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The increase in the world population, the advent of new infections and health issues, and the scarcity of natural biological products have spotlighted the importance of recombinant protein technology and its large-scale production in a cost-effective manner. Microalgae have become a significant promising platform with the potential to meet the increasing demand for recombinant proteins and other biologicals. Microalgae are safe organisms that can grow rapidly and are easily cultivated with basic nutrient requirements. Although continuous efforts have led to considerable progress in the algae genetic engineering field, there are still many hurdles to overcome before these microorganisms emerge as a mature expression system. Hence, there is a need to develop efficient expression approaches to exploit microalgae for the production of recombinant proteins at convenient yields. This study aimed to test the ability of the DNA geminiviral vector with Rep-mediated replication to transiently express recombinant proteins in the freshwater microalgal species Chlamydomonas reinhardtii and Chlorella vulgaris using Agrobacterium- mediated transformation. The SARS-CoV-2 receptor binding domain (RBD) and basic fibroblast growth factor (bFGF) are representative antigen proteins and growth factor proteins, respectively, that were subcloned in a geminiviral vector and were used for nuclear transformation to transiently express these proteins in C. reinhardtii and C. vulgaris . The results showed that the geminiviral vector allowed the expression of both recombinant proteins in both algal species, with yields at 48 h posttransformation of up to 1.14 μg/g RBD and 1.61 ng/g FGF in C. vulgaris and 1.61 μg/g RBD and 1.025 ng/g FGF in C. reinhardtii . Thus, this study provides a proof of concept for the use of DNA viral vectors for the simple, rapid, and efficient production of recombinant proteins that repress the difficulties faced in the genetic transformation of these unicellular green microalgae. This concept opens an avenue to explore and optimize green microalgae as an ideal economically valuable platform for the production of therapeutic and industrially relevant recombinant proteins in shorter time periods with significant yields.

The green model microalga Chlamydomonas reinhardtii recently emerged as a sustainable production chassis for the efficient biosynthesis of recombinant proteins and high-value metabolites. Its capacity for scalable, rapid and light-driven growth in minimal salt solutions, its simplicity for genetic manipulation and its “Generally Recognized As Safe” (GRAS) status are key features for its application in industrial biotechnology. Although nuclear transformation has typically resulted in limited transgene expression levels, recent developments now allow the design of powerful and innovative bioproduction concepts. In this review, we summarize the main obstacles to genetic engineering in C. reinhardtii and describe all essential aspects in sequence adaption and vector design to enable sufficient transgene expression from the nuclear genome. Several biotechnological examples of successful engineering serve as blueprints for the future establishment of C. reinhardtii as a green cell factory.

Recently, plants have emerged as a lucrative alternative system for the production of recombinant proteins, as recombinant proteins produced in plants are safer and cheaper than those produced in bacteria and animal cell-based production systems. To obtain high yields in plants, recombinant proteins are produced in chloroplasts using different strategies. The first strategy is based on chloroplast transformation, followed by gene expression and translation in chloroplasts. This has proven to be a powerful approach for the production of proteins at high levels. The second approach is based on nuclear transformation, followed by post-translational import of proteins from the cytosol into chloroplasts. In the nuclear transformation approach, foreign genes are stably integrated into the nuclear genome or transiently expressed in the nucleus by non-integrating T-DNA. Although this approach also has great potential for protein production at high levels, it has not been thoroughly investigated. In this review, we focus on nuclear transformation-based protein expression and its subsequent sequestration in chloroplasts, and summarize the different strategies used for high-level production of recombinant proteins. We also discuss future directions for further improvements in protein production in chloroplasts through nuclear transformation-based gene expression.

D-serine is toxic to plants. D-serine ammonia lyase, which is encoded by the dsdA gene, can attenuate this toxicity with high specificity. In the present study, we explored the function of codon-optimized dsdA with tobacco plastids and rice nuclear transformation system. It was shown that dsdA gene was site-specifically integrated into the tobacco plastid genome and displayed a high level of expression. Genetic analysis of the progenies showed that dsdA gene is maternally inherited and confers sufficient D-serine resistance in tobacco. The effective screening concentrations of D-serine for seed germination, callus regeneration and foliar spray were 10, 30, and 75 mM, respectively. In addition, calluses from homozygous transgenic rice lines also showed significant tolerance to D-serine (up to 75 mM). Our study proves the feasibility of using dsdA gene as a selectable marker in both plastid and nuclear transformation systems.

