Molecular Access Engineering for Microbial Biocatalysis: from Enzyme Tunnels to Microbial Cell Factories

Abstract

From Catalytic Activity to Molecular AccessibilityMicrobial biocatalysis has evolved far beyond isolated enzymes to encompass increasingly integrated systems, including multienzyme assemblies, electroenzymatic platforms, whole-cell biocatalysts, and genetically programmable microbial cell factories [1][2][3].These advances have transformed sustainable biomanufacturing by enabling the conversion of renewable feedstocks into a broad range of biobased products [2, 4, 5], including sustainable aviation fuels, biodegradable polymers, and high-value therapeutics.As these systems become more integrated and complex, the focus of biocatalyst design has increasingly shifted from optimizing individual enzymes [3,6] to coordinating molecular processes that influence system-level performance [7].Over the past three decades, biocatalyst development has been driven by active-site engineering, directed evolution, de novo enzyme design, metabolic engineering, and synthetic biology [1, 8-11].These advances have enabled industrial-scale bioproduction, selective biotransformations [12,13], and sophisticated biosynthetic pathways comprising dozens of catalytic reactions [14].Yet catalytic activity alone rarely guarantees optimal biocatalytic performance, despite continuous improvements in catalytic engineering [15,16].Performance increasingly reflects how efficiently molecules are delivered [17], exchanged [18], and coordinated within integrated systems [19].Meeting this challenge requires strategies that extend beyond catalytic optimization [20][21][22].Despite their diverse biological organization, microbial biocatalytic systems often share a common physicochemical constraint that controls access to catalytic functions.Examples include gas diffusion through enzyme tunnels [23,24], substrate channeling [18], membrane transport [17], and metabolic organization [3, 7], which have traditionally been investigated independently.Although these mechanisms appear distinct, these processes can be viewed as regulating a common underlying property, molecular accessibility.As illustrated in Fig. 1, this shared property may provide the conceptual link connecting seemingly disparate microbial biocatalytic systems.In this Review, we propose molecular access engineering as a conceptual framework for microbial biocatalysis.Rather than replacing established concepts such as diffusion, transport engineering, or Microbial biocatalysis spans biological platforms ranging from purified enzymes and multienzyme assemblies to electroenzymatic systems, whole-cell biocatalysts, and genetically programmable microbial cell factories.Yet despite their biological diversity, functional biocatalytic performance is often influenced by both catalytic activity and molecular accessibility.This recurring constraint highlights molecular accessibility as a common engineering consideration across microbial biocatalysis.Accordingly, this Review presents the concept of molecular access engineering as the rational control of molecular accessibility to improve functional biocatalytic performance across biological scales.The concept encompasses three complementary strategies governing molecular entry, intermediate transfer, and molecular exchange throughout biological systems.Examples from gas-converting enzymes, multienzyme assemblies, electroenzymatic systems, catalytic cascades, whole-cell biocatalysts, and microbial cell factories illustrate how engineering molecular accessibility can improve catalytic robustness, pathway efficiency, biological compatibility, and systems-level productivity.These examples therefore suggest that molecular access engineering has the potential to broaden the design space for microbial biocatalysis by complementing catalytic engineering.