An Exploratory Review of the Potential of Lytic Proteins and Bacteriophages for the Treatment of Tuberculosis.

Tuberculosis (TB) is a highly prevalent infectious disease that causes more than 1.5 million deaths a year. More than 25% of TB deaths occur in Africa, and TB is South Africa's leading cause of death, with about 89,000 people dying of it yearly. The emergence of multidrug-resistant TB (MDR-TB) poses a significant threat to health security and could reverse the positive gains already made in the fight against TB. Antibiotic treatments are available, but side effects and the alarming increase in the prevalence of drug-resistant strains of Mycobacterium tuberculosis (Mtb) will compromise the control of the spread and treatment of the disease. A promising option is to employ specialized enzymes encoded by bacteriophages, which destroy bacterial cell membranes and walls to treat tuberculosis. Phage therapy against bacteria is a known treatment that is now reemerging with lytic proteins. These proteins provide an alternative means to treat infectious diseases where conventional antibiotic regimens do not meet the requirements. This review explores and discusses the potential of lytic protein therapy as an antimicrobial strategy against M. tuberculosis and multidrug-resistant tuberculosis.

South Africa's indigenous microbial diversity for industrial applications: A review of the current status and opportunities.

The unique metagenomic, metaviromic libraries and indigenous micro diversity within Southern Africa have the potential for global beneficiation in academia and industry. Microorganisms that flourish at high temperatures, adverse pH conditions, and high salinity are likely to have enzyme systems that function efficiently under those conditions. These attributes afford researchers and industries alternative approaches that could replace existing chemical processes. Thus, a better understanding of African microbial/genetic diversity is crucial for the development of "greener" industries. A concerted drive to exploit the potential locked in biological resources has been previously seen with companies such as Diversa Incorporated and Verenium (Badische Anilin-und SodaFabrik-BASF) both building business models that pioneered the production of high-performance specialty enzymes for a variety of different industrial applications. The market potential and accompanying industry offerings have not been fully exploited in South Africa, nor in Africa at large. Utilization of the continent's indigenous microbial repositories could create long-lasting, sustainable growth in various production sectors, providing economic growth in resource-poor regions. By bolstering local manufacture of high-value bio-based products, scientific and engineering discoveries have the potential to generate new industries which in turn would provide employment avenues for many skilled and unskilled laborers. The positive implications of this could play a role in altering the face of business markets on the continent from costly import-driven markets to income-generating export markets. This review focuses on identifying microbially diverse areas located in South Africa while providing a profile for all associated microbial/genetically derived libraries in this country. A comprehensive list of all the relevant researchers and potential key players is presented, mapping out existing research networks for the facilitation of collaboration. The overall aim of this review is to facilitate a coordinated journey of exploration, one which will hopefully realize the value that South Africa's microbial diversity has to offer.

Engineering enhanced thermostability into the Geobacillus pallidus nitrile hydratase.

Nitrile hydratases (NHases) are important biocatalysts for the enzymatic conversion of nitriles to industrially-important amides such as acrylamide and nicotinamide. Although thermostability in this enzyme class is generally low, there is not sufficient understanding of its basis for rational enzyme design. The gene expressing the Co-type NHase from the moderate thermophile, Geobacillus pallidus RAPc8 (NRRL B-59396), was subjected to random mutagenesis. Four mutants were selected that were 3 to 15-fold more thermostable than the wild-type NHase, resulting in a 3.4-7.6 â€‹kJ/mol increase in the activation energy of thermal inactivation at 63 â€‹°C. High resolution X-ray crystal structures (1.15-1.80 â€‹Å) were obtained of the wild-type and four mutant enzymes. Mutant 9E, with a resolution of 1.15 â€‹Å, is the highest resolution crystal structure obtained for a nitrile hydratase to date. Structural comparisons between the wild-type and mutant enzymes illustrated the importance of salt bridges and hydrogen bonds in enhancing NHase thermostability. These additional interactions variously improved thermostability by increased intra- and inter-subunit interactions, preventing cooperative unfolding of α-helices and stabilising loop regions. Some hydrogen bonds were mediated via a water molecule, specifically highlighting the significance of structured water molecules in protein thermostability. Although knowledge of the mutant structures makes it possible to rationalize their behaviour, it would have been challenging to predict in advance that these mutants would be stabilising.

