Production Of Foreign Proteins Research Articles

Improving transient expression in N. benthamiana by suppression of the Nb-SABP2 and Nb-COI1 plant defence response related genes.

Currently transient expression is one of the preferred plant-based technologies for recombinant protein manufacturing, particularly in respect to pharmaceutically relevant products. Modern hybrid transient expression systems combine the features of Agrobacterium tumefaciens and viral vectors. However, host plant reaction to Agrobacterium-mediated delivery of gene of interest can negatively affect foreign protein accumulation. In this study, we investigated whether the modulation of plant immune response through knockdown of the Nb-SABP2 and Nb-COI1 N. benthamiana genes could improve recombinant protein yield. In plants, the SABP2 and COI1 proteins are involved in the salicylic acid and jasmonic acid metabolism, respectively. We exemplified the utility of this approach with the green fluorescence (GFP) and β nerve growth factor (βNGF) proteins: compared to the tobacco mosaic virus (TMV)-based vector the Nb-SABP2 and Nb-COI1-suppressed plants provided an increased recombinant protein accumulation. We also show that this strategy is extendable to the expression systems utilizing potato virus X (PVX) as the vector backbone: the enhanced amounts of βNGF were detected in the Nb-SABP2 and Nb-COI1-depleted leaves co-infiltrated with the PVX-βNGF. These findings suggest that modulating host plant reaction to agrodelivery of expression vectors could be useful for improving transient foreign protein production in N. benthamiana.

'Spikeopathy': COVID-19 Spike Protein Is Pathogenic, from Both Virus and Vaccine mRNA.

The COVID-19 pandemic caused much illness, many deaths, and profound disruption to society. The production of 'safe and effective' vaccines was a key public health target. Sadly, unprecedented high rates of adverse events have overshadowed the benefits. This two-part narrative review presents evidence for the widespread harms of novel product COVID-19 mRNA and adenovectorDNA vaccines and is novel in attempting to provide a thorough overview of harms arising from the new technology in vaccines that relied on human cells producing a foreign antigen that has evidence of pathogenicity. This first paper explores peer-reviewed data counter to the 'safe and effective' narrative attached to these new technologies. Spike protein pathogenicity, termed 'spikeopathy', whether from the SARS-CoV-2 virus or produced by vaccine gene codes, akin to a 'synthetic virus', is increasingly understood in terms of molecular biology and pathophysiology. Pharmacokinetic transfection through body tissues distant from the injection site by lipid-nanoparticles or viral-vector carriers means that 'spikeopathy' can affect many organs. The inflammatory properties of the nanoparticles used to ferry mRNA; N1-methylpseudouridine employed to prolong synthetic mRNA function; the widespread biodistribution of the mRNA and DNA codes and translated spike proteins, and autoimmunity via human production of foreign proteins, contribute to harmful effects. This paper reviews autoimmune, cardiovascular, neurological, potential oncological effects, and autopsy evidence for spikeopathy. With many gene-based therapeutic technologies planned, a re-evaluation is necessary and timely.

YBac: A novel and simple baculovirus system for heterologous protein production in Spodoptera frugiperda 9 (Sf9) cells.

The baculovirus expression vector system (BEVS) has been widely used for heterologous protein expression due to its powerful functionality and easy access to commercial expression vectors. Currently, most laboratories prefer two strategies for protein production using BEVS. One is recombinant bacmid based on transposition in Escherichia coli (e.g., Bac-to-Bac), and another is homologous recombination in insect cells (e.g., flashBac). In this manuscript, a rapid and simple YBac system was established. This novel system uses an Ac99KO bacmid as a virus vector and co-transfected into Spodoptera frugiperda 9 (Sf9) cells with a donor plasmid capable of recombination into Ac42 loci that carry the genes of interest (GOIs) along with the complete Ac99 fragment. Based on the intracellular homologous recombination system, the production of foreign proteins was achieved by the complementation of the Ac99 gene together with the insertion of GOIs. In this study, the human thyroid peroxidase (hTPO) and porcine epidemic diarrhea virus-like particles (PEDV VLPs) were successfully expressed using the YBac system. The entire process was shortened to 10 days, and the components involved in the system could be easily prepared in the laboratory, suggesting that the YBac system may have great potential in the production of heterologous proteins.

Effect of the At-CDC27a gene on Nicotiana benthamiana phenotype and accumulation of recombinant proteins.

