Biology subjects

Lalwani, M. A.

Publications and source records attributed to Lalwani, M. A..

3 recordsLinked to original sources

bioRxiv2025

Balancing doses of EL222 and light improves optogenetic induction of protein production in Komagataella phaffii

Komagataella phaffii, also known as Pichia pastoris, is a powerful host for recombinant protein production, in part due to its exceptionally strong and tightly controlled PAOX1 promoter. Most K. phaffii bioprocesses for recombinant protein production rely on PAOX1 to achieve dynamic control in two-phase processes. Cells are first grown under conditions that repress PAOX1 (growth phase), followed by methanol-induced recombinant protein expression (production phase). In this study, we propose a methanol-free approach for dynamic metabolic control in K. phaffii using optogenetics, which can help enhance input tunability and flexibility in process optimization and control. The light-responsive transcription factor EL222 from Erythrobacter litoralis is used to regulate protein production from the PC120 promoter in K. phaffii with 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 using K. phaffii.

bioengineering↗

bioRxiv2020

Optogenetic Amplification Circuits for Light-Induced Metabolic Control

Dynamic control of microbial metabolism is an effective strategy to improve chemical production in fermentations. While dynamic control is most often implemented using chemical inducers, optogenetics offers an attractive alternative due to the high tunability and reversibility afforded by light. However, a major concern of applying optogenetics in metabolic engineering is the risk of insufficient light penetration at high cell densities, especially in large bioreactors. Here, we present a new series of optogenetic circuits we call OptoAMP, which amplify the transcriptional response to blue light by as much as 21.8-fold compared to the basal circuit (OptoEXP). These circuits show as much as a 41-fold induction between dark and light conditions, efficient activation at light doses as low as ~1%, and strong homogeneous light-induction in bioreactors of at least 5L, with limited illumination at cell densities above 40 OD600. We demonstrate the ability of OptoAMP circuits to control engineered metabolic pathways in novel three-phase fermentations using different light schedules to control enzyme expression and improve production of lactic acid, isobutanol, and naringenin. These circuits expand the applicability of optogenetics to metabolic engineering.

synthetic biology↗

bioRxiv2019

Optogenetic lac operon to control chemical and protein production in Escherichia coli with light

Control of the lac operon with IPTG has been used for decades to regulate gene expression in E. coli for countless applications, including metabolic engineering and recombinant protein production. However, optogenetics offers unique capabilities such as easy tunability, reversibility, dynamic induction strength, and spatial control that are difficult to obtain with chemical inducers. We developed an optogenetic lac operon in a series of circuits we call OptoLAC. With these circuits, we control gene expression from various IPTG-inducible promoters using only blue light. Applying them to metabolic engineering improves mevalonate and isobutanol production by 24% and 27% respectively, compared to IPTG induction, in light-controlled fermentations scalable to at least 2L bioreactors. Furthermore, OptoLAC circuits enable light control of recombinant protein production, reaching yields comparable to IPTG induction, but with enhanced tunability of expression and spatial control. OptoLAC circuits are potentially useful to confer light controls over other cell functions originally engineered to be IPTG-inducible.

synthetic biology↗