Production of therapeutic proteins in algae,analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii

Recombinant proteins are widely used today in many industries, including the biopharmaceutical industry, and can be expressed in bacteria, yeasts, mammalian and insect cell cultures, or in transgenic plants and animals. In addition, transgenic algae have also been shown to support recombinant protein expression, both from the nuclear and chloroplast genomes. However, to date, there are only a few reports on recombinant proteins expressed in the algal chloroplast. It is unclear whether this is because of few attempts or of limitations of the system that preclude expression of many proteins. Thus, we sought to assess the versatility of transgenic algae as a recombinant protein production platform. To do this, we tested whether the algal chloroplast could support the expression of a diverse set of current or potential human therapeutic proteins. Of the seven proteins chosen, >50% expressed at levels sufficient for commercial production. Three expressed at 2%–3% of total soluble protein, while a forth protein accumulated to similar levels when translationally fused to a well‐expressed serum amyloid protein. All of the algal chloroplast‐expressed proteins are soluble and showed biological activity comparable to that of the same proteins expressed using traditional production platforms. Thus, the success rate, expression levels, and bioactivity achieved demonstrate the utility of Chlamydomonas reinhardtii as a robust platform for human therapeutic protein production.     

Algae as protein factories: expression of a human antibody and the respective antigen in the diatom Phaeodactylum tricornutum

Microalgae are thought to offer great potential as expression system for various industrial, therapeutic and diagnostic recombinant proteins as they combine high growth rates with all benefits of eukaryotic expression systems. Moreover, microalgae exhibit a phototrophic lifestyle like land plants, hence protein expression is fuelled by photosynthesis, which is CO(2)-neutral and involves only low production costs. So far, however, research on algal bioreactors for recombinant protein expression is very rare calling for further investigations in this highly promising field. In this study, we present data on the expression of a monoclonal human IgG antibody against the Hepatitis B surface protein and the respective antigen in the diatom Phaeodactylum tricornutum. Antibodies are fully-assembled and functional and accumulate to 8.7% of total soluble protein, which complies with 21 mg antibody per gram algal dry weight. The Hepatitis B surface protein is functional as well and is recognized by algae-produced and commercial antibodies.     

<Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> as a viable platform for the production of recombinant proteins: current status and perspectives

Chlamydomonas reinhardtii has many advantages compared with traditional systems for the molecular farming of recombinant proteins. These include low production costs, rapid scalability at pilot level, absence of human pathogens and the ability to fold and assemble complex proteins accurately. Currently, the successful expression of several proteins with pharmaceutical relevance has been reported from the nuclear and the chloroplastic genome of this alga, demonstrating its usefulness for biotechnological applications. However, several factors affect the level of recombinant protein expression in Chlamydomonas such as enhancer elements, codon dependency, sensitivity to proteases and transformation-associated genotypic modification. The present review outlines a number of strategies to increase protein yields and summarizes recent achievements in algal protein production including biopharmaceuticals such as vaccines, antibodies, hormones and enzymes with implications on health-related approaches. The current status of bioreactor developments for algal culture and the challenges of scale-up and optimization processes are also discussed.     

Plant seeds as bioreactors for recombinant protein production

Plants are attractive expression systems for the economic production of recombinant proteins. Among the different plant-based systems, plant seed is the leading platform and holds several advantages such as high protein yields and stable storage of target proteins. Significant advances in using seeds as bioreactors have occurred in the past decade, which include the first commercialized plant-derived recombinant protein. Here we review the current progress on seeds as bioreactors, with focus on the different food crops as production platforms and comprehensive strategies in optimizing recombinant protein production in seeds.     

Strategies to enhance production of microalgal biomass and lipids for biofuel feedstock

ABSTRACT

Microalgae have enormous potential as feedstock for biofuel production compared with other sources, due to their high areal productivity, relatively low environmental impact, and low impact on food security. However, high production costs are the major limitation for commercialization of algal biofuels. Strategies to maximize biomass and lipid production are crucial for improving the economics of using microalgae for biofuels. Selection of suitable algal strains, preferably from indigenous habitats, and further improvement of those ‘platform strains’ using mutagenesis and genetic engineering approaches are desirable. Conventional approaches to improve biomass and lipid productivity of microalgae mainly involve manipulation of nutritional (e.g. nitrogen and phosphorus) and environmental (e.g. temperature, light and salinity) factors. Approaches such as the addition of phytohormones, genetic and metabolic engineering, and co-cultivation of microalgae with yeasts and bacteria are more recent strategies to enhance biomass and lipid productivity of microalgae. Improvement in culture systems and the use of a hybrid system (i.e. a combination of open ponds and photobioreactors) is another strategy to optimize algal biomass and lipid production. In addition, the use of low-cost substrates such as agri-industrial wastewater for the cultivation of microalgae will be a smart strategy to reduce production costs. Such systems not only generate high algal biomass and lipid productivity, but are also useful for bioremediation of wastewater and bioremoval of waste CO₂. The aim of this review is to highlight the advances in the use of various strategies to enhance production of algal biomass and lipids for biofuel feedstock.     

