Efficient targeting of recombinant proteins to the thylakoid lumen in Chlamydomonas reinhardtii using a bacterial Tat signal peptide

Zedler, Julie A Z; Mullineaux, Conrad W.; Robinson, Colin

doi:10.1016/j.algal.2016.07.007

Cited by 6 publications

(5 citation statements)

References 23 publications

(11 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In most cases these are single subunit proteins and therefore involve the introduction of only a single transgene, although there have been a few examples of multigenic engineering of the plastome [32,33]. To date, all of the therapeutic proteins that have been reported are soluble and accumulate in the chloroplast stroma, save for a single report of the targeting of an antibody fragment to the thylakoid lumen [34], although membrane-anchored proteins have been successfully produced in the algal chloroplast [35].…”

Section: Biopharmaceuticals Made In the C Reinhardtii Chloroplastmentioning

confidence: 99%

The algal chloroplast as a synthetic biology platform for production of therapeutic proteins

2018

View full text Add to dashboard Cite

The chloroplast of Chlamydomonas reinhardtii and other microalgae represents an attractive new platform for the synthesis of recombinant therapeutics using synthetic biology (synbio) approaches. Transgenes can be designed in silico, assembled from validated DNA parts and inserted at precise and predetermined locations within the chloroplast genome to give stable synthesis of a desired recombinant protein. Numerous recent examples of different therapeutic proteins produced successfully in the C. reinhardtii chloroplast highlight the potential of this green alga as a simple, low-cost and benign host. Furthermore, some of the features of the alga may offer additional advantages over more-established microbial, mammalian or plant-based systems. These include efficient folding and accumulation of the product in the chloroplast; a lack of contaminating toxins or infectious agents; reduced downstream processing requirements; the possibility to make complex therapeutics such as immunotoxins; and the opportunity to use the whole alga as a low-cost oral vaccine. In this paper we review the current status of algal chloroplast engineering with respect to therapeutic proteins. We also consider future advances in synbio tools, together with improvements to recipient strains, which will allow the design of bespoke strains with high levels of productivity.

show abstract

Section: Biopharmaceuticals Made In the C Reinhardtii Chloroplastmentioning

confidence: 99%

The algal chloroplast as a synthetic biology platform for production of therapeutic proteins

2018

View full text Add to dashboard Cite

show abstract

“…To investigate the impact on growth of dsRNA expression in the PYP strain, the growth of the strain was analyzed in comparison to a previously generated transformant that has the pSRSapI vector integrated in its chloroplast genome without a gene of interest (strain termed SR) 15 . This strain was chosen as a control since the TN72 strain used for transformation cannot grow phototrophically.…”

Section: Resultsmentioning

confidence: 99%

“…The growth of the generated C. reinhardtii strain expressing dsRNA-YHV (PYP) was analyzed in direct comparison to a control strain (TN72 transformed with the pSRSapI plasmid) previously generated 15 . Pre-cultures of both strains were prepared as described in Section 2.1 and 10 mL of each strain were re-inoculated into 400 mL TAP medium with continuous shaking at 100 rpm and 50 µmol photons m −2 s −1 illumination at 25 °C.…”

Section: Methodsmentioning

confidence: 99%

Generation of microalga Chlamydomonas reinhardtii expressing shrimp antiviral dsRNA without supplementation of antibiotics

Charoonnart

Worakajit

Zedler

et al. 2019

Sci Rep

Self Cite

View full text Add to dashboard Cite

RNA interference (RNAi) is an effective way of combating shrimp viruses by using sequence-specific double-stranded (dsRNA) designed to knock down key viral genes. The aim of this study was to use microalgae expressing antiviral dsRNA as a sustainable feed supplement for shrimp offering viral protection. In this proof of concept, we engineered the chloroplast genome of the green microalga Chlamydomonas reinhardtii for the expression of a dsRNA cassette targeting a shrimp yellow head viral gene. We used a previously described chloroplast transformation approach that allows for the generation of stable, marker-free C. reinhardtii transformants without the supplementation of antibiotics. The generated dsRNA-expressing microalgal strain was then used in a shrimp feeding trial to evaluate the efficiency of the algal RNAi-based vaccine against the virus. Shrimps treated with dsRNA-expressed algal cells prior to YHV infection had 50% survival at 8 day-post infection (dpi), whereas 84.1% mortality was observed in control groups exposed to the YHV virus. RT-PCR using viral specific primers revealed a lower infection rate in dsRNA-expressing algae treated shrimp (55.6 ± 11.1%) compared to control groups (88.9 ± 11.1% and 100.0 ± 0.0%, respectively). Our results are promising for using microalgae as a novel, sustainable alternative as a nutritious, anti-viral protective feedstock in shrimp aquaculture.

show abstract

“…The authors demonstrated the usefulness of synthetic promoters to drive gene expression as a complementary method for the production of recombinant proteins. With another method, bacterial Tat export signal peptides were applied to translocate recombinant proteins within the thylakoid lumen of the C. reinhardtii chloroplast, and the result presented an innovative approach of using thylakoid lumen as a new compartment for producing recombinant proteins, which provided more protective environments for delicate proteins [ 30 ].…”

Section: Emerging Trends In Genetic Engineering Of Microalgae For Com...mentioning

confidence: 99%

Emerging Trends in Genetic Engineering of Microalgae for Commercial Applications

Grama

Liu

2022

Marine Drugs

View full text Add to dashboard Cite

Recently, microalgal biotechnology has received increasing interests in producing valuable, sustainable and environmentally friendly bioproducts. The development of economically viable production processes entails resolving certain limitations of microalgal biotechnology, and fast evolving genetic engineering technologies have emerged as new tools to overcome these limitations. This review provides a synopsis of recent progress, current trends and emerging approaches of genetic engineering of microalgae for commercial applications, including production of pharmaceutical protein, lipid, carotenoids and biohydrogen, etc. Photochemistry improvement in microalgae and CO2 sequestration by microalgae via genetic engineering were also discussed since these subjects are closely entangled with commercial production of the above mentioned products. Although genetic engineering of microalgae is proved to be very effective in boosting performance of production in laboratory conditions, only limited success was achieved to be applicable to industry so far. With genetic engineering technologies advancing rapidly and intensive investigations going on, more bioproducts are expected to be produced by genetically modified microalgae and even much more to be prospected.

show abstract

Efficient targeting of recombinant proteins to the thylakoid lumen in Chlamydomonas reinhardtii using a bacterial Tat signal peptide

Cited by 6 publications

References 23 publications

The algal chloroplast as a synthetic biology platform for production of therapeutic proteins

The algal chloroplast as a synthetic biology platform for production of therapeutic proteins

Generation of microalga Chlamydomonas reinhardtii expressing shrimp antiviral dsRNA without supplementation of antibiotics

Emerging Trends in Genetic Engineering of Microalgae for Commercial Applications

Contact Info

Product

Resources

About