Biology subjects

Garcia-Garcia, J. D.

Publications and source records attributed to Garcia-Garcia, J. D..

6 recordsLinked to original sources

bioRxiv2022

Directed evolution of aerotolerance in sulfide-dependent thiazole synthases

Sulfide-dependent THI4 thiazole synthases could potentially be used to replace plant cysteine-dependent suicide THI4s, whose high protein turnover rates make thiamin synthesis exceptionally energy-expensive. However, sulfide-dependent THI4s are anaerobic or microoxic enzymes and hence unadapted to the aerobic conditions in plants; they are also slow enzymes (kcat <1 h-1). To improve aerotolerance and activity, we applied continuous directed evolution under aerobic conditions in the yeast OrthoRep system to two sulfide-dependent bacterial THI4s. Six beneficial single mutations were identified, of which five lie in the active-site cleft predicted by structural modeling and two recapitulate features of naturally aerotolerant THI4s. That single mutations gave substantial improvements suggests that further advance under selection will be possible by stacking mutations. This proof-of-concept study established that the performance of sulfide-dependent THI4s in aerobic conditions is evolvable and, more generally, that yeast OrthoRep provides a plant-like bridge to adapt nonplant enzymes to work better in plants.

synthetic biology↗

bioRxiv2021

Using Continuous Directed Evolution to Improve Enzymes for Plant Applications

Continuous directed evolution of enzymes and other proteins in microbial hosts is capable of outperforming classical directed evolution by executing hypermutation and selection concurrently in vivo, at scale, with minimal manual input. Provided that a target enzymes activity can be coupled to growth of the host cells, the activity can be improved simply by selecting for growth. Like all directed evolution, the continuous version requires no prior mechanistic knowledge of the target. Continuous directed evolution is thus a powerful new way to modify plant or non-plant enzymes for use in plant metabolic research and engineering. Here, we first describe the basic features of the Saccharomyces cerevisiae OrthoRep system for continuous directed evolution and compare it briefly with other systems. We then give a step-by-step account of three ways in which OrthoRep can be deployed to evolve primary metabolic enzymes, using a THI4 thiazole synthase as an example and illustrating the mutational outcomes obtained. We close by outlining applications of OrthoRep that serve growing demands (i) to change the characteristics of plant enzymes destined for return to plants, and (ii) to adapt ( plantize) enzymes from prokaryotes - especially exotic prokaryotes - to function well in mild, plant-like conditions. One-sentence summaryContinuous directed evolution using the yeast OrthoRep system is a powerful new way to improve enzymes for use in plant engineering as illustrated by plantizing a bacterial thiamin synthesis enzyme..

synthetic biology↗

bioRxiv2021

Structure and function of aerotolerant, multiple-turnover THI4 thiazole synthases

Plant and fungal THI4 thiazole synthases produce the thiamin thiazole moiety in aerobic conditions via a single-turnover suicide reaction that uses an active-site Cys residue as sulfur donor. Multipleturnover (i.e. catalytic) THI4s lacking an active-site Cys (non-Cys THI4s) that use sulfide as sulfur donor have been characterized - but only from archaeal methanogens that are anaerobic, O2-sensitive hyperthermophiles from sulfide-rich habitats. These THI4s prefer iron as cofactor. A survey of prokaryote genomes uncovered non-Cys THI4s in aerobic mesophiles from sulfide-poor habitats, suggesting that multiple-turnover THI4 operation is possible in aerobic, mild, low-sulfide conditions. This was confirmed by testing 23 representative non-Cys THI4s for complementation of an Escherichia coli {Delta}thiG thiazole auxotroph in aerobic conditions. Sixteen were clearly active, and more so when intracellular sulfide level was raised by supplying Cys, demonstrating catalytic function in the presence of O2 at mild temperatures and indicating use of sulfide or a sulfide metabolite as sulfur donor. Comparative genomic evidence linked non-Cys THI4s with proteins from families that bind, transport, or metabolize cobalt or other heavy metals. The crystal structure of the aerotolerant bacterial Thermovibrio ammonificans THI4 was determined to probe the molecular basis of aerotolerance. The structure suggested no large deviations compared to the structures of THI4s from O2-sensitive methanogens, but is consistent with an alternative catalytic metal. Together with complementation data, the use of cobalt rather than iron was supported. We conclude that catalytic THI4s can indeed operate aerobically and that the metal cofactor inserted is a likely natural determinant of aerotolerance.

