 x 10A Da with a polydispersity index of 2.
In conclusion, this work successfully demonstrates a robust strategy for engineering S.
cerevisiae into an efficient cell factory for producing high-quality bioplastics from a globally relevant agricultural waste The high titers, yields, and superior polymer properties achieved present a significant advancement towards establishing an economica lly viable and sustainable process for bioplastic production within a circular Introduction This model champions the use of The 21st century is defined by the interconnected renewable biological resources, such as agricultural global challenges of climate change, environmental and forestry residues, not as waste but as valuable degradation, and resource depletion, largely stemming from a deeply entrenched linear economic model biochemicals, and biomaterials, thereby creating predicated on "take-make-dispose" principles.
1 In closed-loop response, the concept of a circular bioeconomy has environmental impact.
2 At the heart of this paradigm emerged as a critical paradigm for sustainable lies lignocellulosic biomass, the most abundant and renewable form of terrestrial carbon on Earth, naturally synthesized by numerous microorganisms representing a cornerstone for the next generation of as intracellular carbon and energy storage granules.
industrial biotechnology.
Their material properties are highly tunable, ranging Lignocellulosic biomass is a complex composite making them suitable for a wide spectrum of Through applications, including packaging films, medical conversion processes, the carbohydrate polymersAi implants, and consumer goods.
The commercialization cellulose and hemicelluloseAican be hydrolyzed into of PHAs has been historically constrained by high production costs, which are intrinsically linked to the predominantly the C6 sugar glucose and the C5 sugar price of sterile, refined substrates like glucose or These sugars serve as a versatile platform for microbial fermentation to produce a diverse portfolio lignocellulosic waste as a feedstock is therefore a critical strategy for hemicellulose, and from rigid and brittle to flexible and elastomeric, value-added products.
bottleneck in However, a lignocellulosic biorefineries is the efficient co-utilization of both glucose and xylose.
The low-cost, making PHAs non-food fossil-fuel-derived Many industrially relevant microorganisms either This study aims to develop and optimize a robust cannot naturally metabolize xylose or exhibit strong Saccharomyces cerevisiae cell factory for the high- carbon catabolite repression, where the presence of efficiency conversion of lignocellulosic waste into high- glucose inhibits the uptake and metabolism of other value bioplastics.
The novelty of this research lies in sugars, leading to an incomplete and inefficient the integrated, multi-level metabolic engineering valorization of the feedstock.
strategy employed.
We combine the introduction of Saccharomyces cerevisiae, colloquially known as yeast, is the undisputed workhorse efficient glucose and xylose co-utilization machinery with the high-level expression of a heterologous PHB industrial biotechnology.
Its long history of safe use synthesis pathway and, (GRAS statu.
, exceptional tolerance to industrial elimination of major carbon-competing pathways inhibitors found in hydrolysates, and amenability to using precise CRISPR-Cas9 genome editing.
This genetic manipulation make it a premier host for large- holistic approach is designed to channel the maximum scale fermentation processes.
The primary limitation possible carbon flux from rice straw-derived sugars wild-type PHB.
Over the past two decades, significant breakthroughs compared to previous studies include: .
the targeted in synthetic biology and metabolic engineering have knockout of both ethanol (ADH.
and glycerol (GPD.
The The applications is its innate inability to metabolize xylose.
critically, the xylose-utilizing yeast
PHB
introduction of heterologous xylose isomerase or production, a combination designed for maximal oxidoreductase pathways has enabled the engineering precursor redirection.
the utilization of locally of yeast strains capable of efficiently co-fermenting sourced rice straw from Palembang.
Indonesia, glucose and xylose, unlocking the full potential of demonstrating the adaptability of the technology to lignocellulosic feedstocks.
regionally specific feedstocks.
a detailed Among the vast array of potential bioproducts, characterization of the resulting polymer, revealing superior molecular weight properties that enhance its interest as a sustainable solution to the global crisis of plastic pollution.
Polyhydroxyalkanoates (PHA.
are a synthetic biology tools with optimized bioprocess class of bio-based and biodegradable polyesters engineering, we present a compelling blueprint for the sustainable contributing to the advancement of Saccharomyces CEN.
PK2-1C
(MATa.
leu2-3,112.
MAL2-8C.
SUC.
was used as the parental strain for all genetic Standard yeast cultivation was performed Methods in YPD medium .
g/L yeast extract, 20 g/L peptone.
