What is the role of microbial biotechnology and genetic engineering in medicine?

Microbial products are essential for developing various therapeutic agents, including antibiotics, anticancer drugs, vaccines, and therapeutic enzymes. Genetic engineering techniques, functional genomics, and synthetic biology unlock previously uncharacterized natural products. This review highlights major advances in microbial biotechnology, focusing on gene-based technologies for medical applications.

Genome-scale metabolic model of the versatile bacterium Paracoccus denitrificans Pd1222.

A genome scale metabolic model of the bacterium Paracoccus denitrificans has been constructed. The model containing 972 metabolic genes, 1,371 reactions, and 1,388 unique metabolites has been reconstructed. The model was used to carry out quantitative predictions of biomass yields on 10 different carbon sources under aerobic conditions. Yields on C1 compounds suggest that formate is oxidized by a formate dehydrogenase O, which uses ubiquinone as redox co-factor. The model also predicted the threshold methanol/mannitol uptake ratio, above which ribulose biphosphate carboxylase has to be expressed in order to optimize biomass yields. Biomass yields on acetate, formate, and succinate, when NO3- is used as electron acceptor, were also predicted correctly. The model reconstruction revealed the capability of P. denitrificans to grow on several non-conventional substrates such as adipic acid, 1,4-butanediol, 1,3-butanediol, and ethylene glycol. The capacity to grow on these substrates was tested experimentally, and the experimental biomass yields on these substrates were accurately predicted by the model.IMPORTANCEParacoccus denitrificans has been broadly used as a model denitrifying organism. It grows on a large portfolio of carbon sources, under aerobic and anoxic conditions. These characteristics, together with its amenability to genetic manipulations, make P. denitrificans a promising cell factory for industrial biotechnology. This paper presents and validates the first functional genome-scale metabolic model for P. denitrificans, which is a key tool to enable P. denitrificans as a platform for metabolic engineering and industrial biotechnology. Optimization of the biomass yield led to accurate predictions in a broad scope of substrates.

Characterization of the Keratinolytic Activity of Three Streptomyces Strains and Impact of Their Co-Cultivation on This Activity.

In this study, we describe the characterization of three efficient chicken feather-degrading Streptomyces bacteria isolated from honeybee samples and assess the impact of their co-cultivation on this activity and antistaphylococcal activity. Streptomyces griseoaurantiacus AD2 was the strain showing the highest keratinolytic activity (4000 U × mL-1), followed by Streptomyces albidoflavus AN1 and Streptomyces drozdowiczii AD1, which both generated approximately 3000 U × mL-1. Moreover, a consortium constituted of these three strains was able to use chicken feathers as its sole nutrient source and growth in such conditions led to a significant increase in antibiotic production. S. griseoaurantiacus AD2 was the only strain that exhibited weak antimicrobial activity against Staphylococcus aureus. UPLC analyses revealed that a significant number of peaks detected in the extracts of co-cultures of the three strains were missing in the extracts of individual cultures. In addition, the production of specialized metabolites, such as undecylprodigiosin and manumycin A, was clearly enhanced in co-culture conditions, in agreement with the results of the antimicrobial bioassays against S. aureus. Our results revealed the benefits of co-cultivation of these bacterial species in terms of metabolic wealth and antibiotic production. Our work could thus contribute to the development of novel microbial-based strategies to valorize keratin waste.

Screening Enzymes That Can Depolymerize Commercial Biodegradable Polymers: Heterologous Expression of Fusarium solani Cutinase in Escherichia coli.

In recent years, a number of microbial enzymes capable of degrading plastics have been identified. Biocatalytic depolymerization mediated by enzymes has emerged as a potentially more efficient and environmentally friendly alternative to the currently employed methods for plastic treatment and recycling. However, the functional and systematic study of depolymerase enzymes with respect to the degradation of a series of plastic polymers in a single work has not been widely addressed at present. In this study, the ability of a set of enzymes (esterase, arylesterase and cutinase) to degrade commercial biodegradable polymers (PBS, PBAT, PHB, PHBH, PHBV, PCL, PLA and PLA/PCL) and the effect of pre-treatment methods on their degradation rate was assessed. The degradation products were identified and quantified by HPLC and LC-HRMS analysis. Out of the three enzymes, Fusarium solani cutinase (FsCut) showed the highest activity on grinded PBAT, PBS and PCL after 7 days of incubation. FsCut was engineered and heterologous expressed in Escherichia coli, which conferred the bacterium the capability of degrading solid discs of PBAT and to grow in PBS as the sole carbon source of the medium.