Light and nutrients regulate energy transfer through benthic and pelagic food chains

The amount of energy flowing to top trophic levels depends on primary production and the efficiency at which it is converted to production at each trophic level. In aquatic systems, algal production is often limited by light and nutrients, and the nutritional quality of algae depends on the relative balance of these two resources. In this study, we used a mesocosm experiment to examine how light and nutrient variation affected food chain efficiency (FCE, defined as the proportion of primary production converted to top trophic level production), using a food web with benthic and pelagic food chains. We also related variation in benthic and pelagic efficiencies to the nutritional quality of primary producers, i.e. carbon:nitrogen:phosphorus stoichiometry. As predicted, pelagic and benthic FCEs were highest under low light/high nutrient conditions, the treatment with the best algal food quality, i.e. the lowest C:nutrient ratios. Pelagic FCE and pelagic herbivore efficiency (HE_P) were more responsive than benthic FCE to variation in light and nutrients. Furthermore, pelagic FCE and HE_P were highly correlated with algal C:P, suggesting ‘carryover effects’ of algal food quality on carnivores (larval fish) via effects on herbivore (zooplankton) quality. Benthic (tadpole) production was primarily explained by primary production rate, suggesting food quantity rather than quality drives their production. However, benthic FCE was also highest at low light/high nutrients and was significantly correlated with food quality. The stronger effect of food quality in mediating pelagic compared to benthic efficiencies, is consistent with differences in the stoichiometric mismatches between algae and consumers. Pelagic FCE and HE_P were more likely to be P‐limited, whereas benthic FCE was more likely N‐limited. This study is the first to examine both pelagic and benthic FCE within the same system, and highlights the importance of differential consumer needs in determining how food quality affects energy transfer efficiency.     

Two-stage heterotrophic and phototrophic culture strategy for algal biomass and lipid production

A two-stage heterotrophic and phototrophic culture strategy for algal biomass and lipid production was studied, wherein high density heterotrophic cultures of Chlorellasorokiniana serve as seed for subsequent phototrophic growth. The data showed growth rate, cell density and productivity of heterotrophic C.sorokiniana were 3.0, 3.3 and 7.4 times higher than phototrophic counterpart, respectively. Hetero- and phototrophic algal seeds had similar biomass/lipid production and fatty acid profile when inoculated into phototrophic culture system. To expand the application, food waste and wastewater were tested as feedstock for heterotrophic growth, and supported cell growth successfully. These results demonstrated the advantages of using heterotrophic algae cells as seeds for open algae culture system. Additionally, high inoculation rate of heterotrophic algal seed can be utilized as an effective method for contamination control. This two-stage heterotrophic phototrophic process is promising to provide a more efficient way for large scale production of algal biomass and biofuels.     

An engineered diatom acting like a plasma cell secreting human IgG antibodies with high efficiency

ABSTRACT: BACKGROUND: Although there are many different expression systems for recombinant production of pharmaceutical proteins, many of these suffer from drawbacks such as yield, cost, complexity of purification, and possible contamination with human pathogens. Microalgae have enormous potential for diverse biotechnological applications and currently attract much attention in the biofuel sector. Still underestimated, though, is the idea of using microalgae as solar-fueled expression system for the production of recombinant proteins. RESULTS: In this study, we show for the first time that completely assembled and functional human IgG antibodies can not only be expressed to high levels in algal systems, but also secreted very efficiently into the culture medium. We engineered the diatom Phaeodactylum tricornutum to synthesize and secrete a human IgG antibody against the Hepatitis B Virus surface protein. As the diatom P. tricornutum is not known to naturally secrete many endogenous proteins, the secreted antibodies are already very pure making extensive purification steps redundant and production extremely cost efficient. CONCLUSIONS: Microalgae combine rapid growth rates with all the advantages of eukaryotic expression systems, and offer great potential for solar-powered, low cost production of pharmaceutical proteins.     

