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Chlamydomonas reinhardtii has many attractive features for use as a model organism for both fundamental studies and as a biotechnological platform. Nonetheless, despite the many molecular tools and resources that have been developed, there are challenges for its successful engineering, in particular to obtain reproducible and high levels of transgene expression. Here we describe a synthetic biology approach to screen several hundred independent transformants using standardised parts to explore different parameters that might affect transgene expression. We focused on terminators and, using a standardised workflow and quantitative outputs, tested 9 different elements representing three different size classes of native terminators to determine their ability to support high level expression of a GFP reporter gene. We found that the optimal size reflected the median size of element found in the C. reinhardtii genome. The behaviour of the terminator parts was similar with different promoters, in different host strains and with different transgenes. This approach is applicable to the systematic testing of other genetic elements, facilitating comparison to determine optimal transgene design.

Anaerobic digestion plays a pivotal role in the modern circular economy, as it offers a sustainable solution for converting organic waste into biogas (methane). It also results in a nutrient-rich liquid stream, referred to as digestate. This digestate is extensively applied to agricultural land as fertilizer due to its high macronutrient (N, P) content, but the bioactive micronutrients it contains and their significance for downstream applications remain largely unknown. Here, we investigate whether digestate generated from a vitamin B 12 -deficient substrate (fruit and vegetable waste) can be enriched in this vitamin through anaerobic digestion, and explore the capability of the microalga Chlorella vulgaris to grow in this medium and bioaccumulate B 12. Our findings uncover, for the first time, that substantial amounts of B 12 are synthesized during anaerobic digestion, and that C. vulgaris can effectively be enriched with this vitamin when grown in the digestate (10.6 μg Β 12 ·g−1 dry weight). Additionally, we identified that pH-induced ammonia toxicity was the main inhibitor when growing microalgae in the digestate, which allowed us to significantly enhance productivity at lab- and pilot-scale through pH control. The case of B 12 synthesis in digestate and accumulation in microalgae highlights the potential for enhancing the value of these waste streams through the identification and utilization of bioactive compounds. [Display omitted] • Anaerobic digestion produces substantial amounts of vitamin B 12 , a bioactive compound. • Chlorella vulgaris effectively grows and accumulates B 12 when cultivated in digestate. • pH control boosts microalgae productivity in digestate, overcoming ammonia toxicity. • Digestate-grown Chlorella vulgaris can be a high-value feed in aquaculture. • Utilizing bioactive compounds like vitamin B 12 expands the applications of digestate. [ABSTRACT FROM AUTHOR]