All experimental work was conducted at the Eureka 20 g/L glucos.
For selection of yeast transformants.
Research Laboratory in Palembang.
Indonesia.
Rice synthetic complete (SC) drop-out medium was used, lacking the appropriate amino acid or nucleotide for lignocellulosic feedstock, was sourced from local auxotrophic selection.
Escherichia coli DH5 was used paddy fields in the Ogan Ilir district.
South Sumatra, for all plasmid cloning and propagation and was Indonesia.
Upon collection, the straw was extensively cultivated in LB medium .
g/L tryptone, 5 g/L yeast washed with tap water to remove soil and debris, sun- extract, 10 g/L NaC.
supplemented with 100 AAg/mL dried for 72 hours, and then oven-dried at 60AC to a ampicillin where required.
All plasmids, genetic parts, constant weight.
The dried straw was subsequently and primers used in this study are detailed in Table 1.
milled using a Wiley mill and sieved to obtain a A stepwise metabolic engineering strategy was uniform particle size of 1Ae2 mm for subsequent employed to construct the final production strain.
All (Oryza A two-stage dilute acid hydrolysis protocol was homologous recombination, and marker-less gene deletions were achieved using the CRISPR-Cas9 hemicellulose and cellulose from the rice straw.
Stage The xylose oxidoreductase pathway was 1 (Hemicellulose Hydrolysi.
: The milled rice straw was chosen for xylose assimilation.
The genes encoding mixed with 1.
5% .
sulfuric acid at a solid-to-liquid xylose reductase (XR) and xylitol dehydrogenase (XDH) ratio of 1:10 .
The slurry was autoclaved at 121AC were sourced from Scheffersomyces stipitis, and the for 60 minutes.
After cooling, the xylose-rich liquid endogenous xylulokinase (XK) gene from S.
hydrolysate was separated from the solid residue by was overexpressed.
All genes were codon-optimized for Stage 2 (Cellulose Hydrolysi.
: The cellulose- expression in yeast.
The three genes, under the control enriched solid residue from the first stage was of strong constitutive promoters .
TDH3 for XR, resuspended in 2.
5% .
sulfuric acid at a 1:10 ratio pTEF1 for XDH, pPGK1 for XK), were assembled into a and subjected to a more severe hydrolysis condition at single expression cassette and integrated into the 150AC for 30 minutes.
The resulting glucose-rich yeast genome at the HIS3 locus, yielding strain YL- XYL-01.
Detoxification: The hydrolysates from both stages were The PHB biosynthesis pathway from Cupriavidus This combined hydrolysate was detoxified necator H16 was introduced.
This pathway consists of to remove microbial inhibitors .
HMF, acetic three enzymes: -ketothiolase .
ncoded by phaA), aci.
by overliming.
The pH was raised to 10.
0 by the acetoacetyl-CoA reductase .
ncoded by phaB), and slow addition of solid calcium hydroxide (Ca(OH)CC) PHA synthase .
ncoded by phaC).
The genes were under constant stirring, followed by incubation at codon-optimized and placed under the control of 60AC for 30 minutes.
The mixture was then filtered to strong glycolytic promoters .
TEF1 for phaA, pPGK1 remove the precipitate, and the pH of the detoxified for phaB, and pTDH3 for phaC) and terminators liquid hydrolysate .
ADH1 and tCYC.
The three expression cassettes concentrated sulfuric acid.
The final hydrolysate was were integrated into the yeast genome at the URA3, filter-sterilized through a 0.
22 AAm membrane prior to LEU2, and TRP1 loci of strain YL-XYL-01, resulting in use in fermentation media.
strain YL-PHB-01.
adjusted to To redirect carbon flux from native fermentation nearly depleted .
ndicated by a sharp spike in byproducts towards the PHB pathway precursor, dissolved oxyge.
, a pre-programmed exponential acetyl-CoA, two key genes were targeted for complete feeding strategy was initiated to maintain a high cell knockout using the CRISPR-Cas9 system.
The primary alcohol dehydrogenase gene.
ADH1, was deleted from concentrated, detoxified rice straw hydrolysate .
the genome of YL-PHB-01 to block ethanol formation, g/L glucose, 130 g/L xylos.
and was delivered at a creating strain YL-PHB-03.
The primary glycerol-3- rate designed to keep the residual total sugar metabolism and substrate inhibition.
The temperature glycerol formation, resulting in the final strain.
YL- was controlled at 30AC, and the pH was maintained at PHB-05.
