Intron-containing algal transgenes mediate efficient recombinant gene expression in the green microalga Chlamydomonas reinhardtii

Abstract

Among green freshwater microalgae, Chlamydomonas reinhardtii has the most comprehensive and developed molecular toolkit, however, advanced genetic and metabolic engineering driven from the nuclear genome is generally hindered by inherently low transgene expression levels. Progressive strain development and synthetic promoters have improved the capacity of transgene expression; however, the responsible regulatory mechanisms are still not fully understood. Here, we elucidate the sequence specific dynamics of native regulatory element insertion into nuclear transgenes. Systematic insertions of the first intron of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit 2 (rbcS2i1) throughout codon-optimized coding sequences (CDS) generates optimized algal transgenes which express reliably in C. reinhardtii. The optimal rbcS2i1 insertion site for efficient splicing was systematically determined and improved gene expression rates were shown using a codon-optimized sesquiterpene synthase CDS. Sequential insertions of rbcS2i1 were found to have a step-wise additive effect on all levels of transgene expression, which is likely correlated to a synergy of transcriptional machinery recruitment and mimicking the short average exon lengths natively found in the C. reinhardtii genome. We further demonstrate the value of this optimization with five representative transgene examples and provide guidelines for the design of any desired sequence with this strategy.

Figure 1.

Gene design and splicing efficiency analysis of artificial rbcS2i1 insertion sites in the shble coding sequence represented by the transformation efficiency of C. reinhardtii. (A) Antibiotic selection cassette adopted from the pOptimized vector toolkit (19) containing the shble gene and an N-terminal linker which includes an additional SmaI restriction enzyme cleavage site and a representative TAP agar plate showing regenerated mutants in the presence of 10 mg L⁻¹ zeocin. (B) A frameshifted selection cassette with a single additional nucleotide results in no viable transformants. (C) The shble selection cassette including an amplified rbcS2i1 (145 bp) with modified exon boundaries. In total 16 combinations of the four nucleotides guanine (G), adenine (A), thymine (T) and cytosine (C) surrounding the intron insertion site were tested for effective splicing by colony survival in the presence of zeocin. The given percentage reflects the mean number of obtained colonies per pmol DNA in relation to the intron-less control. Equal amounts of plasmid DNA were used and linearized prior to transformation (Supplementary Figure S2) H R i – the HSP70/RBCS2i1 promoter, 3′ UTR – 3′ untranslated region of the rbcS2 gene. shble – Streptoalloteichus hindustanus phleomycin resistance gene, N – denotes nucleotide positions modified in each construct.

Figure 2.

Effects of rbcS2i1 addition on transgene expression levels of the codon-optimized P. cablin patchoulol synthase (PcPs) gene. (A) Screening method of regenerated mutants—fluorescence microscopy for the YFP reporter was conducted directly on the transformation plate as previously described (35). Cultivation was conducted in microtiter plates and mutants were pooled according to their respective cell densities prior to analysis e.g. flow cytometry. (B) A–F Expression cassettes of PcPs rbcS2i1 intron variants cloned in the pOpt_mVenus_Paro vector. The relative expression levels for each construct are shown as relative abundance of mRNA (RTqPCR), protein titer (WB – western blot, α-GFP with Coomassie Brilliant Blue (CBB) as loading control) and mean fluorescence cell⁻¹ (flow cytometry). (C) Patchoulol productivity was quantified by pooled mutant cultivation after 5 days in shake-flasks using 5% (v/v) dodecane overlay followed by GC–MS as previously described (35). (D) Vector set with alternative PsaD promoter sequence (38) and respective transformant PcPs_YFP expression levels analyzed by WB. Error bars represent standard deviations from mean of triplicate measurements for pooled populations, pre-selected by YFP fluorescence. H R i – the HSP70/RBCS2i1 promoter, 3′ UTR – 3′ untranslated region of the rbcS2 gene.

Figure 3.

Analysis of position effects of different insertion sites of the rbcS2i1 in the codon-optimized PcPs CDS. Vector G is a modification of vector C wherein the actin intron 3 (95 bp, NCBI: D50838.1) has been inserted into the empty third position. In vector H the rbcS2i1 was placed upstream of the first insertion site. Vectors C* and D* are shown for construct and expression efficiency comparison. Vector I contains the original third rbcS2i1 position as well as the additional upstream insertion found in vector H. In addition, the rbcS2i2 located in the mVenus reporter was replaced by a rbcS2i1 (vector J). Transformants were generated as above and the relative expression levels per construct were quantified by RTqPCR as the relative abundance of transcript mRNA normalized to vector D. UVM4 parental strain is shown as a negative control. Error bars represent standard deviations from mean of triplicate measurements for pooled populations, pre-selected by YFP fluorescence.

Figure 4.

Design of selected gene of interest sequences as optimized algal transgenes for enhanced nuclear expression in C. reinhardtii: (A) Escherichia coli TesA (NCBI accession no: MH004289, (50)), Anaerococcus tetradius Tes3 (MH004290, (51)), Santalum album santalene synthase SaSs (MH004288, (52)), Mentha spicata limonene synthase MsLs (MH004287, (53)), P. cablin patchoulol synthase PcPs (KX097887, (54)) and A. grandis bisabolene synthase AgBs (MG052654.1, (55)). Genes were designed in silico followed by gene synthesis, cloned into the pOpt_mVenus_Paro vector backbone and expressed in UVM4. (B) Protein samples from expressing mutants transformed with the different optimized transgenes were separated by SDS-PAGE prior to western blot analysis (α-GFP). (C) Relative numbers of transformants from 300 randomly picked C. reinhardtii mutants per construct, which express detectible levels of YFP fusion proteins. Relative expression rates across this population are indicated with different colour intensities. Vector diagrams: light green boxes indicate the 36 amino acid PsaD chloroplast target peptide used on some constructs for subcellular targeting. H R i – the HSP70/RBCS2i1 promoter, 3′ UTR – 3′ untranslated region of the rbcS2 gene. shble bleomycin/zeocin resistance gene.