Abstract

As products of multiple-step enzymatic reactions, the production of polyhydroxyalkanoate (PHA) can be strongly influenced by the expression level of each gene related to these reactions. Taking polyhydroxybutyrate (PHB) as an example, its synthesis involves at least three genes and their products: PhbC, PhbA, PhbB. The expression levels of these genes can affect the accumulation rates and patterns. Ribosomal Binding Sites (RBS) are genetic regulation elements that regulate gene expression at translational level. It was shown that different RBSs can lead to dramatically different protein production levels due to their different affinity to ribosomes. Here we successfully constructed a plasmid library with PHB synthesis genes PhaC, PhaA and PhaB with their RBSs randomly picked from a library of RBSs with different translation initiation rate (TIR) calculated in-silico, . The resulting plasmids were transformed into E. coli to generate a strain library in which each member has different combinations of three RBSs for three genes. The library was then screened in a high-throughput way. PHA was stained with a fluorescence dye called Bodipy, enabling quantitative measurement and screening of single cells with different PHA contents using Flow Cytometry. Approximately a hundred of strains with different PHA accumulation rates were isolated from the library. Each of them contains a plasmid with the same PHB synthesis genes but different RBSs in front of them. Among them, the PHA accumulation under particular culture conditions varied from 0% to more than over 95%. Using the original phaCAB operon from R. entropha (which is widely used for PHA production in E. coli) as a control, selected strains of the library led to stable, higher accumulation of PHB, different molecular weights of PHB, different PHA granule sizes and granule number in a single cell. PHB production curve during cultivation also differed among these strains.