BUSCADOR RECOLECTA

Understanding the functionality of immobilized enzymes with spatiotemporal resolution and under operando conditions is an unmet need in applied biocatalysis, as well as priceless information to guide the optimization of heterogeneous biocatalysts for industrial purposes. Unfortunately, enzyme immobilization still relies on trial-and-error approximations that prevail over rational designs. Hence, a modern fabrication process to achieve efficient and robust heterogeneous biocatalysts demands comprehensive characterization techniques to track and understand the immobilization process at the protein-material interface. Recently, our group has developed a new generation of self-sufficient heterogeneous biocatalysts based on alcohol dehydrogenases co-immobilized with nicotinamide cofactors on agarose porous microbeads. Harnessing the autofluorescence of NAD+(P)H and using time-lapse fluorescence microscopy, enzyme activity toward the redox cofactors can be monitored inside the beads. To analyze these data, herein we present an image analytical tool to quantify the apparent Michaelis-Menten parameters of alcohol dehydrogenases co-immobilized with NAD(P)+/H at the single-particle level. Using this tool, we found a strong negative correlation between the apparent catalytic performance of the immobilized enzymes and the bead radius when using exogenous bulky substrates in reduction reactions. Therefore, applying image analytics routines to microscopy studies, we can directly unravel the functional heterogeneity of different heterogeneous biocatalyst samples tested under different reaction conditions.

[EN] Background
CRISPR-based programmable transcriptional activators (PTAs) are used in plants for rewiring gene networks. Better tuning of their activity in a time and dose-dependent manner should allow precise control of gene expression. Here, we report the optimization of a Copper Inducible system called CI-switch for conditional gene activation in Nicotiana benthamiana. In the presence of copper, the copper-responsive factor CUP2 undergoes a conformational change and binds a DNA motif named copper-binding site (CBS).

Results
In this study, we tested several activation domains fused to CUP2 and found that the non-viral Gal4 domain results in strong activation of a reporter gene equipped with a minimal promoter, offering advantages over previous designs. To connect copper regulation with downstream programmable elements, several copper-dependent configurations of the strong dCasEV2.1 PTA were assayed, aiming at maximizing activation range, while minimizing undesired background expression. The best configuration involved a dual copper regulation of the two protein components of the PTA, namely dCas9:EDLL and MS2:VPR, and a constitutive RNA pol III-driven expression of the third component, a guide RNA with anchoring sites for the MS2 RNA-binding domain. With these optimizations, the CI/dCasEV2.1 system resulted in copper-dependent activation rates of 2,600-fold and 245-fold for the endogenous N. benthamiana DFR and PAL2 genes, respectively, with negligible expression in the absence of the trigger.

Conclusions
The tight regulation of copper over CI/dCasEV2.1 makes this system ideal for the conditional production of plant-derived metabolites and recombinant proteins in the field., This work has been funded by EU Horizon 2020 Project Newcotiana Grant 760331, PID2019-108203RB-100 Plan Nacional I + D, Spanish Ministry of Economy and Competitiveness and 'ERACoBioTech' Project SUSPHIRE Grant No. 722361. Vazquez-Vilar, M. is recipient of APOSTD/2020/096 (Generalitat Valenciana and Fondo Social Europeo post-doctoral grant). Garcia-Perez, E. is recipient of ACIF-2020 fellowship (Generalitat Valenciana). Diego-Martin, B. and Moreno-Gimenez, E. are recipients of FPU fellowships. Selma, S. is recipient of FPI fellowship.

Three enzymatic routes toward γ-hydroxy-α-amino acids by tandem aldol addition-transamination one-pot two-step reactions are reported. The approaches feature an enantioselective aldol addition of pyruvate to various nonaromatic aldehydes catalyzed by trans - o -hydroxybenzylidene pyruvate hydratase-aldolase (HBPA) from Pseudomonas putida. This affords chiral 4-hydroxy-2-oxo acids, which were subsequently enantioselectively aminated using S -selective transaminases. Three transamination processes were investigated involving different amine donors and transaminases: (i) -Ala as an amine donor with pyruvate recycling, (ii) a benzylamine donor using benzaldehyde lyase from Pseudomonas fluorescens Biovar I (BAL) to transform the benzaldehyde formed into benzoin, minimizing equilibrium limitations, and (iii) -Glu as an amine donor with a double cascade comprising branched-chain α-amino acid aminotransferase (BCAT) and aspartate amino transferase (AspAT), both from E. coli, using -Asp as a substrate to regenerate -Glu. The γ-hydroxy-α-amino acids thus obtained were transformed into chiral α-amino-γ-butyrolactones, structural motifs found in many biologically active compounds and valuable intermediates for the synthesis of pharmaceutical agents.

