Heterologous Production of Glycine Betaine Using Synechocystis sp. PCC 6803-Based Chassis Lacking Native Compatible Solutes

Eunice A Ferreira; Catarina C Pacheco; João S Rodrigues; Filipe Pinto; Pedro Lamosa; David Fuente; Javier Urchueguía; Paula Tamagnini

doi:10.3389/fbioe.2021.821075

Heterologous Production of Glycine Betaine Using Synechocystis sp. PCC 6803-Based Chassis Lacking Native Compatible Solutes

Front Bioeng Biotechnol. 2022 Jan 7:9:821075. doi: 10.3389/fbioe.2021.821075. eCollection 2021.

Authors

Eunice A Ferreira^{1

2

3}, Catarina C Pacheco^{1

2}, João S Rodrigues^{1

2

4}, Filipe Pinto^{1

2}, Pedro Lamosa⁵, David Fuente⁶, Javier Urchueguía⁶, Paula Tamagnini^{1

2

4}

Affiliations

¹ I3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.
² IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal.
³ ICBAS-Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal.
⁴ Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Porto, Portugal.
⁵ Instituto de Tecnologia Química e Biológica António Xavier, ITQB NOVA, Oeiras, Portugal.
⁶ Instituto de Aplicaciones de las Tecnologías de la Información y de las Comunicaciones Avanzadas, Universitat Politècnica de València, València, Spain.

Abstract

Among compatible solutes, glycine betaine has various applications in the fields of nutrition, pharmaceuticals, and cosmetics. Currently, this compound can be extracted from sugar beet plants or obtained by chemical synthesis, resulting in low yields or high carbon footprint, respectively. Hence, in this work we aimed at exploring the production of glycine betaine using the unicellular cyanobacterium Synechocystis sp. PCC 6803 as a photoautotrophic chassis. Synechocystis mutants lacking the native compatible solutes sucrose or/and glucosylglycerol-∆sps, ∆ggpS, and ∆sps∆ggpS-were generated and characterized. Under salt stress conditions, the growth was impaired and accumulation of glycogen decreased by ∼50% whereas the production of compatible solutes and extracellular polymeric substances (capsular and released ones) increased with salinity. These mutants were used as chassis for the implementation of a synthetic device based on the metabolic pathway described for the halophilic cyanobacterium Aphanothece halophytica for the production of the compatible solute glycine betaine. Transcription of ORFs comprising the device was shown to be stable and insulated from Synechocystis' native regulatory network. Production of glycine betaine was achieved in all chassis tested, and was shown to increase with salinity. The introduction of the glycine betaine synthetic device into the ∆ggpS background improved its growth and enabled survival under 5% NaCl, which was not observed in the absence of the device. The maximum glycine betaine production [64.29 µmol/gDW (1.89 µmol/mg protein)] was reached in the ∆ggpS chassis grown under 3% NaCl. Taking into consideration this production under seawater-like salinity, and the identification of main key players involved in the carbon fluxes, this work paves the way for a feasible production of this, or other compatible solutes, using optimized Synechocystis chassis in a pilot-scale.

Keywords: Synechocystis; compatible solutes; cyanobacteria; glucosylglycerol; glycine betaine; salt stress; sucrose; synthetic biology.