Red Fluorescent Protein Variant with a Dual-Peak Emission of Fluorescence.

The marine environment is a rich reservoir of diverse biological entities, many of which possess unique properties that are of immense value to biotechnological applications. One such example is the red fluorescent protein derived from the coral Discosoma sp. This protein, encoded by the DsRed gene, has been the subject of extensive research due to its potential applications in various fields. In the study, a variant of the red fluorescent protein was generated through random mutagenesis using the DsRed2 gene as a template. The process employed error-prone PCR (epPCR) to introduce random mutations, leading to the isolation of twelve gene variants. Among these, one variant stood out due to its unique spectral properties, exhibiting dual fluorescence emission at both 480 nm (green) and 550 nm (red). This novel variant was expressed in both Escherichia coli and zebrafish (Danio rerio) muscle, confirming the dual fluorescence emission in both model systems. One of the immediate applications of this novel protein variant is in ornamental aquaculture. The dual fluorescence can serve as a unique marker or trait, enhancing the aesthetic appeal of aquatic species in ornamental settings.

Application of Rhizomucor miehei lipase-displaying Pichia pastoris whole cell for biodiesel production using agro-industrial residuals as substrate.

This work aimed the application of a new biocatalyst for biodiesel production from residual agro-industrial fatty acids. A recombinant Pichia pastoris displaying lipase from Rhizomucor miehei (RML) on the cell surface, using the PIR-1 anchor system, were prepared using glycerol as the carbon source. The biocatalyst, named RML-PIR1 showed optimum temperature of 45 °C (74.0 U/L). The stability tests resulted in t1/2 of 3.49 and 2.15 h at 40 and 45 °C, respectively. RML-PIR1 was applied in esterification reactions using industrial co-products as substrates, palm fatty acid distillate (PFAD) and soybean fatty acid distillate (SFAD). The highest productivity was observed for SFAD after 48 h presenting 79.1% of conversion using only 10% of biocatalyst and free-solvent system. This is about ca. eight times higher than commercial free RML in the same conditions. The stabilizing agents study revealed that the treatment using glutaraldehyde (GA) and poly(ethylene glycol) (PEG) enabled increased stability and reuse of biocatalyst. It was observed by SEM analysis that the treatment modified the cell morphology. RML-PIR1-GA presented 87.9% of the initial activity after 6 reuses, whilst the activity of unmodified RML-PIR decreased by 40% after the first use. These results were superior to those obtained in the literature, making this new biocatalyst promising for biotechnological applications, such as the production of biofuels on a large scale.

Benchmarking recombinant Pichia pastoris for 3-hydroxypropionic acid production from glycerol.

The use of the methylotrophic yeast Pichia pastoris (Komagataella phaffi) to produce heterologous proteins has been largely reported. However, investigations addressing the potential of this yeast to produce bulk chemicals are still scarce. In this study, we have studied the use of P. pastoris as a cell factory to produce the commodity chemical 3-hydroxypropionic acid (3-HP) from glycerol. 3-HP is a chemical platform which can be converted into acrylic acid and to other alternatives to petroleum-based products. To this end, the mcr gene from Chloroflexus aurantiacus was introduced into P. pastoris. This single modification allowed the production of 3-HP from glycerol through the malonyl-CoA pathway. Further enzyme and metabolic engineering modifications aimed at increasing cofactor and metabolic precursors availability allowed a 14-fold increase in the production of 3-HP compared to the initial strain. The best strain (PpHP6) was tested in a fed-batch culture, achieving a final concentration of 3-HP of 24.75 g l-1 , a product yield of 0.13 g g-1 and a volumetric productivity of 0.54 g l-1  h-1 , which, to our knowledge, is the highest volumetric productivity reported in yeast. These results benchmark P. pastoris as a promising platform to produce bulk chemicals for the revalorization of crude glycerol and, in particular, to produce 3-HP.

Identification and recombinant expression of an antimicrobial peptide (cecropin B-like) from soybean pest Anticarsia gemmatalis.