Transient proteolysis reduction of Nicotiana benthamiana-produced CAP256 broadly neutralizing antibodies using CRISPR/Cas9.

The hypersensitive response is elicited by Agrobacterium infiltration of Nicotiana benthamiana, including the induction and accumulation of pathogenesis-related proteins, such as proteases. This includes the induction of the expression of several cysteine proteases from the C1 (papain-like cysteine protease) and C13 (legumain-like cysteine protease) families. This study demonstrates the role of cysteine proteases: NbVPE-1a, NbVPE-1b, and NbCysP6 in the proteolytic degradation of Nicotiana benthamiana (glycosylation mutant ΔXTFT)-produced anti-human immunodeficiency virus broadly neutralizing antibody, CAP256-VRC26.25. Three putative cysteine protease cleavage sites were identified in the fragment crystallizable region. We further demonstrate the transient coexpression of CAP256-VRC26.25 with CRISPR/Cas9-mediated genome editing vectors targeting the NbVPE-1a, NbVPE-1b, and NbCysP6 genes which resulted in a decrease in CAP256-VRC26.25 degradation. No differences in structural features were observed between the human embryonic kidney 293 (HEK293)-produced and ΔXTFT broadly neutralizing antibodies produced with and without the coexpression of genome-editing vectors. Furthermore, despite the presence of proteolytically degraded fragments of plant-produced CAP256-VRC26.25 without the coexpression of genome editing vectors, no influence on the in vitro functional activity was detected. Collectively, we demonstrate an innovative in planta strategy for improving the quality of the CAP256 antibodies through the transient expression of the CRISPR/Cas9 vectors.

Recombinant Expression, Purification and PEGylation of DNA Ligases.

BACKGROUND: Reagent proteins such as DNA ligases play a central role in the global reagents market. DNA ligases are commonly used and are vital in academic and science research environments. Their major functions include sealing nicks by linking the 5'-phosphorylated end to a 3'-hydroxyl end on the phosphodiester backbone of DNA, utilizing ATP or NADP molecules as an energy source. OBJECTIVE: The current study sought to investigate the role of PEGylation on the biological activity of purified recombinant DNA ligases. METHODS: We produced two recombinant DNA ligases (Ligsv081 and LigpET30) using E. coli expression system and subsequently purified using affinity chromatography. The produced proteins wereconjugated to site specific PEGylation or non-specific PEGylation. FTIR and UV-VIS spectroscopy were used to analyze secondary structures of the PEG conjugated DNA ligases. Differential scanning fluorimetry was employed to assess the protein stability when subjected to various PEGylation conditions. RESULTS: In this study, both recombinant DNA ligases were successfully expressed and purified as homogenous proteins. Protein PEGylation enhanced ligation activity, increased transformation efficiency by 2-foldfor plasmid ligations and reduced the formation of protein aggregates. CONCLUSION: Taken together, site-specific PEGylation can potentially be explored to enhance the biological activity and stability of reagent proteins such as ligases.

A plant-biotechnology approach for producing highly potent anti-HIV antibodies for antiretroviral therapy consideration.

Despite a reduction in global HIV prevalence the development of a pipeline of new therapeutics or pre-exposure prophylaxis to control the HIV/AIDS epidemic are of high priority. Antibody-based therapies offer several advantages and have been shown to prevent HIV-infection. Plant-based production is efficient for several biologics, including antibodies. We provide a short review on the work by Singh et al., 2020 who demonstrated the transient production of potent CAP256-VRC26 broadly neutralizing antibodies. These antibodies have engineered posttranslational modifications, namely N-glycosylation in the fragment crystallizable region and O-sulfation of tyrosine residues in the complementary-determining region H3 loop. The glycoengineered Nicotiana benthamiana mutant (ΔXTFT) was used, with glycosylating structures lacking ß1,2-xylose and/or α1,3-fucose residues, which is critical for enhanced effector activity. The CAP256-VRC26 antibody lineage targets the first and second variable region of the HIV-1 gp120 envelope glycoprotein. The high potency of this lineage is mediated by a protruding O-sulfated tyrosine in the CDR H3 loop. Nicotiana benthamiana lacks human tyrosyl protein sulfotransferase 1, the enzyme responsible for tyrosine O-sulfation. The transient coexpression of the CAP256-VRC26 antibodies with tyrosyl protein sulfotransferase 1 in planta had restored the efficacy of these antibodies through the incorporation of the O-sulfation modification. This approach demonstrates the strategic incorporation of posttranslational modifications in production systems, which may have not been previously considered. These plant-produced CAP256-VRC26 antibodies have therapeutic as well as topical and systemic pre-exposure prophylaxis potential in enabling the empowerment of young girls and women given that gender inequalities remain a major driver of the epidemic.

