Algal chloroplast produced camelid VH H antitoxins are capable of neutralizing botulinum neurotoxin.

We have produced three antitoxins consisting of the variable domains of camelid heavy chain-only antibodies (VH H) by expressing the genes in the chloroplast of green algae. These antitoxins accumulate as soluble proteins capable of binding and neutralizing botulinum neurotoxin. Furthermore, they accumulate at up to 5% total soluble protein, sufficient expression to easily produce these antitoxins at scale from algae. The genes for the three different antitoxins were transformed into Chlamydomonas reinhardtii chloroplasts and their products purified from algae lysates and assayed for in vitro biological activity using toxin protection assays. The produced antibody domains bind to botulinum neurotoxin serotype A (BoNT/A) with similar affinities as camelid antibodies produced in Escherichia coli, and they are similarly able to protect primary rat neurons from intoxication by BoNT/A. Furthermore, the camelid antibodies were produced in algae without the use of solubilization tags commonly employed in E. coli. These camelid antibody domains are potent antigen-binding proteins and the heterodimer fusion protein containing two VH H domains was capable of neutralizing BoNT/A at near equimolar concentrations with the toxin. Intact antibody domains were detected in the gastrointestinal (GI) tract of mice treated orally with antitoxin-producing microalgae. These findings support the use of orally delivered antitoxins produced in green algae as a novel treatment for botulism.

Bioprospecting of microalgae for integrated biomass production and phytoremediation of unsterilized wastewater and anaerobic digestion centrate.

Eighteen microalgae, including two local isolates, were evaluated for their ability to grow and remove nutrients from unsterilized primary or secondary wastewater effluents as well as wastewater supplemented with nutrient-rich anaerobic digester centrate (ADC). Most of the tested species except several phylogenetically clustered Chlorella sorokiniana including local isolates and Scenedesmus strains were unable to grow efficiently. This may reflect the presence of certain genetic traits important for robust growth in the unsterilized wastewater. The maximum algal-specific growth rates and biomass density obtained in these bacterial-contaminated cultures were in the range of 0.8-1 day(-1) and 250-350 mg L(-1), respectively. ADC supplementation was especially helpful to biologically treated secondary effluent with its lower initial macronutrient and micronutrient content. As a result of algal growth, total nitrogen and orthophosphate levels were reduced by as much as 90 and 70 %, respectively. Biological assimilation was estimated to be the main mechanism of nitrogen removal in primary and secondary effluents with ammonia volatilization and bacterial nitrification-denitrification contributing for cultures supplemented with ADC. Assimilation by algae served as the principal mechanism of orthophosphate remediation in secondary wastewater cultures, while chemical precipitation appeared also to be important for orthophosphate removal in primary wastewater. Overall, cultivation of microalgae in primary and primary + 5 % ADC may be more favorable from an economical and sustainability perspective due to elimination of the costly and energy-intensive biological treatment step. These findings demonstrate that unsterilized wastewater and ADC can serve as critical nutrient sources for biomass generation and that robust microalgae can be potent players in wastewater phytoremediation.

Generation of a phage-display library of single-domain camelid VH H antibodies directed against Chlamydomonas reinhardtii antigens, and characterization of VH Hs binding cell-surface antigens.

Single-domain antibodies (sdAbs) are powerful tools for the detection, quantification, purification and subcellular localization of proteins of interest in biological research. We have generated camelid (Lama pacos) heavy chain-only variable VH domain (VH H) libraries against antigens in total cell lysates from Chlamydomonas reinhardtii. The sdAbs in the sera from immunized animals and VH H antibody domains isolated from the library show specificity to C. reinhardtii and lack of reactivity to antigens from four other algae: Chlorella variabilis, Coccomyxa subellipsoidea, Nannochloropsis oceanica and Thalassiosira pseudonana. Antibodies were produced against a diverse representation of antigens as evidenced by sera ELISA and protein-blot analyses. A phage-display library consisting of the VH H region contained at least 10(6) individual transformants, and thus should represent a wide range of C. reinhardtii antigens. The utility of the phage library was demonstrated by using live C. reinhardtii cells to pan for VH H clones with specific recognition of cell-surface epitopes. The lead candidate VH H clones (designated B11 and H10) bound to C. reinhardtii with EC50 values ≤ 0.5 nm. Treatment of cells with VH H B11 fused to the mCherry or green fluorescent proteins allowed brilliant and specific staining of the C. reinhardtii cell wall and analysis of cell-wall genesis during cell division. Such high-complexity VH H antibody libraries for algae will be valuable tools for algal researchers and biotechnologists.