Characterization of Bacillus Strains from Natural Honeybee Products with High Keratinolytic Activity and Antimicrobial Potential.

Two efficient feather-degrading bacteria were isolated from honeybee samples and identified as Bacillus sonorensis and Bacillus licheniformis based on 16S rRNA and genome sequencing. The strains were able to grow on chicken feathers as the sole carbon and nitrogen sources and degraded the feathers in a few days. The highest keratinase activity was detected by the B. licheniformis CG1 strain (3800 U × mL-1), followed by B. sonorensis AB7 (1450 U × mL-1). Keratinase from B. licheniformis CG1 was shown to be active across a wide range of pH, potentially making this strain advantageous for further industrial applications. All isolates displayed antimicrobial activity against Micrococcus luteus; however, only B. licheniformis CG1 was able to inhibit the growth of Mycobacterium smegmatis. In silico analysis using BAGEL and antiSMASH identified gene clusters associated with the synthesis of non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKSs) and/or ribosomally synthesized and post-translationally modified peptides (RiPPs) in most of the Bacillus isolates. B. licheniformis CG1, the only strain that inhibited the growth of the mycobacterial strain, contained sequences with 100% similarity to lichenysin (also present in the other isolates) and lichenicidin (only present in the CG1 strain). Both compounds have been described to display antimicrobial activity against distinct bacteria. In summary, in this work, we have isolated a strain (B. licheniformis CG1) with promising potential for use in different industrial applications, including animal nutrition, leather processing, detergent formulation and feather degradation.

Genome sequence analysis and characterization of Bacillus altitudinis B12, a polylactic acid- and keratin-degrading bacterium.

Keratin-rich wastes, mainly in the form of feathers, are recalcitrant residues generated in high amounts as by-products in chicken farms and food industry. Polylactic acid (PLA) is the second most common biodegradable polymer found in commercial plastics, which is not easily degraded by microbial activity. This work reports the 3.8-Mb genome of Bacillus altitudinis B12, a highly efficient PLA- and keratin-degrading bacterium, with potential for environmental friendly biotechnological applications in the feed, fertilizer, detergent, leather, and pharmaceutical industries. The whole genome sequence of B. altitudinis B12 revealed that this strain (which had been previously misclassified as Bacillus pumilus B12) is closely related to the B. altitudinis strains ER5, W3, and GR-8. A total of 4056 coding sequences were annotated using the RAST server, of which 2484 are core genes of the pan genome of B. altitudinis and 171 are unique to this strain. According to the sequence analysis, B. pumilus B12 has a predicted secretome of 353 proteins, among which a keratinase and a PLA depolymerase were identified by sequence analysis. The presence of these two enzymes could explain the characterized PLA and keratin biodegradation capability of the strain.

Modulation of Multiple Gene Clusters' Expression by the PAS-LuxR Transcriptional Regulator PteF.

PAS-LuxR transcriptional regulators are conserved proteins governing polyene antifungal biosynthesis. PteF is the regulator of filipin biosynthesis from Streptomyces avermitilis. Its mutation drastically abates filipin, but also oligomycin production, a macrolide ATP-synthase inhibitor, and delays sporulation; thus, it has been considered a transcriptional activator. Transcriptomic analyses were performed in S. avermitilis DpteF and its parental strain. Both strains were grown in a YEME medium without sucrose, and the samples were taken at exponential and stationary growth phases. A total of 257 genes showed an altered expression in the mutant, most of them at the exponential growth phase. Surprisingly, despite PteF being considered an activator, most of the genes affected showed overexpression, thereby suggesting a negative modulation. The affected genes were related to various metabolic processes, including genetic information processing; DNA, energy, carbohydrate, and lipid metabolism; morphological differentiation; and transcriptional regulation, among others, but were particularly related to secondary metabolite biosynthesis. Notably, 10 secondary metabolite gene clusters out of the 38 encoded by the genome showed altered expression profiles in the mutant, suggesting a regulatory role for PteF that is wider than expected. The transcriptomic results were validated by quantitative reverse-transcription polymerase chain reaction. These findings provide important clues to understanding the intertwined regulatory machinery that modulates antibiotic biosynthesis in Streptomyces.