In this study the anaphase promoting complex subunit CDC27a from Arabidopsis thaliana was introduced in the genome of Nicotiana benthamiana by Agrobacterium tumefaciens. The presence of the At-CDC27a gene facilitates plant biomass production. Compared to wild type N. benthamiana the leaf mass fraction of the best performing transgenic line At-CDC27a-29 was increased up to 154%. The positive effect of the At-CDC27a expression on leaf biomass accumulation was accompanied by an enlarged total leaf area. Furthermore, the ectopic expression of the At-CDC27a also affected cellular conditions for the production of foreign proteins delivered by the TRBO vector. In comparison to the non-transgenic control, the protein accumulation in the At-CDC27a-29 plant host increased up to 146% for GFP and up to 181% for scFv-TM43-E10. Collectively, the modified N. benthamiana plants developed in this study might be useful to improve the yield of recombinant proteins per biomass unit in closed facilities.

Establishment of a Novel Baculovirus-Silkworm Expression System.

The baculovirus vector expression system is a well-established tool for foreign protein production and gene delivery. In this study, we constructed a recombinant baculovirus vector system. The UAS promotor region and Bombyx mori nucleopolyhedrovirus (BmNPV) polyhedrin coding region were ligated into a pFastBac Dual vector to obtain a BmBac-UPS recombinant bacmid. The recombinant bacmid BmBac-Gal4 was generated by the same strategy which has a Gal4 coding region controlled by the IE2 promoter. BmBac-UPS and BmBac-IGal4 were co-infected into silkworm BmN cells to confirm the ability of the UAS/Gal4 system to form polyhedrons in B. mori cells. Furthermore, the recombinant viruses were tested for infection efficiency and the ability to generate polyhedra in transgenic B. mori cell line BmE. The results showed that recombinant viruses have the ability to form polyhedrons and gain raised pathogenicity when orally infected B. mori larvae and are applied as the preferred tool for foreign gene delivery and expression

Evaluation of protein production in rice seedlings under dark conditions

Although plants have several advantages for foreign protein production, cultivation of transgenic plants in artificial plant growth facilities involves the use of a great amount of electricity for lightning and air conditioning, reducing cost-effectiveness. Protein production in plants grown in darkness can overcome this problem, but the amount of protein produced in the dark is unknown. In this study, the total amount of soluble protein produced in rice seedlings germinated and grown in light or darkness were examined at several time points after germination and under different temperature, nutritional, and seedling density conditions. Our results indicate that rice seedlings grown in darkness produce a comparable amount of total soluble protein to those grown in light. Furthermore, we found that the best conditions for protein production in dark-grown rice seedlings are large seeds germinated and grown for 10–12 days at 28 °C supplemented with Murashige and Skoog medium and 30 g/l sucrose in dense planting. Therefore, our results suggest that foreign proteins can be produced in rice seedlings in the dark, with a reduced electricity use and an increase in cost-effectiveness.

Cloning and functional analysis of the promoter of allergen gene Ara h 1 from peanut

Peanut seeds are ideal bioreactors for the production of foreign recombinant proteins or secondary metabolites. Seed-specific promoters (SSPs) can direct the expression of genes specifically in seeds to avoid undesirable effects associated with constitutive expression. However, few SSPs have been identified in peanut. Previous studies have shown that some allergen-encoding genes encode seed storage proteins or exhibit seed-specific/preferential expression. In this study, we characterized allergen-encoding genes from across the genomes of Arachis species to explore seed-specific genes. We found that at least 9 out of 16 identified peanut allergen-encoding genes were expressed specifically in the seeds or were preferentially expressed. A 1493-bp promoter fragment of allergen gene Ara h 1 (we named it AHSSP6) was isolated from cultivated peanut genome. cis-element analysis showed that three RY repeat elements which usually exsisted in seed or embryo specific promoter sequence were also present in AHSSP6 sequence. Histochemical analysis showed AHSSP6 could drive the expression of a β-glucuronidase (GUS) reporter gene specifically in the seeds or cotyledon tissue of transgenic Arabidopsis, while not in other tissues. These findings indicated that these promoters of allergen genes were candidate SSPs, and AHSSP6 was a novel SSP which could be potentially utilized in peanut improvement.

