The membrane associated accessory protein is an adeno-associated viral egress factor.

Adeno-associated viruses (AAV) rely on helper viruses to transition from latency to lytic infection. Some AAV serotypes are secreted in a pre-lytic manner as free or extracellular vesicle (EV)-associated particles, although mechanisms underlying such are unknown. Here, we discover that the membrane-associated accessory protein (MAAP), expressed from a frameshifted open reading frame in the AAV cap gene, is a novel viral egress factor. MAAP contains a highly conserved, cationic amphipathic domain critical for AAV secretion. Wild type or recombinant AAV with a mutated MAAP start site (MAAPΔ) show markedly attenuated secretion and correspondingly, increased intracellular retention. Trans-complementation with MAAP restored secretion of multiple AAV/MAAPΔ serotypes. Further, multiple processing and analytical methods corroborate that one plausible mechanism by which MAAP promotes viral egress is through AAV/EV association. In addition to characterizing a novel viral egress factor, we highlight a prospective engineering platform to modulate secretion of AAV vectors or other EV-associated cargo.

Antitumor Effect of IL-2 and TRAIL Proteins Expressed by Recombinant Salmonella in Murine Bladder Cancer Cells.

BACKGROUND/AIMS: Cancer is the second most deadly disease in the world. The bladder cancer is one of the most aggressive types and shows a continuous increase in the number of cases. The use of bacteria as live vectors to deliver molecules directly to the tumor is a promising tool and has been used as an adjuvant treatment against several types of cancer. The aim of this study was to investigate the antitumor effect of Interleukin 2 (IL-2), TNF-related apoptosis-inducing ligand (TRAIL) and protein MIX against murine bladder cancer cells, lineage MB49. METHODS: The attenuated Salmonella strain SL3261 was transformed by inserting the IL-2 and TRAIL genes. The effects of proteins on cell viability (MTT method), cell morphology (optical microscopy), cell recovery (clonogenic assay), cell membrane (lactate dehydrogenase release - LDH), on oxidative stress pathway (levels of nitric oxide, NO) and apoptosis (flow cytometry and high resolution epifluorescence images) were evaluated at intervals of 24 and 48 hours of action. RESULTS: The results showed that there was a decrease in cell viability via damage to the cell membrane, alteration of cell morphology, non-recovery of cells, increase in the production of NO and incubate for of cells in the state of apoptosis in the two periods analyzed. CONCLUSION: The data presented suggest that IL-2, TRAIL and their MIX proteins in MB49 cells have cytotoxic potential and that this is associated with oxidative stress and apoptosis pathways. These results may contribute to the development of new therapeutic strategies for bladder cancer.

Anillin/Mid1p interacts with the ESCRT-associated protein Vps4p and mitotic kinases to regulate cytokinesis in fission yeast.

Cytokinesis is the final stage of the cell cycle which separates cellular constituents to produce two daughter cells. Using the fission yeast Schizosaccharomyces pombe we have investigated the role of various classes of proteins involved in this process. Central to these is anillin/Mid1p which forms a ring-like structure at the cell equator that predicts the site of cell separation through septation in fission yeast. Here we demonstrate a direct physical interaction between Mid1p and the endosomal sorting complex required for transport (ESCRT)-associated protein Vps4p, a genetic interaction of the mid1 and vps4 genes essential for cell viability, and a requirement of Vps4p for the correct cellular localization of Mid1p. Furthermore, we show that Mid1p is phosphorylated by aurora kinase, a genetic interaction of the mid1 and the aurora kinase ark1 genes is essential for cell viability, and that Ark1p is also required for the correct cellular localization of Mid1p. We mapped the sites of phosphorylation of Mid1p by human aurora A and the polo kinase Plk1 and assessed their importance in fission yeast by mutational analysis. Such analysis revealed serine residues S332, S523 and S531 to be required for Mid1p function and its interaction with Vps4p, Ark1p and Plo1p. Combined these data suggest a physical interaction between Mid1p and Vps4p important for cytokinesis, and identify phosphorylation of Mid1p by aurora and polo kinases as being significant for this process.

