Sethuram, H. | Search bioRxiv

bioRxiv2024

Description of a novel extremophile green algae, Chlamydomonas pacifica, and its potential as a biotechnology host

We present the comprehensive characterization of a newly identified microalga, Chlamydomonas pacifica, originally isolated from a soil sample in San Diego, CA, USA. This species showcases remarkable biological versatility, including a broad pH range tolerance (6-11.5), high thermal tolerance (up to 42{degrees}C), and salinity resilience (up to 2% NaCl). Its amenability to genetic manipulation and sexual reproduction via mating, particularly between the two opposing strains CC-5697 & CC-5699, now publicly available through the Chlamydomonas Resource Center, underscores its potential as a biotechnological chassis. The biological assessment of C. pacifica revealed versatile metabolic capabilities, including diverse nitrogen assimilation capability, motility and phototaxis. Genomic and transcriptomic analyses identified 17,829 genes within a 121 Mb genome, featuring a GC content of 61%. The codon usage of C. pacifica closely mirrors that of C. reinhardtii, indicating a conserved genetic architecture that supports a trend in codon preference with minor variations. Phylogenetic analyses position C. pacifica within the core-Reinhardtinia clade yet distinct from known Volvocales species. The lipidomic data revealed an abundance of triacylglycerols (TAGs), promising for biofuel applications and lipids for health-related benefits. Our investigation lays the groundwork for exploiting C. pacifica in biotechnological applications, from biofuel generation to synthesizing biodegradable plastics, positioning it as a versatile host for future bioengineering endeavors. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/611117v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1ead226org.highwire.dtl.DTLVardef@18af631org.highwire.dtl.DTLVardef@291c2borg.highwire.dtl.DTLVardef@f860ac_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

molecular biology↗

bioRxiv2024

Bioinformatic prediction and high throughput in vivo screening to identify cis-regulatory elements for the development of algal synthetic promoters

Algae biotechnology holds immense promise for revolutionizing the bioeconomy through the sustainable and scalable production of various bioproducts. However, its development has been hindered by the lack of advanced genetic tools. This study introduces a synthetic biology approach to develop such tools, focusing on the construction and testing of synthetic promoters. By analyzing conserved DNA motifs within the promoter regions of highly expressed genes across six different algal species, we identified cis-regulatory elements (CREs) associated with high transcriptional activity. Combining the algorithms POWRS, STREME and PhyloGibbs, we predicted 1511 CREs and inserted them into a minimal synthetic promoter sequence in 1, 2 or 3 copies, resulting in 4533 distinct synthetic promoters. These promoters were evaluated in vivo for their capacity to drive the expression of a transgene in a high-throughput manner through next-generation sequencing post antibiotic selection and fluorescence-activated cell sorting. To validate our approach, we sequenced hundreds of transgenic lines showing high GFP expression. Further, we individually tested fourteen identified promoters, revealing substantial increases in GFP expression--up to nine times higher than the baseline synthetic promoter, with five matching or even surpassing the performance of the native AR1 promoter. As a result of this study, we identified a catalog of CREs that can now be used to build superior synthetic algal promoters. More importantly, here we present a validated pipeline to generate building blocks for innovative synthetic genetic tools applicable to any algal species with a sequenced genome and transcriptome dataset. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/585996v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1869cd4org.highwire.dtl.DTLVardef@a25d8dorg.highwire.dtl.DTLVardef@1abd96forg.highwire.dtl.DTLVardef@1501bc8_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