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Alper, H.

Publications and source records attributed to Alper, H..

2 recordsLinked to original sources

Real-time, in situ fluorescence and optical density measurements of liquid cultures in simulated microgravity

As human space exploration expands to the Moon, Mars, and beyond, there is a growing need to study the effects of altered gravity on the microbial systems that we will bring with us for life support. Because spaceflight experiment opportunities are rare and resource-intensive, most space biology experiments are conducted using ground-based simulators. The most common microgravity simulator for microbial experiments, the rotating wall vessel, can approximate the low-shear and low-turbulence conditions that characterize microgravity. However, current designs do not allow for real-time measurement of growth or metabolic activity during rotation: experiments require destructive sampling or disruption of the microgravity simulation conditions. Here, we describe the development of an in situ spectroscopy system compatible with the Cell Spinpod rotating wall vessel, which enables measurement of both optical absorbance and fluorescence with high temporal resolution, producing growth curves similar to those from an off-the-shelf plate reader. These results are validated using two common microbial hosts: Escherichia coli and Saccharomyces cerevisiae. The Spinpod Optical System has the potential to diversify the types of microbiology experiments possible in simulated microgravity, allowing the measurement of not only growth curve parameters but also metabolic activity, gene expression, or community dynamics. It thus has the potential to improve the quality of experiments seeking to characterize microbial responses to spaceflight conditions.

microbiology↗

Synthetic microbial sensing and biosynthesis of amaryllidaceae alkaloids

A major challenge to achieving industry-scale biomanufacturing of therapeutic alkaloids is the slow process of biocatalyst engineering. Amaryllidaceae alkaloids, such as the Alzheimers medication galantamine, are complex plant secondary metabolites with recognized therapeutic value. Due to their difficult synthesis they are regularly sourced by extraction and purification from low-yielding plants, including the wild daffodil Narcissus pseudonarcissus. Engineered biocatalytic methods have the potential to stabilize the supply chain of amaryllidaceae alkaloids. Here, we propose a highly efficient biosensor-AI technology stack for biocatalyst development, which we apply to engineer amaryllidaceae alkaloid production in Escherichia coli. Directed evolution is used to develop a highly sensitive (EC50= 20 uM) and specific biosensor for the key amaryllidaceae alkaloid branchpoint 4-OMethylnorbelladine. A machine learning model (MutComputeX) was subsequently developed and used to generate activity-enriched variants of a plant methyltransferase, which were rapidly screened with the biosensor. Functional enzyme variants were identified that yielded a 60% improvement in product titer, 17-fold reduced remnant substrate, and 3-fold lower off-product regioisomer formation.

synthetic biology↗