Light-Controlled Fermentations for Microbial Chemical and Protein Production

Optogenetics has been used to achieve higher titers of several chemicals and proteins compared to typical inducers. Using this protocol, others can enhance their own processes with light-based control. Optogenetics control is noninvasive, highly tuneable, and reversible.

These qualities allow for a streamlined optimization of microbial processes and even open unique opportunities, like three-phase fermentations and multi-chromatic control. For any new chemical, the optimal light conditions will differ. So if low production is observed using the parameters in this protocol, different light conditions should be tested.

Begin by obtaining a Saccharomyces cerevisiae strain with HIS3 oxytrophy, a marker necessary for OptoINVRT plasmids. If seeking to control a gene native to Saccharomyces cerevisiae, ensure that the endogenous copy of the gene is deleted before proceeding. To begin the strain construction, linearize the plasmid containing the OptoINVRT7 circuit, such as EZL439.

If using EZL439, which contains the components to repress the PIGA one promoter in the light and activate it in the dark, linearize at the Pme1 restriction site. Using standard lithium acetate transformation methods, integrate the plasmid into a HIS3 auxotrophic strain. Following the transformation, centrifuge the cells at 150 G's for one minute.

Gently resuspend the cells in 200 microliters of fresh SC-HIS medium. Plate the entire cell volume onto an SC-HIS agar plate and incubate at 30 degree Celsius for two to three days until colonies appear. Prepare competent cells using standard lithium acetate transformation protocols.

Transform the cells with the plasmid containing the genes that you wish to optogenetically control downstream of either the PIGA1M or PIGA1S promoters. After transformation, centrifuge the culture at 150 G's for one minute and gently resuspend the cell pellet in 200 microliters of fresh SC drop-out medium. Plate the entire cell volume on a yeast extract peptone dextrose agar plate, if integrating into Delta sites or an SC drop-out plate if transforming with a plasmid containing a selection marker.

Incubate the cells at 30 degree Celsius for 16 hours under constant blue light to keep the optogenetically controlled gene repressed. Use any light source of the wavelength of 465 nanometers and place an LED panel approximately 40 centimeters above the plate such that the light intensity is approximately 80 to 110 micromoles per square meter per second. Measure the intensity using a quantum meter.

If integrating into Delta sites, replicate the plate onto yeast extract peptone dextrose plates containing a range of Zeocin concentrations between 400 micrograms per milliliter and 1, 200 micrograms per milliliter to select for a variety of integration copy numbers. Incubate the replica plates at 30 degree Celsius under constant or pulsed blue light for two to three days until the colonies appear. To perform the preliminary screening, select eight colonies from each plate and use them to inoculate one milliliter of SC-HIS medium supplemented with 2%glucose in individual wells of a 24 well plate.

Grow the cells overnight at 30 degree Celsius shaking at 200 revolutions per minute under constant blue light illumination. The next day, dilute each culture in a fresh SC-HIS medium with 2%glucose to obtain the optical density values ranging from 0.01 to 0.3 at 600 nanometers and grow the cultures in shaking condition 200 revolutions per minute under constant or pulsed light at 30 degrees Celsius until the optical density reaches between two and nine. Then, wrap the plates with aluminum foil, turn off the light panel, and incubate the plates in the dark for four hours at 30 degrees Celsius with 200 revolutions per minute.

Centrifuge the cultures in the 24 well plate at 234 G's for five minutes and resuspend cells in one milliliter of fresh synthetic complete drop-out medium with 2%glucose. Seal the plates to prevent evaporation of the desired product using sterile microplate sealing tape. Ferment the sealed plates in the dark for 48 hours at 30 degree Celsius shaking at 200 revolutions per minute.

To harvest the fermentations, centrifuge the plates for five minutes at 234 G's and transfer 800 microliters of the supernatant into a 1.5 milliliter microcentrifuge tube. Depending on the chemical of interest, analyze using high performance liquid chromatography, gas chromatography mass spectrometry, or another analytical method using the sample preparation technique best suited for the instrument used. Select eight colonies from each plate and use them to inoculate one milliliter of SC-HIS medium with 2%glucose in individual wells of a 24 well plate.

Grow the cells overnight in the dark at 30 degree Celsius with 200 revolutions per minute shaking. The next morning, dilute each culture in a fresh SC-HIS medium with 2%glucose to 0.1 optical density. Wrap the plates in aluminum foil to prevent exposure to light and grow the culture in the dark at 30 degree Celsius with 200 revolutions per minute shaking until they reach an optical density of three.

Then, incubate the plates under pulsed light for 12 hours at 30 degree Celsius with 200 revolutions per minute shaking. Centrifuge the cultures at 234 G's for five minutes and resuspend in fresh SC-HIS medium with 2%glucose. Seal the plates to prevent evaporation of the desired product using sterile microplate sealing tape.

Ferment the sealed plates in light for 48 hours at 30 degrees Celsius shaking at 200 revolutions per minute. After performing optogenetic fermentations, chemical production was tested at a range of cell densities at the time of induction with two circuits. The OptoINVRT7 circuit demonstrated high titers of lactic acid and isobutanol with an optimal cell density of induction value of 7.0 and 8.75 compared to the OptoINVRT1 and 2 circuits.

Fermentations using the OptoAMP circuits were studied in three phases defined by different light duty cycles. Lactic acid production can be optimized using a pulsed growth phase and fully illuminated induction and production phases. Isobutanol production was optimized using a pulsed growth phase, fully illuminated induction phase, and pulsed production phase.

Whereas naringenin biosynthesis was best optimized using a pulsed growth fully illuminated induction and dark production phase. The OptoLAC system has been used in E.Coli to produce transcription factor FDER at titers that are comparable to or higher than those achieved using chemical induction with IPDG. OptoLAC circuits have been applied to produce mevalonate and the production exceeds titers achieved using IPDG induction.

Mevalonate production at the bioreactor level demonstrates the scalability and tunability of optogenetic regulation for chemical and protein production in bacteria beyond microplate levels. It's important to ensure that the light intensity is in the proper range for the illuminated steps and that light contamination is avoided for steps that require darkness. This technique paved the way to achieve record titers of isobutanol in yeast, stabilized consortia populations with light, and even control metabolic flux using synthetic light controlled organelles.