BACKGROUND: Insects can be found in numerous diverse environments, being exposed to pathogenic organisms like fungi and bacteria. Once these pathogens cross insect physical barriers, the innate immune system operates through cellular and humoral responses. Antimicrobial peptides are small molecules produced by immune signaling cascades that develop an important and generalist role in insect defenses against a variety of microorganisms. In the present work, a cecropin B-like peptide (AgCecropB) sequence was identified in the velvetbean caterpillar Anticarsia gemmatalis and cloned in a bacterial plasmid vector for further heterologous expression and antimicrobial tests. METHODS: AgCecropB sequence (without the signal peptide) was cloned in the plasmid vector pET-M30-MBP and expressed in the Escherichia coli BL21(DE3) expression host. Expression was induced with IPTG and a recombinant peptide was purified using two affinity chromatography steps with Histrap column. The purified peptide was submitted to high-resolution mass spectrometry (HRMS) and structural analyses. Antimicrobial tests were performed using gram-positive (Bacillus thuringiensis) and gram-negative (Burkholderia kururiensis and E. coli) bacteria. RESULTS: AgCecropB was expressed in E. coli BL21 (DE3) at 28°C with IPTG 0.5 mM. The recombinant peptide was purified and enriched after purification steps. HRMS confirmed AgCrecropB molecular mass (4.6 kDa) and circular dichroism assay showed α-helix structure in the presence of SDS. AgCrecropB inhibited almost 50% of gram-positive B. thuringiensis bacteria growth. CONCLUSIONS: The first cecropin B-like peptide was described in A. gemmatalis and a recombinant peptide was expressed using a bacterial platform. Data confirmed tertiary structure as predicted for the cecropin peptide family. AgCecropB was capable to inhibit B. thuringiensis growth in vitro.

Identification and recombinant expression of an antimicrobial peptide (cecropin B-like) from soybean pest Anticarsia gemmatalis

Insects can be found in numerous diverse environments, being exposed to pathogenic organisms like fungi and bacteria. Once these pathogens cross insect physical barriers, the innate immune system operates through cellular and humoral responses. Antimicrobial peptides are small molecules produced by immune signaling cascades that develop an important and generalist role in insect defenses against a variety of microorganisms. In the present work, a cecropin B-like peptide (AgCecropB) sequence was identified in the velvetbean caterpillar Anticarsia gemmatalis and cloned in a bacterial plasmid vector for further heterologous expression and antimicrobial tests. Methods AgCecropB sequence (without the signal peptide) was cloned in the plasmid vector pET-M30-MBP and expressed in the Escherichia coli BL21(DE3) expression host. Expression was induced with IPTG and a recombinant peptide was purified using two affinity chromatography steps with Histrap column. The purified peptide was submitted to high-resolution mass spectrometry (HRMS) and structural analyses. Antimicrobial tests were performed using gram-positive (Bacillus thuringiensis) and gram-negative (Burkholderia kururiensis and E. coli) bacteria. Results AgCecropB was expressed in E. coli BL21 (DE3) at 28°C with IPTG 0.5 mM. The recombinant peptide was purified and enriched after purification steps. HRMS confirmed AgCrecropB molecular mass (4.6 kDa) and circular dichroism assay showed α-helix structure in the presence of SDS. AgCrecropB inhibited almost 50% of gram-positive B. thuringiensis bacteria growth. Conclusions The first cecropin B-like peptide was described in A. gemmatalis and a recombinant peptide was expressed using a bacterial platform. Data confirmed tertiary structure as predicted for the cecropin peptide family. AgCecropB was capable to inhibit B. thuringiensis growth in vitro.(AU)

Construction of wild-type Yarrowia lipolytica IMUFRJ 50682 auxotrophic mutants using dual CRISPR/Cas9 strategy for novel biotechnological approaches.