To understand the origin and character of individual radioactive emissions accompanying nuclear transformation processes, it is essential to understand what an unstable nucleus is, what its motivation is to transform, and how the nucleus selects the best way to transform. The following chapter represents part I of a series of two articles. It introduces the composition of stable atomic nuclei, the meaning of “isotopes”, the discrepancy between the mass of the nucleus and the sum of the mass of the nucleons it is composed of (the “mass defect”), and the parameter of “mean nucleon binding energy”. It is the fate of those unstable nuclei, that they transform to more stable ones. Part I will focus on the “primary” pathways of those transformations, i.e., those, where the nucleon composition of an unstable nucleus is modified to gain mean nucleon binding energy. Those transformation processes are the origin of certain radioactive emissions, and part I will discuss those originating from the primary transformation pathways, i.e., mainly β- and α-emissions.

Plant photosynthesis and growth are often limited by the activity of the CO₂-fixing enzyme Rubisco. The broad kinetic diversity of Rubisco in nature is accompanied by differences in the composition and compatibility of the ancillary proteins needed for its folding, assembly, and metabolic regulation. Variations in the protein folding needs of catalytically efficient red algae Rubisco prevent their production in plants. Here, we show this impediment does not extend to Rubisco from Rhodobacter sphaeroides (RsRubisco)—a red-type Rubisco able to assemble in plant chloroplasts. In transplastomic tobRsLS lines expressing a codon optimized Rs-rbcLS operon, the messenger RNA (mRNA) abundance was ∼25%of rbcL transcript and RsRubisco ∼40% the Rubisco content in WT tobacco. To mitigate the low activation status of RsRubisco in tobRsLS (∼23% sites active under ambient CO₂), the metabolic repair protein RsRca (Rs-activase) was introduced via nuclear transformation. RsRca production in the tobRsLS::X progeny matched endogenous tobacco Rca levels (∼1 μmol protomer·m²) and enhanced RsRubisco activation to 75% under elevated CO₂ (1%, vol/vol) growth. Accordingly, the rate of photosynthesis and growth in the tobRsLS::X lines were improved >twofold relative to tobRsLS. Other tobacco lines producing RsRubisco containing alternate diatom and red algae S-subunits were nonviable as CO₂-fixation rates (kcat c) were reduced >95%and CO₂/O₂ specificity impaired 30–50%. We show differences in hybrid and WT RsRubisco biogenesis in tobacco correlated with assembly in Escherichia coli advocating use of this bacterium to preevaluate the kinetic and chloroplast compatibility of engineered RsRubisco, an isoform amenable to directed evolution.

Agriculture is experiencing a technological inflection point in its history, while also facing unprecedented challenges posed by human population growth and global climate changes. Key advancements in precise genome editing and new methods for rapid generation of bioengineered crops promise to both revolutionize the speed and breadth of breeding programmes and increase our ability to feed and sustain human population growth. Although genome editing enables targeted and specific modifications of DNA sequences, several existing barriers prevent the widespread adoption of editing technologies for basic and applied research in established and emerging crop species. Inefficient methods for the transformation and regeneration of recalcitrant species and the genotype dependency of the transformation process remain major hurdles. These limitations are frequent in monocotyledonous crops, which alone provide most of the calories consumed by human populations. Somatic embryogenesis and de novo induction of meristems — pluripotent groups of stem cells responsible for plant developmental plasticity — are essential strategies to quickly generate transformed plants. Here we review recent discoveries that are rapidly advancing nuclear transformation technologies and promise to overcome the obstacles that have so far impeded the widespread adoption of genome editing in crop species.This Perspective reviews recent advances in crop transformation technologies that promise to lead to the widespread adoption of genome editing in crop species.