Stoichiometric impacts of increased carbon dioxide on a planktonic herbivore

The partial pressure of carbon dioxide (pCO₂) in lake ecosystems varies over four orders of magnitude and is affected by local and global environmental perturbations associated with both natural and anthropogenic processes. Little is known, however, about how changes in pCO₂ extend into the function and structure of food webs in freshwater ecosystems. To fill this gap, we performed laboratory experiments using the ecologically important planktonic herbivore Daphnia and its algal prey under a natural range of pCO₂ with low light and phosphorus supplies. The experiment showed that increased pCO₂ stimulated algal growth but reduced algal P : C ratio. When feeding on algae grown under high pCO₂, herbivore growth decreased regardless of algal abundance. Thus, high CO₂‐raised algae were poor food for Daphnia. Short‐term experimental supplementation of PO₄ raised the P content of the high CO₂‐raised algae and improved Daphnia growth, indicating that low Daphnia growth rates under high pCO₂ conditions were due to lowered P content in the algal food. These results suggest that, in freshwater ecosystems with low nutrient supplies, natural processes as well as anthropogenic perturbations resulting in increased pCO₂ enhance algal production but reduce energy and mass transfer efficiency to herbivores by decreasing algal nutritional quality.     

Food quality triggers the reproductive mode in the cyclical parthenogen <Emphasis Type="Italic">Daphnia</Emphasis> (Cladocera)

Cyclic parthenogenesis (heterogony) is a widespread reproductive mode found in diverse taxa such as digenean trematodes, gall wasps, gall midges, aphids, cladocerans and rotifers. It is of particular interest as it combines the advantages of asexual reproduction (rapid population growth) and sexual reproduction (recombination). Usually sexual reproduction is initiated when, or slightly before, environmental conditions deteriorate, and often results in the production of resting stages. The optimal timing of diapause induction must thus be under strong natural selection. Using the cladoceran Daphnia as a model system, we show here for the first time that the switch from parthenogenetic to sexual reproduction in a cyclical parthenogenetic organism can be influenced by the chemical composition of food. Under crowding conditions Daphnia reproduced parthenogenetically with subitaneous eggs when fed the algal species Cryptomonas sp., but started the production of resting eggs when fed with the green algal species Scenedesmus obliquus. Supplementation experiments with lipids and especially proteins showed that the induction of resting egg production in two clones of different Daphnia species was due to a dietary deficiency in the green alga. Hence, the low food quality induced a switch in the reproductive mode that may contribute to optimal timing of the sexual reproduction of Daphnia in nature. Furthermore, our results have two other major implications: first, they suggest that protein compounds should be added to the list of diet constituents potentially limiting or influencing Daphnia reproduction. Second, we show that the role of food quality goes far beyond the up to now documented effects of food quality on somatic growth and trophic transfer efficiency of herbivores: due to its effects on sexual reproduction and the production of resting eggs, food quality might influence genetic diversity and long-term persistence of Daphnia in lakes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Life-history patterns of populations of the limpet Patella granularis: the dominant roles of food supply and mortality rate

Summary Rates of algal production were measured at 6 rocky intertidal sites on the southwestern Cape coast, South Africa, where populations of the limpet Patella granularis occur. Rates of algal production and limpet mortality were recorded at each site and both factors affected limpet reproductive fitness. Limpet growth rates and biomass at different sites were significantly correlated with rates of algal production, and limpet mortality was related to the density of African Black Oystercatchers, important predators of P. granularis. Life-time gametic output of a hypothetical cohort of limpets at each site was modelled using the measurements of growth, reproduction and mortality made at each site. Limpet cohorts at sites with rapid rates of algal production were predicted to have a larger life-time production of gametic material than cohorts at sites with slow algal production rates, except in instances of acute predatory pressure. In the light of the overriding influence of food supply on the expression of limpet life-history parameters, it is imperative that researchers consider food availability before assuming that local, population-specific differences in life-history patterns are due to genetic differences.     

Role of cultivation media in the development of yeast strains for large scale industrial use

Pure, soluble and functional proteins are of high demand in modern biotechnology. Natural protein sources rarely meet the requirements for quantity, ease of isolation or price and hence recombinant technology is often the method of choice. Recombinant cell factories are constantly employed for the production of protein preparations bound for downstream purification and processing. Eschericia coli is a frequently used host, since it facilitates protein expression by its relative simplicity, its inexpensive and fast high density cultivation, the well known genetics and the large number of compatible molecular tools available. In spite of all these qualities, expression of recombinant proteins with E. coli as the host often results in insoluble and/or nonfunctional proteins. Here we review new approaches to overcome these obstacles by strategies that focus on either controlled expression of target protein in an unmodified form or by applying modifications using expressivity and solubility tags.     

