light-controlled-fermentations-for-microbial-chemical-protein

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, ...

Princeton University

The use of optogenetics in metabolic engineering for light-controlled microbial chemical production raises the prospect of utilizing control and optimization techniques routinely deployed in traditional chemical manufacturing. However, such mechanisms require well-characterized, customizable tools that respond fast enough to be used as real-time inputs during fermentations. Here, we present OptoINVRT7, a new rapid optogenetic inverter circuit to control gene expression in . The circuit induces gene expression in only 0.6 h after switching cells from light to darkness, which is at least 6 times faster than previous OptoINVRT optogenetic circuits used for chemical production. In addition, we introduce an engineered inducible  promoter (P), which is stronger than any constitutive or inducible promoter commonly used in yeast. Combining OptoINVRT7 with P achieves strong and light-tunable levels of gene expression with as much as 132.9 ± 22.6-fold induction in darkness. The high performance of this new optogenetic circuit in controlling metabolic enzymes boosts production of lactic acid and isobutanol by more than 50% and 15%, respectively. The strength and controllability of OptoINVRT7 and P open the door to applying process control tools to engineered metabolisms to improve robustness and yields in microbial fermentations for chemical production.

Design and Characterization of Rapid Optogenetic Circuits for Dynamic Control in Yeast Metabolic Engineering.

Future expansion of corn-derived ethanol raises concerns of sustainability and competition with the food industry. Therefore, cellulosic biofuels derived from agricultural waste and dedicated energy crops are necessary. To date, slow and incomplete saccharification as well as high enzyme costs have hindered the economic viability of cellulosic biofuels, and while approaches like simultaneous saccharification and fermentation (SSF) and the use of thermotolerant microorganisms can enhance production, further improvements are needed. Cellulosic emulsions have been shown to enhance saccharification by increasing enzyme contact with cellulose fibers. In this study, we use these emulsions to develop an emulsified SSF (eSSF) process for rapid and efficient cellulosic biofuel production and make a direct three-way comparison of ethanol production between S. cerevisiae, O. polymorpha, and K. marxianus in glucose and cellulosic media at different temperatures.

Cellulosic biofuel production using emulsified simultaneous saccharification and fermentation (eSSF) with conventional and thermotolerant yeasts.

Microbial co-culture fermentations can improve chemical production from complex biosynthetic pathways over monocultures by distributing enzymes across multiple strains, thereby reducing metabolic burden, overcoming endogenous regulatory mechanisms, or exploiting natural traits of different microbial species. However, stabilizing and optimizing microbial subpopulations for maximal chemical production remains a major obstacle in the field. In this study, we demonstrate that optogenetics is an effective strategy to dynamically control populations in microbial co-cultures. Using a new optogenetic circuit we call OptoTA, we regulate an endogenous toxin-antitoxin system, enabling tunability of  growth using only blue light. With this system we can control the population composition of co-cultures of  and . When introducing in each strain different metabolic modules of biosynthetic pathways for isobutyl acetate or naringenin, we found that the productivity of co-cultures increases by adjusting the population ratios with specific light duty cycles. This study shows the feasibility of using optogenetics to control microbial consortia populations and the advantages of using light to control their chemical production.

Article	Year
Design and Characterization of Rapid Optogenetic Circuits for Dynamic Control in Yeast Metabolic Engineering. ACS synthetic biology\| PubMed ID: 33232598	2020
Cellulosic biofuel production using emulsified simultaneous saccharification and fermentation (eSSF) with conventional and thermotolerant yeasts. Biotechnology for biofuels\| PubMed ID: 34274018	2021
Optogenetic Control of Microbial Consortia Populations for Chemical Production. ACS synthetic biology\| PubMed ID: 34351122	2021

Shannon M. Hoffman

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