AUTHOR=Xiao Yubei , Tan Xuemei , He Qiaoning , Yang Shihui 

TITLE=Systematic metabolic engineering of Zymomonas mobilis for β-farnesene production

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=12

YEAR=2024

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2024.1392556

DOI=10.3389/fbioe.2024.1392556

ISSN=2296-4185

ABSTRACT=<p><italic>Zymomonas mobilis</italic> is an ethanologenic bacterium that can produce hopanoids using farnesyl pyrophosphate (FPP), which can be used as the precursor by β-farnesene synthase for β-farnesene production. To explore the possibility and bottlenecks of developing <italic>Z. mobilis</italic> for β-farnesene production, five heterologous β-farnesene synthases were selected and screened, and <italic>AaBFS</italic> from <italic>Artemisia annua</italic> had the highest β-farnesene titer. Recombinant strains with <italic>AaBFS</italic> driven by the strong constitutive promoter P<italic>gap</italic> (P<italic>gap</italic>–<italic>AaBFS</italic>) doubled its β-farnesene production to 25.73 ± 0.31 mg/L compared to the recombinant strain with <italic>AaBFS</italic> driven by P<italic>tet</italic> (P<italic>tet</italic>–<italic>AaBFS</italic>), which can be further improved by overexpressing the P<italic>gap</italic>–<italic>AaBFS</italic> construct using the strategies of multiple plasmids (41.00 ± 0.40 mg/L) or genomic multi-locus integration (48.33 ± 3.40 mg/L). The effect of cofactor NADPH balancing on β-farnesene production was also investigated, which can be improved only in <italic>zwf</italic>-overexpressing strains but not in <italic>ppnK</italic>-overexpressing strains, indicating that cofactor balancing is important and sophisticated. Furthermore, the β-farnesene titer was improved to 73.30 ± 0.71 mg/L by overexpressing <italic>dxs</italic>, <italic>ispG</italic>, and <italic>ispH</italic>. Finally, the β-farnesene production was increased to 159.70 ± 7.21 mg/L by fermentation optimization, including the C/N ratio, flask working volume, and medium/dodecane ratio, which was nearly 13-fold improved from the parental strain. This work thus not only generated a recombinant β-farnesene production <italic>Z. mobilis</italic> strain but also unraveled the bottlenecks to engineer <italic>Z. mobilis</italic> for farnesene production, which will help guide the future rational design and construction of cell factories for terpenoid production in non-model industrial microorganisms.</p>