Standardisation of
genetic parts has become a topic of increasing
interest over the last decades. The promise of simplifying molecular
cloning procedures, while at the same time making them more predictable
and reproducible has led to the design of several biological standards,
one of which is modular cloning (MoClo). The Yeast MoClo toolkit provides
a large library of characterised genetic parts combined with a comprehensive
and flexible assembly strategy. Here we aimed to (1) simplify the
adoption of the standard by providing a simple design tool for including
new parts in the MoClo library, (2) characterise the toolkit further
by demonstrating the impact of a BglII site in promoter parts on protein
expression, and (3) expand the toolkit to enable efficient construction
of gRNA arrays, marker-less integration cassettes and combinatorial
libraries. These additions make the toolkit more applicable for common
engineering tasks and will further promote its adoption in the yeast
biological engineering community.
Background: The sesquiterpenoid abscisic acid (ABA) is mostly known for regulating developmental processes and abiotic stress responses in higher plants. Recent studies show that ABA also exhibits a variety of pharmacological activities. Affordable and sustainable production will be required to utilize the compound in agriculture and as a potential pharmaceutical. Saccharomyces cerevisiae is an established workhorse for the biotechnological production of chemicals. In this study, we constructed and characterised an ABA-producing S. cerevisiae strain using the ABA biosynthetic pathway from Botrytis cinerea. Results: Expression of the B. cinerea genes bcaba1, bcaba2, bcaba3 and bcaba4 was sufficient to establish ABA production in the heterologous host. We characterised the ABA-producing strain further by monitoring ABA production over time and, since the pathway contains two cytochrome P450 enzymes, by investigating the effects of overexpressing the native S. cerevisiae or the B. cinerea cytochrome P450 reductase. Both, overexpression of the native or heterologous cytochrome P450 reductase, led to increased ABA titres. We were able to show that ABA production was not affected by precursor or NADPH supply, which suggested that the heterologous enzymes were limiting the flux towards the product. The B. cinerea cytochrome P450 monooxygenases BcABA1 and BcABA2 were identified as pathway bottlenecks and balancing the expression levels of the pathway enzymes resulted in 4.1-fold increased ABA titres while reducing by-product formation. Conclusion: This work represents the first step towards a heterologous ABA cell factory for the commercially relevant sesquiterpenoid.
The electronic properties
of the long-known cationic 1,2,4-triazoliumylidene 3a have been determined. The 77Se NMR chemical
shift of its Se adduct 9 (δ = 138 ppm) indicates
that 3a is only moderately π-acidic. M(CO)2Cl complexes of 3a (M = Rh, Ir) allowed the IR
spectroscopic determination of a TEP value of 2073 cm–1, the highest value known to date for a N-heterocyclic carbene (NHC).
The properties of cationic 3a were compared to those
of the related neutral triazolylidene 5, which was prepared
for comparison. Density functional theory calculations support the
experimental findings. Overall, the cationic carbene 3a has to be considered a very poor σ-donor. Nevertheless, 3a is able to form di- (19) and even tricationic
bis-NHC complexes (20 and 21).
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