SPOTLIGHT
&Enzyme Stabilization by Removing Unstable
Surface Loop
Thermostability of enzymes is a prerequisite for their
industrial application. Development of methods for engi-
neering of thermostable enzymes converges towards semi-
rational approaches favored for reduced library size
combined with necessary diversity. Still, the advancement
of protein crystallography and structure modeling should
further increase the efficiency of enzyme engineering. In this
regard, Damnjanovic et al. came up with a new structure-
derived strategy for enzyme thermostabilization based on the
removal of a dynamic, unstable surface loop from the enzyme
molecule. Applying this strategy, the thermostability of
phospholipase D (PLD) used for synthesis of phosphatidy-
linositol (PI) has been increased to the point where the
thermostable variant showed 11.7 times increased inactiva-
tion half-life at 70 C compared to the parent enzyme and
afforded twofold higher PI yield. The target loop of PLD was
identified as unstable by structural analyses, which previously
led the authors to randomly mutate its most dynamic residue
and observe stabilizing effects. Page 674
DOI: 10.1002/bit.25045
&A High-Performing TALEN System for Genome
Engineering
Transcription activator-like effector nucleases (TALENs)
have rapidly emerged as a powerful tool for genome
engineering. The site-specific DNA double-strand breaks
generated by TALENs can induce homologous recombina-
tion or non-homologous end joining, resulting in desired
genomic modifications in various organisms and cell types.
In this work, Sun and coworkers have isolated a high-
performing TALEN variant through high-throughput screen-
ing in yeast. The corresponding TALEN variant, named
SunnyTALEN, has significantly improved genome editing
efficacy. The SunnyTALEN system increases the rate of
genetic modification at all the 13 tested loci of human
genome and is compatible with heterodimer TALEN
architectures. This enhanced TALEN variant represents a
novel second-generation TALEN system and has great
potential for biological and therapeutic applications. Page 683
DOI: 10.1002/bit.25044
Published online in Wiley Online Library
(wileyonlinelibrary.com).
ß 2014 Wiley Periodicals, Inc.
&Transcriptional Engineering Tools for E. coli
Classical strain engineering methods have long been
employed in industry to enhance cell performance under
various stresses. However, due to the complexity of metabolic
networks and unknown genotype–phenotype information, it
is often very difficult to achieve optimal results within a short
period of time. In recent years, transcriptional engineering
has started to attract attention in strain engineering as it
doesn’t require detailed metabolic or genetic information.
Here, the Jiang group has successfully improved E. coli
isobutanol tolerance by engineering its global regulator
cAMP receptor protein (CRP), which can regulate more than
400 genes in E. coli. Other publications from the Jiang group,
including improving E. coli tolerance towards oxidative
stress, acetate, osmotic pressure, biofuels, and various organic
solvents, have demonstrated that CRP engineering is an
efficient strain engineering approach for E. coli. Page 700
DOI: 10.1002/bit.25042
&A Model-Driven Quantitative Metabolomics
Analysis for E. coli
The advent of model-enabled workflows in systems biology
allows for the integration of experimental data types with
genome-scale models to discover new features of biology. The
authors demonstrate such a workflow applied to study the
metabolomic differences of Escherichia coli growing anaerobi-
cally and aerobically. First, the authors utilized constraint-
based modeling to deduce a target list of influential
compounds for which analytical and experimental method-
ologies were developed. Next, the authors assayed the
metabolite levels of anaerobic and aerobic E. coli using a
custom-made rapid sampling apparatus, and validated the
data by comparison to previous small-scale studies. Finally,
the authors integrated the data with the E. coli genome-scale
metabolic model (GEM) via a thermodynamic sensitivity
analysis. The analysis reconciled network usage inconsisten-
cies and identified key biochemical differences between
anaerobic and aerobic growth. The demonstrated model-
enabled workflow can be extended to different organisms and
growth conditions of interest. Page 803
DOI: 10.1002/bit.25043
Biotechnology and Bioengineering, Vol. 111, No. 4, April, 2014