Resveratrol Production in Yeast Hosts: Current Status and Perspectives.

Resveratrol is a plant secondary metabolite known for its therapeutic applications as an antioxidant, anti-cancer, anti-inflammatory, anti-aging, cardio-protective, and neuroprotective agent. Topical formulas of resveratrol are also used for skin disease management and in cosmetic industries. Due to its importance, high resveratrol production is urgently required. Since the last decade, intensive efforts have been devoted to obtaining resveratrol from microorganisms by pathway and metabolic engineering. Yeasts were proven to be excellent host candidates for resveratrol production. In addition to the similar intracellular compartments between yeasts and plants, yeasts exhibit the ability to express genes coding for plant-derived enzymes and to perform post-translational modification. Therefore, this review summarizes the attempts to use yeasts as a platform for resveratrol synthesis as the next promising route in producing high titers of resveratrol from genetically engineered strains.

Function Enhancement of a Metabolic Module via Endogenous Promoter Replacement for Pseudomonas sp. JY-Q to Degrade Nicotine in Tobacco Waste Treatment.

Nicotine-degrading Pseudomonas sp. JY-Q is a preferred strain utilized in reconstituted tobacco process for tobacco waste treatment. However, its efficiency of nicotine metabolism still requires to be improved via genomic technology such as promoter engineering based on genomic information. Concerning upstream module of nicotine metabolic pathway, we found that two homologous genes of nicotine dehydrogenase (nicA2 and nox) coexisted in strain JY-Q. However, the transcriptional amount of nox was 20-fold higher than that of nicA2. Thus, the nicA2 expression required improvement. Combinatorial displacement was accomplished for two predicted endogenous promoters, named as PnicA2 and Pnox for nicA2 and nox, respectively. The mutant with Pnox as the promoters for both nicA2 and nox exhibited the best nicotine metabolic capacity which increased by 66% compared to the wild type. These results suggested that endogenous promoter replacement is also feasible for function improvement of metabolic modules and strain enhancement of biodegradation capacity to meet real environment demand.

Plasmid-Free System and Modular Design for Efficient 5-Aminolevulinic Acid Production by Engineered Escherichia coli.

5-Aminolevulinic acid (ALA) is an essential intermediate for many organisms and has been considered for the applications of medical especially in photodynamic therapy of cancer recently. However, ALA production via chemical approach is complicated; hence, microbial manufacturing has received more attentions. In this study, a modular design to simultaneously express ALA synthase from Rhodobacter sphaeroides (RshemA), a non-specific ALA exporter (RhtA), and chaperones was first developed and discussed. The ALA production was significantly increased by coexpressing RhtA and RshemA. Besides, ALA was enhanced by the cofactor pyridoxal phosphate (PLP) which was supplied by expressing genes of pdxK and pdxY or direct addition. However, inclusion bodies of RshemA served as an obstacle; thus, chaperones DnaK and GroELS were introduced to reform the conformation of proteins and successfully improved ALA production. Finally, a plasmid-free strain RrGI, as the robust chassis, was established and a 6.23-fold enhancement on ALA biosynthesis and led to 7.47 g/L titer and 0.588 g/L/h productivity under the optimal cultural condition.

Bioelectronic control of a microbial community using surface-assembled electrogenetic cells to route signals.

We developed a bioelectronic communication system that is enabled by a redox signal transduction modality to exchange information between a living cell-embedded bioelectronics interface and an engineered microbial network. A naturally communicating three-member microbial network is 'plugged into' an external electronic system that interrogates and controls biological function in real time. First, electrode-generated redox molecules are programmed to activate gene expression in an engineered population of electrode-attached bacterial cells, effectively creating a living transducer electrode. These cells interpret and translate electronic signals and then transmit this information biologically by producing quorum sensing molecules that are, in turn, interpreted by a planktonic coculture. The propagated molecular communication drives expression and secretion of a therapeutic peptide from one strain and simultaneously enables direct electronic feedback from the second strain, thus enabling real-time electronic verification of biological signal propagation. Overall, we show how this multifunctional bioelectronic platform, termed a BioLAN, reliably facilitates on-demand bioelectronic communication and concurrently performs programmed tasks.

