Tong Zhang,Shubin Li,Lei Chen,Weiwen Zhang,Tao Sun
Cyanobacteria are known to be photoautotrophic cell factories capable of converting CO2 into valuable chemicals. The newly discovered marine cyanobacterium Synechococcus sp. PCC 11901 (hereafter PCC 11901) offers several advantages like rapid growth, high biomass, and high salinity tolerance, representing a promising chassis. To promote its application, we developed genetic toolboxes applicable to PCC 11901 in this study. First, a cobalamin (VB12)-independent chassis was constructed, allowing for cheaper cultivation. Second, genome copy numbers and transformation methods were, respectively, measured and optimized. Then, 14 neutral sites were identified and characterized within the genome PCC 11901, providing locations for genetic integration of exogenous cassettes. Subsequently, promoter libraries were developed, reaching an expression range of approximately 800 folds for constitutive promoters and an induction fold of up to approximately 400 for inducible promotors, respectively. As a proof of concept, natural production of the total lipid and phycocyanin was investigated using VB12-independent chassis, which realized an increase of 14.91% with lipid content compared with that of the wild-type strain. Further, we engineered the synthetic pathways of glucosylglycerol (GG) into PCC 11901 using the established toolboxes, reaching 590.41 ± 21.48 mg/L for GG production and self-sedimentation in photoreactors with the highest OD750 nm at 17.57 ± 0.77. Finally, the GG-producing strain grew well in seawater, reaching 324.50 ± 5.34 mg/L in shaking flask, which provided new strategies for cyanobacteria cultivation and production. Our work here made it possible to develop the fast-growing PCC 11901 as efficient carbon-neutral cell factory in the future.
O-Glycosylation of polyols attracts great interest in manufacturing chiral polyol glucosides available in cosmetics and detergent industries. Achieving synchronously controlled regio- and stereoselectivities in glycosylation of polyols remains challenging. Here, we discovered that the glucosylglycerol phosphorylase from Marinobacter adhaerens (MaGGP) presented broad catalytic promiscuity in glycosylation of various polyols, particularly for high carbon 1,2-diols, with excellent regioselectivity and stereoselectivity. Then, we overcame the well-known activity–stability trade-off in enzyme engineering and simultaneously improved thermostability and catalytic efficiency of MaGGP by 1200- and 13.7-fold, respectively. Crystal structure analysis and molecular dynamics simulation revealed the origin of enhanced thermostability and catalytic efficiency. Furthermore, we presented an ATP-free and thermodynamically favorable multienzyme system for polyol glycosylation with inexpensive starch as a glycosyl donor and successfully synthesized several polyol glucosides with high conversion yield. This efficient synthetic platform overcomes the reaction equilibrium and low atom economies of conventional biocatalytic methods and provides the promise in manufacturing other diversified valuable chiral 1,2-cis-glycosides.
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