Application of molecular farming based on barley plant (Hordeum vulgare L.) platform: A Review

Historically, humans have used plants as a source of medicine since ancient times. But the use of transgenic plants in molecular farming is a major source of molecular drugs. The use of molecular farming offers a promising prospect for producing of large-scale, low-cost pharmaceutical and industrial compounds. The results of this research include the production of plasma proteins, enzymes, growth factors, vaccines and antibodies. As plants can offer an inexpensive and convenient platform for the large-scale production of recombinant proteins with various functions, the driven force from the giant market for recombinant protein pharmaceuticals and industrial enzymes makes this subject grow and advance very quickly. With an increased demand for biopharmaceuticals, high costs, and inefficiency of current production systems, transgenic plants have received more attention as a new generation of bioreactors; but determining the best plant platform for the expression of recombinant proteins is one of the critical issues in molecular farming. So far, several expression platforms on this basis for the production of foreign proteins have been evaluated. Among these, the use of dry cereal seeds is one of the most efficient methods used to accumulate recombinant proteins. On the other hand, barley is a right candidate for recombinant protein expression platforms due to its easy cultivation management, high protein content, and low protein diversity, high adaptability to climatic conditions and low production costs. Besides, the highly self-pollinating nature of barley, along with its inability to produce fertile hybrids with related crops or wild species, creates a genetic mechanism, which practically prevents the unwanted dispersion of genetically modified germplasm under field conditions. Iran is also one of the centers of barley diversity in the Middle East, and the vast genetic variety, and nativeness of barley in this region is of particular importance to breeders.

Increased mRNA Stability and Expression Level of Croceibacter atlanticus Lipase Gene Developed through Molecular Evolution Process.

In order to use an enzyme industrially, it is necessary to increase the activity of the enzyme and optimize the reaction characteristics through molecular evolution techniques. We used the error-prone PCR method to improve the reaction characteristics of LipCA lipase discovered in Antarctic Croceibacter atlanticus. Recombinant Escherichia coli colonies showing large halo zones were selected in tributyrin-containing medium. The lipase activity of one mutant strain (M3-1) was significantly increased, compared to the wild-type (WT) strain. M3-1 strain produced about three times more lipase enzyme than did WT strain. After confirming the nucleotide sequence of the M3-1 gene to be different from that of the WT gene by four bases (73, 381, 756, and 822), the secondary structures of WT and M3-1 mRNA were predicted and compared by RNAfold web program. Compared to the mean free energy (MFE) of WT mRNA, that of M3-1 mRNA was lowered by 4.4 kcal/mol, and the MFE value was significantly lowered by mutations of bases 73 and 756. Site-directed mutagenesis was performed to find out which of the four base mutations actually affected the enzyme expression level. Among them, one mutant enzyme production decreased as WT enzyme production when the base 73 was changed (T→C). These results show that one base change at position 73 can significantly affect protein expression level, and demonstrate that changing the mRNA sequence can increase the stability of mRNA, and can increase the production of foreign protein in E. coli.

Mitigation of deleterious phenotypes in chloroplast-engineered plants accumulating high levels of foreign proteins

BackgroundThe global demand for functional proteins is extensive, diverse, and constantly increasing. Medicine, agriculture, and industrial manufacturing all rely on high-quality proteins as major active components or process additives. Historically, these demands have been met by microbial bioreactors that are expensive to operate and maintain, prone to contamination, and relatively inflexible to changing market demands. Well-established crop cultivation techniques coupled with new advancements in genetic engineering may offer a cheaper and more versatile protein production platform. Chloroplast-engineered plants, like tobacco, have the potential to produce large quantities of high-value proteins, but often result in engineered plants with mutant phenotypes. This technology needs to be fine-tuned for commercial applications to maximize target protein yield while maintaining robust plant growth.ResultsHere, we show that a previously developed Nicotiana tabacum line, TetC-cel6A, can produce an industrial cellulase at levels of up to 28% of total soluble protein (TSP) with a slight dwarf phenotype but no loss in biomass. In seedlings, the dwarf phenotype is recovered by exogenous application of gibberellic acid. We also demonstrate that accumulating foreign protein represents an added burden to the plants’ metabolism that can make them more sensitive to limiting growth conditions such as low nitrogen. The biomass of nitrogen-limited TetC-cel6A plants was found to be as much as 40% lower than wildtype (WT) tobacco, although heterologous cellulase production was not greatly reduced compared to well-fertilized TetC-cel6A plants. Furthermore, cultivation at elevated carbon dioxide (1600 ppm CO2) restored biomass accumulation in TetC-cel6A plants to that of WT, while also increasing total heterologous protein yield (mg Cel6A plant−1) by 50–70%.ConclusionsThe work reported here demonstrates that well-fertilized tobacco plants have a substantial degree of flexibility in protein metabolism and can accommodate considerable levels of some recombinant proteins without exhibiting deleterious mutant phenotypes. Furthermore, we show that the alterations to protein expression triggered by growth at elevated CO2 can help rebalance endogenous protein expression and/or increase foreign protein production in chloroplast-engineered tobacco.

