Biology subjects

Caro-Astorga, J.

Publications and source records attributed to Caro-Astorga, J..

5 recordsLinked to original sources

bioRxiv2025

Continuous evolution of a halogenase enzyme with improved solubility and activity for sustainable bioproduction

Halogenation enhances the stability and function of pharmaceuticals, biomaterials, and industrial compounds. However, chemical halogenation lacks stereoselectivity and requires the use of toxic or expensive chemicals. Although enzymatic halogenation can improve selectivity and reduce environmental impact, current halogenases are inefficient and insoluble, leading to low yields that limit their applications. Here, we develop RebHEvo4, a soluble and highly active tryptophan halogenase, containing 12 mutations that confer 37-fold and 44-fold increases in 7-chloro and 7-bromotryptophan production respectively, in vivo. To create RebHEvo4, we devised an aminoacyl tRNA synthetase based halogenase biosensor and conducted over 500 hours of phage-assisted continuous evolution (PACE). Use of RebHEvo4 in a bioreactor resulted in the production of 2.7 g/L of halogenated tryptophan. When coupled with a downstream enzyme, RebHEvo4 allowed 36-fold increased yields of halogenated tryptamines compared to the wild-type enzyme. Additionally, RebHEvo4 enabled efficient production of genetically encoded antimicrobial halogenated peptides. The efficient, site-specific halogenation by our evolved halogenase will accelerate sustainable biomanufacturing of halogenated drugs.

biochemistry↗

bioRxiv2024

CapP mediates the structural formation of biofilm-specific pili in the opportunistic human pathogen Bacillus cereus

Polymeric proteinaceous filaments are structural scaffolds that diversify the functionality of the bacterial extracellular matrix. Here, we report a previously uncharacterized bacterial factor called bc1280 that is exclusive to B. cereus group and indispensable for the establishment of a biofilm lifestyle. We propose that BC1280 is an essential chaperone for the assembly of the filamentous platform that tightly controls the polymerization of heteropili containing CalY and TasA as major subunits in a concentration-dependent manner. Additionally, BC1280 modulates the expression of EPS via an uncharacterized pathway that is activated by a protease and an ECF-type sigma factor. The pilus biogenesis system described in this work highlights the complexity of extracellular matrix assembly in B. cereus and introduces a singular three-part structuration mechanism during biofilm formation and maturation. Graphical abstractDuring the process of biofilm formation in B. cereus, TasA, CapP, and CalY serve as the main components, in addition to other factors, ensuring that the extracellular matrix correctly assembles. The three proteins are produced and processed by the signal peptidase SipW and subsequently secreted through the Sec pathway in an unfolded conformation. At early biofilm formation stages, CapP is initially present at low levels, associates with the cell wall and is secreted into the ECM where it interacts with unfolded subunits of CalY, facilitating CalY folding and initiating fibril growth. CalY serves as a nucleator for incorporating TasA subunits into the pilus, forming TasA-CalY heteropolymers. Once the ECM scaffold is established, CapP levels increase, forming stable complexes with CalY that prevent its folding and arrest filament growth. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/582368v2_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@df9d9dorg.highwire.dtl.DTLVardef@e4b712org.highwire.dtl.DTLVardef@1af2421org.highwire.dtl.DTLVardef@16d02c7_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2021

Komagataeibacter tool kit (KTK): a modular cloning system for multigene constructs and programmed protein secretion from cellulose producing bacteria

Bacteria proficient at producing cellulose are an attractive synthetic biology host for the emerging field of Engineered Living Materials (ELMs). Species from the Komagataeibacter genus produce high yields of pure cellulose materials in a short time with minimal resources, and pioneering work has shown that genetic engineering in these strains is possible and can be used to modify the material and its production. To accelerate synthetic biology progress in these bacteria, we introduce here the Komagataeibacter tool kit (KTK), a standardised modular cloning system based on Golden Gate DNA assembly that allows DNA parts to be combined to build complex multigene constructs expressed in bacteria from plasmids. Working in Komagataeibacter rhaeticus, we describe basic parts for this system, including promoters, fusion tags and reporter proteins, before showcasing how the assembly system enables more complex designs. Specifically, we use KTK cloning to reformat the Escherichia coli curli amyloid fibre system for functional expression in K. rhaeticus, and go on to modify it as a system for programming protein secretion from the cellulose producing bacteria. With this toolkit, we aim to accelerate modular synthetic biology in these bacteria, and enable more rapid progress in the emerging ELMs community.

synthetic biology↗

bioRxiv2020

Bacterial cellulose spheroids as building blocks for 2D and 3D engineered living materials

Engineered living materials (ELMs) based on bacterial cellulose (BC) offer a promising avenue for cheap-to-produce materials that can be programmed with genetically encoded functionalities. Here we explore how ELMs can be fabricated from millimetre-scale balls of cellulose occasionally produced by Acetobacteriacea species, which we call BC spheroids. We define a reproducible protocol to produce BC spheroids and demonstrate their potential for use as building blocks to grow ELMs in 2D and 3D shapes. These BC spheroids can be genetically functionalized and used as the method to make and grow patterned BC-based ELMs to design. We further demonstrate the use of BC spheroids for the repair and regeneration of BC materials, and measure the survival of the BC-producing bacteria in the material over time. This work forwards our understanding of BC spheroid formation and showcases their potential for creating and repairing engineered living materials.

bioengineering↗

bioRxiv2019

Biofilm formation displays intrinsic offensive and defensive features of Bacillus cereus

BackgroundBiofilm formation is a strategy of many bacterial species to adapt to a variety of stresses and has become a part of infections, contaminations or beneficial interactions. We previously observed that B. cereus ATCC 14579 (CECT148), formed a thick biomass of cells firmly adhered to abiotic surfaces.\n\nResultsIn this study, we combined two techniques, RNAseq and iTRAQ mass spectrometry, to demonstrate the profound physiological changes that permit Bacillus cereus to switch from a floating to a sessile lifestyle, to undergo further maturation of the biofilm, and to differentiate into offensive or defensive populations. The rearrangement of nucleotides, sugars, amino acids and energy metabolism lead to changes promoting reinforcement of the cell wall, activation of ROS detoxification strategies or secondary metabolite production, all oriented to defend biofilm cells from external aggressions. However, floating cells maintain a fermentative metabolic status along with a higher aggressiveness against hosts, evidenced by the production of toxins and other virulent factors.\n\nConclusionsWe show that biofilm-associated cells seem to direct the energy to the individual and global defense against external aggressions and competitors. By contrary, floating cells are more aggressive against hosts. The maintenance of the two distinct subpopulations is an effective strategy to face changeable environmental conditions found in the life cycle of B. cereus.

microbiology↗