4 research outputs found
Asymmetric Ene-Reduction of α,β-Unsaturated Compounds by F<sub>420</sub>-Dependent Oxidoreductases A Enzymes from Mycobacterium smegmatis
The stereoselective reduction of alkenes conjugated to
electron-withdrawing
groups by ene-reductases has been extensively applied to the commercial
preparation of fine chemicals. Although several different enzyme families
are known to possess ene–reductase activity, the old yellow
enzyme (OYE) family has been the most thoroughly investigated. Recently,
it was shown that a subset of ene-reductases belonging to the flavin/deazaflavin
oxidoreductase (FDOR) superfamily exhibit enantioselectivity that
is generally complementary to that seen in the OYE family. These enzymes
belong to one of several FDOR subgroups that use the unusual deazaflavin
cofactor F420. Here, we explore several enzymes of the
FDOR-A subgroup, characterizing their substrate range and enantioselectivity
with 20 different compounds, identifying enzymes (MSMEG_2027 and MSMEG_2850)
that could reduce a wide range of compounds stereoselectively. For
example, MSMEG_2027 catalyzed the complete conversion of both isomers
of citral to (R)-citronellal with 99% ee, while MSMEG_2850
catalyzed complete conversion of ketoisophorone to (S)-levodione with 99% ee. Protein crystallography combined with computational
docking has allowed the observed stereoselectivity to be mechanistically
rationalized for two enzymes. These findings add further support for
the FDOR and OYE families of ene-reductases displaying general stereocomplementarity
to each other and highlight their potential value in asymmetric ene-reduction
Integration of Yeast Episomal/Integrative Plasmid Causes Genotypic and Phenotypic Diversity and Improved Sesquiterpene Production in Metabolically Engineered <i>Saccharomyces cerevisiae</i>
The variability in phenotypic outcomes
among biological
replicates
in engineered microbial factories presents a captivating mystery.
Establishing the association between phenotypic variability and genetic
drivers is important to solve this intricate puzzle. We applied a
previously developed auxin-inducible depletion of hexokinase 2 as
a metabolic engineering strategy for improved nerolidol production
in Saccharomyces cerevisiae, and biological
replicates exhibit a dichotomy in nerolidol production of either 3.5
or 2.5 g L–1 nerolidol. Harnessing Oxford Nanopore’s
long-read genomic sequencing, we reveal a potential genetic causethe
chromosome integration of a 2μ sequence-based yeast episomal
plasmid, encoding the expression cassettes for nerolidol synthetic
enzymes. This finding was reinforced through chromosome integration
revalidation, engineering nerolidol and valencene production strains,
and generating a diverse pool of yeast clones, each uniquely fingerprinted
by gene copy numbers, plasmid integrations, other genomic rearrangements,
protein expression levels, growth rate, and target product productivities.
Τhe best clone in two strains produced 3.5 g L–1 nerolidol and ∼0.96 g L–1 valencene. Comparable
genotypic and phenotypic variations were also generated through the
integration of a yeast integrative plasmid lacking 2μ sequences.
Our work shows that multiple factors, including plasmid integration
status, subchromosomal location, gene copy number, sesquiterpene synthase
expression level, and genome rearrangement, together play a complicated
determinant role on the productivities of sesquiterpene product. Integration
of yeast episomal/integrative plasmids may be used as a versatile
method for increasing the diversity and optimizing the efficiency
of yeast cell factories, thereby uncovering metabolic control mechanisms
Integration of Yeast Episomal/Integrative Plasmid Causes Genotypic and Phenotypic Diversity and Improved Sesquiterpene Production in Metabolically Engineered <i>Saccharomyces cerevisiae</i>
The variability in phenotypic outcomes
among biological
replicates
in engineered microbial factories presents a captivating mystery.
Establishing the association between phenotypic variability and genetic
drivers is important to solve this intricate puzzle. We applied a
previously developed auxin-inducible depletion of hexokinase 2 as
a metabolic engineering strategy for improved nerolidol production
in Saccharomyces cerevisiae, and biological
replicates exhibit a dichotomy in nerolidol production of either 3.5
or 2.5 g L–1 nerolidol. Harnessing Oxford Nanopore’s
long-read genomic sequencing, we reveal a potential genetic causethe
chromosome integration of a 2μ sequence-based yeast episomal
plasmid, encoding the expression cassettes for nerolidol synthetic
enzymes. This finding was reinforced through chromosome integration
revalidation, engineering nerolidol and valencene production strains,
and generating a diverse pool of yeast clones, each uniquely fingerprinted
by gene copy numbers, plasmid integrations, other genomic rearrangements,
protein expression levels, growth rate, and target product productivities.
Τhe best clone in two strains produced 3.5 g L–1 nerolidol and ∼0.96 g L–1 valencene. Comparable
genotypic and phenotypic variations were also generated through the
integration of a yeast integrative plasmid lacking 2μ sequences.
Our work shows that multiple factors, including plasmid integration
status, subchromosomal location, gene copy number, sesquiterpene synthase
expression level, and genome rearrangement, together play a complicated
determinant role on the productivities of sesquiterpene product. Integration
of yeast episomal/integrative plasmids may be used as a versatile
method for increasing the diversity and optimizing the efficiency
of yeast cell factories, thereby uncovering metabolic control mechanisms
Integration of Yeast Episomal/Integrative Plasmid Causes Genotypic and Phenotypic Diversity and Improved Sesquiterpene Production in Metabolically Engineered <i>Saccharomyces cerevisiae</i>
The variability in phenotypic outcomes
among biological
replicates
in engineered microbial factories presents a captivating mystery.
Establishing the association between phenotypic variability and genetic
drivers is important to solve this intricate puzzle. We applied a
previously developed auxin-inducible depletion of hexokinase 2 as
a metabolic engineering strategy for improved nerolidol production
in Saccharomyces cerevisiae, and biological
replicates exhibit a dichotomy in nerolidol production of either 3.5
or 2.5 g L–1 nerolidol. Harnessing Oxford Nanopore’s
long-read genomic sequencing, we reveal a potential genetic causethe
chromosome integration of a 2μ sequence-based yeast episomal
plasmid, encoding the expression cassettes for nerolidol synthetic
enzymes. This finding was reinforced through chromosome integration
revalidation, engineering nerolidol and valencene production strains,
and generating a diverse pool of yeast clones, each uniquely fingerprinted
by gene copy numbers, plasmid integrations, other genomic rearrangements,
protein expression levels, growth rate, and target product productivities.
Τhe best clone in two strains produced 3.5 g L–1 nerolidol and ∼0.96 g L–1 valencene. Comparable
genotypic and phenotypic variations were also generated through the
integration of a yeast integrative plasmid lacking 2μ sequences.
Our work shows that multiple factors, including plasmid integration
status, subchromosomal location, gene copy number, sesquiterpene synthase
expression level, and genome rearrangement, together play a complicated
determinant role on the productivities of sesquiterpene product. Integration
of yeast episomal/integrative plasmids may be used as a versatile
method for increasing the diversity and optimizing the efficiency
of yeast cell factories, thereby uncovering metabolic control mechanisms