CRISPR/Cas technology promotes the various application of Dunaliella salina system.

Dunaliella salina (D. salina) has been widely applied in various fields because of its inherent advantages, such as the study of halotolerant mechanism, wastewater treatment, recombinant proteins expression, biofuel production, preparation of natural materials, and others. However, owing to the existence of low yield or in the laboratory exploration stage, D. salina system has been greatly restricted for practical production of various components. In past decade, significant progresses have been achieved for research of D. salina in these fields. Among them, D. salina as a novel expression system demonstrated a bright prospect, especially for large-scale production of foreign proteins, like the vaccines, antibodies, and other therapeutic proteins. Due to the low efficiency, application of traditional regulation tools is also greatly limited for exploration of D. salina system. The emergence of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system offers a precise editing tool to overcome the obstacles of D. salina system. This review not only comprehensively summarizes the recent progresses of D. salina in domain of gene engineering but also gives a deep analysis of problems and deficiencies in different fields of D. salina. Moreover, further prospects of CRISPR/Cas system and its significant challenges have been discussed in various aspects of D. salina. It provides a great referencing value for speeding up the maturity of D. salina system, and also supplies practical guiding significance to expand the new application fields for D. salina. KEY POINTS: • The review provides recent research progresses of various applications of D. salina. • The problems and deficiencies in different fields of D. salina were deeply analyzed. • The further prospects of CRISPR/Cas technology in D. salina system were predicted. • CRISPR/Cas system will promote the new application fields and maturity for D. salina.

Development of a novel enhanced baculovirus expression vector via promoter combination

Abstract Baculovirus expression systems (BESs) have been used to express many foreign proteins. BESs are known to be very suitable for foreign protein production due to the strong gene expression ability of the polyhedrin promoter. However, expression of foreign proteins using the polyhedrin promoter begins at a very late phase, in the last stage of viral infection. This causes insufficient production of foreign proteins due to death of the host cells. Therefore, our study aimed to construct a vector that can enable not only earlier production of foreign recombinant proteins but also increased production. For this purpose, a novel overexpression vector based on the polyhedrin promoter was constructed by using transcriptional enhancers (hr3 and repeated burst sequences (BSs)) and an additional promoter capable of inducing earlier gene expression than the polyhedrin promoter. Among the vectors with additional promoters that were evaluated, the overexpression vector with the p6.9 promoter showed the highest foreign protein expression efficiency. This vector showed approximately 94 times greater and one-day-earlier expression of the foreign protein than the control vector containing only the polyhedrin promoter. In order of greatest to least influence, the factors that increased the expression efficiency of the polyhedrin promoter were the repeated BSs, the p6.9 promoter and hr3.

CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation

The introduction of insertion-deletions (INDELs) by non-homologous end-joining (NHEJ) pathway underlies the mechanistic basis of CRISPR-Cas9-directed genome editing. Selective gene ablation using CRISPR-Cas9 is achieved by installation of a premature termination codon (PTC) from a frameshift-inducing INDEL that elicits nonsense-mediated decay (NMD) of the mutant mRNA. Here, by examining the mRNA and protein products of CRISPR targeted genes in a cell line panel with presumed gene knockouts, we detect the production of foreign mRNAs or proteins in ~50% of the cell lines. We demonstrate that these aberrant protein products stem from the introduction of INDELs that promote internal ribosomal entry, convert pseudo-mRNAs (alternatively spliced mRNAs with a PTC) into protein encoding molecules, or induce exon skipping by disruption of exon splicing enhancers (ESEs). Our results reveal challenges to manipulating gene expression outcomes using INDEL-based mutagenesis and strategies useful in mitigating their impact on intended genome-editing outcomes.

