A structural basis for the assembly and functions of a viral polymer that inactivates multiple tumor suppressors.

Evolution of minimal DNA tumor virus' genomes has selected for small viral oncoproteins that hijack critical cellular protein interaction networks. The structural basis for the multiple and dominant functions of adenovirus oncoproteins has remained elusive. E4-ORF3 forms a nuclear polymer and simultaneously inactivates p53, PML, TRIM24, and MRE11/RAD50/NBS1 (MRN) tumor suppressors. We identify oligomerization mutants and solve the crystal structure of E4-ORF3. E4-ORF3 forms a dimer with a central ß core, and its structure is unrelated to known polymers or oncogenes. E4-ORF3 dimer units coassemble through reciprocal and nonreciprocal exchanges of their C-terminal tails. This results in linear and branched oligomer chains that further assemble in variable arrangements to form a polymer network that partitions the nuclear volume. E4-ORF3 assembly creates avidity-driven interactions with PML and an emergent MRN binding interface. This reveals an elegant structural solution whereby a small protein forms a multivalent matrix that traps disparate tumor suppressors.

The mechanism of prion inhibition by HET-S.

HET-S (97% identical to HET-s) has an N-terminal globular domain that exerts a prion-inhibitory effect in cis on its own prion-forming domain (PFD) and in trans on HET-s prion propagation. We show that HET-S fails to form fibrils in vitro and that it inhibits HET-s PFD fibrillization in trans. In vivo analyses indicate that beta-structuring of the HET-S PFD is required for HET-S activity. The crystal structures of the globular domains of HET-s and HET-S are highly similar, comprising a helical fold, while NMR-based characterizations revealed no differences in the conformations of the PFDs. We conclude that prion inhibition is not encoded by structure but rather in stability and oligomerization properties: when HET-S forms a prion seed or is incorporated into a HET-s fibril via its PFD, the beta-structuring in this domain induces a change in its globular domain, generating a molecular species that is incompetent for fibril growth.

Preparation and Characterization of Stable α-Synuclein Lipoprotein Particles.

Multiple neurodegenerative diseases are caused by the aggregation of the human α-Synuclein (α-Syn) protein. α-Syn possesses high structural plasticity and the capability of interacting with membranes. Both features are not only essential for its physiological function but also play a role in the aggregation process. Recently it has been proposed that α-Syn is able to form lipid-protein particles reminiscent of high-density lipoproteins. Here, we present a method to obtain a stable and homogeneous population of nanometer-sized particles composed of α-Syn and anionic phospholipids. These particles are called α-Syn lipoprotein (nano)particles to indicate their relationship to high-density lipoproteins formed by human apolipoproteins in vivo and of in vitro self-assembling phospholipid bilayer nanodiscs. Structural investigations of the α-Syn lipoprotein particles by circular dichroism (CD) and magic angle solid-state nuclear magnetic resonance (MAS SS-NMR) spectroscopy establish that α-Syn adopts a helical secondary structure within these particles. Based on cryo-electron microscopy (cryo-EM) and dynamic light scattering (DLS) α-Syn lipoprotein particles have a defined size with a diameter of ∼23 nm. Chemical cross-linking in combination with solution-state NMR and multiangle static light scattering (MALS) of α-Syn particles reveal a high-order protein-lipid entity composed of ∼8-10 α-Syn molecules. The close resemblance in size between cross-linked in vitro-derived α-Syn lipoprotein particles and a cross-linked species of endogenous α-Syn from SH-SY5Y human neuroblastoma cells indicates a potential functional relevance of α-Syn lipoprotein nanoparticles.

BMP-9 enhances fibroblast growth factor 21 expression and suppresses obesity.

Although BMP-9 has been reported to induce browning of white adipose tissues (WATs) and suppress high fat diet-induced obesity, detailed molecular mechanism needs to be further elucidated. We report here that administration of MB109, a recombinant derivative of human BMP-9, into obese mice enhanced gene expression of fibroblast growth factor 21 (FGF21), a metabolic regulator, and alleviates a spectrum of pathological symptoms due to high fat diet-induced obesity. In addition, periodical injection of MB109 (500µg/kg/week) reduced an amount of lipid droplets in the liver, serum levels of alanine aminotransferase (ALT), and total cholesterol. These results indicate that MB109 is also effective to treat obesity-mediated non-alcoholic fatty liver disease (NAFLD).

