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ABSTRACT Komagataella phaffii, also known asPichia pastoris, is a powerful host for recombinant protein production, in part due to its exceptionally strong and tightly controlled P_AOX1promoter. MostK. phaffiibioprocesses for recombinant protein production rely on P_AOX1to achieve dynamic control in two‐phase processes. Cells are first grown under conditions that repress P_AOX1(growth phase), followed by methanol‐induced recombinant protein expression (production phase). In this study, we propose a methanol‐free approach for dynamic metabolic control inK. phaffiiusing optogenetics, which can help enhance input tunability and flexibility in process optimization and control. The light‐responsive transcription factor EL222 fromErythrobacter litoralisis used to regulate protein production from the P_C120promoter inK. phaffiiwith blue light. We used two system designs to explore the advantages and disadvantages of coupling or decoupling EL222 integration with that of the gene of interest. We investigate the relationship between EL222 gene copy number and light dosage to improve production efficiency for intracellular and secreted proteins. Experiments in lab‐scale bioreactors demonstrate the feasibility of the outlined optogenetic systems as potential alternatives to conventional methanol‐inducible bioprocesses usingK. phaffii. 

Temporal gradient estimation is a pervasive phenomenon in natural biological systems and holds great promise for synthetic counterparts with broad-reaching applications. Here, we advance the concept of BioSD (Biomolecular Signal Differentiators) by introducing a novel biomolecular topology, termed Autocatalytic-BioSD or AC-BioSD. Its structure allows for insensitivity to input signal changes and high precision in terms of signal differentiation, even when operating far from nominal conditions. Concurrently, disruptive high-frequency signal components are effectively attenuated. In addition, the usefulness of our topology in biological regulation is highlighted via a PID (Proportional-Integral-Derivative) bio-control scheme with set point weighting and filtered derivative action in both the deterministic and stochastic domains. 

In recent years, light-responsive systems from the field of optogenetics have been applied to several areas of metabolic engineering with remarkable success. By taking advantage of light's high tunability, reversibility, and orthogonality to host endogenous processes, optogenetic systems have enabled unprecedented dynamical controls of microbial fermentations for chemical production, metabolic flux analysis, and population compositions in co-cultures. In this article, we share our opinions on the current state of this new field of metabolic optogenetics.We make the case that it will continue to impact metabolic engineering in increasingly new directions, with the potential to challenge existing paradigms for metabolic pathway and strain optimization as well as bioreactor operation. 

Optogenetics has been used in a variety of microbial engineering applications, such as chemical and protein production, studies of cell physiology, and engineered microbe–host interactions. These diverse applications benefit from the precise spatiotemporal control that light affords, as well as its tunability, reversibility, and orthogonality. This combination of unique capabilities has enabled a surge of studies in recent years investigating complex biological systems with completely new approaches. We briefly describe the optogenetic tools that have been developed for microbial engineering, emphasizing the scientific advancements that they have enabled. In particular, we focus on the unique benefits and applications of implementing optogenetic control, from bacterial therapeutics to cybergenetics. Finally, we discuss future research directions, with special attention given to the development of orthogonal multichromatic controls. With an abundance of advantages offered by optogenetics, the future is bright in microbial engineering.