Phytoplankton food quality control of planktonic food web processes

We developed a mechanistic model of nutrient, phytoplankton, zooplankton and fish interactions to test the effects of phytoplankton food quality for herbivorous zooplankton on planktonic food web processes. When phytoplankton food quality is high strong trophic cascades suppress phytoplankton biomass, the zooplankton can withstand intense zooplanktivory, and energy is efficiently transferred through the food web sustaining higher trophic level production. Low food quality results in trophic decoupling at the plant-animal interface, with phytoplankton biomass determined primarily by nutrient availability, zooplankton easily eliminated by fish predation, and poor energy transfer through the food web. At a given nutrient availability, food quality and zooplanktivory interact to determine zooplankton biomass which in turn determines algal biomass. High food quality resulted in intense zooplankton grazing which favored fast-growing phytoplankton taxa, whereas fish predation favored slow-growing phytoplankton. These results suggest algal food quality for herbivorous zooplankton can strongly influence the nature of aquatic food web dynamics, and can have profound effects on water quality and fisheries production. Handling editor: D. Hamilton     

Do Inactivated Microbial Preparations Improve Life History Traits of the Copepod <Emphasis Type="Italic">Acartia tonsa</Emphasis>?

We have tested a microbial preparation with probiotic effects (PSI; Sorbial A/S DANISCO) on the calanoid copepod Acartia tonsa (Dana) development time and reproduction effectiveness in culture. The hypotheses were that PSI increases the productivity and quality of copepods in culture (increased egg production and hatching success, HS). This was carried out because the use of copepods as live prey in aquaculture could increase the number of fish successfully raised through their entire life cycle. However, the availability of copepods is limited by their difficulty to be effectively raised. Our results show that the addition of PSI to the algal food increased the individual size of the adult females and their egg production. The PSI, together with Rhodomonas salina, also increased the HS of the eggs produced by PSI-treated females. These effects were observed despite that the biochemical analysis of the PSI revealed that it is a nutritionally poor food lacking essential fatty acids, and hence it cannot be used alone to raise copepods but instead as a food additive. This is the first demonstration that the effectiveness of copepod culturing can be improved using microbial preparations as a food additive.     

Micro-algae come of age as a platform for recombinant protein production

A complete set of genetic tools is still being developed for the micro-alga Chlamydomonas reinhardtii. Yet even with this incomplete set, this photosynthetic single-celled plant has demonstrated significant promise as a platform for recombinant protein expression. In recent years, techniques have been developed that allow for robust expression of genes from both the nuclear and plastid genome. With these advances, many research groups have examined the pliability of this and other micro-algae as biological machines capable of producing recombinant peptides and proteins. This review describes recent successes in recombinant protein production in Chlamydomonas, including production of complex mammalian therapeutic proteins and monoclonal antibodies at levels sufficient for production at economic parity with existing production platforms. These advances have also shed light on the details of algal protein production at the molecular level, and provide insight into the next steps for optimizing micro-algae as a useful platform for the production of therapeutic and industrially relevant recombinant proteins.     

Transient protein expression systems in plants and their applications

The production of recombinant proteins is important in academic research to identify protein functions. Moreover, recombinant enzymes are used in the food and chemical industries, and high-quality proteins are required for diagnostic, therapeutic, and pharmaceutical applications. Though many recombinant proteins are produced by microbial or mammalian cell-based expression systems, plants have been promoted as alternative, cost-effective, scalable, safe, and sustainable expression systems. The development and improvement of transient expression systems have significantly reduced the period of protein production and increased the yield of recombinant proteins in plants. In this review, we consider the importance of plant-based expression systems for recombinant protein production and as genetic engineering tools.     

Cross-reconstitution of various extrinsic proteins and photosystem II complexes from cyanobacteria, red algae and higher plants

Photosystem II (PSII) contains different extrinsic proteins required for oxygen evolution among different organisms. Cyanobacterial PSII contains the 33 kDa, 12 kDa proteins and cytochrome (cyt) c-550; red algal PSII contains a 20 kDa protein in addition to the three homologous cyanobacterial proteins; whereas higher plant PSII contains the 33 kDa, 23 kDa and 17 kDa proteins. In order to understand the binding and functional properties of these proteins, we performed cross-reconstitution experiments with combinations of PSII and extrinsic proteins from three different sources: higher plant (spinach), red alga (Cyanidium caldarium) and cyanobacterium (Synechococcus vulcanus). Among all of the extrinsic proteins, the 33 kDa protein is common to all of the organisms and is totally exchangeable in binding to PSII from any of the three organisms. Oxygen evolution of higher plant and red algal PSII was restored to a more or less similar level by binding of any one of the three 33 kDa proteins, whereas oxygen evolution of cyanobacterial PSII was restored to a larger extent with its own 33 kDa protein than with the 33 kDa protein from other sources. In addition to the 33 kDa protein, the red algal 20 kDa, 12 kDa proteins and cyt c-550 were able to bind to cyanobacterial and higher plant PSII, leading to a partial restoration of oxygen evolution in both organisms. The cyanobacterial 12 kDa protein and cyt c-550 partially bound to the red algal PSII, but this binding did not restore oxygen evolution. The higher plant 23 kDa and 17 kDa proteins bound to the cyanobacterial and red algal PSII only through non-specific interactions. Thus, only the red algal extrinsic proteins are partially functional in both the cyanobacterial and higher plant PSII, which implies a possible intermediate position of the red algal PSII during its evolution from cyanobacteria to higher plants.     

