Metabolic engineering of Synechococcus elongatus for photoautotrophic production of mannitol.

With multiple applications in food, pharmaceutical, and chemical industries as antioxidant or nonmetabolizable sweetener; the bioproduction of d-mannitol is gaining global attention, especially with photosynthetic organisms as hosts. Considering the sustainability prospects, the current work encompasses metabolic engineering of a widely used cyanobacterial strain, Synechococcus elongatus PCC 7942, and two newly isolated fast-growing cyanobacterial strains; S. elongatus PCC 11801 and S. elongatus PCC 11802, for mannitol production. We engineered these strains with a two-step pathway by cloning genes for mannitol-1-phosphate dehydrogenase (mtlD) and mannitol-1-phosphatase (mlp), where the mtlD expression was under the control of different promoters from PCC 7942, namely, Prbc225 , PcpcB300 , PcpcBm1 , PrbcLm17 , and PrbcLm15 . The strains were tested under the "switch conditions," where the growth conditions were switched after the first 3 days, thereby resulting in differential promoter activity. Among the engineered strains of PCC 11801 and PCC 11802, the strains possessing Prbc225 -mtlD module produced relatively high mannitol titers of 401 ± 18 mg/L and 537 ± 18 mg/L, respectively. The highest mannitol titer of 701 ± 15 mg/L (productivity 60 mg/L.d, yield 895 µM/OD730 ) was exhibited by the engineered strain of PCC 7942 expressing PcpcB300 -mtlD module. It is by far the highest obtained mannitol yield from the engineered cyanobacteria.

Photosynthetic Co-Production of Succinate and Ethylene in A Fast-Growing Cyanobacterium, Synechococcus elongatus PCC 11801.

Cyanobacteria are emerging as hosts for photoautotrophic production of chemicals. Recent studies have attempted to stretch the limits of photosynthetic production, typically focusing on one product at a time, possibly to minimise the additional burden of product separation. Here, we explore the simultaneous production of two products that can be easily separated: ethylene, a gaseous product, and succinate, an organic acid that accumulates in the culture medium. This was achieved by expressing a single copy of the ethylene forming enzyme (efe) under the control of PcpcB, the inducer-free super-strong promoter of phycocyanin ß subunit. We chose the recently reported, fast-growing and robust cyanobacterium, Synechococcus elongatus PCC 11801, as the host strain. A stable recombinant strain was constructed using CRISPR-Cpf1 in a first report of markerless genome editing of this cyanobacterium. Under photoautotrophic conditions, the recombinant strain shows specific productivities of 338.26 and 1044.18 µmole/g dry cell weight/h for ethylene and succinate, respectively. These results compare favourably with the reported productivities for individual products in cyanobacteria that are highly engineered. Metabolome profiling and 13C labelling studies indicate carbon flux redistribution and suggest avenues for further improvement. Our results show that S. elongatus PCC 11801 is a promising candidate for metabolic engineering.