TitleEngineering prokaryotic transcriptional activators as metabolite biosensors in yeast io-based production of chemicals and fuels is an attractive avenue to reduce dependence on petroleum. For bio-based production, biocatalysts must often be genetically modified to increase production. However, the current efficiency of genomeengineering methods and parts prospecting allows for unprecedented genotype diversity that vastly outstrips our ability to screen for best cell performance 1,2 .To meet current demand, bioengineers have started to develop genetically encoded devices and systems that enable screening and selection of better-performing biocatalysts in higher throughput. Genetic devices including oscillators, amplifiers and recorders, which have been developed based on fine-tuned relationships between input and output signals, are promising tools for programming and controlling gene expression in living cells [3][4][5] . These devices sense extracellular or intracellular perturbations and actuate cellular decision-making processes akin to logic gates in electrical circuits. Hence, from a diverse set of inputs, molecular gating components such as RNA aptamers and allosterically regulated transcription factors have been engineered to control outputs for applications such as high-throughput screening, actuation on cellular metabolism and evolution-based selection of optimal cell performance [6][7][8] .A key component in many of the reported devices is a ligandinducible transcriptional regulator. Transcriptional regulators are straightforward and powerful components, with many uses in genetic designs. Owing to their modular structure, transcriptional regulators have proven to be versatile platforms for genetically encoded Boolean logic functions 9,10 . In particular, gene switches based on ligand-binding transcriptional repressors bind to genomic targets in the absence of their cognate ligand and thereby repress gene expression of the downstream gene(s), whereas binding between ligand and repressor causes the release of the repressor from the DNA and thereby a derepression 11 . In such 'NOT' gates, simple steric hindrance of RNA polymerase progression, as in the case of the tetracycline-responsive gene switch TetR, have for decades been used for conditional control of gene expression in both prokaryotic and eukaryotic chassis 12,13 . Transcriptional repressors and other artificial transcriptional regulators can be further engineered, for example, via the addition of nuclear localization signals, destabilization domains and transcriptional activation regions, to repurpose conditional repressors into activators [13][14][15] . Though conceptually intriguing and practically relevant, the repurposing of logic gates can suffer from the inherent need for extensive engineering 9,16,17 .Though most ligand-inducible genetic devices adopted for eukaryotes historically have been founded on transcriptional repressors, a hitherto untapped resource for use in genetic designs is ligand-inducible transcriptional activators. Bac...

Phenylalanine and tyrosine ammonia-lyases form cinnamic acid and p-coumaric acid, which are precursors of a wide range of aromatic compounds of biotechnological interest. Lack of highly active and specific tyrosine ammonia-lyases has previously been a limitation in metabolic engineering approaches. We therefore identified 22 sequences in silico using synteny information and aiming for sequence divergence. We performed a comparative in vivo study, expressing the genes intracellularly in bacteria and yeast. When produced heterologously, some enzymes resulted in significantly higher production of p-coumaric acid in several different industrially important production organisms. Three novel enzymes were found to have activity exclusively for phenylalanine, including an enzyme from the low-GC Gram-positive bacterium Brevibacillus laterosporus, a bacterial-type enzyme from the amoeba Dictyostelium discoideum, and a phenylalanine ammonia-lyase from the moss Physcomitrella patens (producing 230 M cinnamic acid per unit of optical density at 600 nm [OD 600 ]) in the medium using Escherichia coli as the heterologous host). Novel tyrosine ammonia-lyases having higher reported substrate specificity than previously characterized enzymes were also identified. Enzymes from Herpetosiphon aurantiacus and Flavobacterium johnsoniae resulted in high production of p-coumaric acid in Escherichia coli (producing 440 M p-coumaric acid OD 600 unit ؊1 in the medium) and in Lactococcus lactis. The enzymes were also efficient in Saccharomyces cerevisiae, where p-coumaric acid accumulation was improved 5-fold over that in strains expressing previously characterized tyrosine ammonia-lyases. Small organic molecules of biotechnological interest include aromatic structures that are derived from p-coumaric acid (pHCA). pHCA can be formed from phenylalanine either through deamination to cinnamic acid (CA) by phenylalanine ammonialyase (PAL; EC 4.3.1.24) and subsequent hydroxylase activity by trans-cinnamate-4-monooxygenase or through deamination of tyrosine by tyrosine ammonia-lyase (TAL; EC 4.3.1.23) (Fig. 1). Considering that the route from phenylalanine requires activity of a P450 enzyme, which has been shown to be the limiting step in previous engineering strategies (1, 2), deamination of tyrosine for production of pHCA may be preferred in microbial cell factories. Tyrosine ammonia-lyases (TAL) have been employed for the production of plant biochemicals and aromatic compounds, e.g., for pHCA itself (3-5), or in the production of stilbenes, such as resveratrol (6, 7), flavonoids, such as naringenin (8, 9), cinnamoyl anthranilates (10), or plastic precursors, such as p-hydroxystyrene (11, 12), yet the TAL reaction may be the limiting step (10, 13).Due to the significant interest in production of phenolic compounds in biotechnological processes, we aimed at increasing the range of characterized phenylalanine ammonia-lyases (PAL) and tyrosine ammonia-lyases (TAL), and in particular at identifying the optimal TAL for use in microbial cell factories...

