23Control of the lac operon with IPTG has been used for decades to regulate gene expression in E. 24 coli for countless applications, including metabolic engineering and recombinant protein 25 production. However, optogenetics offers unique capabilities such as easy tunability, 26 reversibility, dynamic induction strength, and spatial control that are difficult to obtain with 27 chemical inducers. We developed an optogenetic lac operon in a series of circuits we call 28OptoLAC. With these circuits, we control gene expression from various IPTG-inducible 29 promoters using only blue light. Applying them to metabolic engineering improves mevalonate 30 and isobutanol production by 24% and 27% respectively, compared to IPTG induction, controlled fermentations scalable to at least 2L bioreactors. Furthermore, OptoLAC circuits 32 enable light control of recombinant protein production, reaching yields comparable to IPTG 33 induction, but with enhanced tunability of expression and spatial control. OptoLAC circuits are 34 potentially useful to confer light controls over other cell functions originally engineered to be 35 . 36 37 38 39 40 41 42 43 44 45 a preferred host for many biotechnological applications, including metabolic engineering for 48 chemical production and expression of recombinant proteins. The vast wealth of knowledge on 49 the physiology, genetics, and metabolism of this bacterium, as well as the abundance of 50 molecular tools for its genetic manipulation, have made E. coli an organism of choice across the 51 spectrum of metabolic engineering, from proofs of principle 1 to the development of large-scale 52 industrial processes 2-5 . However, metabolic engineering in E. coli still faces important 53 challenges, especially considering that each pathway presents distinct obstacles, which may be 54 addressed with technological improvements. 55
IPTG-inducible
56A key challenge in metabolic engineering is the need for dynamic control 6 . This need commonly 57 arises when the biosynthesis of the product of interest competes with cell growth, or when the 58 product of interest, or its precursors, are toxic to the host organism, leading to growth inhibition 59 and poor productivity. These challenges are usually addressed by dividing fermentations into a 60 growth phase (in which the biosynthetic pathway of interest is repressed and metabolism is 61 devoted to growing biomass) and a production phase (in which the pathway of interest is 62 chemically induced, frequently at the expense of cell growth). However, pathway productivities 63 can be greatly impacted by the timing, rates, and levels of induction, which can be difficult to 64 control with chemical inducers. We recently showed that light can be an effective alternative to 65 chemicals as an inducing agent for metabolic engineering in S. cerevisiae, enhancing the 66 dynamic control over fermentations by adding tunability, reversibility, and independence from 67 4 medium composition 7 . To achieve this, we devised optogenetic circuits that harn...