5 via the automated addition of 2 M NaOH.
The GPD1, subsequently deleted from YL-PHB-03 The The CRISPR-Cas9 system, consisting of a plasmid dissolved oxygen (DO) level was maintained above 20% co-expressing the Cas9 protein and a gene-specific saturation by cascading the agitation speed .
-900 guide RNA .
RNA), was used for gene knockouts.
A 100
and the aeration rate .
-2 vv.
bp donor DNA fragment consisting of sequences Cell growth was monitored by measuring ODCICACA.
homologous to the regions immediately upstream and Cell dry weight (CDW) was determined by filtering 5 downstream of the target gene's open reading frame mL of culture broth through a pre-weighed 0.
45 AAm filter paper.
The filter was washed twice with deionized co-transformed homology-directed Successful gene knockouts were initially water and dried at 80AC for 24 hours to a constant screened by their inability to grow on selective media The maximum specific growth rate (_ma.
or was calculated during the exponential growth phase confirmed by diagnostic colony PCR using primers using the formula: = .
n(XCC) - ln(XCA)) / .
CC - tCA), where flanking the X is the CDW at times tCA and tCC.
Sanger sequencing of the PCR product to verify the seamless The concentrations of glucose, xylose, ethanol, and Functional confirmation of knockout was glycerol in the culture supernatant .
btained by achieved via HPLC analysis, which showed the centrifugation at 13,000 x g for 5 mi.
were quantified by high-performance liquid chromatography (HPLC) relevant culture conditions.
on a Shimadzu system equipped with a Bio-Rad Initial characterization of all engineered strains Aminex HPX-87H column and a refractive index (RI) was conducted in 250 mL baffled shake flasks The column was operated at 60AC with 5 mM containing 50 mL of SC medium supplemented with HCCSOCE as the mobile phase at a flow rate of 0.
20 g/L glucose and 10 g/L xylose.
Cultures were mL/min.
inoculated to a starting optical density at 600 nm The intracellular PHB content was determined by (ODCICACA) of 0.
1 and incubated at 30AC with vigorous gas chromatography (GC).
Approximately 10-15 mg of shaking at 200 lyophilized cells were subjected to methanolysis with 2 Samples were withdrawn aseptically at regular time points for analysis.
The best-performing strain.
YL-PHB-05, mL of chloroform and 2 mL of methanol containing 3% .
HCCSOCE at 100AC for 4 hours.
This process evaluated in a 2 L stirred-tank bioreactor (Sartorius, converts the PHB polymer into its constituent methyl German.
with an initial working volume of 1 L.
The ester, 3-hydroxybutyrate methyl ester, which was then medium consisted of detoxified rice straw quantified by GC (Agilent 7890B) equipped with a hydrolysate supplemented with 5 g/L yeast extract flame ionization detector (FID).
The PHB content was and 10 g/L peptone.
The fermentation was initiated as expressed as a percentage of the CDW.
For polymer a batch culture.
After the initial batch of sugars was characterization.
PHB was extracted from a larger batch of lyophilized cells using hot chloroform, and bioreactor fermentations, were performed in purified by precipitation in cold methanol, and biological triplicate.
The data are presented as the The weight-average molecular weight (M.
mean A standard deviation.
Statistical significance and polydispersity index (PDI) were determined by gel between different experimental groups was determined permeation chromatography (GPC) using polystyrene using a one-way analysis of variance (ANOVA) followed The chemical structure was confirmed by by Tukey's post-hoc test for multiple comparisons.
AH and AAC nuclear magnetic resonance (NMR) using GraphPad Prism software.
A p-value of < 0.
All experiments, including shake flask was considered statistically significant.
Results and discussion A 1.
5 g/L, respectively, reflecting the successful The optimized two-stage dilute acid hydrolysis and cellulose and hemicellulose.
The subsequent detoxification process yielded a sugar- detoxification by overliming proved effective, reducing rich, low-inhibitor hydrolysate suitable for yeast the concentrations of key inhibitors like furfural and The detailed composition is presented in 5-hydroxymethylfurfural (HMF) to negligible levels .
Table 2.
Glucose and xylose were the most abundant A 0.
02 g/L and 0.
05 A 0.
01 g/L, respectivel.
, well sugars, with concentrations of 65.
4 A 2.
1 g/L and 32.
below their typical inhibitory thresholds for cerevisiae.
A series of engineered strains were .
, then to produce PHB (YL-PHB-.