Expanding the spectral palette of fluorescent proteins for the green microalga Chlamydomonas reinhardtii.

Fluorescent proteins (FPs) have become essential tools for a growing number of fields in biology. However, such tools have not been widely adopted for use in microalgal research. The aim of this study was to express and compare six FPs (blue mTagBFP, cyan mCerulean, green CrGFP, yellow Venus, orange tdTomato and red mCherry) in the popular model microalga Chlamydomonas reinhardtii. To circumvent the transgene silencing that often occurs in C. reinhardtii, the FPs were expressed from the nuclear genome as transcriptional fusions with the sh-ble antibiotic resistance gene, with the foot and mouth disease virus 2A self-cleaving sequence placed between the coding sequences. All ble-2A-FPs tested are well-expressed and efficiently processed to yield mature, unfused FPs that localize throughout the cytoplasm. The fluorescence signals of each FP were detectable in whole cells by fluorescence microplate reader analysis, live-cell fluorescence microscopy, and flow cytometry. Furthermore, we report a comparative analysis of fluorescence levels relative to auto-fluorescence for the chosen FPs. Finally, we demonstrate that the ble-2A expression vector may be used to fluorescently label an endogenous protein (α-tubulin). We show that the mCerulean-α-tubulin fusion protein localizes to the cytoskeleton and flagella, as expected, and that cells containing this fusion protein had normal cellular function. Overall, our results indicate that, by use of the ble-2A nuclear expression construct, a wide array of FP tools and technologies may be applied to microalgal research, opening up many possibilities for microalgal biology and biotechnology.

A rapid live-cell ELISA for characterizing antibodies against cell surface antigens of Chlamydomonas reinhardtii and its use in isolating algae from natural environments with related cell wall components.

BACKGROUND: Cell walls are essential for most bacteria, archaea, fungi, algae and land plants to provide shape, structural integrity and protection from numerous biotic and abiotic environmental factors. In the case of eukaryotic algae, relatively little is known of the composition, structure or mechanisms of assembly of cell walls in individual species or between species and how these differences enable algae to inhabit a great diversity of environments. In this paper we describe the use of camelid antibody fragments (VHHs) and a streamlined ELISA assay as powerful new tools for obtaining mono-specific reagents for detecting individual algal cell wall components and for isolating algae that share a particular cell surface component. RESULTS: To develop new microalgal bioprospecting tools to aid in the search of environmental samples for algae that share similar cell wall and cell surface components, we have produced single-chain camelid antibodies raised against cell surface components of the single-cell alga, Chlamydomonas reinhardtii. We have cloned the variable-region domains (VHHs) from the camelid heavy-chain-only antibodies and overproduced tagged versions of these monoclonal-like antibodies in E. coli. Using these VHHs, we have developed an accurate, facile, low cost ELISA that uses live cells as a source of antigens in their native conformation and that requires less than 90 minutes to perform. This ELISA technique was demonstrated to be as accurate as standard ELISAs that employ proteins from cell lysates and that generally require >24 hours to complete. Among the cloned VHHs, VHH B11, exhibited the highest affinity (EC50 < 1 nM) for the C. reinhardtii cell surface. The live-cell ELISA procedure was employed to detect algae sharing cell surface components with C. reinhardtii in water samples from natural environments. In addition, mCherry-tagged VHH B11 was used along with fluorescence activated cell sorting (FACS) to select individual axenic isolates of presumed wild relatives of C. reinhardtii and other Chlorphyceae from the same environmental samples. CONCLUSIONS: Camelid antibody VHH domains provide a highly specific tool for detection of individual cell wall components of algae and for allowing the selection of algae that share a particular cell surface molecule from diverse ecosystems.

The effect of iron on growth, lipid accumulation, and gene expression profile of the freshwater microalga Chlorella sorokiniana.

The effects of iron on the growth, lipid accumulation, and gene expression profiles of the limnetic Chlorella sorokiniana CCTCC M209220 under photoautotrophy were investigated. The addition of iron up to 10(-5) mol l(-l) increased final cell densities by nearly 2-fold at 2.3 × 10(7) cells/ml, growth rate by 2-fold, and the length of the exponential phase by 5 days as compared to unsupplemented controls while 10(-3) mol l(-1) iron was toxic. The lipid content increased from 12 % for unsupplemented cultures to 33 % at 10(-4) mol l(-1) iron while the highest overall lipid yield reached 179 mg l(-1). A genefishing and qPCR comparison between the C. sorokiniana at low and high iron levels indicated increases in the expression of several genes, including carbonic anhydrase involved in microalgal cell growth, as well as acc1 and choline transporter related to lipid synthesis. This study provides insights into changes in gene expression and metabolism that accompany iron supplementation to Chlorella as well as potential metabolic engineering targets for improving growth and lipid synthesis in microalgae.