Enhanced foreign protein accumulation in Nicotiana benthamiana leaves co-infiltrated with a TMV vector and plant cell cycle regulator genes.

In this short communication, we report that the cell cycle checkpoint genes At-CycD2 and At-CDC27a from Arabidopsis thaliana enhance the transient heterologous protein expression in Nicotiana benthamiana. We selected a well-studied and widely used virus expression vector based on TMV for the delivery of recombinant proteins into the host plant. Co-infiltration of TMV-gfp and binary expression vectors carrying the At-CycD2 and At-CDC27a genes, respectively, resulted in enhanced GFP fluorescence in agroinoculated leaves. These findings corresponded with the observation of (1) higher mRNA levels for TMV and gfp and (2) increased GFP protein accumulation. Furthermore, by co-delivery of the TMV-scFv-TM43-E10 and At-CycD2/At-CDC27a expressing constructs we observed an enhanced amount of the scFv-TM43-E10 antibody fragment compared to the delivery of the TMV-scFv-TM43-E10 alone. We anticipate that this finding might be adapted for enhancing foreign protein production in N. benthamiana as the host plant.

Identification of sugar response complex in the metallothionein OsMT2b gene promoter for enhancement of foreign protein production in transgenic rice.

A 146-bp sugar response complex MTSRC is identified in the promoter of rice metallothionein OsMT2b gene conferring high-level expression of luciferase reporter gene and bioactive recombinant haFGF in transgenic rice. A rice subfamily type 2 plant metallothionein (pMT) gene, OsMT2b, encoding a reactive oxygen species (ROS) scavenger protein, has been previously shown to exhibit the most abundant gene expression in young rice seedling. Expression of OsMT2b was found to be regulated negatively by ethylene and hydrogen peroxide in rice stem node under flooding stress, but little is known about its response to sugar depletion. In this study, transient expression assay and transgenic approach were employed to characterize the regulation of the OsMT2b gene expression in rice. We found that the expression of OsMT2b gene is induced by sugar starvation in both rice suspension cells and germinated embryos. Deletion analysis and functional assay of the OsMT2b promoter revealed that the 5'-flanking region of the OsMT2b between nucleotides - 351 and - 121, which contains the sugar response complex (- 266 to - 121, designated MTSRC) is responsible for high-level promoter activity under sugar starvation. It was also found that MTSRC significantly enhances the Act1 promoter activity in transgenic rice cells and seedlings. The modified Act1 promoter, Act1-MTSRC, was used to produce the recombinant human acidic fibroblast growth factor (haFGF) in rice cells. Our result shows that the bioactive recombinant haFGF is stably produced in transformed rice cell culture and yields are up to 2% of total medium proteins. Our studies reveal that MTSRC serves as a strong transcriptional activator and the Act1-MTSRC promoter can be applicable in establishing an efficient expression system for the high-level production of foreign proteins in transgenic rice cells and seedlings.

H-102 The potential role of “defective” proviruses in the pathogenesis of HIV-1 infection

A pool of CD4+T cells harboring HIV-1 proviruses is established early during HIV-1 infection and persists despite years of suppressive combination antiretroviral therapy (cART). Greater than 95% of the proviruses in these pools are “defective” due to large internal deletions and lethal genetic mutations. Although unable to encode intact viruses, these “defective” proviruses are biologically active, transcribe HIV-RNA and produce HIV-1 proteins in vivo. We hypothesize that ongoing production of foreign proteins from these translationally-competent yet defective HIV-1 proviruses contribute to the persistent immune activation and systemic inflammation during suppressive ART.

Genome-wide mining seed-specific candidate genes from peanut for promoter cloning