Effect of turbulence on the feeding and moulting of the cirripede Balanus Balanoides (L.) given an algal diet

The shell growth, moulting, and faecal pellet production of Balanus balanoides (L.) have been investigated experimentally in turbulent and quiet conditions. In turbulent conditions an algal food level of 50% was sufficient to maintain the same shell growth as at the 100% level, whereas at this level in quiet conditions growth was less than at 50% with turbulence. At the 10% food level growth was markedly reduced in quiet conditions. On the assumption that production of faeces is a measure of feeding activity, the latter is stimulated by turbulence as is the moulting rate. The relation between production of faeces and moulting is, however, independent of water movement. Faecal pellet production continues at higher algal food levels after the growth rate has become constant indicating that when assimilation is maximal there is no feed-back mechanism regulating food intake.     

Protein secretion in Lactococcus lactis : an efficient way to increase the overall heterologous protein production

Lactococcus lactis, the model lactic acid bacterium (LAB), is a food grade and well-characterized Gram positive bacterium. It is a good candidate for heterologous protein delivery in foodstuff or in the digestive tract. L. lactis can also be used as a protein producer in fermentor. Many heterologous proteins have already been produced in L. lactis but only few reports allow comparing production yields for a given protein either produced intracellularly or secreted in the medium. Here, we review several works evaluating the influence of the localization on the production yields of several heterologous proteins produced in L. lactis. The questions of size limits, conformation, and proteolysis are addressed and discussed with regard to protein yields. These data show that i) secretion is preferable to cytoplasmic production; ii) secretion enhancement (by signal peptide and propeptide optimization) results in increased production yield; iii) protein conformation rather than protein size can impair secretion and thus alter production yields; and iv) fusion of a stable protein can stabilize labile proteins. The role of intracellular proteolysis on heterologous cytoplasmic proteins and precursors is discussed. The new challenges now are the development of food grade systems and the identification and optimization of host factors affecting heterologous protein production not only in L. lactis, but also in other LAB species.     

Comparison of binding and functional properties of two extrinsic components,Cyt c550 and a 12 kDa protein,in cyanobacterial PSII with those in red algal PSII

Cyt c550 and 12 kDa protein are two extrinsic proteins of photosystem II (PSII) found in cyanobacteria and some eukaryotic algae. The binding patterns of these two extrinsic proteins are different between cyanobacterial (Thermosynechococcus vulcanus) and red algal (Cyanidium caldarium) PSIIs [Shen and Inoue (1993) Biochemistry 32: 1825; Enami et al. (1998) Biochemistry 39: 2787]. In order to elucidate the possible causes responsible for these differences, we first cloned the psbV gene encoding Cyt c550 from a red alga, Cyanidium caldarium, which was compared with the homologous sequences from other organisms. Cross-reconstitution experiments were then performed with different combinations of the extrinsic proteins and the cyanobacterial or red algal PSII. (1). Both the cyanobacterial and red algal Cyt c550 bound directly to the cyanobacterial PSII, whereas none of them bound directly to the red algal PSII, indicating that direct binding of Cyt c550 to PSII principally depends on the structure of PSII intrinsic proteins but not that of Cyt c550 itself. (2). Cyt c550 was functionally exchangeable between the red algal and the cyanobacterial PSII, and the red algal 12 kDa protein functionally bound to the cyanobacterial PSII, whereas the cyanobacterial 12 kDa protein did not bind to the red algal PSII. (3). The antibody against the cyanobacterial or red algal 12 kDa protein reacted with its original one but not with the homologous protein from the other organism, whereas the antibody against the red algal Cyt c550 reacted with both cyanobacterial and red algal Cyt c550. These results imply that the structure and function of Cyt c550 have been largely conserved, whereas those of the 12 kDa protein have been changed, in the two organisms studied here.