Yarrowia lipolytica IMUFRJ 50682 is a Brazilian wild-type strain with potential application in bioconversion processes which can be improved through synthetic biology. In this study, we focused on a combinatorial dual cleavage CRISPR/Cas9-mediated for construction of irreversible auxotrophic mutants IMUFRJ 50682, which genomic information is not available, thought paired sgRNAs targeting upstream and downstream sites of URA3 gene. The disruption efficiency ranged from 5 to 28 % for sgRNAs combinations closer to URA3's start and stop codon and the auxotrophic mutants lost about 970 bp containing all coding sequence, validating this method for genomic edition of wild-type strains. In addition, we introduced a fluorescent phenotype and achieved cloning rates varying from 80 to 100 %. The ura3Δ strains IMUFRJ 50682 were also engineered for ß-carotene synthesis as proof of concept. Carotenoid-producing strains exhibited a similar growth profile compared to the wild-type strain and were able to synthesized 30.54-50.06 mg/L (up to 4.8 mg/g DCW) of ß-carotene in YPD and YNB flask cultures, indicating a promisor future of the auxotrophic mutants IMUFRJ 50682 as a chassis for production of novel value-added chemicals.

Environmentally friendly rhamnolipid production for petroleum remediation.

Biosurfactants have potential applications in the remediation of petroleum-contaminated sites. Several strategies can be used to reduce the production costs of these surfactants and make the process more environmentally friendly. In this study, we combined some of these strategies to produce the rhamnolipid-type biosurfactant, including the use of the genetically modified strain Pseudomonas aeruginosa-estA, an industrial coproduct as a carbon source, a simple and low-cost medium, and a simple downstream process. The process resulted in a high yield (17.6 g L-1), even using crude glycerin as the carbon source, with substrate in product conversion factor (YRML/s) of 0.444. The cell-free supernatant (CFS) was not toxic to Artemia salina and selected mammalian cell lineages, suggesting that it can be used directly in the environment without further purification steps. Qualitative analysis showed that CFS has excellent dispersion in the oil-displacement test, emulsifying (IE24 = 65.5%), and tensoactive properties. When salinity, temperature and pressure were set to seawater conditions, the values for interfacial tension between crude oil and water were below 1.0 mN m-1. Taken together, these results demonstrate that it is possible to obtain a nontoxic crude rhamnolipid product, with high productivity, to replace petroleum-based surfactants in oil spill cleanups and other environmental applications.

Increase of Candida antarctica lipase B production under PGK promoter in Pichia pastoris: effect of multicopies.

The effect of gene dosage on the production of Candida antarctica lipase B (CalB) in the methylotrophic yeast Komagataella phaffii, at high densities in a simple medium containing crude glycerin as the sole carbon source, is described. The use of crude glycerin, the main by-product of biodiesel production from vegetable oils, will reduce the production cost of the bioprocess. Two K. phaffii strains were constructed with one or three copies of LipB, an optimized version of the gene encoding CalB under the control of the constitutive PPGK1 promoter. These two constructs were tested and compared on batches using minimal-salts medium with crude glycerin. The strain with three copies achieved a higher enzyme yield (48,760 U/L, 2.3-fold higher than the one-copy strain), with 42 g/L biomass, with no effects on growth.

Expression, purification, and characterization of asparaginase II from Saccharomyces cerevisiae in Escherichia coli.

l-asparaginase catalyzes the conversion of l-asparagine to l-aspartate and ammonium. This protein is an important therapeutic enzyme used for the treatment of acute lymphoblastic leukemia. In this study, the asparaginase II-encoding gene ASP3 from Saccharomyces cerevisiae was cloned into the expression vector pET28a in-fusion with a 6x histidine tag and was expressed in Escherichia coli BL21 (DE3) cells. The protein was expressed at a high level (225.6 IU/g cells) as an intracellular and soluble molecule and was purified from the supernatant by nickel affinity chromatography. The enzyme showed very low activity against l-glutamine. The denaturing electrophoresis analysis indicated that the recombinant protein had a molecular mass of ∼38 kDa. The native enzyme was a tetramer with a molecular mass of approximately 178 kDa. The enzyme preparation showed antitumor activity against the K562 and Jurkat cell lines comparable or even superior to the E. coli commercial asparaginase.

Enhanced rhamnolipid production by Pseudomonas aeruginosa overexpressing estA in a simple medium.