, and finally, constructed to systematically channel carbon flux competing byproduct pathways for ethanol (YL-PHB- from sugars to PHB.
The genotypes of these strains are summarized in Table 3.
The parental strain.
CEN.
PK2-
knocked out.
and glycerol (YL-PHB-.
were sequentially 1C, was first engineered to co-utilize xylose (YL-XYL- To assess the metabolic burden imposed by these ethanol production from 9.
5 A 0.
5 g/L to 0.
4 A 0.
1 g/L extensive modifications, the maximum specific growth and significantly increased the PHB titer to 2.
1 A 0.
rate (_ma.
of each strain was determined in shake g/L .
< 0.
The final knockout of GPD1 in strain flasks on SC medium with glucose (Table .
While the YL-PHB-05 completely abolished glycerol production introduction of the heterologous pathways led to a and further boosted the PHB titer to 3.
5 A 0.
3 g/L, minor reduction in growth rate, even the final strain, which was significantly higher than all other strains .
YL-PHB-05, maintained over 85% of the parental < 0.
These results unequivocally demonstrate strain's growth rate, indicating a well-tolerated that blocking the major carbon-draining pathways is a metabolic load.
The engineered strains were evaluated highly effective strategy for redirecting carbon flux in shake flask cultures to assess the efficacy of the towards the heterologous PHB pathway.
Based on its genetic modifications (Figure .
As described in Figure superior performance, strain YL-PHB-05 was selected 1, all engineered strains demonstrated efficient co- for process optimization in a controlled bioreactor consumption of glucose and xylose, with glucose being environment using a fed-batch strategy with detoxified rice straw hydrolysate.
The introduction of the PHB fermentation is depicted in Figure 2.
The fermentation pathway in YL-PHB-01 resulted in a basal PHB profile .
escribed in Figure .
shows an initial batch production of 0.
8 A 0.
1 g/L.
The subsequent knockout phase of rapid growth, followed by a prolonged fed- of ADH1 in strain YL-PHB-03 dramatically reduced batch phase .
tarting at 24 .
characterized by engineered yeast.
The time-course of sustained cell growth and PHB accumulation.
The g/L.
This corresponds to a remarkable intracellular controlled feeding strategy successfully maintained PHB content of 45.
0 A 1.
2% of CDW.
The overall yield low residual sugar levels, enabling the culture to reach of PHB from consumed sugars .
lucose and xylos.
a high final cell density of 33.
8 A 1.
5 g/L after 132 was calculated to be 0.
28 g/g, which is 68% of the theoretical maximum yield from acetyl-CoA.
PHB
associated, accumulating to a final titer of 15.
2 A 0.
The PHB produced by strain YL-PHB-05 was lignocellulosic waste.
By systematically re-wiring the extracted, purified, and its material properties were intricate metabolic circuitry of yeast, we have created NMR spectroscopy confirmed the chemical a robust and resilient strain capable of converting a structure as poly.
-hydroxybutyrat.
GPC
Indonesian weight (M.
2 x 10A Da and a number-average hydroxybutyrat.
(PHB).
The achieved titers, yields, molecular weight (M.
7 x 10AA Da, resulting in a and polymer characteristics are not only highly polydispersity index (PDI) of 2.
These properties competitive but represent a significant and tangible step towards the industrial viability of sustainable thermoplastic processing and are superior to many bioplastics, providing a compelling blueprint for the PHB types produced in native bacterial systems.
This principles of a circular bioeconomy in action.
study successfully demonstrates the profound efficacy cornerstone of this work was the targeted, stepwise of a rational, multi-tiered metabolic engineering redirection of carbon flux, a strategy predicated on a deep understanding of high-quality efficient Saccharomyces cerevisiae cell factory for the high-value straw into derived from analysis revealed a very high weight-average molecular The yeast's native metabolic PHB high-quality poly.
- from Cupriavidus necator into a xylose-utilizing yeast factory on the outskirts of a bustling metabolic city but chassis .
train YL-PHB-.
yielded only modest PHB had not yet constructed the highways to supply it with This outcome was anticipated, as the raw materials.
The cell's primary imperative, honed by newly installed pathway, despite being driven by millennia of evolution, remained the rapid conversion strong promoters, was forced to compete for the of sugars to ethanol for energy generation and redox central metabolic balance, leaving only a trickle of carbon available for precursor, acetyl-CoA, against our engineered pathway.
11,12
fermentation pathways.
