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Microorganisms
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Production of Recombinant Molecules in Algal Chloroplasts
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Production of Recombinant Molecules in Algal Chloroplasts

A special issue of Microorganisms (ISSN 2076-2607). This special issue belongs to the section "Microbial Biotechnology".

Deadline for manuscript submissions: closed (30 April 2023) | Viewed by 23463

Special Issue Editor

Prof. Dr. Saul Purton

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Guest Editor
Institute of Structural and Molecular Biology, Darwin Building, University College London, Gower Street, London WC1E 6BT, UK
Interests: algal biotechnology; synthetic biology; chloroplast genetic engineering; oral vaccines and anti-microbials; photosynthesis
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

There is a growing interest in the use of microalgae as cell factories for the light-driven synthesis of recombinant products. These include recombinant proteins and RNAs that have potential application in areas such as therapeutics, and novel metabolites and biopolymers that have bioactive properties or could serve as feedstocks for green technologies. Key to the successful synthesis of a recombinant molecule is the robust expression of the transgene(s) and the hyper-accumulation of the product within the cell. In this regard, the algal chloroplast represents an attractive sub-cellular target as it possesses a small genetically tractable genome, and is able to serve as a storage compartment for proteins, polymers and lipids. Furthermore, the GRAS status of key microalgal species makes possible the idea of whole cell therapeutics in which live or dried cells serve as bioencapsulation devices for oral delivery of bioactive compounds. Although the model species Chlamydomonas reinhardtii is the most developed platform for algal chloroplast transgenics, the technology is now spreading to other industrially relevant species. It is, therefore, timely to launch this Special Issue, and I invite original reports or review articles covering basic or applied topics in the field—for example, methodologies for genetic engineering of algal chloroplasts (using either chloroplast or nuclear transformation approaches), novel recombinants produced in the algal chloroplast, and new biotechnological applications of engineered strains. 

Prof. Dr. Saul Purton
Guest Editor

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Keywords

  • microalgae
  • chloroplast
  • plastome
  • metabolic engineering
  • recombinant product
  • algal biotechnology

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Published Papers (7 papers)