The effects of a single intravenous injection of novel activin A/BMP-2 (AB204) on toxicity and the respiratory and central nervous systems.

The purpose of this study was to determine the effects of a single intravenous injection of a novel osteoinductive material, activin A/BMP-2 (AB204), to rodents on toxicity and their respiratory functions and central nervous system (CNS). A single intravenous injection of AB204 was given to Sprague-Dawley (SD) rats in doses of 0, 0.625, 2.5 and 10 mg/kg to observe the mortality rate, the general symptoms for 14 days. The experimental groups were also given 0.2, 0.4 and 0.8 mg/kg of AB204, respectively, and the respiration rate, the tidal volume and the minute volume were measured for 240 min. The experimental groups of imprinting control region (ICR) mice were given a single intravenous injection of 0.2, 0.4 and 0.8 mg/kg of AB204, respectively. Their body temperature was taken and general behaviors were observed to evaluate the effect of AB204 on the CNS for 240 min. The study on toxicity of a single intravenous injection found no death or abnormal symptoms, abnormal findings from autopsy, or abnormal body weight gain or loss in all the experimental groups. No abnormal variation associated with the test substance was observed in the respiration rate, the tidal volume, the minute volume, body temperature or the general behaviors. On the basis of these results, the approximate lethal dose of AB204 for a single intravenous injection exceeds 10 mg/kg for SD rats and a single intravenous injection of ≤0.8 mg/kg AB204 has no effect on their respiratory system for SD rat and no effect on their CNS for ICR mice.

Solution NMR structure and functional analysis of the integral membrane protein YgaP from Escherichia coli.

The solution NMR structure of the α-helical integral membrane protein YgaP from Escherichia coli in mixed 1,2-diheptanoyl-sn-glycerol-3-phosphocholine/1-myristoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol) micelles is presented. In these micelles, YgaP forms a homodimer with the two transmembrane helices being the dimer interface, whereas the N-terminal cytoplasmic domain includes a rhodanese-fold in accordance to its sequence homology to the rhodanese family of sulfurtransferases. The enzymatic sulfur transfer activity of full-length YgaP as well as of the N-terminal rhodanese domain only was investigated performing a series of titrations with sodium thiosulfate and potassium cyanide monitored by NMR and EPR. The data indicate the thiosulfate concentration-dependent addition of several sulfur atoms to the catalytic Cys-63, which process can be reversed by the addition of potassium cyanide. The catalytic reaction induces thereby conformational changes within the rhodanese domain, as well as on the transmembrane α-helices of YgaP. These results provide insights into a potential mechanism of YgaP during the catalytic thiosulfate activity in vivo.

Designer nodal/BMP2 chimeras mimic nodal signaling, promote chondrogenesis, and reveal a BMP2-like structure.

Nodal, a member of the TGF-ß superfamily, plays an important role in vertebrate and invertebrate early development. The biochemical study of Nodal and its signaling pathway has been a challenge, mainly because of difficulties in producing the protein in sufficient quantities. We have developed a library of stable, chemically refoldable Nodal/BMP2 chimeric ligands (NB2 library). Three chimeras, named NB250, NB260, and NB264, show Nodal-like signaling properties including dependence on the co-receptor Cripto and activation of the Smad2 pathway. NB250, like Nodal, alters heart looping during the establishment of embryonic left-right asymmetry, and both NB250 and NB260, as well as Nodal, induce chondrogenic differentiation of human adipose-derived stem cells. This Nodal-induced differentiation is shown to be more efficient than BPM2-induced differentiation. Interestingly, the crystal structure of NB250 shows a backbone scaffold similar to that of BMP2. Our results show that these chimeric ligands may have therapeutic implications in cartilage injuries.

In Situ Formation of an Azo Bridge on Proteins Controllable by Visible Light.

Optical modulation of proteins provides superior spatiotemporal resolution for understanding biological processes, and photoswitches built on light-sensitive proteins have been significantly advancing neuronal and cellular studies. Small molecule photoswitches could complement protein-based switches by mitigating potential interference and affording high specificity for modulation sites. However, genetic encodability and responsiveness to nonultraviolet light, two desired properties possessed by protein photoswitches, are challenging to be engineered into small molecule photoswitches. Here we developed a small molecule photoswitch that can be genetically installed onto proteins in situ and controlled by visible light. A pentafluoro azobenzene-based photoswitchable click amino acid (F-PSCaa) was designed to isomerize in response to visible light. After genetic incorporation into proteins via the expansion of the genetic code, F-PSCaa reacts with a nearby cysteine within the protein generating an azo bridge in situ. The resultant bridge is switchable by visible light and allows conformation and binding of CaM to be regulated by such light. This photoswitch should prove valuable in optobiology for its minimal interference, site flexibility, genetic encodability, and response to the more biocompatible visible light.