Microalgae comprise a biodiverse group of photosynthetic organisms that reside in water sources and sediments. The green microalgae Chlamydomonas reinhardtii was adopted as a useful model organism for studying various physiological systems. Its ability to grow under both photosynthetic and heterotrophic conditions allows efficient growth of non-photosynthetic mutants, making Chlamydomonas a useful genetic tool to study photosynthesis. In addition, this green alga can grow as haploid or diploid cells, similar to yeast, providing a powerful genetic system. As a result, easy and efficient transformation systems have been developed for Chlamydomonas, targeting both the chloroplast and nuclear genomes. Since microalgae comprise a rich repertoire of species that offer variable advantages for biotech and biomed industries, gene transfer technologies were further developed for many microalgae to allow for the expression of foreign proteins of interest. Expressing foreign genes in the chloroplast enables the targeting of foreign DNA to specific sites by homologous recombination. Chloroplast transformation also allows for the introduction of genes encoding several enzymes from a complex pathway, possibly as an operon. Expressing foreign proteins in the chloroplast can also be achieved by introducing the target gene into the nuclear genome, with the protein product bearing a targeting signal that directs import of the transgene-product into the chloroplast, like other endogenous chloroplast proteins. Integration of foreign genes into the nuclear genome is mostly random, resulting in large variability between different clones, such that extensive screening is required. The use of different selection modalities is also described, with special emphasis on the use of herbicides and metabolic markers which are considered to be friendly to the environment, as compared to drug-resistance genes that are commonly used. Finally, despite the development of a wide range of transformation tools and approaches, expression of foreign genes in microalgae suffers from low efficiency. Thus, novel tools have appeared in recent years to deal with this problem. Finally, while C. reinhardtii was traditionally used as a model organism for the development of transformation systems and their subsequent improvement, similar technologies can be adapted for other microalgae that may have higher biotechnological value.

Taxadiene, the first committed precursor to taxol, is synthesized from geranylgeranyl pyrophosphate (GGPP) by action of taxadiene synthase (TS). Heterologous production of taxadiene could potentially rely on both cytosolic mevalonic acid (MVA) pathway and the plastidic methylerythritol phosphate (MEP) pathway. We suggest the compartmentalized engineering in chloroplast as an efficient approach for taxadiene production. In this study, we directly introduced the TS gene from Taxus brevifolia into the tobacco chloroplast genome and found that the transplastomic plants accumulated a low content of taxadiene, ~ 5.6 μg/g dry weight (DW). Moreover, we tried a combination of MEP and MVA pathways for taxadiene synthesis by nuclear transformation with a truncated version of TS (without encoding a transit peptide) into the transplastomic plants. However, this did not further improve the taxadiene production. In contrast, we found that taxadiene could be produced up to 87.8 μg/g DW in leaves of transgenic plants expressing TS with a chloroplast transit peptide, which was significantly higher than that in leaves of transplastomic plants. Thus, this study highlights the importance of TS importation into chloroplast for production of taxadiene.

Nannochloropsis oceanica is a unicellular oleaginous microalga of emerging biotechnological interest with a sequenced, annotated genome, available transcriptomic and proteomic data, and well-established basic molecular tools for genetic engineering. To establish N. oceanica as a eukaryotic host for recombinant protein synthesis and develop molecular technology for vaccine production, we chose the viral surface protein 2 (VP2) of a pathogenic fish virus that causes infectious pancreatic necrosis as a model vaccine. Upon stable nuclear transformation of N. oceanica strain CCMP1779 with the codon-optimized VP2 gene, a Venus reporter fusion served to evaluate the strength of different endogenous promoters in transformant populations by qPCR and flow cytometry. The highest VP2 yields were achieved for the elongation factor promoter, with enhancer effects by its N-terminal leader sequence. Individual transformants differed in their production capability of reporter-free VP2 by orders of magnitude. When subjecting the best candidates to kinetic analyses of growth and VP2 production in photobioreactors, recombinant protein integrity was maintained until the early stationary growth phase, and a high yield of 4.4% VP2 of total soluble protein was achieved. The maximum yield correlated with multiple integrations of the expression vector into the nuclear genome. The results demonstrate that N. oceanica was successfully engineered to constitute a robust platform for high-level production of a model subunit vaccine. The molecular methodology established here can likely be adapted in a straightforward manner to the production of further vaccines in the same host, allowing their distribution to fish, vertebrates, or humans via a microalgae-containing diet. Key points • We engineered N. oceanica for recombinant protein production. • The antigenic surface protein 2 of IPN virus could indeed be expressed in the host. • A high yield of 4.4% VP2 of total soluble protein was achieved in N. oceanica.