Understanding how the immobilization of enzymes on solid carriers affects their performance is paramount for the design of highly efficient heterogeneous biocatalysts. An efficient supply of substrates onto the solid phase is one of the main challenges to maximize the activity of the immobilized enzymes. Herein, we apply advanced single-particle analysis to decipher the optimal design of an immobilized NADH oxidase (NOX) whose activity depends both on O2 and NADH concentrations. Carrier physicochemical properties and its functionality along with the enzyme distribution across the carrier were implemented as design variables to study the effects of the intraparticle concentration of substrates (O2 and NADH) on the activity. Intraparticle O2-sensing analysis revealed the superior performance of the enzyme immobilized at the outer surface in terms of effective supply of O2. Furthermore, the co-immobilization of NADH and NOX within the tuned surface of porous microbeads increases the effective concentration of NADH in the surroundings of the enzyme. As a result, the optimal spatial organization of NOX and its confinement with NADH allow a 100% recovery of the activity of the soluble enzyme upon the immobilization process. By engineering these variables, we increase the NADH oxidation activity of the heterogeneous biocatalyst by up to 650% compared to NOX immobilized under suboptimal conditions. In conclusion, this work highlights the rational design and engineering of the enzyme–carrier interface to maximize the efficiency of heterogeneous biocatalysts., A.I.B.-M and F.L.G. are grateful to MINECO (RTI2018-094398-B-I00). ERC-Co (METACELL-878089)) and ERA-CoBioTech (Project ID: 61 HOMBIOCAT/ PCI2018-092984) projects are acknowledged for funding F.L.G. F.L.G. also thanks the IKERBASQUE Foundation. C.H. acknowledges ERASMUS+ for the student exchange grant from the University of Graz to the University of Zaragoza. J.M.B. acknowledges funding from the Government of Community of Madrid (2018-T1/BIO-10200)., Peer reviewed

A two‐enzyme cascade reaction plus in situ oxidative decarboxylation for the transformation of readily available canonical and non‐canonical l‐α‐amino acids into 2‐substituted 3‐hydroxycarboxylic acid derivatives is described. The biocatalytic cascade consisted of an oxidative deamination of l‐α‐amino acids by an l‐α‐amino acid deaminase from Cosenzaea myxofaciens, rendering 2‐oxoacid intermediates, with an ensuing aldol addition reaction to formaldehyde, catalyzed by metal‐dependent (R)‐ or (S)‐selective carboligases namely 2‐oxo‐3‐deoxy‐l‐rhamnonate aldolase (YfaU) and ketopantoate hydroxymethyltransferase (KPHMT), respectively, furnishing 3‐substituted 4‐hydroxy‐2‐oxoacids. The overall substrate conversion was optimized by balancing biocatalyst loading and amino acid and formaldehyde concentrations, yielding 36–98% aldol adduct formation and 91–98% ee for each enantiomer. Subsequent in situ follow‐up chemistry via hydrogen peroxide‐driven oxidative decarboxylation afforded the corresponding 2‐substituted 3‐hydroxycarboxylic acid derivatives., This project has received funding from Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación, Fondo Europeo de Desarrollo Regional (FEDER) (grants RTI2018‐094637‐B‐I00) and Programación Conjunta Internacional PCI2018‐092937 under the ERACoBioTech (European Union's Horizon 2020 research and innovation programme under grant agreement No [722361]. This research was funded in part by the Austrian Science Fund (FWF) [J 4227‐B21]. M. P. acknowledges funding by the Austrian FWF through an Erwin Schrödinger fellowship.. The PmaLAAD expression plasmid was a generous gift from Prof. Wolfgang Kroutil (University of Graz)., Peer reviewed