Engineering Approaches in Plant Molecular Farming for Global Health.

Since the demonstration of the first plant-produced proteins of medical interest, there has been significant growth and interest in the field of plant molecular farming, with plants now being considered a viable production platform for vaccines. Despite this interest and development by a few biopharmaceutical companies, plant molecular farming is yet to be embraced by 'big pharma'. The plant system offers a faster alternative, which is a potentially more cost-effective and scalable platform for the mass production of highly complex protein vaccines, owing to the high degree of similarity between the plant and mammalian secretory pathway. Here, we identify and address bottlenecks in the use of plants for vaccine manufacturing and discuss engineering approaches that demonstrate both the utility and versatility of the plant production system as a viable biomanufacturing platform for global health. Strategies for improving the yields and quality of plant-produced vaccines, as well as the incorporation of authentic posttranslational modifications that are essential to the functionality of these highly complex protein vaccines, will also be discussed. Case-by-case examples are considered for improving the production of functional protein-based vaccines. The combination of all these strategies provides a basis for the use of cutting-edge genome editing technology to create a general plant chassis with reduced host cell proteins, which is optimised for high-level protein production of vaccines with the correct posttranslational modifications.

Plant-based production of highly potent anti-HIV antibodies with engineered posttranslational modifications.

Broadly neutralising antibodies (bNAbs) against human immunodeficiency virus type 1 (HIV-1), such as CAP256-VRC26 are being developed for HIV prevention and treatment. These Abs carry a unique but crucial post-translational modification (PTM), namely O-sulfated tyrosine in the heavy chain complementarity determining region (CDR) H3 loop. Several studies have demonstrated that plants are suitable hosts for the generation of highly active anti-HIV-1 antibodies with the potential to engineer PTMs. Here we report the expression and characterisation of CAP256-VRC26 bNAbs with posttranslational modifications (PTM). Two variants, CAP256-VRC26 (08 and 09) were expressed in glycoengineered Nicotiana benthamiana plants. By in planta co-expression of tyrosyl protein sulfotransferase 1, we installed O-sulfated tyrosine in CDR H3 of both bNAbs. These exhibited similar structural folding to the mammalian cell produced bNAbs, but non-sulfated versions showed loss of neutralisation breadth and potency. In contrast, tyrosine sulfated versions displayed equivalent neutralising activity to mammalian produced antibodies retaining exceptional potency against some subtype C viruses. Together, the data demonstrate the enormous potential of plant-based systems for multiple posttranslational engineering and production of fully active bNAbs for application in passive immunisation or as an alternative for current HIV/AIDS antiretroviral therapy regimens.

Functional screening of a soil metagenome for DNA endonucleases by acquired resistance to bacteriophage infection.

Endonucleases play a crucial role as reagents in laboratory research and diagnostics. Here, metagenomics was used to functionally screen a fosmid library for endonucleases. A fosmid library was constructed using metagenomic DNA isolated from soil sampled from the unique environment of the Kogelberg Nature Reserve in the Western Cape of South Africa. The principle of acquired immunity against phage infection was used to develop a plate-based screening technique for the isolation of restriction endonucleases from the library. Using next-generation sequencing and bioinformatics tools, sequence data were generated and analysed, revealing 113 novel open reading frames (ORFs) encoding putative endonuclease genes and ORFs of unknown identity and function. One endonuclease designated Endo52 was selected from the putative endonuclease ORFs and was recombinantly produced in Escherichia coli Rosetta™ (DE3) pLysS. Endo52 was purified by immobilised metal affinity chromatography and yielded 0.437 g per litre of cultivation volume. Its enzyme activity was monitored by cleaving lambda DNA and pUC19 plasmid as substrates, and it demonstrated non-specific endonuclease activity. In addition to endonuclease-like genes, the screen identified several unknown genes. These could present new phage resistance mechanisms and are an opportunity for future investigations.

Molecular farming for therapies and vaccines in Africa.

