Optimization of Phycocyanobilin Synthesis in E. coli BL21: Biotechnological Insights and Challenges for Scalable Production.

Phycocyanobilin (PCB) is a small chromophore found in certain phycobiliproteins, such as phycocyanins (PCs) and allophycocyanins (APCs). PCB, along with other phycobilins (PBs) and intermediates such as biliverdin (BV) or phycoerythrobilin (PEB), is attracting increasing biotechnological interest due to its fluorescent and medicinal properties that allow potential applications in biomedicine and the food industry. This study aims to optimize PCB synthesis in Escherichia coli BL21 (DE3) and scale the process to a pre-industrial level. Parameters such as optimal temperature, inducer concentration, initial OD600, and stirring speed were analyzed in shake flask cultures to maximize PCB production. The best results were obtained at a temperature of 28 °C, an IPTG concentration of 0.1 mM, an initial OD600 of 0.5, and an orbital shaking speed of 260 rpm. Furthermore, the optimized protocol was scaled up into a 2 L bioreactor batch, achieving a maximum PCB concentration of 3.8 mg/L. Analysis of the results revealed that biosynthesis of exogenous PBs in Escherichia coli BL21 (DE3) is highly dependent on the metabolic burden of the host. Several scenarios, such as too rapid growth, high inducer concentration, or mechanical stress, can advance entry into the stationary phase. That progressively halts pigment synthesis, leading, in some cases, to its excretion into the growth media and ultimately triggering rapid degradation by the host. These conclusions provide a promising protocol for scalable PCB production and highlight the main biotechnological challenges to increase the yields of the process.

Deepening the knowledge of universal stress proteins in Haloferax mediterranei.

Haloarchaea, like many other microorganisms, have developed defense mechanisms such as universal stress proteins (USPs) to cope with environmental stresses affecting microbial growth. Despite the wide distribution of these proteins in Archaea, their biochemical characteristics still need to be discovered, and there needs to be more knowledge about them focusing on halophilic Archaea. Therefore, elucidating the role of USPs would provide valuable information to improve future biotechnological applications. Accordingly, transcriptional expression of the 37 annotated USPs in the Haloferax mediterranei genome has been examined under different stress conditions. From a global perspective, finding a clear tendency between particular USPs and specific stress conditions was not possible. Contrary, data analysis indicates that there is a recruitment mechanism of proteins with a similar sequence able to modulate the H. mediterranei growth, accelerating or slowing it, depending on their number. In fact, only three of these USPs were expressed in all the tested conditions, pointing to the cell needing a set of USPs to cope with stress conditions. After analysis of the RNA-Seq data, three differentially expressed USPs were selected and homologously overexpressed. According to the growth data, the overexpression of USPs induces a gain of tolerance in response to stress, as a rule. Therefore, this is the only work that studies all the USPs in an archaeon. It represents a significant first base to continue advancing, not only in this important family of stress proteins but also in the field of biotechnology and, at an industrial level, to improve applications such as designing microorganisms resistant to stress situations. KEY POINTS: • Expression of Haloferax mediterranei USPs has been analyzed in stress conditions. • RNA-seq analysis reveals that most of the USPs in H. mediterranei are downregulated. • Homologous overexpression of USPs results in more stress-tolerant strains.

Global Lrp regulator protein from Haloferax mediterranei: Transcriptional analysis and structural characterization.

Haloferax mediterranei, an extreme halophilic archaeon thriving in hypersaline environments, has acquired significant attention in biotechnological and biochemical research due to its remarkable ability to flourish in extreme salinity conditions. Transcription factors, essential in regulating diverse cellular processes, have become focal points in understanding its adaptability. This study delves into the role of the Lrp transcription factor, exploring its modulation of glnA, nasABC, and lrp gene promoters in vivo through ß-galactosidase assays. Remarkably, our findings propose Lrp as the pioneering transcriptional regulator of nitrogen metabolism identified in a haloarchaeon. This study suggests its potential role in activating or repressing assimilatory pathway enzymes (GlnA and NasA). The interaction between Lrp and these promoters is analyzed using Electrophoretic Mobility Shift Assay and Differential Scanning Fluorimetry, highlighting l-glutamine's indispensable role in stabilizing the Lrp-DNA complex. Our research uncovers that halophilic Lrp forms octameric structures in the presence of l-glutamine. The study reveals the three-dimensional structure of the Lrp as a homodimer using X-ray crystallography, confirming this state in solution by Small-Angle X-ray Scattering. These findings illuminate the complex molecular mechanisms driving Hfx. mediterranei's nitrogen metabolism, offering valuable insights about its gene expression regulation and enriching our comprehension of extremophile biology.

Lrp as a potential transcriptional regulator involved in stress response in Haloferax mediterranei.