Application of the CRISPR/Cas9 Gene Editing Method for Modulating Antibody Fucosylation in CHO Cells.

Genetic engineering plays an essential role in the development of cell lines for biopharmaceutical manufacturing. Advanced gene editing tools can improve both the productivity of recombinant cell lines as well as the quality of therapeutic antibodies. Antibody glycosylation is a critical quality attribute for therapeutic biologics because the glycan patterns on the antibody fragment crystallizable (Fc) region can alter its clinical efficacy and safety as a therapeutic drug. As an example, recombinant antibodies derived from Chinese hamster ovary (CHO) cells are generally highly fucosylated; the absence of α1,6-fucose significantly enhances antibody-dependent cell-mediated cytotoxicity (ADCC) against cancer cells. This chapter describes a protocol applying clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) approach with different formats to disrupt the α-1,6-fucosyltransferase (FUT8) gene and subsequently inhibit α-1,6 fucosylation on antibodies expressed in CHO cells.

Physiological evaluation of a new Chlorella sorokiniana isolate for its biomass production and lipid accumulation in photoautotrophic and heterotrophic cultures.

A novel green unicellular microalgal isolate from the freshwater of the Inner Mongolia Province of China and named as CCTCC M209220, grows between pH 6 and 11 and temperatures of 20-35°C with optimal conditions at pH 9 and 30°C. Morphological features and the phylogenetic analysis for the 18S rRNA gene reveal that the isolate is a Chlorella sorokiniana strain. A nitrogen source test reveals that this strain can grow well with nitrate and urea, but not ammonium. The strain can grow heterotrophically with glucose as the carbon source and accumulates lipid content as high as 56% (w/w) dry weight after 7 days in high glucose concentrations compared to 19% lipids achieved in 30 days of photoautotrophic culture. The relative neutral lipid content as a fraction of the total lipid is also much higher in heterotrophic culture as compared to photoautotrophic culture.

The effect of mixotrophy on microalgal growth, lipid content, and expression levels of three pathway genes in Chlorella sorokiniana.

Nannochloropsis oculata CCMP 525, Dunaliella salina FACHB 435, and Chlorella sorokiniana CCTCC M209220 were compared in mixotrophic and photoautotrophic cultures in terms of growth rate, protein, and lipid content. Growth improved in glucose, and the biomass productivities of N. oculata, D. salina, and C. sorokiniana were found to be 1.4-, 2.2- and 4.2-fold that observed photoautotrophically. However, biomass and lipid production decreased at the highest glucose concentrations. Meanwhile, the content of protein and lipid were significantly augmented for mixotrophic conditions at least for some species. C. sorokiniana was found to be well suited for lipid production based on its high biomass production rate and lipid content reaching 51% during mixotrophy. Expression levels of accD (heteromeric acetyl-CoA carboxylase beta subunit), acc1 (homomeric acetyl-CoA carboxylase), rbcL (ribulose 1, 5-bisphosphate carboxylase/oxygenase large subunit) genes in C. sorokiniana were studied by real-time PCR. Increased expression levels of accD reflect the increased lipid content in stationary phase of mixotrophic growth, but expression of the acc1 gene remains low, suggesting that this gene may not be critical to lipid accumulation. Additionally, reduction of expression of the rbcL gene during mixotrophy indicated that utilization of glucose was found to reduce the role of this gene and photosynthesis.

An improved colony PCR procedure for genetic screening of Chlorella and related microalgae.

A colony PCR technique was applied for both genomic and chloroplast DNA in the green microalgae Chlorella. Of five different lysis buffers, Chelex-100 was superior for DNA extraction, PCR and DNA storage. It also was insensitive to variations in cell density. The conditions established for an improved PCR formulation are applicable for screening of genetically-engineered transformants as well as bioprospecting of natural microalgal isolates. Besides multiple Chlorella species, we also demonstrate the efficacy of Chelex-100 for colony PCR with a number of other microalgal strains, including Chlamydomonas reinhardtii, Dunaliella salina, Nannochloropsis sp., Coccomyxa sp., and Thalassiosira pseudonana.

Surveilling cellular vital signs: toward label-free biosensors and real-time viability assays for bioprocessing.