A modified Pseudomonas aeruginosa strain capable of overexpressing the estA gene, an encoding gene for a membrane-bound esterase, was constructed and its rhamnolipid (RML) production was studied. Fermentations using wild-type (WT) and modified P. aeruginosa strains were conducted until exhaustion of glycerol in Medium Salt Production, using two different C/N ratios. At a C/N of 83.2, the modified strain produced up to 3.9 times more RMLs than the WT, yielding a maximum concentration of 14.62 g/L RML when measured by HPLC and 22 g/L by the orcinol assay. Cell-free supernatant from the modified strain reduced surface tension to 29.4 mN/m and had a CMC of 240 mg/L and CMD of 56.05. This is the first report on the construction of an estA-based recombinant strain for RML production.

Heterologous expression of the antimyotoxic protein DM64 in Pichia pastoris.

Snakebite envenomation is a neglected condition that constitutes a public health problem in tropical and subtropical countries, including Brazil. Interestingly, some animals are resistant to snake envenomation due to the presence of inhibitory glycoproteins in their serum that target toxic venom components. DM64 is an acidic glycoprotein isolated from Didelphis aurita (opossum) serum that has been characterized as an inhibitor of the myotoxicity induced by bothropic toxins bearing phospholipase A2 (PLA2) structures. This antitoxic protein can serve as an excellent starting template for the design of novel therapeutics against snakebite envenomation, particularly venom-induced local tissue damage. Therefore, the aim of this work was to produce a recombinant DM64 (rDM64) in the methylotrophic yeast Pichia pastoris and to compare its biological properties with those of native DM64. Yeast fermentation in the presence of Pefabloc, a serine protease inhibitor, stimulated cell growth (~1.5-fold), increased the rDM64 production yield approximately 10-fold and significantly reduced the susceptibility of rDM64 to proteolytic degradation. P. pastoris fermentation products were identified by mass spectrometry and Western blotting. The heterologous protein was efficiently purified from the culture medium by affinity chromatography (with immobilized PLA2 myotoxin) and/or an ion exchange column. Although both native and recombinant DM64 exhibit different glycosylation patterns, they show very similar electrophoretic mobilities after PNGase F treatment. rDM64 formed a noncovalent complex with myotoxin II (Lys49-PLA2) from Bothrops asper and displayed biological activity that was similar to that of native DM64, inhibiting the cytotoxicity of myotoxin II by 92% at a 1:1 molar ratio.

Correction: Recombinant L-Asparaginase from Zymomonas mobilis: A Potential New Antileukemic Agent Produced in Escherichia coli.

[This corrects the article DOI: 10.1371/journal.pone.0156692.].

Recombinant L-Asparaginase from Zymomonas mobilis: A Potential New Antileukemic Agent Produced in Escherichia coli.

L-asparaginase is an enzyme used as a chemotherapeutic agent, mainly for treating acute lymphoblastic leukemia. In this study, the gene of L-asparaginase from Zymomonas mobilis was cloned in pET vectors, fused to a histidine tag, and had its codons optimized. The L-asparaginase was expressed extracellularly and intracellularly (cytoplasmically) in Escherichia coli in far larger quantities than obtained from the microorganism of origin, and sufficient for initial cytotoxicity tests on leukemic cells. The in silico analysis of the protein from Z. mobilis indicated the presence of a signal peptide in the sequence, as well as high identity to other sequences of L-asparaginases with antileukemic activity. The protein was expressed in a bioreactor with a complex culture medium, yielding 0.13 IU/mL extracellular L-asparaginase and 3.6 IU/mL intracellular L-asparaginase after 4 h of induction with IPTG. The cytotoxicity results suggest that recombinant L-asparaginase from Z. mobilis expressed extracellularly in E.coli has a cytotoxic and cytostatic effect on leukemic cells.

