Development of a silicon limitation inducible expression system for recombinant protein production in the centric diatoms Thalassiosira pseudonana and Cyclotella cryptica

Abstract

BackgroundAn inducible promoter for recombinant protein expression provides substantial benefits because under induction conditions cellular energy and metabolic capability can be directed into protein synthesis. The most widely used inducible promoter for diatoms is for nitrate reductase, however, nitrogen metabolism is tied into diverse aspects of cellular function, and the induction response is not necessarily robust. Silicon limitation offers a means to eliminate energy and metabolic flux into cell division processes, with little other detrimental effect on cellular function, and a protein expression system that works under those conditions could be advantageous.ResultsIn this study, we evaluate a number of promoters for recombinant protein expression induced by silicon limitation and repressed by the presence of silicon in the diatoms Thalassiosira pseudonana and Cyclotella cryptica. In addition to silicon limitation, we describe additional strategies to elevate recombinant protein expression level, including inclusion of the 5′ fragment of the coding region of the native gene and reducing carbon flow into ancillary processes of pigment synthesis and formation of photosynthetic storage products. We achieved yields of eGFP to 1.8% of total soluble protein in C. cryptica, which is about 3.6-fold higher than that obtained with chloroplast expression and ninefold higher than nuclear expression in another well-established algal system.ConclusionsOur studies demonstrate that the combination of inducible promoter and other strategies can result in robust expression of recombinant protein in a nuclear-based expression system in diatoms under silicon limited conditions, separating the protein expression regime from growth processes and improving overall recombinant protein yields.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0760-3) contains supplementary material, which is available to authorized users.

Novel endogenous promoters for genetic engineering of the marine microalga Nannochloropsis gaditana CCMP526

“…However, the clonal population did not show uniform mVenus expression (Fig. S4 Heterogeneous transgene expression has been observed in different microalgae species and seems to be a common consequence of random chromosomal integration of the expression cassette [92,93]. Similar phenomena occur in mammalian model systems, where the transgene expression was found to be heterogeneous due to silencing position effects [84].…”

Genome editing in diatoms: achievements and goals

“…However, studies of NR promotor-driven GFP expression in P. tricornutum also indicated that gene expression cannot be completely switched off in the absence of nitrate (Chu et al 2016). Furthermore, nitrate deprivation may affect the photosynthetic capacity, the chlorophyll content, and the accumulation of neutral lipids, leading to possible secondary phenotypes (Valenzuela et al 2012;Alipanah et al 2015;Chu et al 2016;Shrestha and Hildebrand 2017). An alternative system could be derived from the silicon starvation inducible promoters (SSIPs), that are induced in the absence of silica in T. pseudonana and C. cryptica (Shrestha and Hildebrand 2017).…”

“…Furthermore, nitrate deprivation may affect the photosynthetic capacity, the chlorophyll content, and the accumulation of neutral lipids, leading to possible secondary phenotypes (Valenzuela et al 2012;Alipanah et al 2015;Chu et al 2016;Shrestha and Hildebrand 2017). An alternative system could be derived from the silicon starvation inducible promoters (SSIPs), that are induced in the absence of silica in T. pseudonana and C. cryptica (Shrestha and Hildebrand 2017). Under such silica-limited conditions, while both cell division and cell growth are blocked, the energy is channeled into the gene expression process, with little detrimental effect on cellular physiology (Shrestha and Hildebrand 2017).…”

“…An alternative system could be derived from the silicon starvation inducible promoters (SSIPs), that are induced in the absence of silica in T. pseudonana and C. cryptica (Shrestha and Hildebrand 2017). Under such silica-limited conditions, while both cell division and cell growth are blocked, the energy is channeled into the gene expression process, with little detrimental effect on cellular physiology (Shrestha and Hildebrand 2017).…”

Whole synthetic pathway engineering of recombinant protein production

“…Efforts to optimize the chassis-expression cassette interactome (all direct and indirect interactions between the host cell and recombinant DNA [rDNA] or rDNA-derived synthetic intermediates) have focused on reducing the burden that synthetic constructs impose on the host cell. For example, using inducible expression systems, product biosynthesis can be selectively "turned-off" during the early stages of culture to facilitate maximized accumulation of cellular capacity (Møller et al, 2017;Shrestha & Hildebrand, 2017).…”