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Research

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13 pages, 2626 KiB  
Article
Accelerating Chloroplast Engineering: A New System for Rapid Generation of Marker-Free Transplastomic Lines of Chlamydomonas reinhardtii
by Henry N. Taunt, Harry O. Jackson, Ísarr N. Gunnarsson, Rabbia Pervaiz and Saul Purton
Microorganisms 2023, 11(8), 1967; https://doi.org/10.3390/microorganisms11081967 - 31 Jul 2023
Cited by 4 | Viewed by 1874
Abstract
‘Marker-free’ strategies for creating transgenic microorganisms avoid the issue of potential transmission of antibiotic resistance genes to other microorganisms. An already-established strategy for engineering the chloroplast genome (=plastome) of the green microalga Chlamydomonas reinhardtii involves the restoration of photosynthetic function using a recipient [...] Read more.
‘Marker-free’ strategies for creating transgenic microorganisms avoid the issue of potential transmission of antibiotic resistance genes to other microorganisms. An already-established strategy for engineering the chloroplast genome (=plastome) of the green microalga Chlamydomonas reinhardtii involves the restoration of photosynthetic function using a recipient strain carrying a plastome mutation in a key photosynthesis gene. Selection for transformant colonies is carried out on minimal media, such that only those cells in which the mutated gene has been replaced with a wild-type copy carried on the transgenic DNA are capable of phototrophic growth. However, this approach can suffer from issues of efficiency due to the slow growth of C. reinhardtii on minimal media and the slow die-back of the untransformed lawn of cells when using mutant strains with a limited photosensitivity phenotype. Furthermore, such phototrophic rescue has tended to rely on existing mutants that are not necessarily ideal for transformation and targeted transgene insertion: Mutants carrying point mutations can easily revert, and those with deletions that do not extend to the intended transgene insertion site can give rise to a sub-population of rescued lines that lack the transgene. In order to improve and accelerate the transformation pipeline for C. reinhardtii, we have created a novel recipient line, HNT6, carrying an engineered deletion in exon 3 of psaA, which encodes one of the core subunits of photosystem I (PSI). Such PSI mutants are highly light-sensitive allowing faster recovery of transformant colonies by selecting for light-tolerance on acetate-containing media, rather than phototrophic growth on minimal media. The deletion extends to a site upstream of psaA-3 that serves as a neutral locus for transgene insertion, thereby ensuring that all of the recovered colonies are transformants containing the transgene. We demonstrate the application of HNT6 using a luciferase reporter. Full article
(This article belongs to the Special Issue Production of Recombinant Molecules in Algal Chloroplasts)
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14 pages, 2592 KiB  
Article
Transgenic Microalgae Expressing Double-Stranded RNA as Potential Feed Supplements for Controlling White Spot Syndrome in Shrimp Aquaculture
by Patai Charoonnart, Henry Nicholas Taunt, Luyao Yang, Conner Webb, Colin Robinson, Vanvimon Saksmerprome and Saul Purton
Microorganisms 2023, 11(8), 1893; https://doi.org/10.3390/microorganisms11081893 - 26 Jul 2023
Cited by 5 | Viewed by 2438
Abstract
Viral infection of farmed fish and shellfish represents a major issue within the aquaculture industry. One potential control strategy involves RNA interference of viral gene expression through the oral delivery of specific double-stranded RNA (dsRNA). In previous work, we have shown that recombinant [...] Read more.
Viral infection of farmed fish and shellfish represents a major issue within the aquaculture industry. One potential control strategy involves RNA interference of viral gene expression through the oral delivery of specific double-stranded RNA (dsRNA). In previous work, we have shown that recombinant dsRNA can be produced in the chloroplast of the edible microalga Chlamydomonas reinhardtii and used to control disease in shrimp. Here, we report a significant improvement in antiviral dsRNA production and its use to protect shrimp against white spot syndrome virus (WSSV). A new strategy for dsRNA synthesis was developed that uses two convergent copies of the endogenous rrnS promoter to drive high-level transcription of both strands of the WSSV gene element in the chloroplast. Quantitative RT-PCR indicated that ~119 ng dsRNA was produced per liter of culture of the transgenic microalga. This represents an ~10-fold increase in dsRNA relative to our previous report. The engineered alga was assessed for its ability to prevent WSSV infection when fed to shrimp larvae prior to a challenge with the virus. The survival of shrimp given feed supplemented with dried alga containing the dsRNA was significantly enhanced (~69% survival) relative to a negative control (<10% survival). The findings suggest that this new dsRNA production platform could be employed as a low-cost, low-tech control method for aquaculture. Full article
(This article belongs to the Special Issue Production of Recombinant Molecules in Algal Chloroplasts)
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19 pages, 2824 KiB  
Article
Spray Drying Is a Viable Technology for the Preservation of Recombinant Proteins in Microalgae
by Anaëlle Vilatte, Xenia Spencer-Milnes, Harry Oliver Jackson, Saul Purton and Brenda Parker
Microorganisms 2023, 11(2), 512; https://doi.org/10.3390/microorganisms11020512 - 17 Feb 2023
Cited by 13 | Viewed by 3817
Abstract
Microalgae are promising host organisms for the production of encapsulated recombinant proteins such as vaccines. However, bottlenecks in bioprocess development, such as the drying stage, need to be addressed to ensure feasibility at scale. In this study, we investigated the potential of spray [...] Read more.
Microalgae are promising host organisms for the production of encapsulated recombinant proteins such as vaccines. However, bottlenecks in bioprocess development, such as the drying stage, need to be addressed to ensure feasibility at scale. In this study, we investigated the potential of spray drying to produce a recombinant vaccine in microalgae. A transformant line of Chlamydomonas reinhardtii carrying a subunit vaccine against salmonid alphavirus was created via chloroplast engineering. The integrity of the recombinant protein after spray drying and its stability after 27 months storage at –80 °C, +4 °C and room temperature were assessed by immunoblotting. The protein withstood spray drying without significant losses. Long-term storage at +4 °C and room temperature resulted in 50% and 92% degradation, respectively. Optimizing spray drying and storage conditions should minimize degradation and favour short-term storage at positive temperatures. Using data on yield and productivity, the economics of spray drying- and freeze drying-based bioprocesses were compared. The drying stage corresponded to 41% of the total production cost. Process optimization, genetic engineering and new market strategies were identified as potential targets for cost reduction. Overall, this study successfully demonstrates the suitability of spray drying as a process option for recombinant protein production in microalgae at the industrial scale. Full article