On the debate about teleology in biology: the notion of "teleological obstacle" / O debate sobre teleologia na biologia: a noção de "obstáculo teleológico"

Among the epistemological obstacles described by Gaston Bachelard, we contend that unitary and pragmatic knowledge is correlated to the teleological categories of Ernst Mayr and is the basis for prevailing debate on the notion of "function" in biology. Given the proximity of the aspects highlighted by these authors, we propose to associate the role of teleological thinking in biology and the notion of unitary and pragmatic knowledge as an obstacle to scientific knowledge. Thus, teleological thinking persists acting as an epistemological obstacle in biology, according to Bachelardian terminology. Our investigation led us to formulate the "teleological obstacle," which we consider important for the future of biology and possibly other sciences.

Dentre os obstáculos epistemológicos descritos por Gaston Bachelard, propomos que o conhecimento unitário e pragmático se relaciona com as categorias de teleologia propostas por Ernst Mayr e fundamenta as discussões atuais sobre a noção de "função" em biologia. Dada a proximidade dos aspectos salientados por ambos, propomos relacionar o papel do pensamento teleológico na biologia e a noção do conhecimento unitário e pragmático como obstáculo ao conhecimento científico. O pensamento teleológico, portanto, ainda atua como obstáculo epistemológico na biologia, segundo a terminologia bachelardiana. Nossas investigações nos levaram à formulação do "obstáculo teleológico", que entendemos ser importante para o desenvolvimento da biologia e possivelmente para outras ciências.

Displaying Lipase B from Candida antarctica in Pichia pastoris Using the Yeast Surface Display Approach: Prospection of a New Anchor and Characterization of the Whole Cell Biocatalyst.

Yeast Surface Display (YSD) is a strategy to anchor proteins on the yeast cell wall which has been employed to increase enzyme stability thus decreasing production costs. Lipase B from Candida antarctica (LipB) is one of the most studied enzymes in the context of industrial biotechnology. This study aimed to assess the biochemical features of this important biocatalyst when immobilized on the cell surface of the methylotrophic yeast Pichia pastoris using the YSD approach. For that purpose, two anchors were tested. The first (Flo9) was identified after a prospection of the P. pastoris genome being related to the family of flocculins similar to Flo1 but significantly smaller. The second is the Protein with Internal Repeats (Pir1) from P. pastoris. An immunolocalization assay showed that both anchor proteins were able to display the reporter protein EGFP in the yeast outer cell wall. LipB was expressed in P. pastoris fused either to Flo9 (FLOLIPB) or Pir1 (PIRLIPB). Both constructions showed hydrolytic activity towards tributyrin (>100 U/mgdcw and >80 U/mgdcw, respectively), optimal hydrolytic activity around 45°C and pH 7.0, higher thermostability at 45°C and stability in organic solvents when compared to a free lipase.

Are Lipases Still Important Biocatalysts? A Study of Scientific Publications and Patents for Technological Forecasting.

The great potential of lipases is known since 1930 when the work of J. B. S. Haldane was published. After eighty-five years of studies and developments, are lipases still important biocatalysts? For answering this question the present work investigated the technological development of four important industrial sectors where lipases are applied: production of detergent formulations; organic synthesis, focusing on kinetic resolution, production of biodiesel, and production of food and feed products. The analysis was made based on research publications and patent applications, working as scientific and technological indicators, respectively. Their evolution, interaction, the major players of each sector and the main subject matters disclosed in patent documents were discussed. Applying the concept of technology life cycle, S-curves were built by plotting cumulative patent data over time to monitor the attractiveness of each technology for investment. The results lead to a conclusion that the use of lipases as biocatalysts is still a relevant topic for the industrial sector, but developments are still needed for lipase biocatalysis to reach its full potential, which are expected to be achieved within the third, and present, wave of biocatalysis.

On the debate about teleology in biology: the notion of "teleological obstacle".

Among the epistemological obstacles described by Gaston Bachelard, we contend that unitary and pragmatic knowledge is correlated to the teleological categories of Ernst Mayr and is the basis for prevailing debate on the notion of "function" in biology. Given the proximity of the aspects highlighted by these authors, we propose to associate the role of teleological thinking in biology and the notion of unitary and pragmatic knowledge as an obstacle to scientific knowledge. Thus, teleological thinking persists acting as an epistemological obstacle in biology, according to Bachelardian terminology. Our investigation led us to formulate the "teleological obstacle," which we consider important for the future of biology and possibly other sciences.

