Biology subjects

Speight, R.

Publications and source records attributed to Speight, R..

3 recordsLinked to original sources

bioRxiv2024

The integration of tandem gene repeats via a bacterial type-II toxin-antitoxin-mediated gene amplification (ToxAmp) system and stability visualisation in Saccharomyces cerevisiae

Tandem gene repeats naturally occur as important genomic features and determine many traits in living organisms, like human diseases and microbial productivities of target bioproducts. Here, we develop a bacterial type-II toxin-antitoxin-mediated method to manipulate genomic integration of tandem gene repeats in Saccharomyces cerevisiae and further visualise the evolutionary trajectories of gene repeats. We designed a tri-vector system to introduce toxin-antitoxin-driven gene amplification (ToxAmp) modules, and accidentally re-visited the high-level capacity of multi-fragment co-transformation in S. cerevisiae. This system delivered the multi-copy gene integration in the form of tandem gene repeats spontaneously and independently from toxin-antitoxin-mediated selection. Inducing the toxin (RelE) expressing via a copper (II)-inducible CUP1 promoter successfully drove the in-situ gene amplification of the antitoxin (RelB) module, resulting in [~]40 copies of a green fluorescence reporter (GFP) gene per copy of genome. The copy-number changes, increasing and decreasing, and stable maintenance were visualised using the GFP and blue chromoprotein AeBlue as reporters. Copy-number increasing happened spontaneously not depending on a selection pressure and was quickly enriched through toxin-antitoxin-mediated selection. In summary, the bacterial toxin-antitoxin systems provide a flexible mechanism to manipulate gene copy number in eukaryotic cells and can be exploited for synthetic biology and metabolic engineering applications. Table of Contents Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/578080v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@75d1f3org.highwire.dtl.DTLVardef@12aafeforg.highwire.dtl.DTLVardef@184a1a5org.highwire.dtl.DTLVardef@90586a_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Active site mutations of F420-dependent alkene reductases reverse stereoselectivity

Ene-reductases from the Flavin/Deazaflavin Oxidoreductase (FDOR) family have potential value in biocatalysis as they typically exhibit complementary stereoselectivity to the widely utilized Old Yellow Enzyme (OYE) family, yet they are comparatively poorly understood at a mechanistic level. Here, we use a rational design approach to generate a library of 46 active site mutants of two FDORs from Mycobacterium smegmatis and examine the effects on conversion and stereoselectivity against a panel of substrates. Analysis of the effects of these mutations on stereoselectivity across all substrates revealed that the catalytic mechanism is highly sensitive to the polarity of the immediate active site. A conserved active site tyrosine in these enzymes, which does not serve as the proton donor, strongly affects stereochemical outcomes with C- (but not C{beta}-) substituted substrates. Notably, a Tyr-Met mutation at this position reversed the diastereomeric excess (de) with (R)-carvone from 85.3% to -17.3% (cis/trans). Additionally, this mutation significantly increases activity with (1S)- verbenone. Finally, we show that the altered stereoselectivity is not due to a "flipped" substrate binding mode in these mutants, but rather that the hydrogenation mode is altered to favor syn relative to anti addition. These results show that the FDORs are highly engineerable and that, despite their superficial similarity, the OYE and FDOR families differ in crucial mechanistic aspects.

synthetic biology↗

bioRxiv2022

Asymmetric ene-reduction of α,β-unsaturated compounds by F420-dependent oxidoreductases A (FDOR-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, including the complete conversion of both isomers of citral to (R)-citronellel 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 being generally stereocomplementary to each other and highlight their potential value in asymmetric ene-reduction.

biochemistry↗