Genetic context effects can override canonical cis regulatory elements in Escherichia coli.

Recent experiments have shown that in addition to control by cis regulatory elements, the local chromosomal context of a gene also has a profound impact on its transcription. Although this chromosome-position dependent expression variation has been empirically mapped at high-resolution, the underlying causes of the variation have not been elucidated. Here, we demonstrate that 1 kb of flanking, non-coding synthetic sequences with a low frequency of guanosine and cytosine (GC) can dramatically reduce reporter expression compared to neutral and high GC-content flanks in Escherichia coli. Natural and artificial genetic context can have a similarly strong effect on reporter expression, regardless of cell growth phase or medium. Despite the strong reduction in the maximal expression level from the fully-induced reporter, low GC synthetic flanks do not affect the time required to reach the maximal expression level after induction. Overall, we demonstrate key determinants of transcriptional propensity that appear to act as tunable modulators of transcription, independent of regulatory sequences such as the promoter. These findings provide insight into the regulation of naturally occurring genes and an independent control for optimizing expression of synthetic biology constructs.

Production of cellulosic organic acids via synthetic fungal consortia.

Consolidated bioprocessing (CBP) is a potential breakthrough technology for reducing costs of biochemical production from lignocellulosic biomass. Production of cellulase enzymes, saccharification of lignocellulose, and conversion of the resulting sugars into a chemical of interest occur simultaneously within a single bioreactor. In this study, synthetic fungal consortia composed of the cellulolytic fungus Trichoderma reesei and the production specialist Rhizopus delemar demonstrated conversion of microcrystalline cellulose (MCC) and alkaline pre-treated corn stover (CS) to fumaric acid in a fully consolidated manner without addition of cellulase enzymes or expensive supplements such as yeast extract. A titer of 6.87 g/L of fumaric acid, representing 0.17 w/w yield, were produced from 40 g/L MCC with a productivity of 31.8 mg/L/hr. In addition, lactic acid was produced from MCC using a fungal consortium with Rhizopus oryzae as the production specialist. These results are proof-of-concept demonstration of engineering synthetic microbial consortia for CBP production of naturally occurring biomolecules.

Design and characterization of synthetic fungal-bacterial consortia for direct production of isobutanol from cellulosic biomass.

Synergistic microbial communities are ubiquitous in nature and exhibit appealing features, such as sophisticated metabolic capabilities and robustness. This has inspired fast-growing interest in engineering synthetic microbial consortia for biotechnology development. However, there are relatively few reports of their use in real-world applications, and achieving population stability and regulation has proven to be challenging. In this work, we bridge ecology theory with engineering principles to develop robust synthetic fungal-bacterial consortia for efficient biosynthesis of valuable products from lignocellulosic feedstocks. The required biological functions are divided between two specialists: the fungus Trichoderma reesei, which secretes cellulase enzymes to hydrolyze lignocellulosic biomass into soluble saccharides, and the bacterium Escherichia coli, which metabolizes soluble saccharides into desired products. We developed and experimentally validated a comprehensive mathematical model for T. reesei/E. coli consortia, providing insights on key determinants of the system's performance. To illustrate the bioprocessing potential of this consortium, we demonstrate direct conversion of microcrystalline cellulose and pretreated corn stover to isobutanol. Without costly nutrient supplementation, we achieved titers up to 1.88 g/L and yields up to 62% of theoretical maximum. In addition, we show that cooperator-cheater dynamics within T. reesei/E. coli consortia lead to stable population equilibria and provide a mechanism for tuning composition. Although we offer isobutanol production as a proof-of-concept application, our modular system could be readily adapted for production of many other valuable biochemicals.

A versatile active learning workflow for optimization of genetic and metabolic networks.

Optimization of biological networks is often limited by wet lab labor and cost, and the lack of convenient computational tools. Here, we describe METIS, a versatile active machine learning workflow with a simple online interface for the data-driven optimization of biological targets with minimal experiments. We demonstrate our workflow for various applications, including cell-free transcription and translation, genetic circuits, and a 27-variable synthetic CO2-fixation cycle (CETCH cycle), improving these systems between one and two orders of magnitude. For the CETCH cycle, we explore 1025 conditions with only 1,000 experiments to yield the most efficient CO2-fixation cascade described to date. Beyond optimization, our workflow also quantifies the relative importance of individual factors to the performance of a system identifying unknown interactions and bottlenecks. Overall, our workflow opens the way for convenient optimization and prototyping of genetic and metabolic networks with customizable adjustments according to user experience, experimental setup, and laboratory facilities.

Random Chromosomal Integration and Screening Yields E. coli K-12 Derivatives Capable of Efficient Sucrose Utilization.

Chromosomal expression of heterologous genes offers stability and maintenance advantages over episomal expression, yet remains difficult to optimize through site-specific integration. The challenge has in large part been due to the variability of chromosomal gene expression, which has only recently been shown to be affected by multiple factors, including the local genomic context. In this work we utilize Tn5 transposase to randomly integrate a three-gene csc operon encoding nonphosphotransferase sucrose catabolism into the E. coli K-12 chromosome. Isolates from the transposon library yielded a range of growth rates on sucrose as the sole carbon source, including some that were comparable to that of E. coli K-12 on glucose (µmax = 0.70 ± 0.03 h-1). Narrowness of the growth rate distributions and faster growth compared to plasmids indicate that efficient csc expression is attainable. Furthermore, enhanced growth rate upon transduction into strains that underwent adaptive laboratory evolution indicate that sucrose catabolism is not limiting to cellular growth. We also show that transduction of a csc fast-growth locus into an isobutanol production strain yields high titer (7.56 ± 0.25 g/L) on sucrose as the sole carbon source. Our results demonstrate that random integration is an effective strategy for optimizing heterologous expression within the context of cellular metabolism for both fast growth and biochemical production phenotypes.

High-Resolution Mapping of the Escherichia coli Chromosome Reveals Positions of High and Low Transcription.

Recent studies on targeted gene integrations in bacteria have demonstrated that chromosomal location can substantially affect a gene's expression level. However, these studies have only provided information on a small number of sites. To measure position effects on transcriptional propensity at high resolution across the genome, we built and analyzed a library of over 144,000 genome-integrated, standardized reporters in a single mixed population of Escherichia coli. We observed more than 20-fold variations in transcriptional propensity across the genome when the length of the chromosome was binned into broad 4 kbp regions; greater variability was observed over smaller regions. Our data reveal peaks of high transcriptional propensity centered on ribosomal RNA operons and core metabolic genes, while prophages and mobile genetic elements were enriched in less transcribable regions. In total, our work supports the hypothesis that E. coli has evolved gene-independent mechanisms for regulating expression from specific regions of its genome.