Enhancement of undecylprodigiosin production from marine endophytic recombinant strain Streptomyces sp. ALAA-R20 through low-cost induction strategy.

Genetic manipulation of the undecylprodigiosin-producing strains and engineered culture medium approaches were applied as the most economical induction strategy for improving production. The hyper-producing recombinant strain ALAA-R20 was obtained after applying protoplast fusion strategy between the potent producer marine endophytic strains Streptomyces sp. ESRAA-10 (P1) and Streptomyces sp. ESRAA-31 (P2) of Dendronephthya hemprichi. Recombinant strain ALAA-R20 produced undecylprodigiosin yield higher than its parental strains ESRAA-10 and ESRAA-31 by 82.45% and 105.52% under submerged fermentation using modified R2YE medium. In order to reduce the costs of producing undecylprodigiosin, a solid-state fermentation (SSF) was applied. Scaled-up of optimized SSF parameters consisting of groundnut oil cake (GOC) sized to 3 mm, initial moisture content 80% with a mixture of dairy mill and fruit processing wastewaters (1:1), pH 7.0, inoculum size equal to 3 × 105 spores/g dry substrate (gds), incubation temperature 30 °C, and 7-day incubation period yielded the highest yield of 181.78 mg/gds of undecylprodigiosin by the recombinant strain Streptomyces sp. ALAA-R20. Extraction and purification of the pigment using the chromatographic techniques as well as mass spectral analysis exhibited maximum absorbance at 539 nm which is physiological property of the undecylprodigiosin. Undecylprodigiosin was stable over a wide temperature ranged from - 20 to 35 °C even after storage for 6 months. The maximum yield and stability of pigment was obtained at the acidic pH (acidified methanol, pH 4.0). Undecylprodigiosin obtained from the recombinant strain Streptomyces sp. ALAA-R20 demonstrated strong antimicrobial activity against all multidrug-resistant bacterial and fungal strains tested with minimum inhibitory, minimum bactericidal, and minimum fungicidal concentrations ranged between 0.5 and 4.0, 0.5 to 4.0, and 1.0 to 8.0 µg/mL, respectively. It also showed complete inhibition of cancer cells; HCT-116, HepG-2, MCF-7 and A-549 at 5, 8, 4, and 7 µM with IC50 equal to 2.0, 4.7, 1.2, and 2.8 µM, respectively.

The C-terminal region of the oxidoreductase MIA40 stabilizes its cytosolic precursor during mitochondrial import.

BACKGROUND: The mitochondrial intermembrane space (IMS) is home to proteins fulfilling numerous essential cellular processes, particularly in metabolism and mitochondrial function. All IMS proteins are nuclear encoded and synthesized in the cytosol and must therefore be correctly targeted and transported to the IMS, either through mitochondrial targeting sequences or conserved cysteines and the mitochondrial disulfide relay system. The mitochondrial oxidoreductase MIA40, which catalyzes disulfide formation in the IMS, is imported by the combined action of the protein AIFM1 and MIA40 itself. Here, we characterized the function of the conserved highly negatively charged C-terminal region of human MIA40. RESULTS: We demonstrate that the C-terminal region is critical during posttranslational mitochondrial import of MIA40, but is dispensable for MIA40 redox function in vitro and in intact cells. The C-terminal negatively charged region of MIA40 slowed import into mitochondria, which occurred with a half-time as slow as 90 min. During this time, the MIA40 precursor persisted in the cytosol in an unfolded state, and the C-terminal negatively charged region served in protecting MIA40 from proteasomal degradation. This stabilizing property of the MIA40 C-terminal region could also be conferred to a different mitochondrial precursor protein, COX19. CONCLUSIONS: Our data suggest that the MIA40 precursor contains the stabilizing information to allow for postranslational import of sufficient amounts of MIA40 for full functionality of the essential disulfide relay. We thereby provide for the first time mechanistic insights into the determinants controlling cytosolic surveillance of IMS precursor proteins.

