A downstream box fusion allows stable accumulation of a bacterial cellulase in Chlamydomonas reinhardtii chloroplasts

Lubna V Richter,Huijun Yang,Beth A Ahner,Mohammad Yazdani,Maureen R Hanson

doi:10.1186/s13068-018-1127-7

Abstract

BackgroundWe investigated strategies to improve foreign protein accumulation in the chloroplasts of the model algae Chlamydomonas reinhardtii and tested the outcome in both standard culture conditions as well as one pertinent to algal biofuel production. The downstream box (DB) of the TetC or NPTII genes, the first 15 codons following the start codon, was N-terminally fused to the coding region of cel6A, an endoglucanase from Thermobifida fusca. We also employed a chimeric regulatory element, consisting of the 16S rRNA promoter and the atpA 5′UTR, previously reported to enhance protein expression, to regulate the expression of the TetC-cel6A gene. We further investigated the accumulation of TetC-Cel6A under N-deplete growth conditions.ResultsBoth of the DB fusions improved intracellular accumulation of Cel6A in transplastomic C. reinhardtii strains though the TetC DB was much more effective than the NPTII DB. Furthermore, using the chimeric regulatory element, the TetC-Cel6A protein accumulation displayed a significant increase to 0.3% total soluble protein (TSP), whereas NPTII-Cel6A remained too low to quantify. Comparable levels of TetC- and NPTII-cel6A transcripts were observed, which suggests that factors other than transcript abundance mediate the greater TetC-Cel6A accumulation. The TetC-Cel6A accumulation was stable regardless of the growth stage, and the transplastomic strain growth rate was not altered. When transplastomic cells were suspended in N-deplete medium, cellular levels of TetC-Cel6A increased over time along with TSP, and were greater than those in cells suspended in N-replete medium.ConclusionsThe DB fusion holds great value as a tool to enhance foreign protein accumulation in C. reinhardtii chloroplasts and its influence is related to translation or other post-transcriptional processes. Our results also suggest that transplastomic protein production can be compatible with algal biofuel production strategies. Cells displayed a consistent accumulation of recombinant protein throughout the growth phase and nitrogen starvation, a strategy used to induce lipid production in algae, led to higher cellular heterologous protein content. The latter result is contrary to what might have been expected a priori and is an important result for the development of future algal biofuel systems, which will likely require co-products for economic sustainability.

Highlights

We investigated strategies to improve foreign protein accumulation in the chloroplasts of the model algae Chlamydomonas reinhardtii and tested the outcome in both standard culture conditions as well as one pertinent to algal biofuel production
Recombinant constructs and chloroplast transformation Expression cassettes were designed to generate transplastomic C. reinhardtii strains that would express the native T. fusca cel6A gene with no codon optimization because the chloroplast and bacterial genomes share a similar codon bias and the native sequence was successfully employed for plastid expression in tobacco [20]
Plasmids containing the expression cassettes were transformed into the C. reinhardtii chloroplasts by biolistic particle bombardment and the transgenes were integrated at the BamHI site within the intergenic region between the psbA and 5S rRNA genes within the C. reinhardtii chloroplast genome (Fig. 1a)

Summary

Introduction

We investigated strategies to improve foreign protein accumulation in the chloroplasts of the model algae Chlamydomonas reinhardtii and tested the outcome in both standard culture conditions as well as one pertinent to algal biofuel production. C. reinhardtii has been investigated as a host for high-value heterologous protein expression due to its fast reproduction rate, its ability to grow as a heterotroph, and its well-established genetic transformation techniques [2,3,4]; examples include proteins with value as biopharmaceuticals [5] and in agricultural applications [6]. Chloroplast transformation has several advantages for recombinant protein expression over nuclear transformation These include precise transgene integration, feasibility to express multiple genes simultaneously, the absence of gene silencing, and the presence of 80–100 identical plastomes per cell resulting in a high transgene copy number [7,8,9]. The choice of the 3′UTR sequence has been shown to have a minor impact on mRNA stability and protein accumulation [12], whereas the 5′UTR was more influential [12, 13]

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