Florentino, L. H. | Search bioRxiv

bioRxiv2025

Cell-Free production of soybean leghemoglobins and non-symbiotic hemoglobin

Hemoglobins are heme proteins and are present in some microorganisms, higher plants and mammals. In legume nodules there are two types: leghemoglobin (LegH) or symbiotic and non-symbiotic (nsHb). LegHs are present in high amounts at legumes roots and are responsible together with bacteroides for the nitrogen fixation process. Non-symbiotic hemoglobins Class 1 protein have very high affinity for O2 and are found in monocotyledons and legumes. LegH has aroused great interest in the vegetable meat industry due to its organoleptic and nutritional properties. Here, we demonstrated that soybean LegH A, C1, C2, C3 and nsHb are produced by E. coli-based cell-free protein synthesis (CFPS) and correctly synthesized in its amino acids sequence. In addition, it was also possible to reproduce some post-translational modifications confirmed by LC/MS analysis. All LegHs produced in this system showed peroxidase activity and heme binding correlated with its concentration in the assays. Furthermore, all proteins were readily digested by pepsin within 1 minute in analog digestion conditions. Therefore, LegHs and nsHb proteins were synthesized using cell-free systems (CFSs), maintaining their functionality and being digestible. These findings suggest that they could serve as viable alternative food additives for plant-based meat. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/643390v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@504374org.highwire.dtl.DTLVardef@17c8d73org.highwire.dtl.DTLVardef@2aad12org.highwire.dtl.DTLVardef@1c8a3e9_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Development of Int-Plex@ binary memory switch system: plant genome modulation driven by large serine-integrases.

The comprehension of virus-host interactions has allowed numerous advances in developing biotechnological methodologies for plant genome editions, constituting a promising path for plant genetic engineering. Among these advancements, phage- encoded large serine-integrases have emerged as noteworthy tools to modulate plant metabolic pathways by inserting, excising, or inverting DNA stretches in a reversible and specific way. The present work shows the foundation of the Int-Plex@ (INTegrase PLant EXpression) binary memory switch system, which consists of the application of four distinct orthogonal prophage large serine-integrases (Int) (BxB1, phiC31, Int13, and Int9) as an input trigger mechanism for the inversion or excision of genomic DNA. The memory genetic switch is divided into the excision module and the inversion module. The excision module is activated by BxB1 or phiC31 enzymes (input). In this case, the DNA sequence flanked by its attachment sites is excised from the genome (output). The inversion module is activated by Int9 or Int13 (input). The inverted mgf gene sequence is flipped to its functional coding sequence, and the switch output is mGFP. Moreover, prokaryotic-based cell-free in vitro transcription-translation reactions (TxTl) were used as a fast platform for testing Ints attB/P in tandem site activity. Furthermore different plasmid delivery strategies for plant cell Int heterologous expression were tested: leaf tissue agroinfiltration of Agrobacterium tumefaciens transformed with binary plasmids and a biolistic system. After each treatment, the edited genomic DNA sequences were amplified and verified by Sanger and Nanopore sequencing. Despite the challenges of using Ints, the potential benefits are significant and deserve deeper exploration and development. The Int-Plex@ binary genome memory switch system can be applied to produce genetic circuits combined with omics tools and sgRNAs to engineer and modulate plant metabolic pathways temporally and reversibly.

synthetic biology↗