Synthesis of acridone derivatives via heterologous expression of a plant type III polyketide synthase in Escherichia coli.

BACKGROUND: Acridone alkaloids are heterocyclic compounds that exhibit a broad-range of pharmaceutical and chemotherapeutic activities, including anticancer, antiviral, anti-inflammatory, antimalarial, and antimicrobial effects. Certain plant species such as Citrus microcarpa, Ruta graveolens, and Toddaliopsis bremekampii synthesize acridone alkaloids from anthranilate and malonyl-CoA. RESULTS: We synthesized two acridones in Escherichia coli. Acridone synthase (ACS) and anthraniloyl-CoA ligase genes were transformed into E. coli, and the synthesis of acridone was examined. To increase the levels of endogenous anthranilate, we tested several constructs expressing proteins involved in the shikimate pathway and selected the best construct. To boost the supply of malonyl-CoA, genes coding for acetyl-coenzyme A carboxylase (ACC) from Photorhabdus luminescens were overexpressed in E. coli. For the synthesis of 1,3-dihydroxy-10-methylacridone, we utilized an N-methyltransferase gene (NMT) to supply N-methylanthranilate and a new N-methylanthraniloyl-CoA ligase. After selecting the best combination of genes, approximately 17.3 mg/L of 1,3-dihydroxy-9(10H)-acridone (DHA) and 26.0 mg/L of 1,3-dihydroxy-10-methylacridone (NMA) were synthesized. CONCLUSIONS: Two bioactive acridone derivatives were synthesized by expressing type III plant polyketide synthases and other genes in E. coli, which increased the supplement of substrates. This study showed that is possible to synthesize diverse polyketides in E. coli using plant polyketide synthases.

Blakeslea trispora Photoreceptors: Identification and Functional Analysis.

Blakeslea trispora is an industrial fungal species used for large-scale production of carotenoids. However, B. trispora light-regulated physiological processes, such as carotenoid biosynthesis and phototropism, are not fully understood. In this study, we isolated and characterized three photoreceptor genes, btwc-1a, btwc-1b, and btwc-1c, in B. trispora Bioinformatics analyses of these genes and their protein sequences revealed that the functional domains (PAS/LOV [Per-ARNT-Sim/light-oxygen-voltage] domain and zinc finger structure) of the proteins have significant homology to those of other fungal blue-light regulator proteins expressed by Mucor circinelloides and Neurospora crassa The photoreceptor proteins were synthesized by heterologous expression in Escherichia coli The chromogenic groups consisting of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) were detected to accompany BTWC-1 proteins by using high-performance liquid chromatography (HPLC) and fluorescence spectrometry, demonstrating that the proteins may be photosensitive. The absorbance changes of the purified BTWC-1 proteins seen under dark and light conditions indicated that they were light responsive and underwent a characteristic photocycle by light induction. Site-directed mutagenesis of the cysteine residual (Cys) in BTWC-1 did not affect the normal expression of the protein in E. coli but did lead to the loss of photocycle response, indicating that Cys represents a flavin-binding domain for photon detection. We then analyzed the functions of BTWC-1 proteins by complementing btwc-1a, btwc-1b, and btwc-1c into the counterpart knockout strains of M. circinelloides for each mcwc-1 gene. Transformation of the btwc-1a complement into mcwc-1a knockout strains restored the positive phototropism, while the addition of btwc-1c complement remedied the deficiency of carotene biosynthesis in the mcwc-1c knockout strains under conditions of illumination. These results indicate that btwc-1a and btwc-1c are involved in phototropism and light-inducible carotenogenesis. Thus, btwc-1 genes share a conserved flavin-binding domain and act as photoreceptors for control of different light transduction pathways in B. trisporaIMPORTANCE Studies have confirmed that light-regulated carotenogenesis is prevalent in filamentous fungi, especially in mucorales. However, few investigations have been done to understand photoinduced synthesis of carotenoids and related mechanisms in B. trispora, a well-known industrial microbial strains. In the present study, three photoreceptor genes in B. trispora were cloned, expressed, and characterized by bioinformatics and photoreception analyses, and then in vivo functional analyses of these genes were constructed in M. circinelloides The results of this study will lead to a better understanding of photoreception and light-regulated carotenoid synthesis and other physiological responses in B. trispora.

