Evaluation of strategies for improving the transgene expression in an oleaginous microalga Scenedesmus acutus

Abstract

BackgroundGenetic transformation of microalgae has been hampered by inefficient transgene expression, limiting the progress of microalgal biotechnology. Many vector tools and strategies have been developed in recent years to improve transgene expression in the model microalga Chlamydomonas, but these were hardly applied to other microalgae. In this work, naturally-isolated oleaginous microalgae were accessed for genetic transformation, and various expression systems were evaluated in a selected microalga to circumvent inefficient transgene expression.ResultsInitially, a strain of Scenedesmus acutus was selected from the oleaginous microalgal collection based on its highest transformation rate and transgene stability. This strain, which had very low or no GFP reporter expression, was first tested to improve transgene expression by using intron-containing constructs and the transcript fusion using ble::E2A. The intron-containing constructs yielded 2.5–7.5% of transformants with 2–4-fold fluorescence signals, while the majority of the transformants of the transcript fusion had the fluorescence signals up to 10-fold. Subsequently, three UV-induced S. acutus mutants were isolated with moderate increases in the level and frequency of transgene expression (2–3-fold and 10–12%, respectively). Finally, a transcript fusion system was developed using psy white mutants with an expression vector containing PSY::E2A for complementation and light selection. Transformants with green colonies were selected under light exposure, and the transgene expression was detected at protein levels. Although the improvement using PSY::E2A was only minor (1–2-fold increase and ~ 7% of transformants), this system provides an alternative selectable marker that is compatible with large-scale culture.ConclusionsHere, the overall improvement of transgene expression using the Chlamydomonas tools was moderate. The most effective tool so far is the transcript fusion using ble::E2A system. This work demonstrates that, so far, genetic engineering of non-model microalgae is still a challenging task. Further development of tools and strategies for transgene expression in microalgae are critically needed.Electronic supplementary materialThe online version of this article (10.1186/s12896-018-0497-z) contains supplementary material, which is available to authorized users.

Doing synthetic biology with photosynthetic microorganisms

“…Electroporation is preferred, because of higher transformation efficiency, whereas particle bombardment is useful for transformation of mitochondria or chloroplast genomes (Wang et al, 2019). Similar methods have been used for the transformation of Chlorella (Yang et al, 2016), Coccomyxa (Kania et al, 2020;Kasai et al, 2018), and for Scenedesmus/Acutodesmus species (Dautor et al, 2014;Guo et al, 2013;Muñoz et al, 2018;Suttangkakul et al, 2019). In addition, an E. coli-mediated conjugation was reported for transformation of Acutodesmus obliquus (Muñoz et al, 2019).…”

“…Some members of the Chlorophyta are readily transformable. However, the members of Chlorophyta have thick cell walls and many strains need significant method optimization or cannot be transformed at all (Dehghani et al, 2018; Kania et al, 2020; Suttangkakul et al, 2019). Various methods are applicable for transformation of C. reinhardtii , including the glass bead method, particle bombardment, Agrobacterium ‐mediated conjugation, and electroporation being the main methods employed (Figure 2).…”

“…A marker recycling system and marker-free engineering have also been reported (Kasai et al, 2017(Kasai et al, , 2018. In the Scenedesmaceae family, the RBCS2-HSP70Ap promoter, and the CaMV35S promoter have been used (Guo et al, 2013;Muñoz et al, 2018Muñoz et al, , 2019Suttangkakul et al, 2019).…”

Grand Challenges in Microalgae Domestication

“…Genomes of S. obliquus (Nag Dasgupta et al, 2018 ; Astafyeva et al, 2020 ) and H. pluvialis (Luo et al, 2019 ; Morimoto et al, 2020 ) are available. Transformation of S. obliquus via A. tumefasciens is feasible (Suttangkakul et al, 2019 ). Both nuclear and chloroplastic transformations have been described for H. pluvialis (Yuan et al, 2019 ; Wang et al, 2020 ; Cui et al, 2021 ).…”

Pursuit of chlorovirus genetic transformation and CRISPR/Cas9-mediated gene editing

“…Although, most of these techniques have been proven to work with great success in such model algal strains as Chlamydomonas reinhardtii , Phaeodactylum tricornutum , Scenedesmus , Ankistrodesmus and some Chlorella sp. [ 8 , 9 ], there is a lack of efficient and stable transformation techniques that can be applied to a broader range of microalgae strains.…”