(585ao) Accelerating Build and Test of Microbial Libraries Via Integration of Synthetic Biology, Robotic Automation and Mass Spectrometry

Whereas synthetic biology initially focuses on modular assembly of standardized ‘function’ units for predicable phenotypes, the importance of standardized ‘physical’ processes in constructing biological systems has been increasingly recognized. Here we demonstrated synergistic integration of both genetic design and workflow/instrument development are indispensable to accelerate the design-build-test-learn cycle in microbial engineering.

For the first example of optimizing Saccharomyces cerevisiae as a microbial cell factory, an automated engineering workflow was established using an integrated robotic system (Illinois Biological Foundry for Advanced Bioengineering, iBioFAB). On the genetic aspect, as robotic automation excels at iterative execution of defined processes, it is desirable to use the same procedure to introduce different genetic modifications. Therefore, we chose to apply trans-acting elements for genetic modulation so that mutations can be created independent of the target chromosomal sequences using standardized vectors. Repetitive genomic sequences were selected as vectors because scalable accumulation of combinatorial mutations can be achieved during repeated integration of trans-acting donors. On the instrument aspect, as the yeast engineering workflow includes several process modules including heat-shock transformation, cell cultivation, on-line spectrophotometric monitoring of cell growth, and sample preparation for off-line analyses, a flexible framework is needed to complete all the distinct tasks on a single platform for full automation. To achieve such flexibility, we enumerate all common unit operations in microbial engineering, including liquid handling, centrifugation, temperature control, and so on, and match them with component instruments in iBioFAB. Then any process module in the yeast workflow can be converted into a custom sequence of unit operations, which is then executed using robotic arms to transfer samples among component instruments. An integrated computational framework is also developed to orchestrate physical operation and data acquisition. With the optimized genetic constructs and process parameters, yeast strain libraries were iteratively created and subjected to serial transfer under different HAc stresses. After three automated engineering cycles in ~1 month, a mutant strain was isolated harboring 26 trans-acting elements from a genome-scale modulation collection for both overexpression and knockdown mutations in yeast. The mutant strain can ferment 2% glucose in the presence of 1.1% (v/v) HAc within 4 days, the highest HAc resistance ever reported in yeast to the best of our knowledge.

In the second example of engineering natural product analogs in Escherichia coli, a structure-based screening method was developed called optically-guided matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). On the instrument aspect, MALDI-MS provides label-free characterization of target molecules with high specificity, but individual samples need to be processed and deposited into defined arrays on MALDI target plates at low throughput. To overcome this limitation, we integrated machine vision for automated mass spectra acquisition at randomly distributed colonies on agar. However, such instrumental design creates additional constrains on the genetic aspect for direct MS analysis of colonies. For a peptidic antibiotic plantazolicin, pathway refactoring was performed to enhance production so that sufficient analytes can be accumulated in a single colony. For another peptidic antibiotic haloduracin, inducible self-lysis of the host cells was programmed for extracellular release of target analytes produced in cytosol. To acquire high-resolution mass spectra only obtainable currently at a low molecular range (<6,000 Da), protease expression was compartmentalized in periplasm so that proteolytic processing cannot be initiated until cell lysis, as the protease activity will interfere with haloduracin biosynthesis.

Together, we found continuing crosstalk between synthetic biology and instrument/workflow development is critical to establish high-throughput technologies in microbial engineering. In addition to rapid isolation of mutants with desirable traits, accelerating build and test helps to collect large data sets of a target biological system. Development of computational tools for data processing will be the future focus to complete the design-build-test-learn cycle.