Improving synthetic methylotrophy via dynamic formaldehyde regulation of pentose phosphate pathway genes and redox perturbation.

Escherichia coli is an ideal choice for constructing synthetic methylotrophs capable of utilizing the non-native substrate methanol as a carbon and energy source. All current E. coli-based synthetic methylotrophs require co-substrates. They display variable levels of methanol-carbon incorporation due to a lack of native regulatory control of biosynthetic pathways, as E. coli does not recognize methanol as a proper substrate despite its ability to catabolize it. Here, using the E. coli formaldehyde-inducible promoter Pfrm, we implement dynamic expression control of select pentose-phosphate genes in response to the formaldehyde produced upon methanol oxidation. Genes under Pfrm control exhibited 8- to 30-fold transcriptional upregulation during growth on methanol. Formaldehyde-induced episomal expression of the B. methanolicus rpe and tkt genes involved in the regeneration of ribulose 5-phosphate required for formaldehyde fixation led to significantly improved methanol assimilation into intracellular metabolites, including a 2-fold increase of 13C-methanol into glutamate. Using a simple strategy for redox perturbation by deleting the E. coli NAD-dependent malate dehydrogenase gene maldh, we demonstrate 5-fold improved biomass formation of cells growing on methanol in the presence of a small concentration of yeast extract. Further improvements in methanol utilization are achieved via adaptive laboratory evolution and heterologous rpe and tkt expression. A short-term in vivo13C-methanol labeling assay was used to determine methanol assimilation activity for Δmaldh strains, and demonstrated dramatically higher labeling in intracellular metabolites, including a 6-fold and 1.8-fold increase in glycine labeling for the rpe/tkt and evolved strains, respectively. The combination of formaldehyde-controlled pentose phosphate pathway expression and redox perturbation with the maldh knock-out greatly improved both growth benefit with methanol and methanol carbon incorporation into intracellular metabolites.

Efficient biosynthesis of antitumor ganoderic acid HLDOA using a dual tunable system for optimizing the expression of CYP5150L8 and a Ganoderma P450 reductase.

Ganoderic acid 3-hydroxy-lanosta-8,24-dien-26-oic acid (GA-HLDOA), an antitumor triterpenoid from the traditional Chinese medicinal higher fungus Ganoderma lucidum, is considered as a key precursor for biosynthesizing other ganoderic acids (GAs) with superior antitumor activities. Our previous study identified CYP5150L8 from G. lucidum as a lanosterol oxidase, and achieved heterologous biosynthesis of GA-HLDOA in Saccharomyces cerevisiae. However, low production of GA-HLDOA in either G. lucidum or heterologous host hindered its further investigation and application. In this study, we constructed a dual tunable system for balancing the expression of CYP5150L8 and a Ganoderma P450 reductase iGLCPR, and performed a comprehensive optimization of CYP5150L8 expression, iGLCPR expression, and glycerol usage. Then, we investigated the fermentation behavior of the best strain in optimized condition in flask and achieved 154.45 mg/L GA-HLDOA production, which was 10.7-fold higher compared with previous report. This study may facilitate the wide-spread application of GA-HLDOA and the discovery of unknown cytochrome P450s in downstream GAs biosynthesis.

Tat-Dependent Heterologous Secretion of Recombinant Tyrosinase by Pseudomonas fluorescens Is Aided by a Translationally Fused Caddie Protein.

