Recombinant Synechococcus elongatus PCC 7942 for improved zeaxanthin production under natural light conditions

Abstract

Cyanobacteria are globally recognized as potential photosynthetic platform cell factories for production of value-added chemicals. Carotenoids are industrially important fine chemicals used in food, pharmaceutical and health-care products. Zeaxanthin has emerged as a high value xanthophyll carotenoid that exhibits superior antioxidant properties over many other carotenoids. Traditionally sourced from plants like marigold flowers wherein, zeaxanthin co-occurs with lutein. Therefore, there is a need for isolated production system for zeaxanthin on account of difficulties in separation of zeaxanthin from lutein due to their very similar molecular structures. Synechococcus elongatus PCC 7942 (PCC 7942) is a cyanobacterium that is known to synthesize zeaxanthin as one of the predominant cellular carotenoids and does not possess pathway genes for lutein production. In order to construct a system capable of industrial production of zeaxanthin, we genetically modified PCC 7942 (Synechococcus 79R48) to improve β‑carotene flux towards zeaxanthin synthesis by cloning CrtR (β‑carotene oxygenase) gene from Synechococcus elongatus PCC 7002 through homologous recombination. This strategy effectively enhanced the yield (mg/g DCW) of zeaxanthin in the transformants. Moreover, to increase zeaxanthin titer (mg/L), an operon construct was synthesized where a heterologous gene GalP (Hexose-H+ symporter) from E. coli MG1655 was cloned downstream of CrtR (Synechococcus 79RG48) which facilitated inherently obligate photoautotrophic WT PCC 7942 cells to uptake extracellular glucose in transformants, thereby increasing biomass productivity. Autotrophically grown cells yielded 9.02 ± 1.10 mg/g DCW of zeaxanthin while under mixotrophy the yield was 8.09 ± 0.19 mg/g DCW, which was 2-fold improvement over the wild type. The volumetric productivities of the transformants were 1.18 ± 0.17 mg/L·d and 1.8 ± 0.06 mg/L·d under autotrophic and mixotrophic conditions, respectively while WT produced 0.58 ± 0.02 mg/L·d. The work was successfully able to demonstrate that the modifications resulted in, enhanced production of lutein-free zeaxanthin in the cyanobacterial system.