Aliivibrio fischeri L-Asparaginase production by engineered Bacillus subtilis: a potential new biopharmaceutical.

L-Asparaginase (L-ASNase) is an enzyme applied in the treatment of lymphoid malignancies. However, an innovative L-ASNase with high yield and lower side effects than the commercially available preparations are still a market requirement. Here, a new-engineered Bacillus subtilis strain was evaluated for Aliivibrio fischeri L-ASNase II production, being the bioprocess development and the enzyme characterization studied. The pBS0E plasmid replicative in Bacillus sp and containing PxylA promoter inducible by xylose and its repressive molecule sequence (XylR) was used for the genetic modification. Initially, cultivations were carried out in orbital shaker, and then the process was scaled up to stirred tank bioreactor (STB). After the bioprocess, the cells were recovered and submitted to ultrasound sonication for cells disruption and intracellular enzyme recovery. The enzymatic extract was characterized to assess its biochemical, kinetic and thermal properties using L-Asparagine and L-Glutamine as substrates. The results indicated the potential enzyme production in STB achieving L-ASNase activity up to 1.539 U mL-1. The enzymatic extract showed an optimum pH of 7.5, high L-Asparagine affinity (Km = 1.2275 mmol L-1) and low L-Glutaminase activity (0.568-0.738 U mL-1). In addition, thermal inactivation was analyzed by two different Kinect models to elucidate inactivation mechanisms, low kinetic thermal inactivation constants for 25 ºC and 37 ºC (0.128 and 0.148 h-1, respectively) indicate an elevated stability. The findings herein show that the produced recombinant L-ASNase has potential to be applied for pharmaceutical purposes.

Expanding the Functionality of an Autoinduction Device for Repression of Gene Expression in Bacillus subtilis.

Autonomous control of gene expression through engineered quorum-sensing processes is broadly applicable to biosynthetic pathways, including simultaneous control of different genes. It is also a powerful tool for balancing growth and production. We had previously engineered a modular autoinduction device for the control of gene expression in B. subtilis. Now, we expand its functionality to repress gene expression autonomously. The engineered R8 promoter responds to AHL accumulation in the culture medium. In a riboflavin-producing strain, the AHL-Lux complex exerts 5-fold repression on the R8-driven expression of the flavokinase/FAD synthetase gene ribC, resulting in a higher titer of the vitamin. We engineered a strain able to autonomously induce and repress different genes simultaneously, demonstrating the potential of the device for use in metabolic engineering.

L-asparaginase production review: bioprocess design and biochemical characteristics.

In the past decades, the production of biopharmaceuticals has gained high interest due to its great sensitivity, specificity, and lower risk of negative effects to patients. Biopharmaceuticals are mostly therapeutic recombinant proteins produced through biotechnological processes. In this context, L-asparaginase (L-asparagine amidohydrolase, L-ASNase (E.C. 3.5.1.1)) is a therapeutic enzyme that has been abundantly studied by researchers due to its antineoplastic properties. As a biopharmaceutical, L-ASNase has been used in the treatment of acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), and other lymphoid malignancies, in combination with other drugs. Besides its application as a biopharmaceutical, this enzyme is widely used in food processing industries as an acrylamide mitigation agent and as a biosensor for the detection of L-asparagine in physiological fluids at nano-levels. The great demand for L-ASNase is supplied by recombinant enzymes from Escherichia coli and Erwinia chrysanthemi. However, production processes are associated to low yields and proteins associated to immunogenicity problems, which leads to the search for a better enzyme source. Considering the L-ASNase pharmacological and food importance, this review provides an overview of the current biotechnological developments in L-ASNase production and biochemical characterization aiming to improve the knowledge about its production. KEY POINTS: • Microbial enzyme applications as biopharmaceutical and in food industry • Biosynthesis process: from the microorganism to bioreactor technology • Enzyme activity and kinetic properties: crucial for the final application.

Signal Amplification for Cell-Free Biosensors, an Analog-to-Digital Converter.

Toehold switches are biosensors useful for the detection of endogenous and environmental RNAs. They have been successfully engineered to detect virus RNAs in cell-free gene expression reactions. Their inherent sequence programmability makes engineering a fast and predictable process. Despite improvements in the design, toehold switches suffer from leaky translation in the OFF state, which compromises the fold change and sensitivity of the biosensor. To address this, we constructed and tested signal amplification circuits for three toehold switches triggered by Dengue and SARS-CoV-2 RNAs and an artificial RNA. The serine integrase circuit efficiently contained leakage, boosted the expression fold change from OFF to ON, and decreased the detection limit of the switches by 3-4 orders of magnitude. Ultimately, the integrase circuit converted the analog switches' signals into digital-like output. The circuit is broadly useful for biosensors and eliminates the hard work of designing and testing multiple switches to find the best possible performer.

Preventing Production Escape Using an Engineered Glucose-Inducible Genetic Circuit.

A proper balance of metabolic pathways is crucial for engineering microbial strains that can efficiently produce biochemicals on an industrial scale while maintaining cell fitness. High production loads can negatively impact cell fitness and hinder industrial-scale production. To address this, fine-tuning gene expression using engineered promoters and genetic circuits can promote control over multiple targets in pathways and reduce the burden. We took advantage of the robust carbon catabolite repression system of Bacillus subtilis to engineer a glucose-inducible genetic circuit that supports growth and production. The circuit is resilient, enabling a quick switch in the production status when exposed to the correct carbon source. By performing serial cultivations for 61 generations under repressive conditions, we preserved the production capacity of the cells, which could be fully accessed by switching to glucose in the next cultivation step. Switching to glucose after 61 generations resulted in 34-fold activation and generated 70% higher production in comparison to standard cultivation in glucose. Conversely, serial cultivation under permanent induction resulted in 62% production loss after 67 generations alongside an increase in the culture growth rate. As a pathway-independent circuit activated by the preferred carbon source, our engineered glucose-inducible genetic circuit is broadly useful and imposes no additional cost to traditional production processes.