(This article belongs to the Special Issue Production of Recombinant Molecules in Algal Chloroplasts)
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15 pages, 2600 KiB  
Article
A Chloroplast-Localised Fluorescent Protein Enhances the Photosynthetic Action Spectrum in Green Algae
by Julio V. Suarez, Elisabeth A. Mudd and Anil Day
Microorganisms 2022, 10(9), 1770; https://doi.org/10.3390/microorganisms10091770 - 1 Sep 2022
Cited by 3 | Viewed by 2341
Abstract
Green microalgae are important sources of natural products and are attractive cell factories for manufacturing high-value products such as recombinant proteins. Increasing scales of production must address the bottleneck of providing sufficient light energy for photosynthesis. Enhancing the photosynthetic action spectrum of green [...] Read more.
Green microalgae are important sources of natural products and are attractive cell factories for manufacturing high-value products such as recombinant proteins. Increasing scales of production must address the bottleneck of providing sufficient light energy for photosynthesis. Enhancing the photosynthetic action spectrum of green algae to improve the utilisation of yellow light would provide additional light energy for photosynthesis. Here, we evaluated the Katushka fluorescent protein, which converts yellow photons to red photons, to drive photosynthesis and growth when expressed in Chlamydomonas reinhardtii chloroplasts. Transplastomic algae expressing a codon-optimised Katushka gene accumulated the active Katushka protein, which was detected by excitation with yellow light. Removal of chlorophyll from cells, which captures red photons, led to increased Katushka fluorescence. In yellow light, emission of red photons by fluorescent Katushka increased oxygen evolution and photosynthetic growth. Utilisation of yellow photons increased photosynthetic growth of transplastomic cells expressing Katushka in light deficient in red photons. These results showed that Katushka was a simple and effective yellow light-capturing device that enhanced the photosynthetic action spectrum of C. reinhardtii. Full article
(This article belongs to the Special Issue Production of Recombinant Molecules in Algal Chloroplasts)
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17 pages, 2592 KiB  
Article
Overcoming Poor Transgene Expression in the Wild-Type Chlamydomonas Chloroplast: Creation of Highly Mosquitocidal Strains of Chlamydomonas reinhardtii
by Obed W. Odom, Seongjoon Kang, Caleb Ferguson, Carrie Chen and David L. Herrin
Microorganisms 2022, 10(6), 1087; https://doi.org/10.3390/microorganisms10061087 - 25 May 2022
Cited by 1 | Viewed by 2435
Abstract
High-level expression of transgenes in the chloroplast of wild-type Chlamydomonas reinhardtii (C. reinhardtii) remains challenging for many genes (e.g., the cry toxin genes from Bacillus thuringiensis israelensis). The bottleneck is presumed to be post-transcriptional and mediated by the 5′ element [...] Read more.
High-level expression of transgenes in the chloroplast of wild-type Chlamydomonas reinhardtii (C. reinhardtii) remains challenging for many genes (e.g., the cry toxin genes from Bacillus thuringiensis israelensis). The bottleneck is presumed to be post-transcriptional and mediated by the 5′ element and the coding region. Using 5′ elements from highly expressed photosynthesis genes such as atpA did not improve the outcome with cry11A regardless of the promoter. However, when we employed the 5′ UTR from mature rps4 mRNA with clean fusions to promoters, production of the rCry11A protein became largely promoter-dependent. The best results were obtained with the native 16S rrn promoter (−91 to −1). When it was fused to the mature 5′ rps4 UTR, rCry11A protein levels were ~50% higher than was obtained with the inducible system, or ~0.6% of total protein. This level was sufficient to visualize the 73-kDa rCry11A protein on Coomassie-stained gels of total algal protein. In addition, analysis of the expression of these transgenes by RT-PCR indicated that RNA levels roughly correlated with protein production. Live cell bioassays using the best strains as food for 3rd instar Aedes aegypti larvae showed that most larvae were killed even when the cell concentration was as low as 2 × 104 cells/mL. Finally, the results indicate that these highly toxic strains are also quite stable, and thus represent a key milestone in using C. reinhardtii for mosquito control. Full article
(This article belongs to the Special Issue Production of Recombinant Molecules in Algal Chloroplasts)
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17 pages, 2077 KiB  
Article
Overview of tRNA Modifications in Chloroplasts
by Maxime Fages-Lartaud and Martin Frank Hohmann-Marriott
Microorganisms 2022, 10(2), 226; https://doi.org/10.3390/microorganisms10020226 - 20 Jan 2022
Cited by 2 | Viewed by 4135
Abstract
The chloroplast is a promising platform for biotechnological innovation due to its compact translation machinery. Nucleotide modifications within a minimal set of tRNAs modulate codon–anticodon interactions that are crucial for translation efficiency. However, a comprehensive assessment of these modifications does not presently exist [...] Read more.
The chloroplast is a promising platform for biotechnological innovation due to its compact translation machinery. Nucleotide modifications within a minimal set of tRNAs modulate codon–anticodon interactions that are crucial for translation efficiency. However, a comprehensive assessment of these modifications does not presently exist in chloroplasts. Here, we synthesize all available information concerning tRNA modifications in the chloroplast and assign translation efficiency for each modified anticodon–codon pair. In addition, we perform a bioinformatics analysis that links enzymes to tRNA modifications and aminoacylation in the chloroplast of Chlamydomonas reinhardtii. This work provides the first comprehensive analysis of codon and anticodon interactions of chloroplasts and its implication for translation efficiency. Full article
(This article belongs to the Special Issue Production of Recombinant Molecules in Algal Chloroplasts)
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Review