Tyrosinase is a monooxygenase that catalyzes both the hydroxylation of p-hydroxyphenyl moieties to o-catechols and the oxidation of o-catechols to o-quinones. Apart from its critical functionality in melanogenesis and the synthesis of various neurotransmitters, this enzyme is also used in a variety of biotechnological applications, most notably mediating covalent cross-linking between polymers containing p-hydroxyphenyl groups, forming a hydrogel. Tyrosinases from the genus Streptomyces are usually secreted as a complex with their caddie protein. In this study, we report an increased secretion efficiency observed when the Streptomyces antibioticus tyrosinase gene melC2 was introduced into Pseudomonas fluorescens along with its caddie protein gene melC1, which has the DNA sequence for the Tat (twin-arginine translocation) signal.IMPORTANCE We observed that the S. antibioticus extracellular tyrosinase secretion level was even higher in its nonnatural translationally conjugated fusion protein form than in the natural complex of two separated polypeptides. The results of this study demonstrate that tyrosinase-expressing P. fluorescens can be a stable source of bacterial tyrosinase through exploiting the secretory machinery of P. fluorescens.

High level production of flavonoid rhamnosides by metagenome-derived Glycosyltransferase C in Escherichia coli utilizing dextrins of starch as a single carbon source.

Flavonoids exert a wide variety of biological functions that are highly attractive for the pharmaceutical and healthcare industries. However, their application is often limited by low water solubility and poor bioavailability, which can generally be relieved through glycosylation. Glycosyltransferase C (GtfC), a metagenome-derived, bacterial glycosyltransferase, was used to produce novel and rare rhamnosides of various flavonoids, including chrysin, diosmetin, biochanin A, and hesperetin. Some of them are to our knowledge firstly described within this work. In our study we deployed a new metabolic engineering approach to increase the rhamnosylation rate in Escherichia coli whole cell biotransformations. The coupling of maltodextrin metabolism to glycosylation was developed in E. coli MG1655 with the model substrate hesperetin. The process proved to be highly dependent on the availability of maltodextrins. Maximal production was achieved by the deletion of the phosphoglucomutase (pgm) and UTP-glucose-1-phosphate uridyltransferase (galU) genes and simultaneous overexpression of the dTDP-rhamnose synthesis genes (rmlABCD) as well as glucan 1,4-alpha-maltohexaosidase for increased maltodextrin degradation next to GtfC in E. coli UHH_CR5-A. These modifications resulted in a 3.2-fold increase of hesperetin rhamnosides compared to E. coli MG1655 expressing GtfC in 24 h batch fermentations. Furthermore, E. coli UHH-CR_5-A was able to produce a final product titer of 2.4 g/L of hesperetin-3'-O-rhamnoside after 48 h. To show the versatility of the engineered E. coli strain, biotransformations of quercetin and kaempferol were performed, leading to production of 4.3 g/L quercitrin and 1.9 g/L afzelin in a 48 h time period, respectively. So far, these are the highest published yields of flavonoid rhamnosylation using a biotransformation approach. These results clearly demonstrate the high potential of the engineered E. coli production host as a platform for the high level biotransformation of flavonoid rhamnosides.

Biotechnological production of glycolic acid and ethylene glycol: current state and perspectives.

Glycolic acid (GA) and ethylene glycol (EG) are versatile two-carbon organic chemicals used in multiple daily applications. GA and EG are currently produced by chemical synthesis, but their biotechnological production from renewable resources has received a substantial interest. Several different metabolic pathways by using genetically modified microorganisms, such as Escherichia coli, Corynebacterium glutamicum and yeast have been established for their production. As a result, the yield of GA and EG produced from sugars has been significantly improved. Here, we describe the recent advancement in metabolic engineering efforts focusing on metabolic pathways and engineering strategies used for GA and EG production.

High-yield production of L-serine from glycerol by engineered Escherichia coli.

