Unlocking the potential of cyanobacteria: a high-throughput strategy for enhancing biocatalytic performance through genetic optimization

Whole-cell biocatalyst expression libraries were characterized in cyanobacteria using a ‘sort and sequence’ approach.Improved expression directly correlated with improved biocatalytic activity.Stable and strong expressing strains were identified for the ketoreductase LfSDR150, the enoate reductase YqjM, and the Baeyer–Villiger monooxygenase CHMOmut.This high-throughput characterization strategy can be transferred to other expression hosts, any genetic tools, or other enzymes. Cyanobacteria show promise as hosts for whole-cell biocatalysis. Their photoautotrophic metabolism can be leveraged for a sustainable production process. Despite advancements, performance still lags behind heterotrophic hosts. A key challenge is the limited ability to overexpress recombinant enzymes, which also hinders their biocatalytic efficiency. To address this, we generated large-scale expression libraries and developed a high-throughput method combining fluorescence-activated cell sorting (FACS) and deep sequencing in Synechocystis sp. PCC 6803 (Syn. 6803) to screen and optimize its genetic background. We apply this approach to enhance expression and biocatalyst performance for three enzymes: the ketoreductase LfSDR1M50, enoate reductase YqjM, and Baeyer–Villiger monooxygenase (BVMO) CHMOmut. Diverse genetic combinations yielded significant improvements: optimizing LfSDR1M50 expression showed a 17-fold increase to 39.2 U gcell dry weight (CDW)–1. In vivo activity of Syn. YqjM was improved 16-fold to 58.7 U gCDW–1 and, for Syn. CHMOmut, a 1.5-fold increase to 7.3 U gCDW–1 was achieved by tailored genetic design. Thus, this strategy offers a pathway to optimize cyanobacteria as expression hosts, paving the way for broader applications in other cyanobacteria strains and larger libraries. [Display omitted] We present a robust strategy for characterizing a large expression library within cyanobacteria, demonstrated on three enzymes. The applicability of the ‘sort and sequence’ technique, initially developed for Escherichia coli, to cyanobacteria, underscores its versatility. The strategy is scalable, based on a one-pot approach (one-pot cloning/enzyme, one-pot characterization/enzyme). Consequently, it is adept at managing sizable expression libraries. Challenges concerning the molecular toolbox include limited availability of orthogonal, tightly regulated, and strong promoters. Considering the characterization, stability issues of the tested proteins impacted the accuracy. To enhanc