Cell viability is an essential facet of mammalian and microbial bioprocessing. While robust methods of monitoring cellular health remain critically important to biomanufacturing and biofabrication, the complexity of advanced cell culture platforms often poses challenges for conventional viability assays. This review surveys novel approaches to discern the metabolic, morphological, and mechanistic hallmarks of living systems - spanning subcellular and multicellular scales. While fluorescent probes coupled with 3D image analysis generate rapid results with spatiotemporal detail, molecular techniques like viability PCR can distinguish live cells with genetic specificity. Notably, label-free biosensors can detect nuanced attributes of cellular vital signs with single-cell resolution via optical, acoustic, and electrical signals. Ultimately, efforts to integrate these modalities with automation, machine learning, and high-throughput workflows will lead to exciting new vistas across the cell viability landscape.

Accelerating vein-to-vein cell therapy workflows with new bioanalytical strategies.

Cell therapies represent a new era of treatment modalities for cancer. Through agile bioprocessing and bioengineering, patient-derived T-cells can be directed toward cancer biomarkers to impart a more robust and targeted immune response. In order to avoid delays in critical treatment timeframes, new bioanalytical tools are needed to accelerate, streamline, and maximize the throughput of T-cell bioprocessing. This review offers a survey of recent biotechnological advances supporting enhanced and expedited biomanufacturing workflows for autologous and allogeneic cell therapies, ranging from novel genetic engineering techniques and cell sorting platforms to stem cells and tumor organoid models. Collectively, these methods can increase the clinical impact of cancer therapeutics by improving the specificity, efficacy, and timely delivery of cell-based products.

Structure and synthesis of polyisoprenoids used in N-glycosylation across the three domains of life.

N-linked protein glycosylation was originally thought to be specific to eukaryotes, but evidence of this post-translational modification has now been discovered across all domains of life: Eucarya, Bacteria, and Archaea. In all cases, the glycans are first assembled in a step-wise manner on a polyisoprenoid carrier lipid. At some stage of lipid-linked oligosaccharide synthesis, the glycan is flipped across a membrane. Subsequently, the completed glycan is transferred to specific asparagine residues on the protein of interest. Interestingly, though the N-glycosylation pathway seems to be conserved, the biosynthetic pathways of the polyisoprenoid carriers, the specific structures of the carriers, and the glycan residues added to the carriers vary widely. In this review we will elucidate how organisms in each basic domain of life synthesize the polyisoprenoids that they utilize for N-linked glycosylation and briefly discuss the subsequent modifications of the lipid to generate a lipid-linked oligosaccharide.

A green light for engineered algae: redirecting metabolism to fuel a biotechnology revolution.

Microalgae have the potential to revolutionize biotechnology in a number of areas including nutrition, aquaculture, pharmaceuticals, and biofuels. Although algae have been commercially cultivated for over 50 years, metabolic engineering now seems necessary in order to achieve their full processing capabilities. Recently, the development of a number of transgenic algal strains boasting recombinant protein expression, engineered photosynthesis, and enhanced metabolism encourage the prospects of designer microalgae. Given the vast contributions that these solar-powered, carbon dioxide-sequestering organisms can provide to current global markets and the environment, an intensified focus on microalgal biotechnology is warranted. Ongoing advances in cultivation techniques coupled with genetic manipulation of crucial metabolic networks will further promote microalgae as an attractive platform for the production of numerous high-value compounds.

Application of the CRISPR/Cas9 Gene Editing Method for Modulating Antibody Fucosylation in CHO Cells.

Genetic engineering plays an essential role in the development of cell lines for biopharmaceutical manufacturing. Advanced gene editing tools can improve both the productivity of recombinant cell lines as well as the quality of therapeutic antibodies. Antibody glycosylation is a critical quality attribute for therapeutic biologics because the glycan patterns on the antibody fragment crystallizable (Fc) region can alter its clinical efficacy and safety as a therapeutic drug. As an example, recombinant antibodies derived from Chinese hamster ovary (CHO) cells are generally highly fucosylated; the absence of fucose significantly enhances antibody dependent cell-mediated cytotoxicity (ADCC) against cancer cells. This chapter describes a protocol applying clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) to disrupt the α-1,6-fucosyltranferase (FUT8) gene and subsequently inhibit α-1,6-fucosylation on antibodies expressed in CHO cells.

Pac-Man for biotechnology: co-opting degrons for targeted protein degradation to control and alter cell function.