Facile backbone structure determination of human membrane proteins by NMR spectroscopy.

Although nearly half of today's major pharmaceutical drugs target human integral membrane proteins (hIMPs), only 30 hIMP structures are currently available in the Protein Data Bank, largely owing to inefficiencies in protein production. Here we describe a strategy for the rapid structure determination of hIMPs, using solution NMR spectroscopy with systematically labeled proteins produced via cell-free expression. We report new backbone structures of six hIMPs, solved in only 18 months from 15 initial targets. Application of our protocols to an additional 135 hIMPs with molecular weight <30 kDa yielded 38 hIMPs suitable for structural characterization by solution NMR spectroscopy without additional optimization.

The toxicological evaluation of repetitive 2- and 4-week intravenous injection of Activin A/BMP-2 chimera (AB204) into rats.

The subchronic (28-days) toxicity of an Activin A/BMP-2 chimera (AB204) was assessed in rats. Sprague-Dawley rats received repetitive intravenous injection of AB204 in doses of 0, 0.25 and 0.5 mg/kg for two weeks and in doses of 0, 0.08, 0.16 and 0.32 mg/kg/day for four weeks. No animal was dead and no change caused by the AB204 was observed in general symptoms, weight variation, and food and water intake as well as blood test and autopsy findings. In conclusion, the no observed adverse effects level (NOAEL) of the AB204 on rats was determined to be 0.32 mg/kg/day.

Cotranslational cis-phosphorylation of the COOH-terminal tail is a key priming step in the maturation of cAMP-dependent protein kinase.

cAMP-dependent protein kinase A (PKA), ubiquitously expressed in mammalian cells, regulates a plethora of cellular processes through its ability to phosphorylate many protein substrates, including transcription factors, ion channels, apoptotic proteins, transporters, and metabolic enzymes. The PKA catalytic subunit has two phosphorylation sites, a well-studied site in the activation loop (Thr(197)) and another site in the C-terminal tail (Ser(338)) for which the role of phosphorylation is unknown. We show here, using in vitro studies and experiments with S49 lymphoma cells, that cis-autophosphorylation of Ser(338) occurs cotranslationally, when PKA is associated with ribosomes and precedes posttranslational phosphorylation of the activation loop Thr(197). Ser(338) phoshorylation is not required for PKA activity or formation of the holoenzyme complex; however, it is critical for processing and maturation of PKA, and it is a prerequisite for phosphorylation of Thr(197). After Thr(197) and Ser(338) are phosphorylated, both sites are remarkably resistant to phosphatases. Phosphatase resistance of the activation loop, a unique feature of both PKA and PKG, reflects the distinct way that signal transduction dynamics are controlled by cyclic nucleotide-dependent PKs.

Drug screening strategy for human membrane proteins: from NMR protein backbone structure to in silica- and NMR-screened hits.

About 8000 genes encode membrane proteins in the human genome. The information about their druggability will be very useful to facilitate drug discovery and development. The main problem, however, consists of limited structural and functional information about these proteins because they are difficult to produce biochemically and to study. In this paper we describe the strategy that combines Cell-free protein expression, NMR spectroscopy, and molecular DYnamics simulation (CNDY) techniques. Results of a pilot CNDY experiment provide us with a guiding light towards expedited identification of the hit compounds against a new uncharacterized membrane protein as a potentially druggable target. These hits can then be further characterized and optimized to develop the initial lead compound quicker. We illustrate such "omics" approach for drug discovery with the CNDY strategy applied to two example proteins: hypoxia-induced genes HIGD1A and HIGD1B.

An Activin A/BMP2 chimera, AB215, blocks estrogen signaling via induction of ID proteins in breast cancer cells.

