High‐yield secretion of recombinant proteins from the microalga Chlamydomonas reinhardtii

Abstract

SummaryMicroalga‐based biomanufacturing of recombinant proteins is attracting growing attention due to its advantages in safety, metabolic diversity, scalability and sustainability. Secretion of recombinant proteins can accelerate the use of microalgal platforms by allowing post‐translational modifications and easy recovery of products from the culture media. However, currently, the yields of secreted recombinant proteins are low, which hampers the commercial application of this strategy. This study aimed at expanding the genetic tools for enhancing secretion of recombinant proteins in Chlamydomonas reinhardtii, a widely used green microalga as a model organism and a potential industrial biotechnology platform. We demonstrated that the putative signal sequence from C. reinhardtii gametolysin can assist the secretion of the yellow fluorescent protein Venus into the culture media. To increase the secretion yields, Venus was C‐terminally fused with synthetic glycomodules comprised of tandem serine (Ser) and proline (Pro) repeats of 10 and 20 units [hereafter (SP)n, wherein n = 10 or 20]. The yields of the (SP)n‐fused Venus were higher than Venus without the glycomodule by up to 12‐fold, with the maximum yield of 15 mg/L. Moreover, the presence of the glycomodules conferred an enhanced proteolytic protein stability. The Venus‐(SP)n proteins were shown to be glycosylated, and a treatment of the cells with brefeldin A led to a suggestion that glycosylation of the (SP)n glycomodules starts in the endoplasmic reticulum (ER). Taken together, the results demonstrate the utility of the gametolysin signal sequence and (SP)n glycomodule to promote a more efficient biomanufacturing of microalgae‐based recombinant proteins.

Engineering ‘designer’ glycomodules for boosting recombinant protein secretion in tobacco hairy root culture and studying hydroxyproline‐O‐glycosylation process in plants

“…In addressing the low protein productivity challenge, we engineered designer HypGP modules as molecular carriers to boost secreted protein yields. This technology was previously developed in tobacco cell culture, and most recently demonstrated in microalgae culture, to dramatically increase secreted protein yields (Ramos-Martinez et al, 2017;Xu et al, 2007Xu et al, , 2010Zhang et al, 2016b). However, the broader application of this technology in differentiated plant organs, such as hairy roots or even whole plants, has yet to be explored.…”

“…The function of the engineered HypGPs as a molecular carrier was hypothesized to promote efficient transport of the fused proteins across the plasmalemma and for protecting the proteins from proteolytic degradation (Zhang et al, 2016b). Besides higher plant cells, engineering HypGPs comprised of a (SP) n motif (n = 10, 20) in green microalgae (Chlamydomonas reinhardtii) was recently reported to increase the secreted yields of a reporter protein by up to 12-fold (Ramos-Martinez et al, 2017). However, the precise process for Hyp-O-glycosylation of the designer HypGPs, particularly the AGPs-like modules that undergo Hyp-O-glycosylation with complex polysaccharides in plant cells, is poorly understood (Showalter and Basu, 2016b).…”

Enhanced secretion of human α1-antitrypsin expressed with a novel glycosylation module in tobacco BY-2 cell culture

“…Our previous studies indicated that the glycosylated HypGP module could function as a 'molecular carrier' to boost the secretion of fused proteins, including interferon α2b, human growth hormone and green fluorescent protein, presumably by facilitating efficient transport of the proteins into extracellular space and preventing proteolytic degradation of the proteins [20,21,24]. Most recently, the HypGP engineering technology was extended to green microalgae (Chlamydomonas reinhardtii), where engineering (SP) n tags (n = 10, 20) enhanced the secreted yields of a fused protein by up to 12-fold [25].…”

The Synthetic Biology Toolkit for Photosynthetic Microorganisms

“…Mixotrophic growth, although failing to fully exploit the free carbon and energy available through photosynthesis, improves growth rates and yields in cyanobacteria Matson and Atsumi, 2018) and may be required to reach maximum potential. An underdeveloped opportunity for metabolic engineering applications is the use of consortia, where a photosynthetic microbe provides the sugar or other carbon source for a heterotroph that acts as the production platform (Ducat et al, 2012;Fedeson and Ducat, 2017;Ramos-Martinez et al, 2017). This provides the benefits of both photoautotrophy and engineerability.…”