synthetic biology↗

bioRxiv2020

Potential For Applying Continuous Directed Evolution To Plant Enzymes

Plant evolution has produced enzymes that may not be optimal for maximizing yield and quality in todays agricultural environments and plant biotechnology applications. By improving enzyme performance, it should be possible to alleviate constraints on yield and quality currently imposed by kinetic properties or enzyme instability. Enzymes can be optimized faster than naturally possible by applying directed evolution, which entails mutating a target gene in vitro and screening or selecting the mutated gene products for the desired characteristics. Continuous directed evolution is a more efficient and scalable version that accomplishes the mutagenesis and selection steps simultaneously in vivo via error-prone replication of the target gene and coupling of the host cells growth rate to the target genes function. However, published continuous systems require custom plasmid assembly, and convenient multipurpose platforms are not available. We discuss two systems suitable for continuous directed evolution of enzymes, OrthoRep in Saccharomyces cerevisiae and EvolvR in Escherichia coli, and our pilot efforts to adapt each system for high-throughput plant enzyme engineering. To test our modified systems, we used the thiamin synthesis enzyme THI4, previously identified as a prime candidate for improvement. Our adapted OrthoRep system shows promise for efficient plant enzyme engineering.

synthetic biology↗

bioRxiv2020

Bioinformatic and experimental evidence for suicidal and catalytic plant THI4s

Like fungi and some prokaryotes, plants use a thiazole synthase (THI4) to make the thiazole precursor of thiamin. Fungal THI4s are suicide enzymes that destroy an essential active-site Cys residue to obtain the sulfur atom needed for thiazole formation. In contrast, certain prokaryotic THI4s have no active-site Cys, use sulfide as sulfur donor, and are truly catalytic. The presence of a conserved active-site Cys in plant THI4s and other indirect evidence implies that they are suicidal. To confirm this, we complemented the Arabidopsis tz-1 mutant, which lacks THI4 activity, with a His-tagged Arabidopsis THI4 construct. LC-MS analysis of tryptic peptides of the THI4 extracted from leaves showed that the active-site Cys was predominantly in desulfurated form, consistent with THI4 having a suicide mech-anism in planta. Unexpectedly, transcriptome datamining and deep proteome profiling showed that barley, wheat, and oat have both a widely expressed canonical THI4 with an active-site Cys, and a THI4-like paralog (non-Cys THI4) that has no active-site Cys and is the major type of THI4 in devel-oping grains. Transcriptomic evidence also indicated that barley, wheat, and oat grains synthesize thiamin de novo, implying that their non-Cys THI4s synthesize thiazole. Structure modeling supported this inference, as did demonstration that non-Cys THI4s have significant capacity to complement thia-zole auxotrophy in Escherichia coli. There is thus a prima facie case that non-Cys cereal THI4s, like their prokaryotic counterparts, are catalytic thiazole synthases. Bioenergetic calculations show that, relative to suicide THI4s, such enzymes could save substantial energy during the grain filling period. AbbreviationsDHAla, dehydroalanine; EST, expressed sequence tag; FDR, false discovery rate; IPTG, {beta}-D-1-thiogalactopyranoside

plant biology↗

bioRxiv2019

De novo β-alanine synthesis in α-proteobacteria involves a β-alanine synthase from the uracil degradation pathway

{beta}-alanine synthesis in bacteria occurs by the decarboxylation of L-aspartate as part of the pantothenate synthesis pathway. In the other two domains of life we find different pathways for {beta}-alanine formation, such as sources from spermine in plants, uracil in yeast and by transamination reactions in insects and mammals. There are also promiscuous decarboxylases that can decarboxylate aspartate. Several bioinformatics studies about the conservation of pantothenate synthesis pathway performed on bacteria, archaea and eukaryotes, have shown a partial conservation of the pathway. As a part of our work, we performed an analysis of the prevalence of reported {beta}-alanine synthesis pathways in 204 genomes of alpha-proteobacteria, with a focus on the Rhizobiales order. The aim of this work was to determine the enzyme or pathway used to synthetize {beta}-alanine in Rhizobium etli CFN42. Our bioinformatics analysis showed that this strain encodes the pyrimidine degradation pathway in its genome. We obtained a {beta}-alanine synthase (amaB) mutant that was a {beta}-alanine auxotroph. Complementation with the cloned gene restored the wild type phenotype. Biochemical analysis confirmed that the recombinant AmaB catalyzed the formation of {beta}-alanine from 3-Ureidopropionic acid in vitro. Here we show a different way in bacteria to produce this essential metabolite.\n\nImportanceSince the pioneer studies of Cronan (1980) on {beta}-alanine synthesis in E. coli, it has been assumed that the decarboxilation of aspartate by the L-aspartate--decarboxylase its the main enzymatic reaction for {beta}-alanine synthesis in bacteria. Forty years later, while we were studying the pantothenic acid synthesis in rhizobia, we demonstrate that a numerous and diverse group of bacteria classified as -proteobacteria synthesize {beta}-alanine de novo using {beta}-alanine synthase, the last enzyme from the reductive pathway for uracil degradation.\n\nAdditionally, there is a growing interest in {beta}-amino acid due to its remarkable pharmaceuticals properties as hypoglycemic, antiketogenic and anti-fungal agents.

microbiology↗