BACKGROUND: One in eight women will be affected by breast cancer in her lifetime. Approximately 75% of breast cancers express estrogen receptor alpha (ERα) and/or progesterone receptor and these receptors are markers for tumor dependence on estrogen. Anti-estrogenic drugs such as tamoxifen are commonly used to block estrogen-mediated signaling in breast cancer. However, many patients either do not respond to these therapies (de novo resistance) or develop resistance to them following prolonged treatment (acquired resistance). Therefore, it is imperative to continue efforts aimed at developing new efficient and safe methods of targeting ER activity in breast cancer. METHODS: AB215 is a chimeric ligand assembled from sections of Activin A and BMP2. BMP2's and AB215's inhibition of breast cancer cells growth was investigated. In vitro luciferase and MTT proliferation assays together with western blot, RT_PCR, and mRNA knockdown methods were used to determine the mechanism of inhibition of estrogen positive breast cancer cells growth by BMP2 and AB215. Additionally in vivo xenograft tumor model was used to investigate anticancer properties of AB215. RESULTS: Here we report that AB215, a chimeric ligand assembled from sections of Activin A and BMP2 with BMP2-like signaling, possesses stronger anti-proliferative effects on ERα positive breast cancer cells than BMP2. We further show that AB215 inhibits estrogen signaling by inducing expression of inhibitor of DNA binding proteins (IDs). Specifically, we demonstrate that knockdown of ID proteins attenuates the anti-estrogen effects of AB215. Remarkably, we find that AB215 is more effective than tamoxifen in suppressing tumor growth in a xenograft model. CONCLUSION: This study shows that IDs have profound role to inhibit estrogen signaling in ERα positive breast cancer cells, and that engineered TGF-beta ligands may have high therapeutic value.

MB109 as bioactive human bone morphogenetic protein-9 refolded and purified from E. coli inclusion bodies.

BACKGROUND: The development of chemical refolding of transforming growth factor-beta (TGF-ß) superfamily ligands has been instrumental to produce the recombinant proteins for biochemical studies and exploring the potential of protein therapeutics. The osteogenic human bone morphogenetic protein-2 (hBMP-2) and its Drosophila DPP homolog were the early successful cases of refolding into functional form. Despite the similarity in their three dimensional structure and amino acid sequences, several other TGF-ß superfamily ligands could not be refolded readily by the same methods. RESULTS: Here, we report a comprehensive study on the variables of a rapid-dilution refolding method, including the concentrations of protein, salt, detergent and redox agents, pH, refolding duration and the presence of aggregation suppressors and host-cell contaminants, in order to identify the optimal condition to refold human BMP-9 (hBMP-9). To produce a recombinant form of hBMP-9 in E. coli cells, a synthetic codon-optimized gene was designed to encode the mature domain of hBMP-9 (Ser320 - Arg429) directly behind the first methionine, which we herein referred to as MB109. An effective purification scheme was also developed to purify the refolded MB109 to homogeneity with a final yield of 7.8 mg from 100 mg of chromatography-purified inclusion bodies as a starting material. The chemically refolded MB109 binds to ALK1, ActRIIb and BMPRII receptors with relatively high affinity as compared to other Type I and Type II receptors based on surface plasmon resonance analysis. Smad1-dependent luciferase assay in C2C12 cells shows that the MB109 has an EC50 of 0.61 ng/mL (25 pM), which is nearly the same as hBMP-9. CONCLUSION: MB109 is prone to be refolded as non-functional dimer and higher order multimers in most of the conditions tested, but bioactive MB109 dimer can be refolded with high efficiency in a narrow window, which is strongly dependent on the pH, refolding duration, the presence of aggregation suppressors and the concentrations of protein, salt and detergent. These results add to the current understanding of producing recombinant TGF-ß superfamily ligands in the microbial E. coli system. An application of the technique to produce a large number of synthetic TGF-ß chimeras for activity screen is also discussed.

Mechanism underlying selective regulation of G protein-gated inwardly rectifying potassium channels by the psychostimulant-sensitive sorting nexin 27.

G protein-gated inwardly rectifying potassium (GIRK) channels are important gatekeepers of neuronal excitability. The surface expression of neuronal GIRK channels is regulated by the psychostimulant-sensitive sorting nexin 27 (SNX27) protein through a class I (-X-Ser/Thr-X-Φ, where X is any residue and Φ is a hydrophobic amino acid) PDZ-binding interaction. The G protein-insensitive inward rectifier channel (IRK1) contains the same class I PDZ-binding motif but associates with a different synaptic PDZ protein, postsynaptic density protein 95 (PSD95). The mechanism by which SNX27 and PSD95 discriminate these channels was previously unclear. Using high-resolution structures coupled with biochemical and functional analyses, we identified key amino acids upstream of the channel's canonical PDZ-binding motif that associate electrostatically with a unique structural pocket in the SNX27-PDZ domain. Changing specific charged residues in the channel's carboxyl terminus or in the PDZ domain converts the selective association and functional regulation by SNX27. Elucidation of this unique interaction site between ion channels and PDZ-containing proteins could provide a therapeutic target for treating brain diseases.

