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

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.

Bidirectional Promoter Libraries Enable the Balanced Co-expression of Two Target Genes in E. coli.

In this chapter, we present a bidirectional promoter library toolbox to evaluate fast and efficiently the optimal conditions for the balanced co-expression of two target genes. As a proof-of-concept, we demonstrate the co-expression of CYP505x and the GroEL/ES complex, which resulted in noticeably elevated enzyme activity with one of the de-novo-designed promoters of the library. The new toolbox offers a straightforward one-pot cloning approach and is highly modular. As such, the method presented here should be of great interest to any gene co-expression study.

Mutations Increasing Cofactor Affinity, Improve Stability and Activity of a Baeyer-Villiger Monooxygenase.

The typically low thermodynamic and kinetic stability of enzymes is a bottleneck for their application in industrial synthesis. Baeyer-Villiger monooxygenases, which oxidize ketones to lactones using aerial oxygen, among other activities, suffer particularly from these instabilities. Previous efforts in protein engineering have increased thermodynamic stability but at the price of decreased activity. Here, we solved this trade-off by introducing mutations in a cyclohexanone monooxygenase from Acinetobacter sp., guided by a combination of rational and structure-guided consensus approaches. We developed variants with improved activity (1.5- to 2.5-fold) and increased thermodynamic (+5 °C T m) and kinetic stability (8-fold). Our analysis revealed a crucial position in the cofactor binding domain, responsible for an 11-fold increase in affinity to the flavin cofactor, and explained using MD simulations. This gain in affinity was compatible with other mutations. While our study focused on a particular model enzyme, previous studies indicate that these findings are plausibly applicable to other BVMOs, and possibly to other flavin-dependent monooxygenases. These new design principles can inform the development of industrially robust, flavin-dependent biocatalysts for various oxidations.

Biocatalysis in Green and Blue: Cyanobacteria.

Recently, several studies have proven the potential of cyanobacteria as whole-cell biocatalysts for biotransformation. Compared to heterotrophic hosts, cyanobacteria show unique advantages thanks to their photoautotrophic metabolism. Their ability to use light as energy and CO2 as carbon source promises a truly sustainable production platform. Their photoautotrophic metabolism offers an encouraging source of reducing power, which makes them attractive for redox-based biotechnological purposes. To exploit the full potential of these whole-cell biocatalysts, cyanobacterial cells must be considered in their entirety. With this emphasis, this review summarizes the latest developments in cyanobacteria research with a strong focus on the benefits associated with their unique metabolism. Remaining bottlenecks and recent strategies to overcome them are evaluated for their potential in future applications.