Identification of Antimicrobial Compounds in Two Streptomyces sp. Strains Isolated From Beehives.

The World Health Organization warns that the alarming increase in antibiotic resistant bacteria will lead to 2.7 million deaths annually due to the lack of effective antibiotic therapies. Clearly, there is an urgent need for short-term alternatives that help to alleviate these alarming figures. In this respect, the scientific community is exploring neglected ecological niches from which the prototypical antibiotic-producing bacteria Streptomycetes are expected to be present. Recent studies have reported that honeybees and their products carry Streptomyces species that possess strong antibacterial activity. In this study, we have investigated the antibiotic profile of two Streptomycetes strains that were isolated from beehives. One of the isolates is the strain Streptomyces albus AN1, which derives from pollen, and shows potent antimicrobial activity against Candida albicans. The other isolate is the strain Streptomyces griseoaurantiacus AD2, which was isolated from honey, and displays a broad range of antimicrobial activity against different Gram-positive bacteria, including pathogens such as Staphylococcus aureus and Enterococus faecalis. Cultures of S. griseoaurantiacus AD2 have the capacity to produce the antibacterial compounds undecylprodigiosin and manumycin, while those of S. albus AN1 accumulate antifungal compounds such as candicidins and antimycins. Furthermore, genome and dereplication analyses suggest that the number of putative bioactive metabolites produced by AD2 and AN1 is considerably high, including compounds with anti-microbial and anti-cancer properties. Our results postulate that beehives are a promising source for the discovery of novel bioactive compounds that might be of interest to the agri-food sector and healthcare pharmaceuticals.

Biodegradation of bioplastics under aerobic and anaerobic aqueous conditions: Kinetics, carbon fate and particle size effect.

The biodegradation of PHB, PHBV, PBS, PBAT, PCL, PLA, and a PLA-PCL blend was compared under aerobic and anaerobic aqueous conditions assessing biodegradation kinetics, extent, carbon fate and particle size influence (in the range of 100-1000 µm). Under standard test conditions, PHB and PBHV were biodegraded anaerobically (83.9 ± 1.3% and 81.2 ± 1.7%, respectively) in 77 days or aerobically (83.0 ± 1.6% and 87.4 ± 7.5%) in 117 days, while PCL was only biodegraded (77.6 ± 2.4%) aerobically in 177 days. Apparent biomass growth accounted for 10 to 30.5% of the total initial carbon depending on the bioplastic and condition. Maximum aerobic and anaerobic biodegradation rates were improved up to 331 and 405%, respectively, at the lowest particle size tested (100-250 µm). This study highlights the usefulness of analysing biodegradation kinetics and carbon fate to improve both the development and testing of biodegradable materials, and waste treatments in the context of a circular bioeconomy.

Imaging and modelling of poly(3-hydroxybutyrate) synthesis in Paracoccus denitrificans.

Poly(3-hydroxybutyrate) (PHB) granule formation in Paracoccus denitrificans Pd1222 was investigated by laser scanning confocal microscopy (LSCM) and gas chromatography analysis. Cells that had been starved for 2 days were free of PHB granules but resynthesized them within 30 min of growth in fresh medium with succinate. In most cases, the granules were distributed randomly, although in some cases they appeared in a more organized pattern. The rates of growth and PHB accumulation were analyzed within the frame of a Genome-Scale Metabolic Model (GSMM) containing 781 metabolic genes, 1403 reactions and 1503 metabolites. The model was used to obtain quantitative predictions of biomass yields and PHB synthesis during aerobic growth on succinate as sole carbon and energy sources. The results revealed an initial fast stage of PHB accumulation, during which all of the acetyl-CoA originating from succinate was diverted to PHB production. The next stage was characterized by a tenfold lower PHB production rate and the simultaneous onset of exponential growth, during which acetyl-CoA was predominantly drained into the TCA cycle. Previous research has shown that PHB accumulation correlates with cytosolic acetyl-CoA concentration. It has also been shown that PHB accumulation is not transcriptionally regulated. Our results are consistent with the mentioned findings and suggest that, in absence of cell growth, most of the cellular acetyl-CoA is channeled to PHB synthesis, while during exponential growth, it is drained to the TCA cycle, causing a reduction of the cytosolic acetyl-CoA pool and a concomitant decrease of the synthesis of acetoacetyl-CoA (the precursor of PHB synthesis).