Three enzymatic routes toward γ-hydroxy-α-amino acids by tandem aldol addition–transamination one-pot two-step reactions are reported. The approaches feature an enantioselective aldol addition of pyruvate to various nonaromatic aldehydes catalyzed by trans-o-hydroxybenzylidene pyruvate hydratase-aldolase (HBPA) from Pseudomonas putida. This affords chiral 4-hydroxy-2-oxo acids, which were subsequently enantioselectively aminated using S-selective transaminases. Three transamination processes were investigated involving different amine donors and transaminases: (i) l-Ala as an amine donor with pyruvate recycling, (ii) a benzylamine donor using benzaldehyde lyase from Pseudomonas fluorescens Biovar I (BAL) to transform the benzaldehyde formed into benzoin, minimizing equilibrium limitations, and (iii) l-Glu as an amine donor with a double cascade comprising branched-chain α-amino acid aminotransferase (BCAT) and aspartate amino transferase (AspAT), both from E. coli, using l-Asp as a substrate to regenerate l-Glu. The γ-hydroxy-α-amino acids thus obtained were transformed into chiral α-amino-γ-butyrolactones, structural motifs found in many biologically active compounds and valuable intermediates for the synthesis of pharmaceutical agents., This project has received funding from the Ministerio de Ciencia e Innovación (MICIN), cofinanced by the Fondo Europeo de Desarrollo Regional (FEDER) (grants RTI2018-094637-B-I00 to P.C. and PGC2018-095808-B-I00 to T.P.), and Programación Conjunta Internacional (PCI2018-092937), through the EU initiative ERA CoBioTech under grant agreement No [722361] to P.C and S.J.Ch.) (Tralaminol)., Peer reviewed

During industrial processes, yeasts are exposed to harsh conditions, which eventually lead to adaptation of the strains. In the laboratory, it is possible to use experimental evolution to link the evolutionary biology response to these adaptation pressures for the industrial improvement of a specific yeast strain. In this work, we aimed to study the adaptation of a wine industrial yeast in stress conditions of the high ethanol concentrations present in stopped fermentations and secondary fermentations in the processes of champagne production. We used a commercial Saccharomyces cerevisiae × S. uvarum hybrid and assessed its adaptation in a modified synthetic must (M-SM) containing high ethanol, which also contained metabisulfite, a preservative that is used during wine fermentation as it converts to sulfite. After the adaptation process under these selected stressful environmental conditions, the tolerance of the adapted strain (H14A7-etoh) to sulfite and ethanol was investigated, revealing that the adapted hybrid is more resistant to sulfite compared to the original H14A7 strain, whereas ethanol tolerance improvement was slight. However, a trade-off in the adapted hybrid was found, as it had a lower capacity to ferment glucose and fructose in comparison with H14A7. Hybrid genomes are almost always unstable, and different signals of adaptation on H14A7-etoh genome were detected. Each subgenome present in the adapted strain had adapted differently. Chromosome aneuploidies were present in S. cerevisiae chromosome III and in S. uvarum chromosome VII–XVI, which had been duplicated. Moreover, S. uvarum chromosome I was not present in H14A7-etoh and a loss of heterozygosity (LOH) event arose on S. cerevisiae chromosome I. RNA-sequencing analysis showed differential gene expression between H14A7-etoh and H14A7, which can be easily correlated with the signals of adaptation that were found on the H14A7-etoh genome. Finally, we report alterations in the lipid composition of the membrane, consistent with conserved tolerance mechanisms., M.L.-P. was supported by a FPU contract from Ministerio de Ciencia, Innovación y Universidades (ref. FPU15/01775). This work was supported by projects ERA CoBioTech H2020 MeMBrane (Grant agreement: 722361) to A.Q. and A.G. and PCI2018-093190 (AEI/FEDER, U.E.) and PROMETEO/2020/014 Project from the Generalitat Valenciana to A.Q. and BBSRC (BB/R02152X/1) to A.G. We thank the Genomics Unit of the Central Service for Experimental Research (SCSIE), University of Valencia, for their genome sequencing support., Peer reviewed