In essence, we had built a new The first and most critical intervention was the could now draw from a substantially enlarged pool of surgical knockout of the ADH1 gene, which encodes the primary alcohol dehydrogenase.
This single genetic intervention alone transformed PHB production from modification in strain YL-PHB-03 provided the most a metabolic curiosity into the cell's new primary significant leap fermentation product.
13,14
unequivocally confirms ethanol production as the acetyl-CoA.
This The subsequent knockout of the GPD1 gene in strain YL-PHB-05 represents a further refinement of Under the oxygen-limited conditions of this flux control strategy.
The GPD1 enzyme is high-density responsible for the synthesis of glycerol, a process that serves dual roles in yeast physiology: it acts as a mechanism for regenerating the NADA consumed secondary carbon sink and, crucially, as an outlet for during glycolysis.
By severing this critical metabolic excess NADH generated during biosynthesis, thereby artery, we effectively created a metabolic dam, causing playing a key role in maintaining cellular redox a massive accumulation of precursor metabolites This blockage forced the cell to reroute its significant carbon drain compared to ethanol, its carbon flux, and the primary beneficiary of this elimination has important secondary effects.
rerouting was the heterologous PHB pathway, which preventing the consumption of NADH for glycerol acetaldehyde to While synthesis, the cellular redox balance is subtly but possesses mechanisms to interconvert NADH and significantly altered.
This shift may further improve NADPH, reducing the overall demand on the NADH the availability of acetyl-CoA and, perhaps more pool by eliminating glycerol synthesis may indirectly importantly, ensures a more favorable intracellular increase the availability of NADPH for the PHB environment for the PHA biosynthesis pathway itself.
This led to the observed incremental, yet Specifically, the acetoacetyl-CoA reductase (PhaB) statistically significant, increase in the final PHB enzyme utilizes NADPH as a cofactor.
While yeast Figure 1.
Performance comparison of engineered S.
cerevisiae strains in shake flask cultivations on a mixed -sugar medium .
g/L glucose, 10 g/L xylos.
(A) Time-course of glucose and xylose consumption.
(B) Final PHB titers after 72 hours.
(C) Final ethanol and glycerol byproduct concentrations after 72 hours.
Data are shown as mean A SD for n=3 biological replicates.
This deliberate and sequential strategy of pathway underscores the critical synergy that must exist perfectly embodies the "push-pull" paradigm that is a between advanced strain engineering and pragmatic central tenet of modern metabolic engineering.
The bioprocess optimization.
The final PHB titer of 15.
strong, constitutive expression of the phaA, phaB, g/L, representing an intracellular accumulation of and phaC genes provided a constant thermodynamic 45% of the cell's dry weight, firmly places this work at "pull," creating a robust sink that continuously the forefront of academic and industrial research into PHB acetyl-CoA strain.
YL-PHB-05, in a controlled fed-batch bioreactor Concurrently, the targeted knockouts of ADH1 and GPD1 provided a challenging, real-world feedstocks.
When contextualized against the existing literature, "push," actively forcing carbon away from its native the significance of this achievement becomes even The Our final strain demonstrates a superior or unequivocal success of this strategy highlights how a highly competitive titer and yield when compared to rational, hypothesis-driven approach, based on a other notable studies.
Crucially, many of these fundamental understanding of cellular metabolism, preceding works utilized highly refined, lab-grade can overcome the inherent metabolic inertia of a host sugar media, which are devoid of the potent microbial inhibitorsAisuch as furfural.
HMF, and acetic acidAi The ultimate validation of any cell factory inevitably generated during lies in its performance under industrially relevant hydrolysis of lignocellulosic biomass.
Our ability to The performance of our final engineered achieve these high production metrics using a detoxified but still complex rice straw hydrolysate The demonstrates the inherent robustness of the S.
respectable volumetric productivity of 0.
115 g/L/h, cerevisiae chassis and the resilience of our engineered achieved in a high-cell-density fermentation, further This is a critical point of differentiation, as the strengthens the case for the industrial potential of this cost and environmental impact of extensive feedstock purification are major barriers to the economic Figure 2.
Time course of fed-batch fermentation of the engineered S.
cerevisiae strain YL-PHB-05 using detoxified rice straw hydrolysate.
The graph plots Cell Dry Weight (CDW, g/L).
PHB concentration .
/L).
PHB content (% of CDW), and residual concentrations of glucose and xylose .
/L) over 132 hours.