A metabolic modeling approach reveals promising therapeutic targets and antiviral drugs to combat COVID-19.

In this study we have developed a method based on Flux Balance Analysis to identify human metabolic enzymes which can be targeted for therapeutic intervention against COVID-19. A literature search was carried out in order to identify suitable inhibitors of these enzymes, which were confirmed by docking calculations. In total, 10 targets and 12 bioactive molecules have been predicted. Among the most promising molecules we identified Triacsin C, which inhibits ACSL3, and which has been shown to be very effective against different viruses, including positive-sense single-stranded RNA viruses. Similarly, we also identified the drug Celgosivir, which has been successfully tested in cells infected with different types of viruses such as Dengue, Zika, Hepatitis C and Influenza. Finally, other drugs targeting enzymes of lipid metabolism, carbohydrate metabolism or protein palmitoylation (such as Propylthiouracil, 2-Bromopalmitate, Lipofermata, Tunicamycin, Benzyl Isothiocyanate, Tipifarnib and Lonafarnib) are also proposed.

Inspired by nature: Microbial production, degradation and valorization of biodegradable bioplastics for life-cycle-engineered products.

The global environmental pollution by micro- and macro-plastics reveals the consequences of an extensive use of recalcitrant plastic products together with inappropriate waste management practices that fail to sufficiently recycle the broad types of conventional plastic waste. Biobased and biodegradable plastics are experiencing an uprising as their properties offer alternative waste management solutions for a more circular material economy. However, although the production of such bioplastics has advanced on scale, the end-of-life (EOL) (bio)technologies to promote circularity are lacking behind. While composting and biogas plants are the only managed EOL options today, advanced biotechnological recycling technologies for biodegradable bioplastics are still in an embryonic stage. Thus, developing efficient biotechnologies capable of transforming bioplastic waste into high-value chemical building blocks or into the constituents of the original polymer offers promising routes towards life-cycle-engineered products. This review aims at providing a comprehensive state-of-the-art overview of microbial-based processes involved in the complete lifecycle of bioplastics. The current trends in the bioplastic market, the beginning and EOL scenarios of bioplastics, and a critical discussion on the key factors and mechanisms governing microbial degradation are systematically presented. Also, a critical evaluation of terminology and international standards to quantify polymer biodegradability is provided together with the latest biotechnological recycling strategies, including the use of different pre-treatments for (bio)plastic waste. Finally, the challenges and future perspectives for the development of life-cycle-engineered biobased and biodegradable plastic products are discussed.

Genome sequencing analysis of Streptomyces coelicolor mutants that overcome the phosphate-depending vancomycin lethal effect.