Protein degradation in normal living cells is precisely regulated to match the cells' physiological requirements. The selectivity of protein degradation is determined by an elaborate degron-tagging system. Degron refers to an amino acid sequence that encodes a protein degradation signal, which is oftentimes a poly-ubiquitin chain that can be transferred to other proteins. Current understanding of ubiquitination dependent and independent protein degradation processes has expanded the application of degrons for targeted protein degradation and novel cell engineering strategies. Recent findings suggest that small molecules inducing protein association can be exploited to create degrons that target proteins for degradation. Here, recent applications of degron-based targeted protein degradation in eukaryotic organisms are reviewed. The degron mediated protein degradation represents a rapidly tunable methodology to control protein abundance, which has broad application in therapeutics and cellular function control and monitoring.

Comparison of biomass and lipid production under ambient carbon dioxide vigorous aeration and 3% carbon dioxide condition among the lead candidate Chlorella strains screened by various photobioreactor scales.

Chlorella species from the UTEX collection, classified by rDNA-based phylogenetic analysis, were screened based on biomass and lipid production in different scales and modes of culture. The lead candidate strains of C. sorokiniana UTEX 1230 and C. vulgaris UTEX 395 and 259 were compared between conditions of vigorous aeration with filtered atmospheric air and 3% CO2 shake-flask cultivation. The biomass of UTEX 1230 produced 2 times higher at 652 mg L(-1) dry weight under both ambient CO2 vigorous aeration and 3% CO2 conditions, while UTEX 395 and 259 under 3% CO2 increased to 3 times higher at 863 mg L(-1) dry weight than ambient CO2 vigorous aeration. The triacylglycerol contents of UTEX 395 and 259 increased more than 30 times to 30% dry weight with 3% CO2, indicating that additional CO2 is essential for both biomass and lipid accumulation in UTEX 395 and 259.

Comparative analyses of three Chlorella species in response to light and sugar reveal distinctive lipid accumulation patterns in the Microalga C. sorokiniana.

While photosynthetic microalgae, such as Chlorella, serve as feedstocks for nutritional oils and biofuels, heterotrophic cultivation can augment growth rates, support high cell densities, and increase triacylglycerol (TAG) lipid content. However, these species differ significantly in their photoautotrophic and heterotrophic characteristics. In this study, the phylogeny of thirty Chlorella strains was determined in order to inform bioprospecting efforts and detailed physiological assessment of three species. The growth kinetics and lipid biochemistry of C. protothecoides UTEX 411, C. vulgaris UTEX 265, and C. sorokiniana UTEX 1230 were quantified during photoautotrophy in Bold's basal medium (BBM) and heterotrophy in BBM supplemented with glucose (10 g L-1). Heterotrophic growth rates of UTEX 411, 265, and 1230 were found to be 1.5-, 3.7-, and 5-fold higher than their respective autotrophic rates. With a rapid nine-hour heterotrophic doubling time, Chlorella sorokiniana UTEX 1230 maximally accumulated 39% total lipids by dry weight during heterotrophy compared to 18% autotrophically. Furthermore, the discrete fatty acid composition of each strain was examined in order to elucidate lipid accumulation patterns under the two trophic conditions. In both modes of growth, UTEX 411 and 265 produced 18:1 as the principal fatty acid while UTEX 1230 exhibited a 2.5-fold enrichment in 18:2 relative to 18:1. Although the total lipid content was highest in UTEX 411 during heterotrophy, UTEX 1230 demonstrated a two-fold increase in its heterotrophic TAG fraction at a rate of 28.9 mg L(-1) d(-1) to reach 22% of the biomass, corresponding to as much as 90% of its total lipids. Interestingly, UTEX 1230 growth was restricted during mixotrophy and its TAG production rate was suppressed to 18.2 mg L-1 d-1. This constraint on carbon flow raises intriguing questions about the impact of sugar and light on the metabolic regulation of microalgal lipid biosynthesis.

Achieving high throughput sequencing of a cDNA library utilizing an alternative protocol for the bench top next-generation sequencing system.

The development of next-generation sequencing (NGS) technologies has provided novel tools for genome analysis and expression profiling. A high throughput cDNA sequencing method using a bench top next-generation sequencing system, GS Junior, is now available. Here, we used an alternative protocol to the standard method for generating the cDNA library. This protocol can decrease the number of processing steps to manipulate RNA when constructing a cDNA library from an RNA sample, and does not require mRNA isolation from total RNA. Thus it can decrease the risk of RNA degradation and the cost for preparing a cDNA library. Also, the efficiency of sequencing data obtained with this approach is comparable to the standard method as verified by sequencing characteristics and expression levels of the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).