The feed was initiated at approximately 24 hours.
Data points represent the mean A SD for n=3 independent bioreactor runs.
typically manifest as severely impaired growth, engineering non-native metabolism is the potential for reduced viability, and genetic instability, ultimately imposing a significant metabolic burden on the host compromising the productivity and robustness of the The forced expression of multiple, high-activity cell factory.
heterologous enzymes and the dramatic rerouting of In this study, our engineered strains exhibited a substantial carbon flux can precipitate a cascade of remarkable degree of physiological resilience.
physiological stresses.
These can include the depletion quantified in Table 4, the final production strain.
YL- of essential precursor pools, the creation of severe PHB-05, despite dedicating nearly half of its mass to a foreign polymer and operating with a fundamentally intermediates, and an overwhelming demand on the rewired central metabolism, retained over 85% of the cell's protein synthesis machinery.
These stresses maximum specific growth rate of its wild-type parent.
This indicates that our strain engineering strategyAi endows the material with its strength and toughness.
which relied on the use of well-characterized, strong Therefore, the exceptionally high Mw PHB produced glycolytic promoters and a balanced integration of from our yeast platform is not merely a scientific pathway componentsAiresulted in a metabolic load it is a premium-grade bioplastic, particularly that was surprisingly well-tolerated.
The yeast cell attractive for demanding thermoplastic applications where material performance is paramount.
15,16
to adeptly manage the synthesis and sequestration of PHB granules without succumbing to catastrophic physiological disruption.
The reason for this dramatically higher molecular weight in a eukaryotic yeast host, as opposed to a This resilience is likely attributable to several native prokaryotic host, is likely multifactorial.
The First, the choice of promoters, primarily from primary contributing factor is almost certainly the the upper glycolytic pathway, ensures that the complete absence of a native PHA depolymerase expression of the PHB pathway enzymes is temporally system in S.
In bacteria like C.
coupled with high carbon flux, thereby minimizing the PHB serves as an intracellular carbon and energy Second, final product.
PHB.
Consequently, these organisms possess a suite of naturally self-assembles into discrete, osmotically depolymerase enzymes that are designed to break inert, and insoluble granules within the cytoplasm.
down the PHB granules and release the monomers for This re-entry into central metabolism when external carbon advantage, as it sequesters the product away from the sources are scarce.
This creates a dynamic equilibrium active cellular machinery, preventing the potential of synthesis and degradation, which inherently limits disruptions in viscosity, osmolarity, or enzyme activity the final chain length of the polymer.
In our engineered that can be caused by high-titer production of soluble yeast.
PHB is not a native storage compound but a This trait is highly advantageous for terminal sink product.
Without any endogenous developing a robust industrial process, as it uncouples machinery to degrade it, the PHA synthase (PhaC) can high productivity from cellular toxicity.
Perhaps one of the most significant and impactful uninterrupted, allowing the polymer chains to grow to findings of this study is the superior quality of the their kinetic limit.
bioplastic produced.
The weight-average molecular Furthermore, weight (M.
of the extracted PHB was determined to environment of a eukaryotic cell likely contributes to 2 x 10A Da, a value that is exceptionally high.
For this phenomenon.
The localized concentrations of the comparison.
PHB produced by its most common native monomer substrate .
-hydroxybutyryl-CoA) and the host.
necator, typically exhibits a Mw in the range PHA synthase enzyme at the site of granule formation, 8 x 10A Da.
This is not an incremental combined with the unique cellular redox state it represents a fundamental step- (NADH/NADPH ratio.
and cytosolic pH of yeast, likely change in the material quality achievable from a create kinetic conditions that strongly favor the biological system.
reaction over The mechanical properties of a polymerAiits tensile termination events.
The observed polydispersity index strength, flexibility, elongation at break, and overall (PDI) of 2.
1 indicates a broader distribution of durabilityAiare directly and intimately correlated with compared to the more uniform polymers sometimes translates to longer individual polymer chains, which produced in bacterial systems.
A PDI value closer to in turn leads to a greater degree of chain entanglement 0 signifies a population of polymer chains that are within the polymer matrix.
It is this entanglement that all very similar in length.
Our higher PDI value is a Higher common and expected feature in engineered biological into valuable, environmentally friendly products and systems and likely reflects the inherent biological mitigating the global challenge of plastic pollution.
variability in polymerization rates across a large cell population and throughout the different phases of the cell cycle.
However, for the vast majority of bulk
References