RibM from Streptomyces davawensis is a riboflavin/roseoflavin transporter and may be useful for the optimization of riboflavin production strains.

BACKGROUND: The bacterium Bacillus subtilis, which is not a natural riboflavin overproducer, has been converted into an excellent production strain by classical mutagenesis and metabolic engineering. To our knowledge, the enhancement of riboflavin excretion from the cytoplasm of overproducing cells has not yet been considered as a target for (further) strain improvement. Here we evaluate the flavin transporter RibM from Streptomyces davawensis with respect to improvement of a riboflavin production strain. RESULTS: The gene ribM from S. davawensis, coding for a putative facilitator of riboflavin uptake, was codon optimized (ribMopt) for expression in B. subtilis. The gene ribMopt was functionally introduced into B. subtilis using the isopropyl-ß-thiogalactopyranoside (IPTG)-inducible expression plasmid pHT01: Northern-blot analysis of total RNA from IPTG treated recombinant B. subtilis cells revealed a ribMopt specific transcript. Western blot analysis showed that the his6-tagged heterologous gene product RibM was present in the cytoplasmic membrane. Expression of ribM in Escherichia coli increased [14C]riboflavin uptake, which was not affected by the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Expression of ribMopt supported growth of a B. subtilis ΔribB::Ermr ΔribU::Kanr double mutant deficient in riboflavin synthesis (ΔribB) and also deficient with respect to riboflavin uptake (ΔribU). Expression of ribMopt increased roseoflavin (a toxic riboflavin analog produced by S. davawensis) sensitivity of a B. subtilis ΔribU::Kanr strain. Riboflavin synthesis by a model riboflavin B. subtilis production strain overproducing RibM was increased significantly depending on the amount of the inducer IPTG. CONCLUSIONS: The energy independent flavin facilitator RibM could in principle catalyze riboflavin export and thus may be useful to increase the riboflavin yield in a riboflavin production process using a recombinant RibM overproducing B. subtilis strain (or any other microorganism).

Riboswitch theo/metE as a Transcription Regulation Tool for Xanthomonas citri subsp. citri.

: Xanthomonas citri subsp. citri (X. citri) is the causal agent of Asiatic Citrus Canker (ACC), a disease that affects citrus. ACC has no cure, and growers must rely on special agricultural practices to prevent bacterial spreading. Understanding X. citri basic biology is essential to foresee potential genetic targets to control ACC. Traditionally, microbial genetics use gene deletion/disruption to investigate gene function. However, essential genes are difficult to study this way. Techniques based on small-RNAs and antisense-RNAs are powerful for gene characterization, but not yet fully explored in prokaryotes. One alternative is riboswitches, which derive from bacteria, and can control transcription/translation. Riboswitches are non-coding RNAs able to modulate gene expression in the presence of specific ligands. Here we demonstrate that the riboswitch theo/metE decreases parB expression in X. citri in a platform responsive to theophylline. By monitoring cell respiration, we showed that higher concentrations of the ligand interfered with bacterial viability. Therefore, we determined the safe dose of theophylline to be used with X. citri. Finally, in downstream investigations of parB transcription modulation, we show evidence for the fact that ParB is stable, remains functional throughout the cell cycle, and is inherited by the daughter cells upon cell division.

Engineering Microbial Living Therapeutics: The Synthetic Biology Toolbox.

Microbes can be engineered to act like living therapeutics designed to perform specific actions in the human body. From fighting and preventing infections to eliminating tumors and treating metabolic disorders, engineered living systems are the next generation of therapeutics. In recent years, synthetic biologists have greatly expanded the genetic toolbox for microbial living therapeutics, adding sensors, regulators, memory circuits, delivery devices, and kill switches. These advances have paved the way for successful engineering of fully functional living therapeutics, with sensing, production, and biocontainment devices. However, some important tools are still missing from the box. In this review, we cover the most recent biological parts and approaches developed and describe the missing tools needed to build robust living therapeutics.

The antibiotics roseoflavin and 8-demethyl-8-amino-riboflavin from Streptomyces davawensis are metabolized by human flavokinase and human FAD synthetase.

The non-pathogenic Gram-positive soil bacterium Streptomyces davawensis synthesizes the riboflavin (vitamin B(2)) analogs roseoflavin (RoF) and 8-demethyl-8-amino-riboflavin (AF). Both compounds are antibiotics. Notably, a number of other riboflavin analogs are currently under investigation with regard to the development of novel antiinfectives. As a first step towards understanding the metabolism of riboflavin analogs in humans, the key enzymes flavokinase (EC 2.7.1.26) and FAD synthetase (EC 2.7.7.2) were studied. Human flavokinase efficiently converted RoF and AF to roseoflavin mononucleotide (RoFMN) and 8-demethyl-8-amino-riboflavin mononucleotide (AFMN), respectively. Human FAD synthetase accepted RoFMN but not AFMN as a substrate. Consequently, roseoflavin adenine dinucleotide (RoFAD) was synthesized by the latter enzyme but not 8-demethyl-8-amino-riboflavin adenine dinucleotide (AFAD). The cofactor analogs RoFMN, AFMN and RoFAD have different physicochemical properties as compared to FMN and FAD. Thus, the cofactor analogs have the potential to render flavoenzymes inactive, which may negatively affect human metabolism. RoF, but not AF, was found to inhibit human flavokinase. In summary, we suggest that AF has a lower toxic potential and may be better suited as a lead structure to develop antimicrobial compounds.