The Design–Build–Test–Learn
(DBTL) cycle,
facilitated by exponentially improving capabilities in synthetic biology,
is an increasingly adopted metabolic engineering framework that represents
a more systematic and efficient approach to strain development than
historical efforts in biofuels and biobased products. Here, we report
on implementation of two DBTL cycles to optimize 1-dodecanol production
from glucose using 60 engineered Escherichia coli MG1655 strains. The first DBTL cycle employed a simple strategy
to learn efficiently from a relatively small number of strains (36),
wherein only the choice of ribosome-binding sites and an acyl-ACP/acyl-CoA
reductase were modulated in a single pathway operon including genes
encoding a thioesterase (UcFatB1), an acyl-ACP/acyl-CoA reductase
(Maqu_2507, Maqu_2220, or Acr1), and an acyl-CoA synthetase (FadD).
Measured variables included concentrations of dodecanol and all proteins
in the engineered pathway. We used the data produced in the first
DBTL cycle to train several machine-learning algorithms and to suggest
protein profiles for the second DBTL cycle that would increase production.
These strategies resulted in a 21% increase in dodecanol titer in
Cycle 2 (up to 0.83 g/L, which is more than 6-fold greater than previously
reported batch values for minimal medium). Beyond specific lessons
learned about optimizing dodecanol titer in E. coli, this study had findings of broader relevance across synthetic biology
applications, such as the importance of sequencing checks on plasmids
in production strains as well as in cloning strains, and the critical
need for more accurate protein expression predictive tools.
Background: Extracellular endosulfatases Sulf1 and Sulf2 hydrolyze 6-O-sulfate in heparan sulfate. Results: Disaccharide analysis showed that 2-O-, 6-O-, and N-trisulfated disaccharide units in heparan sulfate were increased to different degrees in different organs in Sulf1 and Sulf2 knock-out mice. Conclusion: Sulfs generate organ-specific sulfation patterns of heparan sulfate. Significance: This may indicate differences in activity between Sulf1 and Sulf2 in vivo.
A fluorine-doped
tin oxide-coated glass electrode modified with
a bilayer film of underlying α-Co(OH)2 and overlying
Mg-intercalated and Co-doped δ-type (layered) MnO2 (Mg|Co-MnO2) preferentially yielded oxygen with a Faradaic
efficiency as high as 79% in the presence of chloride ions at high
concentration (0.5 M). This noble metal-free electrode was fabricated
by cathodic electrolysis of aqueous Co(NO3)2 followed by anodic electrolysis of a mixture of Mn2+,
Co2+, and cetyltrimethylammonium (CTA+) ions
in water. The CTA+ ions accommodated in the interlayer
spaces of Co-doped δ-MnO2 were replaced with Mg2+ by ion exchange. The upper Mg|Co-MnO2 could effectively
block the permeation of Cl– ions and allow only
H2O and O2, while the under α-Co(OH)2 acted as an oxidation catalyst for the H2O penetrated
through the upper coating. Thus, the oxygen evolution reaction (OER)
was preferred to the chlorine evolution reaction (CER). In artificial
seawater (pH 8.3), the blocking
effect against Cl– decreased because of ion exchange
of the intercalated Mg2+ ions with Na+ in solution,
but the OER efficiency still remained at 57%, much higher than that
(28%) without the upper Mg|Co-MnO2. This demonstrates that
the interlayer spaces between MnO2 layers acted as pathways
for H2O molecules to reach the active sites of the underlying
Co(OH)2. Density functional theory (DFT) calculations revealed
that the most stable structure of hydrated Mg2+ ion, in
which a part of coordinated H2O molecules is hydrolyzed,
has less affinity toward Cl– ion than that of hydrated
Na+ ion.
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