Biology subjects

Purton, S.

Publications and source records attributed to Purton, S..

4 recordsLinked to original sources

bioRxiv2025

Chlamydomonas chloroplast genes tolerate compression of the genetic code to just 51 codons

Genome scale engineering has enabled codon compression of the universal genetic code of up to three codons in E. coli, providing the means for genetic code expansion. To go much beyond this number, smaller and simpler genetic systems are needed to avoid significant technical challenges. Chloroplast genomes offer multiple advantages for codon compression and reassignment. Here we report a recoding scheme for the Chlamydomonas reinhardtii chloroplast genome, in which two stop codons and one or more of the codons for arginine, glycine, isoleucine, leucine and serine, all of which have two cognate tRNAs, are absent, compressing the genetic code to 51 codons. Firstly, several recoding strategies were tested on the essential rpoA gene, encoding a subunit of the chloroplast RNA polymerase. A defined compression scheme, which relied on swapping the target codons with the permitted frequent codons, could replace the native protein coding sequence without affecting chloroplast protein expression levels or the strain fitness. The same strategy was successfully used for codon compression of ycf1, encoding a subunit of the chloroplast translocon, psaA and psbA, intron-containing highly expressed genes encoding reaction centres subunits of both photosystems, and an 8.5 kb operon encoding essential and non-essential genes. Finally, we tested degeneracy of the 51-codon genetic code by exploring the combinatorial design for the large subunit of RuBisCO, relying on restoration of photosynthesis in an rbcL mutant strain. More than 70 functional sequences with diverse codon adaptation indices were recovered. In all codon-compressed genes there was no observable penalty on photosynthetic growth. Significance StatementWe demonstrate the use of a chloroplast genome as a synthetic biology platform to test modifications of the universal genetic code. Elimination of 13 of the 64 possible triplet codes (codons) was possible for several essential and/or highly expressed genes without affecting chloroplast function. The technology is mature, paving the way for codon compression of the entire genome and thus enabling further radical rearrangement or expansion of the genetic code.

synthetic biology↗

bioRxiv2025

Structure-based analysis unveils co-origin of LPOR and nitrogenase-like proteins

Structures of nitrogenases, dark-operative protochlorophyllide oxidoreductases, and light-dependent protochlorophyllide oxidoreductases (LPOR) have been resolved. However, their evolutionary relatedness remains elusive. Here, we show, through structural alignment, that all subunits of nitrogenase-like proteins originated from a co-ancestral archaic one-subdomain precursor. LPOR evolved from the BchX/BchY subunits of nitrogenase-like chlorophyllide a oxidoreductase (COR), and the intermediary retinol dehydrogenase through possible genetic recombination. We thus establish previously unknown structural links among key enzymes involved in biological nitrogen-fixation (BNF) and photosynthesis, unraveling structure-guided functional evolution from a single-subunit iron protein to multi-subunit nitrogenase-like COR, and to the single-subunit LPOR for phototrophic metabolism via bacteriochlorophyll, retinal, and chlorophyll. This work also demonstrates structural similarities are imperative for inferring distant origins of functionally divergent proteins, particularly those lacking primary amino-acid sequence identity. Moreover, our findings coupled with AI may be exploited to design innovative light-driven CORs and/or light-utilizing nitrogenases with enhanced efficacy of photosynthesis and BNF. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/634645v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@9f9a2aorg.highwire.dtl.DTLVardef@4243ecorg.highwire.dtl.DTLVardef@796b4corg.highwire.dtl.DTLVardef@17373da_HPS_FORMAT_FIGEXP M_FIG C_FIG Video abstracthttps://www.youtube.com/watch?v=b0qBE1JMaH0 HighlightsO_LILPOR evolves from BchX and BchY subunits of the nitrogenase-like protein (NGLP) COR C_LIO_LIAll subunits of NGLPs can be traced back to the ancestral one-subdomain precursor C_LIO_LIRetinol dehydrogenase acts as a crucial intermediate in the COR to LPOR evolution C_LIO_LIStructure-based design of LUN is showcased to revolutionize protein engineering C_LI

evolutionary biology↗

bioRxiv2022

Enhanced production of antiviral dsRNA in the Chlamydomonas reinhardtii chloroplast via a novel convergent promoter expression system

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. New vectors were designed that allow rapid Golden Gate-mediated assembly of a transformation plasmid in which the dual promoter-WSSV DNA cassette is targeted into the chloroplast genome, with selection based on the restoration of photosynthesis. PCR analysis of transformant lines confirmed the integration of the cassette and homoplasmy of the polyploid genome. Transcribed sense and antisense VP28-RNA were hypothesised to form an RNA duplex in the chloroplast stroma, and quantitative RT-PCR indicated that [~]100 g dsRNA is produced per litre of transgenic microalgae culture. This represents an [~]10,000-fold increase in dsRNA relative to previous reports using convergent psaA promoters. The engineered alga was assessed for its ability to prevent WSSV infection when fed to shrimp larvae prior to a challenge with the virus. Survival of shrimp fed with dsRNA-expressing C. reinhardtii was significantly enhanced (68.3%) relative to the negative control. The study suggests that this new dsRNA production platform is significantly more efficient than that reported previously, and merits further scale-up and downstream processing studies.

molecular biology↗

bioRxiv2018

Genetic transformation of the dinoflagellate chloroplast

Coral reefs are some of the most important and ecologically diverse marine environments. At the base of the reef ecosystem are dinoflagellate algae, living in symbiosis with coral. Efforts to understand the relationship between alga and coral have been greatly hampered by the lack of an appropriate dinoflagellate genetic transformation technology. By making use of the plasmid-like fragmented chloroplast genome, we have introduced novel genetic material into the dinoflagellate chloroplast genome. We have shown that the introduced genes are expressed and confer the expected phenotypes. Genetically modified cultures have been grown for five months with subculturing, maintaining the introduced genes and phenotypes. This indicates that cells continue to divide after transformation and that the transformation is stable.

genetics↗