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Background  Isobutanol is an attractive biofuel with many advantages. Third-generation biorefineries that convert CO 2  into bio-based fuels have drawn considerable attention due to their lower feedstock cost and more ecofriendly refining process. Although autotrophic cyanobacteria have been genetically modified for isobutanol biosynthesis, there is a lack of stable and convenient strategies to improve their production.    Results  In this study, we first engineered  Synechococcus elongatus  for isobutanol biosynthesis by introducing five exogenous enzymes, reaching a production titer of 0.126 g/L at day 20. It was then discovered that high salinity stress could result in a whopping fivefold increase in isobutanol production, with a maximal in-flask titer of 0.637 g/L at day 20. Metabolomics analysis revealed that high salinity stress substantially altered the metabolic profiles of the engineered  S. elongatus . A major reason for the enhanced isobutanol production is the acceleration of lipid degradation under high salinity stress, which increases NADH. The NADH then participates in the engineered isobutanol-producing pathway. In addition, increased membrane permeability also contributed to the isobutanol production titer. A cultivation system was subsequently developed by mixing synthetic wastewater with seawater to grow the engineered cyanobacteria, reaching a similar isobutanol production titer as cultivation in the medium.    Conclusions  High salinity stress on engineered cyanobacteria is a practical and feasible biotechnology to optimize isobutanol production. This biotechnology provides a cost-effective approach to biofuel production, and simultaneously recycles chemical nutrients from wastewater and seawater.    Supplementary Information  The online version contains supplementary material available at 10.1186/s13068-021-02023-8.

Establishment of a resource recycling strategy by optimizing isobutanol production in engineered cyanobacteria using high salinity stress