Fungal antifungal proteins (AFPs) have attracted attention as novel biofungicides. Their exploitation requires safe and cost-effective producing biofactories. Previously, Penicillium chrysogenum and Penicillium digitatum produced recombinant AFPs with the use of a P. chrysogenum-based expression system that consisted of the paf gene promoter, signal peptide (SP)-pro sequence and terminator. Here, the regulatory elements of the afpA gene encoding the highly produced PeAfpA from Penicillium expansum were developed as an expression system for AFP production through the FungalBraid platform. The afpA cassette was tested to produce PeAfpA and P. digitatum PdAfpB in P. chrysogenum and P. digitatum, and its efficiency was compared to that of the paf cassette. Recombinant PeAfpA production was only achieved using the afpA cassette, being P. chrysogenum a more efficient biofactory than P. digitatum. Conversely, P. chrysogenum only produced PdAfpB under the control of the paf cassette. In P. digitatum, both expression systems allowed PdAfpB production, with the paf cassette resulting in higher protein yields. Interestingly, these results did not correlate with the performance of both promoters in a luciferase reporter system. In conclusion, AFP production is a complex outcome that depends on the regulatory sequences driving afp expression, the fungal biofactory and the AFP sequence., This work was supported by grant RTI2018-101115B-C21 funded by MCIN/AEI/ 10.13039/501100011033 and by “ERDF A way of making Europe”, PROMETEO/2018/066 from ‘Conselleria d’Educació’ (Generalitat Valenciana, Comunitat Valenciana, Spain) and by grant PCI2018-092893 funded by MCIN/AEI/ 10.13039/501100011033 and by “The European Union” (SUSPHIRE Project, ERACoBioTech; Horizon 2020 grant agreement No. 722361). EM and MGL were recipients of predoctoral grants FPU18/02019 and FPU19/02066 funded by MCIN/AEI/ 10.13039/501100011033 and by “ESF Investing in your future”., Peer reviewed

One of the most prominent consequences of global climate warming for the wine industry is a clear increase of the sugar content in grapes, and thus the alcohol level in wines. Among the several approaches to address this important issue, this review focuses on biotechnological solu-tions, mostly relying on the selection and improvement of wine yeast strains for reduced ethanol yields. Other possibilities are also presented. Researchers are resorting to both S. cerevisiae and alternative wine yeast species for the lowering of alcohol yields. In addition to the use of selected strains under more or less standard fermentation conditions, aerobic fermentation is increasingly being explored for this purpose. Genetic improvement is also playing a role in the development of biotechnological tools to counter the increase in the wine alcohol levels. The use of recombinant wine yeasts is restricted to research, but its contribution to the advancement of the field is still rele-vant. Furthermore, genetic improvement by non‐GMO approaches is providing some interesting results, and will probably result in the development of commercial yeast strains with a lower alcohol yield in the near future. The optimization of fermentation processes using natural isolates is, anyway, the most probable source of advancement in the short term for the production of wines with lower alcohol contents., The work of the Microwine group is currently funded by the Spanish Government through
grants PID2019-105159RB-I00 and PCI2018-092949 (co-funded by ERA-CoBioTech). JT is funded by
FGCSIC by the COMFUTURO program. AMG’s predoctoral contract is funded by Consejería de
Desarrollo Económico e Innovación de la Comunidad Autónoma de La Rioja.

Amino-polyols represent attractive chemical building blocks but can be challenging to synthesize because of the high density of asymmetric functionalities and the need for extensive protecting-group strategies. Here we present a three-component strategy for the stereoselective enzymatic synthesis of amino-diols and amino-polyols using a diverse set of prochiral aldehydes, hydroxy ketones, and amines as starting materials. We were able to combine biocatalytic aldol reactions, using variants of d-fructose-6-phosphate aldolase (FSA), with reductive aminations catalyzed by IRED-259, identified from a metagenomic library. A two-step process, without the need for intermediate isolation, was developed to avoid cross-reactivity of the carbonyl components. Stereoselective formation of the 2R,3R,4R enantiomers of amino-polyols was observed and confirmed by X-ray crystallography., We kindly acknowledge financial support from the European Research Council (788231-ProgrES-ERC-2017-ADG to S.L.F.; BIO-H-BORROW to N.J.T.). J.R.M. acknowledges the Industrial Biotechnology Innovation Centre (IBioIC) and BBSRC funding through a CASE studentship with Prozomix Ltd. P.C. acknowledges financial support from the project RTI2018-094637-BI00 funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe” and project PCI2018-092937 MCIN/AEI/10.13039/501100011033 cofunded by the UE under the ERACoBioTech Tralaminol (European Union’s Horizon 2020 research and innovation programme grant agreement no. 722361)., Peer reviewed

