Most mono-and co-culture bioprocess applications rely on large-scale suspension fermentation technologies that are not easily portable, reusable, or suitable for on-demand production. Here, we describe a hydrogel system for harnessing the bioactivity of embedded microbes for on-demand small molecule and peptide production in microbial mono-culture and consortia. This platform bypasses the challenges of engineering a multi-organism consortia by utilizing a temperature-responsive, shear-thinning hydrogel to compartmentalize organisms into polymeric hydrogels that control the final consortium composition and dynamics without the need for synthetic control of mutualism. We demonstrate that these hydrogels provide protection from preservation techniques (including lyophilization) and can sustain metabolic function for over 1 year of repeated use. This approach was utilized for the production of four chemical compounds, a peptide antibiotic, and carbohydrate catabolism by using either mono-cultures or co-cultures. The printed microbe-laden hydrogel constructs' efficiency in repeated production phases, both pre-and post-preservation, outperforms liquid culture.

Synthetic biology and metabolic engineering seek to re-engineer microbes into “living foundries” for the production of high value chemicals. Through a “design-build-test” cycle paradigm, massive libraries of genetically engineered microbes can be constructed and tested for metabolite overproduction and secretion. However, library generation capacity outpaces the rate of high-throughput testing and screening. Well plate assays are flexible but with limited throughput, whereas droplet microfluidic techniques are ultrahigh-throughput but require a custom assay for each target. Here we present RNA-aptamers-in-droplets (RAPID), a method that greatly expands the generality of ultrahigh-throughput microfluidic screening. Using aptamers, we transduce extracellular product titer into fluorescence, allowing ultrahigh-throughput screening of millions of variants. We demonstrate the RAPID approach by enhancing production of tyrosine and secretion of a recombinant protein in Saccharomyces cerevisiae by up to 28- and 3-fold, respectively. Aptamers-in-droplets affords a general approach for evolving microbes to synthesize and secrete value-added chemicals.

Background: Resveratrol is a plant secondary metabolite with diverse, potential health-promoting benefits. Due to its nutraceutical merit, bioproduction of resveratrol via microbial engineering has gained increasing attention and provides an alternative to unsustainable chemical synthesis and straight extraction from plants. However, many studies on microbial resveratrol production were implemented with the addition of water-insoluble phenylalanine or tyrosine-based precursors to the medium, limiting in the sustainable development of bioproduction. Results: Here we present a novel coculture platform where two distinct metabolic background species were modularly engineered for the combined total and de novo biosynthesis of resveratrol. In this scenario, the upstream Escherichia coli module is capable of excreting p-coumaric acid into the surrounding culture media through constitutive overexpression of codon-optimized tyrosine ammonia lyase from Trichosporon cutaneum (TAL), feedback-inhibitionresistant 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase (aroG fbr) and chorismate mutase/prephenate dehydrogenase (tyrA fbr) in a transcriptional regulator tyrR knockout strain. Next, to enhance the precursor malonyl-CoA supply, an inactivation-resistant version of acetyl-CoA carboxylase (ACC1 S659A,S1157A) was introduced into the downstream Saccharomyces cerevisiae module constitutively expressing codon-optimized 4-coumarate-CoA ligase from Arabidopsis thaliana (4CL) and resveratrol synthase from Vitis vinifera (STS), and thus further improve the conversion of p-coumaric acid-to-resveratrol. Upon optimization of the initial inoculation ratio of two populations, fermentation temperature, and culture time, this co-culture system yielded 28.5 mg/L resveratrol from glucose in flasks. In further optimization by increasing initial net cells density at a test tube scale, a final resveratrol titer of 36 mg/L was achieved. Conclusions: This is first study that demonstrates the use of a synthetic E. coli-S. cerevisiae consortium for de novo resveratrol biosynthesis, which highlights its potential for production of other p-coumaric-acid or resveratrol derived biochemicals.

Metabolic engineering allows for the rewiring of basic metabolism to overproduce both native and non-native metabolites. Among these biomolecules, nutraceuticals have received considerable interest due to their health-promoting or disease-preventing properties. Likewise, microbial engineering efforts to produce these value-added nutraceuticals overcome traditional limitations of low yield from extractions and complex chemical syntheses. This review covers current strategies of metabolic engineering employed for the production of a few key nutraceuticals with selecting polyunsaturated fatty acids, polyphenolic compounds, carotenoids and non-proteinogenic amino acids as exemplary molecules. We focus on the use of both mono-culture and co-culture strategies to produce these molecules of interest. In each of these cases, metabolic engineering efforts are enabling rapid production of these molecules.

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