Local manufacturing of protein-based vaccines and therapies in Africa is limited and contributes to a trade deficit, security of supply concerns and poor access to biopharmaceuticals by the poor. Plant molecular farming is a potential technology solution that has received growing adoption by African scientists attracted by the potential for the competitive cost of goods, safety and efficacy. Plant-made pharmaceutical technologies for veterinary and human vaccination and treatment of non-communicable and infectious diseases are available at different stages of development in Africa. There is also growth in the translation of these technologies to commercial operations. Africa is poised to benefit from the real-world impact of molecular farming in the next few years.

Plant-produced chimeric virus-like particles - a new generation vaccine against African horse sickness.

BACKGROUND: African horse sickness (AHS) is a severe arthropod-borne viral disease of equids, with a mortality rate of up to 95% in susceptible naïve horses. Due to safety concerns with the current live, attenuated AHS vaccine, alternate safe and effective vaccination strategies such as virus-like particles (VLPs) are being investigated. Transient plant-based expression systems are a rapid and highly scalable means of producing such African horse sickness virus (AHSV) VLPs for vaccine purposes. RESULTS: In this study, we demonstrated that transient co-expression of the four AHSV capsid proteins in agroinfiltrated Nicotiana benthamiana dXT/FT plants not only allowed for the assembly of homogenous AHSV-1 VLPs but also single, double and triple chimeric VLPs, where one capsid protein originated from one AHS serotype and at least one other capsid protein originated from another AHS serotype. Following optimisation of a large scale VLP purification procedure, the safety and immunogenicity of the plant-produced, triple chimeric AHSV-6 VLPs was confirmed in horses, the target species. CONCLUSIONS: We have successfully shown assembly of single and double chimeric AHSV-7 VLPs, as well as triple chimeric AHSV-6 VLPs, in Nicotiana benthamiana dXT/FT plants. Plant produced chimeric AHSV-6 VLPs were found to be safe for administration into 6 month old foals as well as capable of eliciting a weak neutralizing humoral immune response in these target animals against homologous AHSV virus.

Plant-produced Bluetongue chimaeric VLP vaccine candidates elicit serotype-specific immunity in sheep.

Bluetongue (BT) is a hemorrhagic non-contagious, biting midge-transmitted disease of wild and domestic ruminants that is caused by bluetongue virus (BTV). Annual vaccination plays a pivotal role in BT disease control in endemic regions. Due to safety concerns of the current BTV multivalent live attenuated vaccine (LAV), a safe efficacious new generation subunit vaccine such as a plant-produced BT virus-like particle (VLP) vaccine is imperative. Previously, homogenous BTV serotype 8 (BTV-8) VLPs were successfully produced in Nicotiana benthamiana plants and provided protective immunity in sheep. In this study, combinations of BTV capsid proteins from more than one serotype were expressed and assembled to form chimaeric BTV-3 and BTV-4 VLPs in N. benthamiana plants. The assembled homogenous BTV-8, as well as chimaeric BTV-3 and chimaeric BTV-4 VLP serotypes, were confirmed by SDS-PAGE, Transmission Electron microscopy (TEM) and protein confirmation using liquid chromatography-mass spectrometry (LC-MS/MS) based peptide sequencing. As VP2 is the major determinant eliciting protective immunity, the percentage coverage and number of unique VP2 peptides detected in assembled chimaeric BT VLPs were used as a guide to assemble the most appropriate chimaeric combinations. Both plant-produced chimaeric BTV-3 and BTV-4 VLPs were able to induce long-lasting serotype-specific neutralizing antibodies equivalent to the monovalent LAV controls. Antibody levels remained high to the end of the trial. Combinations of homogenous and chimaeric BT VLPs have great potential as a safe, effective multivalent vaccine with the ability to distinguish between vaccinated and infected individuals (DIVA) due to the absence of non-structural proteins.

The study of degradation mechanisms of glyco-engineered plant produced anti-rabies monoclonal antibodies E559 and 62-71-3.