Membrane domain structures of three classes of histidine kinase receptors by cell-free expression and rapid NMR analysis.

NMR structural studies of membrane proteins (MP) are hampered by complications in MP expression, technical difficulties associated with the slow process of NMR spectral peak assignment, and limited distance information obtainable for transmembrane (TM) helices. To overcome the inherent challenges in the determination of MP structures, we have developed a rapid and cost-efficient strategy that combines cell-free (CF) protein synthesis, optimized combinatorial dual-isotope labeling for nearly instant resonance assignment, and fast acquisition of long-distance information using paramagnetic probes. Here we report three backbone structures for the TM domains of the three classes of Escherichia coli histidine kinase receptors (HKRs). The ArcB and QseC TM domains are both two-helical motifs, whereas the KdpD TM domain comprises a four-helical bundle with shorter second and third helices. The interhelical distances (up to 12 A) reveal weak interactions within the TM domains of all three receptors. Determined consecutively within 8 months, these structures offer insight into the abundant and underrepresented in the Protein Data Bank class of 2-4 TM crossers and demonstrate the efficiency of our CF combinatorial dual-labeling strategy, which can be applied to solve MP structures in high numbers and at a high speed. Our results greatly expand the current knowledge of HKR structure, opening the doors to studies on their widespread and pharmaceutically important bacterial signaling mechanism.

Separation of domain contacts is required for heterotetrameric assembly of functional NMDA receptors.

The precise knowledge of the subunit assembly process of NMDA receptors (NMDA-Rs) is essential to understand the receptor architecture and underlying mechanism of channel function. Because NMDA-Rs are obligatory heterotetramers requiring the GluN1 subunit, it is critical to investigate how GluN1 and GluN2 type subunits coassemble into tetramers. By combining approaches in cell biology, biochemistry, single particle electron microscopy, and x-ray crystallography, we report the mechanisms and phenotypes of mutant GluN1 subunits that are defective in receptor maturation. The T110A mutation in the N-terminal domain (NTD) of the GluN1 promotes heterodimerization between the NTDs of GluN1 and GluN2, whereas the Y109C mutation in the adjacent residue stabilizes the homodimer of the NTD of GluN1. The crystal structure of the NTD of GluN1 revealed the mechanism underlying the biochemical properties of these mutants. Effects of these mutations on the maturation of heteromeric NMDA-Rs were investigated using a receptor trafficking assay. Our results suggest that the NTDs of the GluN1 subunit initially form homodimers and the subsequent dimer dissociation is critical for forming heterotetrameric NMDA-Rs containing GluN2 subunits, defining a molecular determinant for receptor assembly. The domain arrangement of the dimeric NTD of GluN1 is unique among the ionotropic glutamate receptors and predicts that the structure and mechanism around the NTDs of NMDA-Rs are different from those of the homologous AMPA and kainate receptors.

Bone morphogenic protein 9 is a novel thermogenic hepatokine secreted in response to cold exposure.