Peanut seeds are ideal bioreactors for the production of foreign recombinant proteins and/or nutrient metabolites. Seed-Specific Promoters (SSPs) are important molecular tools for bioreactor research. However, few SSPs have been characterized in peanut seeds. The mining of Seed-Specific Candidate Genes (SSCGs) is a prerequisite for promoter cloning. Here, we described an approach for the genome-wide mining of SSCGs via comparative gene expression between seed and nonseed tissues. Three hundred thirty-seven SSCGs were ultimately identified, and the top 108 SSCGs were characterized. Gene Ontology (GO) analysis revealed that some SSCGs were involved in seed development, allergens, seed storage and fatty acid metabolism. RY REPEAT and GCN4 motifs, which are commonly found in SSPs, were dispersed throughout most of the promoters of SSCGs. Expression pattern analysis revealed that all 108 SSCGs were expressed specifically or preferentially in the seed. These results indicated that the promoters of the 108 SSCGs may perform functions in a seed-specific and/or seed-preferential manner. Moreover, a novel SSP was cloned and characterized from a paralogous gene of SSCG29 from cultivated peanut. Together with the previously characterized SSP of the SSCG5 paralogous gene in cultivated peanut, these results implied that the method for SSCG identification in this study was feasible and accurate. The SSCGs identified in this work could be widely applied to SSP cloning by other researchers. Additionally, this study identified a low-cost, high-throughput approach for exploring tissue-specific genes in other crop species.

The effects of overexpression of cytoplasmic chaperones on secretory production of hirudin-PA in E. coli

The secretory production of heterologous proteins in E. coli has revolutionized biotechnology. Efficient periplasmic production of foreign proteins in E. coli often requires a signal peptide to direct proteins to the periplasm. However, the presence of attached signal peptide does not guarantee periplasmic expression of target proteins. Overproduction of auxiliary proteins, such as chaperones can be a useful approach to enhance protein export. In the current study, three chaperone plasmid sets, including GroEL–GroES (GroELS), Dnak–Dnaj–GrpE (DnaKJE), and trigger factor (TF), were coexpressed in E. coli BL21 (DE3) in a pairwise manner with two pET22-b vectors carrying the recombinant hirudin-PA (Hir) gene and different signal sequences alkaline phosphatase (PhoA) and l-asparaginase II (l-ASP). Overexpression of cytoplasmic combinations of molecular chaperones containing GroELS and DnaKJE with PhoAHir increased the secretory production of PhoAHir by 2.6fold (p < 0.05) and 3.5fold (p < 0.01) compared with their controls, respectively. By contrast, secretory production of PhoAHir significantly reduced in the presence of overexpressed TF (p = 0.02). Further, periplasmic expression of l-ASP was significantly increased only in the presence of DnaKJE (p = 0.04). These findings suggest that using molecular chaperones can be helpful for improving periplasmic expression of Hir. However, tagged signal peptides may affect the physicochemical properties and secondary and tertiary structures of mature Hir, which may alter their interactions with chaperones. Hence, using overexpressed chaperones has various effects on secretory production of PhoAHir and l-ASPHir.

Somatic Embryogenesis as Key Technology for Shaping the Rubber Tree of the Future.

Worldwide, Hevea producers face the need to replant large surfaces in the coming years. The rubber yield per ha, produced by trees grafted on heterogeneous illegitimate seedling rootstocks, has reached its maximum. For long-standing Hevea clones, as for a lot of other tree species, one of the consequences of physiological aging is reduced in vitro growth and the lack of a proper geotropic (tap) root system. Somatic embryogenesis on young inner seed integument or stamen filaments provides a mean to regain ontogenetic juvenility. The process is limited by irregular germination of the somatic embryos. Nevertheless, with the obtained in vitro plants, juvenile lines have been established of the most important profitable rubber tree clones. Currently they are micropropagated on a commercial scale. Moreover, the produced plants can serve as mother plants for propagation by means of macro-cutting. Somatic embryogenesis enables the production of transgenic Hevea brasiliensis as well. Genes conferring plant disease resistance, abiotic stress tolerance and production of foreign proteins in the lactiferous vessels will further shape the rubber tree of the future.

Chloroplasts as Cellular Factories for the Cost-effective Production of Cellulases.

Chloroplasts are vital photosynthetic organelles in plant cells that carry out several important cellular functions including synthesis of amino acids, fatty acids, and lipids and metabolism of nitrogen, starch, and Sulphur to sustain the homeostasis in plants. These organelles have got their own genome, and related genetic machinery to synthesize required proteins for various plant functions. Genetic manipulations of the chloroplast genome for different biotech applications has been of great interest due to desired features including the availability of operonal mode of gene expression, high copy number, and maternal mode of inheritance (in the most field crops). Their capacity to often express transgenes at high levels make it a cost-effective platform for the production of foreign proteins, particularly high-value targets of industrial importance, at large scale. This article reviews briefly the research work carried out to produce cellulolytic enzymes in higher plant chloroplasts. The challenges and future opportunities for the same are also discussed.