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

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.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1127-7) contains supplementary material, which is available to authorized users.

Overexpression of Orange (OR) and OR mutant protein inChlamydomonas reinhardtiienhances carotenoid and ABA accumulation and increases resistance to abiotic stress

“…reinhardtii cell-wall mutant CR4349 strain and the derived transgenic lines were maintained in TAP medium (20 mM Tris, 17 mM Acetate, 0.68 mM K 2 HPO 4 , 7.26 mM KH 2 PO 4 , and 7.5 mM NH 4 Cl) (Togasaki et al, 1987) with the addition of trace metals as described previously (Hunter, 1946). Experiments were carried out in 50-400 mL batch cultures of TAP medium incubated at 25 °C with constant shaking ( (EX: 340-500 nm, Em: > 665 nm) (Richter et al, 2018).…”

“…The tubes were then incubated at 25 °C with shaking manually once a day under continuous fluorescent white light (300 µmol m -2 s -1 ). The algae cell growth was monitored daily by measuring the auto-fluorescence of chlorophyll a using a 10-AU Fluorometer (Turner Designs, San Jose, CA), equipped with a Daylight White Lamp F4T5D (EX: 340-500 nm, EM: > 665 nm)(Richter et al, 2018).…”

Engineering microalgae through chloroplast transformation to produce high‐value industrial products

“…Although the microalgal platform did provide lower protein yields compared to tobacco chloroplasts, however, the protein was stably expressed. Recently, Yang and coworkers expressed bacterial proteins in C. reinhardtii chloroplasts as a fusion of the foreign protein to highly expressing regulatory elements. For the bacterial Cel6A protein, that was fused to the downstream box of TetC (fragment from the tetanus toxin) promoter and terminator, protein accumulation of about 0.3% TSP could be obtained.…”

Technical Advances in Chloroplast Biotechnology

“…It is an amazing organelle where more than 120 genes from various sources have been integrated and expressed. This organellar genome has well been explored for a wide variety of applications including crops with elevated level of resistance against biotic (insects, bacterial, viral, and fungal diseases) and abiotic stresses (salinity, drought, and cold); phytoremediation of toxic metals, cytoplasmic male sterility [5]; and production of biopharmaceuticals, vaccine antigens, industrial enzymes, biomaterials, and biofuel [6]. Hyperexpression of recombinant protein in plant expression system is only possible through plastid transformation.…”