Microbial engineering often requires fine control over protein expression; for example, to connect genetic circuits 1-7 or control flux through a metabolic pathway 8-13. We have developed a predictive design method for synthetic ribosome binding sites that enables the rational control of a protein's production rate on a proportional scale. Experimental validation of over 100 predictions in Escherichia coli shows that the method is accurate to within a factor of 2.3 over a range of 100,000-fold. The design method also correctly predicts that reusing a ribosome binding site sequence in different genetic contexts can result in different protein expression levels. We demonstrate the method's utility by rationally optimizing a protein's expression level to connect a genetic sensor to a synthetic circuit. The proposed forward engineering approach will accelerate the construction and systematic optimization of large genetic systems.

Methyl halides are used as agricultural fumigants and are precursor molecules that can be catalytically converted to chemicals and fuels. Plants and microorganisms naturally produce methyl halides, but these organisms produce very low yields or are not amenable to industrial production. A single methyl halide transferase (MHT) enzyme transfers the methyl group from the ubiquitous metabolite S-adenoyl methionine (SAM) to a halide ion. Using a synthetic metagenomic approach, we chemically synthesized all 89 putative MHT genes from plants, fungi, bacteria, and unidentified organisms present in the NCBI sequence database. The set was screened in Escherichia coli to identify the rates of CH(3)Cl, CH(3)Br, and CH(3)I production, with 56% of the library active on chloride, 85% on bromide, and 69% on iodide. Expression of the highest activity MHT and subsequent engineering in Saccharomyces cerevisiae results in productivity of 190 mg/L-h from glucose and sucrose. Using a symbiotic co-culture of the engineered yeast and the cellulolytic bacterium Actinotalea fermentans, we are able to achieve methyl halide production from unprocessed switchgrass (Panicum virgatum), corn stover, sugar cane bagasse, and poplar (Populus sp.). These results demonstrate the potential of producing methyl halides from non-food agricultural resources.

The type III secretion system (T3SS) exports proteins from the cytoplasm, through both the inner and outer membranes, to the external environment. Here, a system is constructed to harness the T3SS encoded within Salmonella Pathogeneity Island 1 to export proteins of biotechnological interest. The system is composed of an operon containing the target protein fused to an N-terminal secretion tag and its cognate chaperone. Transcription is controlled by a genetic circuit that only turns on when the cell is actively secreting protein. The system is refined using a small human protein (DH domain) and demonstrated by exporting three silk monomers (ADF-1, -2, and -3), representative of different types of spider silk. Synthetic genes encoding silk monomers were designed to enhance genetic stability and codon usage, constructed by automated DNA synthesis, and cloned into the secretion control system. Secretion rates up to 1.8 mg l À1 h À1 are demonstrated with up to 14% of expressed protein secreted. This work introduces new parts to control protein secretion in Gram-negative bacteria, which will be broadly applicable to problems in biotechnology.

Many signaling proteins are built from simple, modular components, yet display highly complex signal-processing behavior. Here we explore how modular domains can be used to build an ultrasensitive switch--a nonlinear input/output function that is central to many complex biological behaviors. By systematically altering the number and affinity of modular autoinhibitory interactions, we show that we can predictably convert a simple linear signaling protein into an ultrasensitive switch.

Gram-negative bacteria secrete proteins using a type III secretion system (T3SS), which functions as a needle-like molecular machine. The many proteins involved in T3SS construction are tightly regulated due to its role in pathogenesis and motility. Here, starting with the 35 kb Salmonella pathogenicity island 1 (SPI-1), we eliminated internal regulation and simplified the genetics by removing or recoding genes, scrambling gene order and replacing all non-coding DNA with synthetic genetic parts. This process results in a 16 kb cluster that shares no sequence identity, regulation or organizational principles with SPI-1. Building this simplified system led to the discovery of essential roles for an internal start site (SpaO) and small RNA (InvR). Further, it can be controlled using synthetic regulatory circuits, including under SPI-1 repressing conditions. This work reveals an incredible post-transcriptional robustness in T3SS assembly and aids its control as a tool in biotechnology.