Studying the expression of a lipase from Pyrococcus furiosus using response surfaces.

The need to find more stable catalysts has encouraged the study of naturally resilient enzymes, such as those found in extremophile organisms. In the present work, the influence of rare codons on the expression in Escherichia coli of the lipase Pf2001Δ60 from Pyrococcus furiosus was evaluated. Expression was carried out in two E. coli strains, BL21(DE3)pLysS and the rare tRNA supplemented Rosetta(DE3)pLysS. 3(2) factorial design was used to appraise the influence of temperature and inducer concentration on enzyme expression every hour for the 4-h expression period. Four response surfaces were constructed for each time, and the statistical parameters were evaluated. Lipase production was twice as high for Rosetta(DE3)pLysS than for BL21(DE3)pLysS. The factorial design indicated that optimal expression occurred at 30 °C after 4h, with lipase production of 240 U/L. The analysis of statistical parameters during the expression time showed that the velocity at which the enzyme was produced affected cell growth and maximum activity, with a higher speed leading to lower expression and cell growth. The presence of rare tRNAs prevented bottlenecks in lipase expression, and the experimental design was shown to be important for maximizing the production strategies and minimizing the metabolic load to which the host is subjected.

Obstáculos epistemológicos entre pós-graduandos de bioquímica / Epistemological obstacles amongst graduate students in biochemistry

A partir do estudo da epistemologia de Bachelard na disciplina Lógica e Filosofia da Ciência da Pós-Graduação em Bioquímica do Instituto de Química/Universidade Federal do Rio de Janeiro (IQ/UFRJ), buscou-se identificar obstáculos epistemológicos entre pós-graduandos em bioquímica e áreas correlatas. Um questionário com perguntas e excertos de artigos científicos de revistas de alto fator de impacto foi respondido, anonimamente, por pós-graduandos de diferentes cursos da UFRJ e de outras universidades, que nunca cursaram disciplina relacionada à epistemologia. Foi possível identificar concepções vitalistas (animismo) tanto nas respostas às perguntas como na aceitação ou não identificação deste obstáculo nos excertos. O obstáculo pragmático e unitário foi identificado através de uma concepção teleológica dos processos evolutivos, em afirmações como a existência de objetivos/finalidades na adaptação dos organismos. Verificou-se a presença de figuras de linguagem, metáforas e analogias (obstáculo verbal) na explicação da evolução e do sistema imune, também encontradas nos excertos dos artigos. Foram também identificados obstáculos associados à observação primeira e generalização prematura. A partir deste diagnóstico verificou-se a necessidade de enfatizar o caráter objetivo, material, não teleológico da bioquímica, em disciplinas oferecidas desde a graduação.

Characterization of the Recombinant Thermostable Lipase (Pf2001) from Pyrococcus furiosus: Effects of Thioredoxin Fusion Tag and Triton X-100.

In this work, the lipase from Pyrococcus furiosus encoded by ORF PF2001 was expressed with a fusion protein (thioredoxin) in Escherichia coli. The purified enzymes with the thioredoxin tag (TRX-PF2001Δ60) and without the thioredoxin tag (PF2001Δ60) were characterized, and various influences of Triton X-100 were determined. The optimal temperature for both enzymes was 80°C. Although the thioredoxin presence did not influence the optimum temperature, the TRX-PF2001Δ60 presented specific activity twice lower than the enzyme PF2001Δ60. The enzyme PF2001Δ60 was assayed using MUF-acetate, MUF-heptanoate, and MUF-palmitate. MUF-heptanoate was the preferred substrate of this enzyme. The chelators EDTA and EGTA increased the enzyme activity by 97 and 70%, respectively. The surfactant Triton X-100 reduced the enzyme activity by 50% and lowered the optimum temperature to 60°C. However, the thermostability of the enzyme PF2001Δ60 was enhanced with Triton X-100.