The Archaea domain consists of a heterogeneous group of microorganisms with unique physiological properties that occupy a wide variety of niches in nature. Haloferax mediterranei is an extremely halophilic archaeon classified in the Phylum Euryarchaeota, which requires a high concentration of inorganic salts for optimal growth. In haloarchaea, transcription factors play a fundamental role in an adequate adaptation to environmental and nutritional changes, preserving the survival and integrity of the organism. To deepen knowledge of the Lrp/AsnC transcriptional regulator family, a lrp gene (HFX_RS01210) from this family has been studied. Site-directed mutagenesis has allowed us to identify the TATA-box and two potential sites of the transcriptional factor (TF) to its own promoter and autoregulate itself. Several approaches were carried out to elucidate whether this transcriptional regulator is involved in stresses due to heavy metals and limited nitrogen conditions. Characterization of the lrp deletion mutant and the Lrp overexpressed strain, suggests that the level of lrp expression depends on the nitrogen source and the presence of cobalt. The most striking results were obtained in the presence of nitrate as a nitrogen source due to the inability of the deletion mutant to grow. All these results confirm that Lrp is a powerful candidate for a regulatory role in the stress response, particularly under N-limiting conditions and the presence of cobalt.

Analysis of Haloferax mediterranei Lrp Transcriptional Regulator.

Haloferax mediterranei is an extremely halophilic archaeon, able to live in hypersaline environments with versatile nutritional requirements, whose study represents an excellent basis in the field of biotechnology. The transcriptional machinery in Archaea combines the eukaryotic basal apparatus and the bacterial regulation mechanisms. However, little is known about molecular mechanisms of gene expression regulation compared with Bacteria, particularly in Haloarchaea. The genome of Hfx. mediterranei contains a gene, lrp (HFX_RS01210), which encodes a transcriptional factor belonging to Lrp/AsnC family. It is located downstream of the glutamine synthetase gene (HFX_RS01205), an enzyme involved in ammonium assimilation and amino acid metabolism. To study this transcriptional factor more deeply, the lrp gene has been homologously overexpressed and purified under native conditions by two chromatographic steps, namely nickel affinity and gel filtration chromatography, showing that Lrp behaves asa tetrameric protein of approximately 67 kDa. Its promoter region has been characterized under different growth conditions using bgaH as a reporter gene. The amount of Lrp protein was also analyzed by Western blotting in different nitrogen sources and under various stress conditions. To sum up, regarding its involvement in the nitrogen cycle, it has been shown that its expression profile does not change in response to the nitrogen sources tested. Differences in its expression pattern have been observed under different stress conditions, such as in the presence of hydrogen peroxide or heavy metals. According to these results, the Lrp seems to be involved in a general response against stress factors, acting as a first-line transcriptional regulator.

The Survival of Haloferax mediterranei under Stressful Conditions.

Haloarchaea can survive and thrive under exposure to a wide range of extreme environmental factors, which represents a potential interest to biotechnology. Growth responses to different stressful conditions were examined in the haloarchaeon Haloferax mediterranei R4. It has been demonstrated that this halophilic archaeon is able to grow between 10 and 32.5% (w/v) of sea water, at 32-52 °C, although it is expected to grow in temperatures lower than 32 °C, and between 5.75 and 8.75 of pH. Moreover, it can also grow under high metal concentrations (nickel, lithium, cobalt, arsenic), which are toxic to most living beings, making it a promising candidate for future biotechnological purposes and industrial applications. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis quantified the intracellular ion concentrations of these four metals in Hfx. mediterranei, concluding that this haloarchaeon can accumulate Li+, Co2+, As5+, and Ni2+ within the cell. This paper is the first report on Hfx. mediterranei in which multiple stress conditions have been studied to explore the mechanism of stress resistance. It constitutes the most detailed study in Haloarchaea, and, as a consequence, new biotechnological and industrial applications have emerged.

The Role of Stress Proteins in Haloarchaea and Their Adaptive Response to Environmental Shifts.

Over the years, in order to survive in their natural environment, microbial communities have acquired adaptations to nonoptimal growth conditions. These shifts are usually related to stress conditions such as low/high solar radiation, extreme temperatures, oxidative stress, pH variations, changes in salinity, or a high concentration of heavy metals. In addition, climate change is resulting in these stress conditions becoming more significant due to the frequency and intensity of extreme weather events. The most relevant damaging effect of these stressors is protein denaturation. To cope with this effect, organisms have developed different mechanisms, wherein the stress genes play an important role in deciding which of them survive. Each organism has different responses that involve the activation of many genes and molecules as well as downregulation of other genes and pathways. Focused on salinity stress, the archaeal domain encompasses the most significant extremophiles living in high-salinity environments. To have the capacity to withstand this high salinity without losing protein structure and function, the microorganisms have distinct adaptations. The haloarchaeal stress response protects cells against abiotic stressors through the synthesis of stress proteins. This includes other heat shock stress proteins (Hsp), thermoprotectants, survival proteins, universal stress proteins, and multicellular structures. Gene and family stress proteins are highly conserved among members of the halophilic archaea and their study should continue in order to develop means to improve for biotechnological purposes. In this review, all the mechanisms to cope with stress response by haloarchaea are discussed from a global perspective, specifically focusing on the role played by universal stress proteins.