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27 pages, 1116 KiB  
Review
Harnessing the Algal Chloroplast for Heterologous Protein Production
by Edoardo Andrea Cutolo, Giulia Mandalà, Luca Dall’Osto and Roberto Bassi
Microorganisms 2022, 10(4), 743; https://doi.org/10.3390/microorganisms10040743 - 30 Mar 2022
Cited by 20 | Viewed by 5139
Abstract
Photosynthetic microbes are gaining increasing attention as heterologous hosts for the light-driven, low-cost production of high-value recombinant proteins. Recent advances in the manipulation of unicellular algal genomes offer the opportunity to establish engineered strains as safe and viable alternatives to conventional heterotrophic expression [...] Read more.
Photosynthetic microbes are gaining increasing attention as heterologous hosts for the light-driven, low-cost production of high-value recombinant proteins. Recent advances in the manipulation of unicellular algal genomes offer the opportunity to establish engineered strains as safe and viable alternatives to conventional heterotrophic expression systems, including for their use in the feed, food, and biopharmaceutical industries. Due to the relatively small size of their genomes, algal chloroplasts are excellent targets for synthetic biology approaches, and are convenient subcellular sites for the compartmentalized accumulation and storage of products. Different classes of recombinant proteins, including enzymes and peptides with therapeutical applications, have been successfully expressed in the plastid of the model organism Chlamydomonas reinhardtii, and of a few other species, highlighting the emerging potential of transplastomic algal biotechnology. In this review, we provide a unified view on the state-of-the-art tools that are available to introduce protein-encoding transgenes in microalgal plastids, and discuss the main (bio)technological bottlenecks that still need to be addressed to develop robust and sustainable green cell biofactories. Full article
(This article belongs to the Special Issue Production of Recombinant Molecules in Algal Chloroplasts)
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