Rabies is an ancient and neglected zoonotic disease caused by the rabies virus, a neurotropic RNA virus that belongs to the Rhabdoviridae family, genus Lyssavirus. It remains an important public health problem as there are cost and health concerns imposed by the current human post exposure prophylaxis therapy. The use of monoclonal antibodies (mAbs) is therefore an attractive alternative. Rabies mostly affects people that reside in resource-limited areas where there are occasional failures in the cold-chain. These environmental changes may upset the stability of the mAbs. This study focused on mAbs 62-71-3 and E559; their structures, responses to freeze/thaw (F/T) and exposure to reactive oxygen species were therefore studied with the aid of a wide range of biophysical and in silico techniques in order to elucidate their stability and identify aggregation prone regions. E559 was found to be less stable than 62-71-3. The complementarity determining regions (CDR) contributed the most to its instability, more specifically: peptides 99EIWD102 and 92ATSPYT97 found in CDR3, Trp33 found in CDR1 and the oxidised Met34. The constant region "158SWNSGALTGHTFPAVL175" was also flagged by the special aggregation propensity (SAP) tool and F/T experiments to be highly prone to aggregation. The E559 peptides "4LQESGSVL11 from the heavy chain and 4LTQSPSSL11 from the light chain, were also highly affected by F/T. These residues may serve as good candidates for mutation, in the aim to bring forward more stable therapeutic antibodies, thus paving a way to a more safe and efficacious antibody-based cocktail treatment against rabies.

Safety and immunogenicity of plant-produced African horse sickness virus-like particles in horses.

African horse sickness (AHS) is caused by multiple serotypes of the dsRNA AHSV and is a major scourge of domestic equids in Africa. While there are well established commercial live attenuated vaccines produced in South Africa, risks associated with these have encouraged attempts to develop new and safer recombinant vaccines. Previously, we reported on the immunogenicity of a plant-produced AHS serotype 5 virus-like particle (VLP) vaccine, which stimulated high titres of AHS serotype 5-specific neutralizing antibodies in guinea pigs. Here, we report a similar response to the vaccine in horses. This is the first report demonstrating the safety and immunogenicity of plant-produced AHS VLPs in horses.

Exploring Viral Diversity in a Unique South African Soil Habitat.

The Kogelberg Biosphere Reserve in the Cape Floral Kingdom in South Africa is known for its unique plant biodiversity. The potential presence of unique microbial and viral biodiversity associated with this unique plant biodiversity led us to explore the fynbos soil using metaviromic techniques. In this study, metaviromes of a soil community from the Kogelberg Biosphere Reserve has been characterised in detail for the first time. Metaviromic DNA was recovered from soil and sequenced by Next Generation Sequencing. The MetaVir, MG-RAST and VIROME bioinformatics pipelines were used to analyse taxonomic composition, phylogenetic and functional assessments of the sequences. Taxonomic composition revealed members of the order Caudovirales, in particular the family Siphoviridae, as prevalent in the soil samples and other compared viromes. Functional analysis and other metaviromes showed a relatively high frequency of phage-related and structural proteins. Phylogenetic analysis of PolB, PolB2, terL and T7gp17 genes indicated that many viral sequences are closely related to the order Caudovirales, while the remainder were distinct from known isolates. The use of single virome which only includes double stranded DNA viruses limits this study. Novel phage sequences were detected, presenting an opportunity for future studies aimed at targeting novel genetic resources for applied biotechnology.

Evaluation of plant-produced Clostridium perfringens type D epsilon toxoid in a vaccine against enterotoxaemia in sheep.

Enterotoxaemia (pulpy kidney) is a common bacterial disease of sheep caused by Clostridium perfringens type D epsilon toxin. It has mortality rates of up to 30% in non-vaccinated animals. Current vaccines from whole cell cultures are expensive to manufacture and can induce local inflammatory responses in sheep. They usually have reduced immunogenicity because of the difficulty of standardising the inactivation step in vaccine manufacturing. In the current study, we evaluated the safety and potency of a recombinant plant-made epsilon toxoid protein (r-Etox) as an affordable and safer alternative vaccine for developing countries. Results of injection site reactions, rectal temperature and toxin neutralisation test in single and prime- boost inoculations of mice, guinea pigs and sheep suggest that the product is not toxic to animals and could protect sheep against enterotoxaemia.

The Lyssavirus glycoprotein: A key to cross-immunity.

