Constructing a stable mannitol biosynthetic pathway in cyanobacteria via the introduction of heterologous mannitol dehydrogenase

Abstract

Mannitol, a valuable sugar alcohol, finds extensive application in food and medicine industries. The advancements of synthetic biology technology have unlocked the potential of cyanobacteria for directly converting CO₂ into mannitol. Previously, attempts have been made to engineer mannitol synthesizing cyanobacteria strains by introducing a heterologous pathway consisting of mannitol-1-phosphate dehydrogenase (Mtld) and mannitol-1-phosphatase (M1Pase). However, the recombinant strains generally encountered stability and yield issues. In this work, an alternative mannitol synthesizing pathway in which a heterologous mannitol dehydrogenase (Mdh) that converts fructose into mannitol was engineered and evaluated in Synechococcus elongatus PCC 7942, and photosynthetic cell factories simultaneously producing mannitol and fructose were constructed. A salt stress-induced cultivation method was initially used to achieve stable mannitol synthesis by leveraging the property of mannitol as a compatible solute under salt stress. The engineered strain exhibited an improved mannitol yield of 0.95 g/L, accompanied by the synthesis of 1.05 g/L fructose during a 15-day cultivation process under hypersaline induction. By inductively controlling the upstream sucrose metabolism, salt-independent mannitol-fructose co-production can be realized in long-term cultivation. This led to the accumulation of 0.7 g/L mannitol and 0.27 g/L fructose in the recombinant strain, with no spontaneous mutations observed in the mannitol biosynthesis operon during a 31-day cultivation process. This study represents the first instance of constructing a cyanobacterial mannitol cell factory employing the Mdh-driven pathway. The findings in this work provided new strategies for engineering efficient and stable photosynthetic cell factories of mannitol in future, also sheds light on the plasticity of cyanobacterial sugar metabolism networks.