BACKGROUND: Glycopeptide antibiotics inhibit bacterial cell-wall synthesis, and are important for the treatment of infections caused by multi drug-resistant strains of enterococci, streptococci and staphylococci. The main mechanism by which bacteria resist the action of glycopeptides is by producing a modified cell-wall in which the dipeptide D-Alanine-D-Alanine is substituted by D-Alanine-D-Lactate or D-Alanine-D-Serine. Recently, it has been shown that inorganic phosphate (Pi) induces hypersensitivity to vancomycin in Streptomyces coelicolor (which is highly resistant to the antibiotic in low-Pi media). This finding was surprising because the bacterium possesses the entire set of genes responsible for vancomycin resistance (VR); including those coding for the histidine kinase/response regulator pair VanS/VanR that activates the system. RESULTS: This work shows that high Pi amounts in the medium hamper the activation of the van promoters and consequently inhibit VR in S. coelicolor; i.e. the repression effect being stronger when basic or acidic forms of the nutrient are used. In addition, this work shows that lysozyme resistance is also highly regulated by the Pi concentration in the medium. At least five different mutations contribute to the overcoming of this repression effect over VR (but not over lysozyme resistance). Therefore, the interconnection of VR and lysozyme resistance mechanisms might be inexistent or complex. In particular, two kinds of mutant in which Pi control of VR has been lost (one class expresses the van genes in a constitutive manner; the other retains inducibility by vancomycin) have been isolated and further characterized in this study. Sequencing revealed that the first class of mutation conferred a single amino acid substitution in the second transmembrane helix of the VanS protein; whereas the other class hampered the expression or activity of a putative homolog (SCO1213) to the staphylococcal GatD protein. Complementation, phenotypic and bioinformatics analyses identified SCO1213, and its upstream gene (i.e. murT), as relevant genetic determinants involved with VR in S. coelicolor. CONCLUSION: The genomic approach of this study together with other genetic and phenotypic analyses has allowed the identification of the uncharacterized murT-gatD Streptomyces genes and the characterization of their involvement with the Pi control of VR in S. coelicolor.

Unraveling Nutritional Regulation of Tacrolimus Biosynthesis in Streptomyces tsukubaensis through omic Approaches.

Streptomyces tsukubaensis stands out among actinomycetes by its ability to produce the immunosuppressant tacrolimus. Discovered about 30 years ago, this macrolide is widely used as immunosuppressant in current clinics. Other potential applications for the treatment of cancer and as neuroprotective agent have been proposed in the last years. In this review we introduce the discovery of S. tsukubaensis and tacrolimus, its biosynthetic pathway and gene cluster (fkb) regulation. We have focused this work on the omic studies performed in this species in order to understand tacrolimus production. Transcriptomics, proteomics and metabolomics have improved our knowledge about the fkb transcriptional regulation and have given important clues about nutritional regulation of tacrolimus production that can be applied to improve production yields. Finally, we address some points of S. tsukubaensis biology that deserve more attention.

ylmD and ylmE genes are dispensable for growth, cross-wall formation and sporulation in Streptomyces venezuelae.

Streptomycetes are Gram-positive filamentous soil bacteria that grow by tip extension and branching, forming a network of multinucleoid hyphae. These bacteria also have an elaborate process of morphological differentiation, which involves the formation of an aerial mycelium that eventually undergoes extensive septation into chains of uninucleoid cells that further metamorphose into spores. The tubulin-like FtsZ protein is essential for this septation process. Most of the conserved cell division genes (including ftsZ) have been inactivated in Streptomyces without the anticipated lethality, based on studies of many other bacteria. However, there are still some genes of the Streptomyces division and cell wall (dcw) cluster that remain uncharacterized, the most notable example being the two conserved genes immediately adjacent to ftsZ (i.e. ylmDE). Here, for the first time, we made a ylmDE mutant in Streptomyces venezuelae and analysed it using epifluorescence microscopy, scanning electron microscopy (SEM) and atomic force microscopy (AFM). The mutant showed no significant effects on growth, cross-wall formation and sporulation in comparison to the wild type strain, which suggests that the ylmDE genes do not have an essential role in the Streptomyces cell division cycle (at least under the conditions of this study).

A mechanism for FtsZ-independent proliferation in Streptomyces.

The central player in bacterial cell division, FtsZ, is essential in almost all organisms in which it has been tested, with the most notable exception being Streptomyces. Streptomycetes differ from many bacteria in growing from the cell tip and undergoing branching, similar to filamentous fungi. Here we show that limited cell damage, either mechanical or enzymatic, leads to near complete destruction of mycelial microcolonies of a Streptomyces venezuelae ftsZ mutant. This result is consistent with a lack of ftsZ-dependent cross-walls and may be inconsistent with a recently proposed role for membrane structures in the proliferation of ftsZ mutants in other Streptomyces species. Rare surviving fragments of mycelium, usually around branches, appear to be the preferred sites of resealing. Restoration of growth in hyphal fragments of both wild-type and ftsZ mutant hyphae can occur at multiple sites, via branch-like outgrowths containing DivIVA protein at their tips. Thus, our results highlight branching as a means of FtsZ-independent cell proliferation.

