In E. coli, IspH is an essential enzyme in the MEP pathway, crucial to isoprenoid synthesis which helps maintain, stabilize and support core functions such as respiration. It catalyzes the conversion of HMBPP into the common isoprenoid precursors IPP and DMAPP in a single step. And it has been confirmed that significantly inhibiting the IspH activity in E. coli substantially resctricts the bacterial growth (Kumar et al.). To achieve a balance between HMBPP overproduction and growth activity of E. coli, we decided on the sRNA-based down-regulation of IspH instead of the knockout approach.
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The synthetic small RNA (sRNA) sRNA2(IspH) (BBa_K5186002), which targets the gene IspH, was then designed to encode sRNA. Upon expression, sRNA2(IspH) binds to the Hfq protein, aligning its target-binding sequence with the IspH mRNA. This interaction prevents ribosomes from accessing the ribosome binding site (RBS), effectively downregulating IspH expression and allowing for precise control over HMBPP synthesis without compromising the viability of E. coli.
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In our efforts to enhance HMBPP production this year, we have successfully engineered this overexpression cassette for sRNA2(IspH) in E. coli DH5a. By co-expressing this cassette with the MEP overexpression cassette (BBa_K2741008), we have demonstrated about a 1.1-fold increase in HMBPP yield.
