Promiscuity‐Guided Enzyme Evolution via Substrate Multiplexed Screening

An enzyme's ability to react with diverse substrates while maintaining high yield and selectivity, termed "substrate promiscuity," is a highly sought-after property in biocatalysts. Traditional directed evolution typically relies on single-substrate screening, which enables adaptive evolutionary pathways but provides limited information on promiscuity. This approach can inadvertently narrow substrate scope, and it is often unclear whether alternative mutational paths might have facilitated broader reactivity. Here, we review substrate multiplexed screening (SUMS), a strategy that places substrates in direct competition and quantifies multiple products simultaneously. When enzymes operate in competitive environments, such as living cells or multi-enzyme cascades, SUMS directly mirrors the intended use. We discuss how SUMS generates promiscuity information that can guide engineering, even when the final application involves individual substrates. Examples illustrate how SUMS can increase information density relative to parallel screening and, in select cases, reveal activity shifts that are invisible to single-substrate methods. We discuss the strengths and limitations of SUMS and highlight instances where its application led to enzymes with broad or complementary scopes, identified allosteric mutations, or enabled the circumvention of negative epistasis.