L-Serine is widely used in pharmaceutical, food and cosmetic industries, and the direct fermentation to produce L-serine from cheap carbon sources such as glycerol is greatly desired. The production of L-serine by engineered Escherichia coli from glycerol has not been achieved so far. In this study, E. coli was engineered to efficiently produce L-serine from glycerol. To this end, three L-serine deaminase genes were deleted in turn, and all of the deletions caused the maximal accumulation of L-serine at 0.06 g/L. Furthermore, removal of feedback inhibition by L-serine resulted in a titer of 1.1 g/L. Additionally, adaptive laboratory evolution was employed to improve glycerol utilization in combination with the overexpression of the cysteine/acetyl serine transporter gene eamA, leading to 2.36 g/L L-serine (23.6% of the theoretical yield). In 5-L bioreactor, L-serine titer could reach up to 7.53 g/L from glycerol, demonstrating the potential of the established strain and bioprocess.

Filamentous fungi-like secretory pathway strayed in a yeast system: peculiarities of Yarrowia lipolytica secretory pathway underlying its extraordinary performance.

Microbial production of secretory proteins constitutes one of the key branches of current industrial biotechnology, earning billion dollar (USD) revenues each year. That industrial branch strongly relies on fluent operation of the secretory machinery within a microbial cell. The secretory machinery, directing the nascent polypeptide to its final destination, constitutes a highly complex system located across the eukaryotic cell. Numerous molecular identities of diverse structure and function not only build the advanced network assisting folding, maturation and secretion of polypeptides but also serve as sensors and effectors of quality control points. All these events must be harmoniously orchestrated to enable fluent processing of the protein traffic. Availability of these elements is considered to be the limiting factor determining capacity of protein traffic, which is of crucial importance upon biotechnological production of secretory proteins. The main purpose of this work is to review and discuss findings concerning secretory machinery operating in a non-conventional yeast species, Yarrowia lipolytica, and to highlight peculiarities of this system prompting its use as the production host. The reviewed literature supports the thesis that secretory machinery in Y. lipolytica is characterized by significantly higher complexity than a canonical yeast protein secretion pathway, making it more similar to filamentous fungi-like systems in this regard.

De novo biosynthesis of myricetin, kaempferol and quercetin in Streptomyces albus and Streptomyces coelicolor.

Flavonols are a flavonoid subfamily widely distributed in plants, including several ones of great importance in human and animal diet (apple, tomato, broccoli, onion, beans, tea). These polyphenolic nutraceuticals exert potent antimicrobial (membrane potential disruptors), antioxidant (free-radical scavengers), pharmacokinetic (CYP450 modulators), anti-inflammatory (lipoxygenase inhibitors), antiangiogenic (VEGF inhibitors) and antitumor (cyclin inhibitors) activities. Biotechnological production of these nutraceuticals, for example via heterologous biosynthesis in industrial actinomycetes, is favored since in plants these polyphenols appear as inactive glycosylated derivatives, in low concentrations or as part of complex mixtures with other polyphenolic compounds. In this work, we describe the de novo biosynthesis of three important flavonols, myricetin, kaempferol and quercetin, in the industrially relevant actinomycetes Streptomyces coelicolor and S. albus. De novo biosynthesis of kaempferol, myricetin and quercetin in actinomycetes has not been described before.

Polyprotein strategy for stoichiometric assembly of nitrogen fixation components for synthetic biology.

Re-engineering of complex biological systems (CBS) is an important goal for applications in synthetic biology. Efforts have been made to simplify CBS by refactoring a large number of genes with rearranged polycistrons and synthetic regulatory circuits. Here, a posttranslational protein-splicing strategy derived from RNA viruses was exploited to minimize gene numbers of the classic nitrogenase system, where the expression stoichiometry is particularly important. Operon-based nif genes from Klebsiella oxytoca were regrouped into giant genes either by fusing genes together or by expressing polyproteins that are subsequently cleaved with Tobacco Etch Virus protease. After several rounds of selection based on protein expression levels and tolerance toward a remnant C-terminal ENLYFQ-tail, a system with only five giant genes showed optimal nitrogenase activity and supported diazotrophic growth of Escherichia coli This study provides an approach for efficient translation from an operon-based system into a polyprotein-based assembly that has the potential for portable and stoichiometric expression of the complex nitrogenase system in eukaryotic organisms.