OBJECTIVE: Maintaining a constant core body temperature is essential to homeothermic vertebrate survival. Adaptive thermogenesis in brown adipose tissue and skeletal muscle is the primary mechanism of adjustment to an external stimulus such as cold exposure. Recently, several reports have revealed that the liver can play a role as a metabolic hub during adaptive thermogenesis. In this study, we suggest that the liver plays a novel role in secreting thermogenic factors in adaptive thermogenesis. Bone morphogenetic protein 9 (BMP9) is a hepatokine that regulates many biological processes, including osteogenesis, chondrogenesis, hematopoiesis, and angiogenesis. Previously, BMP9 was suggested to affect preadipocyte proliferation and differentiation. However, the conditions and mechanisms underlying hepatic expression and secretion and adipose tissue browning of BMP9 remain largely unknown. In this study, we investigated the physiological conditions for secretion and the regulatory mechanism of hepatic Bmp9 expression and the molecular mechanism by which BMP9 induces thermogenic gene program activation in adipose tissue. Here, we also present the pharmacological effects of BMP9 on a high-fat-induced obese mouse model. METHODS: To investigate the adaptive thermogenic role of BMP9 in vivo, we challenged mice with cold temperature exposure for 3 weeks and then examined the BMP9 plasma concentration and hepatic expression level. The cellular mechanism of hepatic Bmp9 expression under cold exposure was explored through promoter analysis. To identify the role of BMP9 in the differentiation of brown and beige adipocytes, we treated pluripotent stem cells and inguinal white adipose tissue (iWAT)-derived stromal-vascular (SV) cells with BMP9, and brown adipogenesis was monitored by examining thermogenic gene expression and signaling pathways. Furthermore, to evaluate the effect of BMP9 on diet-induced obesity, changes in body composition and glucose tolerance were analyzed in mice administered recombinant BMP9 (rBMP9) for 8 weeks. RESULTS: Hepatic Bmp9 expression and plasma levels in mice were significantly increased after 3 weeks of cold exposure. Bmp9 mRNA expression in the liver was regulated by transcriptional activation induced by cAMP response-element binding protein (CREB) and CREB-binding protein (CBP) on the Bmp9 promoter. Treatment with BMP9 promoted the differentiation of multipotent stem cells and iWAT-derived SV cells into beige adipocytes, as indicated by the increased expression of brown adipocyte and mitochondrial biogenesis markers. Notably, activation of the mothers against decapentaplegic homolog 1 (Smad1) and p44/p42 mitogen-activated protein kinase (MAPK) pathways was required for the induction of uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC1α) expression in BMP9-induced differentiation of SVs into beige adipocytes. The administration of rBMP9 in vivo also induced browning markers in white adipose tissue. In high-fat diet-induced obese mice, rBMP9 administration conferred protection against obesity and enhanced glucose tolerance. CONCLUSIONS: BMP9 is a hepatokine regulated by cold-activated CREB and CBP and enhances glucose and fat metabolism by promoting the activation of the thermogenic gene program in adipocytes. These data implicate BMP9 as a potential pharmacological tool for protecting against obesity and type 2 diabetes.

The desensitization gating of the MthK K+ channel is governed by its cytoplasmic amino terminus.

The RCK-containing MthK channel undergoes two inactivation processes: activation-coupled desensitization and acid-induced inactivation. The acid inactivation is mediated by the C-terminal RCK domain assembly. Here, we report that the desensitization gating is governed by a desensitization domain (DD) of the cytoplasmic N-terminal 17 residues. Deletion of DD completely removes the desensitization, and the process can be fully restored by a synthetic DD peptide added in trans. Mutagenesis analyses reveal a sequence-specific determinant for desensitization within the initial hydrophobic segment of DD. Proton nuclear magnetic resonance ((1)H NMR) spectroscopy analyses with synthetic peptides and isolated RCK show interactions between the two terminal domains. Additionally, we show that deletion of DD does not affect the acid-induced inactivation, indicating that the two inactivation processes are mutually independent. Our results demonstrate that the short N-terminal DD of MthK functions as a complete moveable module responsible for the desensitization. Its interaction with the C-terminal RCK domain may play a role in the gating process.

Transgenic microalgae expressing Escherichia coli AppA phytase as feed additive to reduce phytate excretion in the manure of young broiler chicks.

Microbial phytases are widely used as feed additive to increase phytate phosphorus utilization and to reduce fecal phytates and inorganic phosphate (iP) outputs. To facilitate the process of application, we engineered an Escherichia coli appA phytase gene into the chloroplast genome of the model microalga, Chlamydomonas reinhardtii, and isolated homoplasmic plastid transformants. The catalytic activity of the recombinant E. coli AppA can be directly detected in the whole-cell lysate, termed Chlasate, prepared by freeze-drying the transgenic cell paste with liquid nitrogen. The E. coli AppA in the Chlasate has a pH and temperature optima of 4.5 and 60°C, respectively, which are similar to those described in the literature. The phytase-expressed Chlasate contains 10 phytase units per gram dry matter at pH 4.5 and 37°C. Using this transgenic Chlasate at 500 U/kg of diet for young broiler chicks, the fecal phytate excretion was reduced, and the iP was increased by 43% and 41%, respectively, as compared to those of the chicks fed with only the basal diet. The effectiveness of the Chlasate to break down the dietary phytates is compatible with the commercial Natuphos fungal phytase. Our data provide the first evidence of functional expression of microbial phytase in microalgae and demonstrate the proof of concept of using transgenic microalgae as a food additive to deliver dietary enzymes with no need of protein purification.