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The signal recognition particle (SRP) is conserved in all living organisms, and it cotranslationally delivers proteins to the inner membrane or endoplasmic reticulum. Recently, SRP loss was found not to be lethal in either the eukaryote Saccharomyces cerevisiae or the prokaryote Streptococcus mutans. In Escherichia coli, the role of SRP in mediating inner membrane protein (IMP) targeting has long been studied. However, the essentiality of SRP remains a controversial topic, partly hindered by the lack of strains in which SRP is completely absent. Here we show that the SRP was nonessential in E. coli by suppressor screening. We identified two classes of extragenic suppressors—two translation initiation factors and a ribosomal protein—all of which are involved in translation initiation. The translation rate and inner membrane proteomic analyses were combined to define the mechanism that compensates for the lack of SRP. The primary factor that contributes to the efficiency of IMP targeting is the extension of the time window for targeting by pausing the initiation of translation, which further reduces translation initiation and elongation rates. Furthermore, we found that easily predictable features in the nascent chain determine the specificity of protein targeting. Our results show why the loss of the SRP pathway does not lead to lethality. We report a new paradigm in which the time delay in translation initiation is beneficial during protein targeting in the absence of SRP. IMPORTANCE Inner membrane proteins (IMPs) are cotranslationally inserted into the inner membrane or endoplasmic reticulum by the signal recognition particle (SRP). Generally, the deletion of SRP can result in protein targeting defects in Escherichia coli. Suppressor screening for loss of SRP reveals that pausing at the translation start site is likely to be critical in allowing IMP targeting and avoiding aggregation. In this work, we found for the first time that SRP is nonessential in E. coli. The time delay in initiation is different from the previous mechanism that only slows down the elongation rate. It not only maximizes the opportunity for untranslated ribosomes to be near the inner membrane but also extends the time window for targeting translating ribosomes by decreasing the speed of translation. We anticipate that our work will be a starting point for a more delicate regulatory mechanism of protein targeting.

The enzyme ZHD101 from Clonostachys rosea hydrolyzes and deactivates the mycotoxin zearalenone (ZEN) and its zearalenol (ZOL) derivatives. ZHD101 prefers ZEN to ZOL as its substrate, but ZOL, especially the α-form, shows higher estrogenic toxicity than ZEN. To enhance α-ZOL selectivity, we solved the complex structures of ZHD101 with both ZOLs and modified several lactone-surrounding residues. Among the mutants, V153H maintained activity for ZEN but showed a 3.7-fold increase in specific activity against α-ZOL, with an ∼2.7-fold reduction in substrate affinity but a 5.2-fold higher turnover rate. We then determined two V153H/ZOL complex structures. Here, the α-ZOL lactone ring is hydrogenbonded to the H153 side chain, yielding a larger space for H242 to reconstitute the catalytic triad. In conclusion, structure-based engineering was successfully employed to improve the ZHD101 activity toward the more toxic α-ZOL, with great potential in further industrial applications.

BackgroundYeast transcription factors (TFs) involved in the regulation of multidrug resistance (MDR) were investigated in experiments with deletion mutants, transformants overexpressing synthetic genes encoding TFs, and toxic concentrations of lignocellulose-derived substances added to cultures as complex mixtures or as specific compounds, viz. coniferyl aldehyde, 5-hydroxymethylfurfural, and furfural.ResultsIn the presence of complex mixtures of toxic substances from spruce wood, transformants overexpressing YAP1 and STB5, TFs involved in oxidative stress response, exhibited enhanced relative growth rates amounting to 4.589 ± 0.261 and 1.455 ± 0.185, respectively. Other TFs identified as important for resistance included DAL81, GZF3, LEU3, PUT3, and WAR1. Potential overlapping functions of YAP1 and STB5 were investigated in experiments with permutations of deletions and overexpression of the two genes. YAP1 complemented STB5 with respect to resistance to 5-hydroxymethylfurfural, but had a distinct role with regard to resistance to coniferyl aldehyde as deletion of YAP1 rendered the cell incapable of resisting coniferyl aldehyde even if STB5 was overexpressed.ConclusionsWe have investigated 30 deletion mutants and eight transformants overexpressing MDR transcription factors with regard to the roles the transcription factors play in the resistance to toxic concentrations of lignocellulose-derived substances. This work provides an overview of the involvement of thirty transcription factors in the resistance to lignocellulose-derived substances, shows distinct and complementary roles played by YAP1 and STB5, and offers directions for the engineering of robust yeast strains for fermentation processes based on lignocellulosic feedstocks. Electronic supplementary materialThe online version of this article (10.1186/s12934-017-0811-9) contains supplementary material, which is available to authorized users.

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Gene knockout has been used as a common strategy to improve microbial strains for producing chemicals. Several algorithms are available to predict the target reactions to be deleted. Most of them apply mixed integer bi-level linear programming (MIBLP) based on metabolic networks, and use duality theory to transform bi-level optimization problem of large-scale MIBLP to single-level programming. However, the validity of the transformation was not proved. Solution of MIBLP depends on the structure of inner problem. If the inner problem is continuous, Karush-Kuhn-Tucker (KKT) method can be used to reformulate the MIBLP to a single-level one. We adopt KKT technique in our algorithm ReacKnock to attack the intractable problem of the solution of MIBLP, demonstrated with the genome-scale metabolic network model of E. coli for producing various chemicals such as succinate, ethanol, threonine and etc. Compared to the previous methods, our algorithm is fast, stable and reliable to find the optimal solutions for all the chemical products tested, and able to provide all the alternative deletion strategies which lead to the same industrial objective.

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We report the structures of an iridoid synthase, nepetalactol synthase, in the presence of NAD+, NADPH or NAD+/10-oxogeranial. The 10-oxogeranial substrate binds in a transoid-O1-C3 conformation and can be reduced by hydride addition to form S-10-oxo-citronellal, a by-product of the enzyme. Tyr178 Oζ is 2.5 Å from the substrate O1, providing the second proton required for reaction. Nepetalactol product formation requires rotation about C1-C2 to form the cisoid isomer leading to formation of the cis-enolate, together with rotation about C4-C5, enabling cyclization and lactol production. The structure is similar to that of progesterone-5β-reductase with almost identical positioning of NADP, Lys146(147), Tyr178(179) and F342(343) but only Tyr178 and Phe342 appear to be essential for activity. The transoid 10-oxogeranial structure also serves as a model for β-face hydride attack in progesterone 5β-reductases and is of general interest in the context of asymmetric synthesis.