Self-control of the PHO regulon: the PhoP-dependent protein PhoU controls negatively expression of genes of PHO regulon in Streptomyces coelicolor.

Phosphate control of the biosynthesis of secondary metabolites in Streptomyces is mediated by the two component system PhoR-PhoP. Linked to the phoR-phoP cluster, and expressed in the opposite orientation, is a phoU-like encoding gene with low identity to the phoU gene of Escherichia coli. Expression of this phoU-like gene is strictly dependent on PhoP activation. We have isolated a PhoU-null mutant and used transcriptomic and RNA-sequencing (RNA-seq) procedures to identify its transcription start site and regulation. RNA-seq studies identified two transcription start sites, one upstream of phoU and the second upstream of the mptA gene. Whereas transcription of PhoU is entirely dependent on PhoP, expression of the downstream mtpA gene is only partially dependent on PhoP activation. The phoU mutant grows more slowly than the parental strain, sporulates poorly and the spores lack pigmentation. Production of actinorhodin and undecylprodigiosin decreased in the phoU mutant, indicating that PhoU has a positive modulating effect on production of these antibiotics. Indeed, transcriptional studies of expression of the actII-ORF4 and redD genes indicated that the PhoU protein activates expression of these antibiotic regulators. Using the glpQ1 promoter as in vivo reporter of the activity of the PHO regulon genes, we observed that expression of glpQ1 is negatively modulated by PhoU. These results were confirmed by reverse transcription-PCR studies of three genes of the PHO regulon; that is, glpQ1, pstS and phoR. In conclusion, PhoU acts as a negative modulator of expression of the PHO regulon genes and as phoU expression is strictly dependent on PhoP activation, this mechanism appears to work as a feed-back control mechanism (self-regulation).The Journal of Antibiotics advance online publication, 1 November 2017; doi:10.1038/ja.2017.130.

Streptomyces tsukubaensis as a new model for carbon repression: transcriptomic response to tacrolimus repressing carbon sources.

In this work, we identified glucose and glycerol as tacrolimus repressing carbon sources in the important species Streptomyces tsukubaensis. A genome-wide analysis of the transcriptomic response to glucose and glycerol additions was performed using microarray technology. The transcriptional time series obtained allowed us to compare the transcriptomic profiling of S. tsukubaensis growing under tacrolimus producing and non-producing conditions. The analysis revealed important and different metabolic changes after the additions and a lack of transcriptional activation of the fkb cluster. In addition, we detected important differences in the transcriptional response to glucose between S. tsukubaensis and the model species Streptomyces coelicolor. A number of genes encoding key players of morphological and biochemical differentiation were strongly and permanently downregulated by the carbon sources. Finally, we identified several genes showing transcriptional profiles highly correlated to that of the tacrolimus biosynthetic pathway regulator FkbN that might be potential candidates for the improvement of tacrolimus production.

Analysis of the Pho regulon in Streptomyces tsukubaensis.

Phosphate regulation of antibiotic biosynthesis in Streptomyces has been studied due to the importance of this genus as a source of secondary metabolites with biological activity. Streptomyces tsukubaensis is the main producer of tacrolimus (or FK506), an immunosuppressant macrolide that generates important benefits for the pharmaceutical market. However, the production of tacrolimus is under a negative control by phosphate and, therefore, is important to know the molecular mechanism of this regulation. Despite its important role, there are no reports about the Pho regulon in S. tsukubaensis. In this work we combined transcriptional studies on the response to phosphate starvation with the search for PHO boxes in the whole genome sequence of S. tsukubaensis. As a result, we identified a set of genes responding to phosphate starvation and containing PHO boxes that include common Pho regulon members but also new species-specific candidates. In addition, we demonstrate for the first time the functional activity of PhoP from S. tsukubaensis through complementation studies in a Streptomyces coelicolor ΔphoP strain. For this purpose, we developed an anhydrotetracycline inducible system that can be applied to the controlled expression of target genes.