Analysis of pheromone content in T2 generation of SPv1.0 plants., European Commission: ERA CoBioTech - Cofund on Biotechnologies (722361)., Peer reviewed

The purpose of this assay is to define the volatilome of SxPv1.0, v1.2 and WT Nicotiana benthamiana plant leaves, focusing on the differences between them, by GC-MS., European Commission: ERA CoBioTech - Cofund on Biotechnologies (722361)., Peer reviewed

Analysis of pheromone content in T1 generation of SPv1.0 plants., European Commission: ERA CoBioTech - Cofund on Biotechnologies (722361)., Peer reviewed

The purpose of this study is to screen the population of T2 transgenic generation of SxPv1.2, to analyze the production of moth sex pheromones in these plants and be able to choose from them the most interesting ones in terms of production for further RNASeq analysis., European Commission: ERA CoBioTech - Cofund on Biotechnologies (722361)., Peer reviewed

The effect of EaDAct in pheromone production was studied by agroinfiltrating in Nicotiana benthamiana leaves the first two enzymes (AtrD11 and HarFAR) with or without the last enzyme EaDAct. P19 was also agroinfiltrated as negative control., European Commission: ERA CoBioTech - Cofund on Biotechnologies (722361)., Peer reviewed

Analysis of pheromone content in T3 generation of SPv1.0 plants, European Commission: ERA CoBioTech - Cofund on Biotechnologies (722361)., Peer reviewed

Stable transformation of Nicotiana benthamiana plants with the three enzymes for moth pheromone production (AtrD11, HarFAR, EaDAct) expressed constitutively., European Commission: ERA CoBioTech - Cofund on Biotechnologies (722361)., Peer reviewed

The constructs for SxPv1.1 and SxPv1.2 were tested transiently in Nicotiana benthamiana leaves. Pheromone content was analysed via GC-MS., European Commission: ERA CoBioTech - Cofund on Biotechnologies (722361)., Peer reviewed

The purpose of this assay is to test the efficiency of conversion of Z11-16OH to Z11-16OAc, in the search of a more efficient alternative to the already used EaDact acetyltransferase gene, chosen for the generation of the SxPv10 and SxPv12. The expression is assayed in a transient expression assay on WT Nicotiana benthamiana plants, agroinfiltrated with the combination of the two previous genes required in the metabolic pathway of moth sex pheromones plus the assayed acetyltransferase gene. Samples were finally measured by GC-MS., European Commission: ERA CoBioTech - Cofund on Biotechnologies (722361)., Peer reviewed

Although optical pure amino alcohols are in high demand due to their widespread applicability, they still remain challenging to synthesize, since commonly elaborated protection strategies are required. Here, a multi-enzymatic methodology is presented that circumvents this obstacle furnishing enantioenriched 1,3-amino alcohols out of commodity chemicals. A Type I aldolase forged the carbon backbone with an enantioenriched aldol motif, which was subsequently subjected to enzymatic transamination. A panel of 194 TAs was tested on diverse nine aldol products prepared through different nucleophiles and electrophiles. Due to the availability of (R)- and (S)-selective TAs, both diastereomers of the 1,3-amino alcohol motif were accessible. A two-step process enabled the synthesis of the desired amino alcohols with up to three chiral centers with de up to >97 in the final products. (Figure presented.)., This research was funded in part by the Austrian Science Fund (FWF) [J 4242-B21]. M. P. acknowledges funding by the Austrian FWF through an Erwin Schrödinger fellowship and by grants PID2021-122166OB−I00 and PCI2018-092937 funded by MCIN/AEI/10.13039/501100011033, and by “ERDF A way of making Europe” and under the ERACoBioTech (European Union's Horizon 2020 research and innovation programme under grant agreement No [722361]. The authors wish to thank Goran Djordjic for technical assistance and Klaus Zangger for NMR measurements and valuable discussions. The University of Graz and BioHealth Graz are acknowledged for financial support., Peer reviewed