Rabies is an acute viral encephalomyelitis in warm-blooded vertebrates, caused by viruses belonging to Rhabdovirus family and genus Lyssavirus. Although rabies is categorised as a neglected disease, the rabies virus (RABV) is the most studied amongst Lyssaviruses which show nearly identical infection patterns. In efforts to improving post-exposure prophylaxis, several anti-rabies monoclonal antibodies (mAbs) targeting the glycoprotein (G protein) sites I, II, III and G5 have been characterized. To explore cross-neutralization capacity of available mAbs and discover new possible B-cell epitopes, we have analyzed all available glycoprotein sequences from Lyssaviruses with a focus on sequence variation and conservation. This information was mapped on the structure of a representative G protein. We proposed several possible cross-neutralizing B-cell epitopes (GUVTTTF, WLRTV, REECLD and EHLVVEEL) in complement to the already well-characterized antigenic sites. The research could facilitate development of novel cross-reactive mAbs against RABV and even more broad, against possibly all Lyssavirus members.

Efficient In Vitro and In Vivo Activity of Glyco-Engineered Plant-Produced Rabies Monoclonal Antibodies E559 and 62-71-3.

Rabies is a neglected zoonotic disease that has no effective treatment after onset of illness. However the disease can be prevented effectively by prompt administration of post exposure prophylaxis which includes administration of passive immunizing antibodies (Rabies Immune Globulin, RIG). Currently, human RIG suffers from many restrictions including limited availability, batch-to batch inconsistencies and potential for contamination with blood-borne pathogens. Anti-rabies monoclonal antibodies (mAbs) have been identified as a promising alternative to RIG. Here, we applied a plant-based transient expression system to achieve rapid, high level production and efficacy of the two highly potent anti-rabies mAbs E559 and 62-71-3. Expression levels of up to 490 mg/kg of recombinant mAbs were obtained in Nicotiana benthamiana glycosylation mutants by using a viral based transient expression system. The plant-made E559 and 62-71-3, carrying human-type fucose-free N-glycans, assembled properly and were structurally sound as determined by mass spectrometry and calorimetric density measurements. Both mAbs efficiently neutralised diverse rabies virus variants in vitro. Importantly, E559 and 62-71-3 exhibited enhanced protection against rabies virus compared to human RIG in a hamster model post-exposure challenge trial. Collectively, our results provide the basis for the development of a multi-mAb based alternative to RIG.

Application of termite hindgut metagenome derived carboxyl ester hydrolases in the modification of cephalosporin substrates.

In the pharmaceutical industry, de-acetylated cephalosporins are highly valuable starting materials for producing semi-synthetic ß-lactam antibiotics. In this study a fosmid metagenome library from termite hindgut symbionts was screened for carboxyl ester hydrolases capable of de-acetylating cephalosporins. Recombinant Escherichia coli clones with esterolytic phenotypes on tributyrin agar plates were selected and further tested for de-acetylating activity against Cephalothin and 7-aminocephalosporanic acid (7-ACA). Two clones displaying de-acetylating activity were sequenced and the corresponding two carboxyl ester hydrolase encoding genes (axeA and axeB) belonging to the carbohydrate esterase family 7 (CE7) were identified. The primary structure of both the axeA and axeB revealed the presence of G-X-S-X-G sequence motif and respective subunit molecular masses of 40 kDa. In addition to de-acetylating cephalosporin based molecules, the two enzymes were also shown to be true esterases based on their preferences for short chain length fatty acid esters.

Engineering color variants of green fluorescent protein (GFP) for thermostability, pH-sensitivity, and improved folding kinetics.

A number of studies have been conducted to improve chromophore maturation, folding kinetics, thermostability, and other traits of green fluorescent protein (GFP). However, no specific work aimed at improving the thermostability of the yellow fluorescent protein (YFP) and of the pH-sensitive, yet thermostable color variants of GFP has so far been done. The protein variants reported in this study were improved through rational multiple site-directed mutagenesis of GFP (ASV) by introducing up to ten point mutations including the mutations near and at the chromophore region. Therefore, we report the development and characterization of fast folder and thermo-tolerant green variant (FF-GFP), and a fast folder thermostable yellow fluorescent protein (FFTS-YFP) endowed with remarkably improved thermostability and folding kinetics. We demonstrate that the fluorescence intensity of this yellow variant is not affected by heating at 75 °C. Moreover, we have developed a pH-unresponsive cyan variant AcS-CFP, which has potential use as part of in vivo imaging irrespective of intracellular pH. The combined improved properties make these fluorescent variants ideal tools to study protein expression and function under different pH environments, in mesophiles and thermophiles. Furthermore, coupling of the FFTS-YFP and AcS-CFP could potentially serve as an ideal tool to perform functional analysis of live cells by multicolor labeling.