This study describes the construction of two flavonoid biosensors, which can be applied for metabolic engineering of Escherichia coli strains. The biosensors are based on transcriptional regulators combined with autofluorescent proteins. The transcriptional activator FdeR from Herbaspirillum seropedicae SmR1 responds to naringenin, while the repressor QdoR from Bacillus subtilis is inactivated by quercetin and kaempferol. Both biosensors showed over a 7-fold increase of the fluorescent signal after addition of their specific effectors, and a linear correlation between the fluorescence intensity and externally added flavonoid concentration. The QdoR-biosensor was successfully applied for detection of kaempferol production in vivo at the single cell level by fluorescence-activated cell sorting. Furthermore, the amount of kaempferol produced highly correlated with the specific fluorescence of E. coli cells containing a flavonol synthase from Arabidopsis thaliana (fls1). We expect the designed biosensors to be applied for isolation of genes involved in flavonoid biosynthetic pathways.

Recombineering in bacteria is a powerful technique for genome reconstruction, but until now, it was not generally applicable for development of small-molecule producers because of the inconspicuous phenotype of most compounds of biotechnological relevance. Here, we establish recombineering for Corynebacterium glutamicum using RecT of prophage Rac and combine this with our recently developed nanosensor technology, which enables the detection and isolation of productive mutants at the single-cell level via fluorescence-activated cell sorting (FACS). We call this new technology RecFACS, which we use for genomic site-directed saturation mutagenesis without relying on pre-constructed libraries to directly isolate l-lysine-producing cells. A mixture of 19 different oligonucleotides was used targeting codon 81 in murE of the wild-type, at a locus where one single mutation is known to cause l-lysine production. Using RecFACS, productive mutants were screened and isolated. Sequencing revealed 12 different amino acid exchanges in the targeted murE codon, which caused different l-lysine production titers. Apart from introducing a rapid genome construction technology for C. glutamicum, the present work demonstrates that RecFACS is suitable to simply create producers as well as genetic diversity in one single step, thus establishing a new general concept in synthetic biology.

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An ultra-high-throughput screening system for NADPH-dependent enzymes, such as stereospecific alcohol dehydrogenases, was established. It is based on the [2Fe-2S] cluster-containing transcriptional regulator SoxR of Escherichia coli that activates expression of soxS in the oxidized but not in the reduced state of the cluster. As SoxR is kept in its reduced state by NADPH-dependent reductases, an increased NADPH demand of the cell counteracts SoxR reduction and increases soxS expression. We have taken advantage of these properties by placing the eyfp gene under the control of the soxS promoter and analyzed the response of E. coli cells expressing an NADPH-dependent alcohol dehydrogenase from Lactobacillus brevis (LbAdh), which reduces methyl acetoacetate to (R)-methyl 3-hydroxybutyrate. Under suitable conditions, the specific fluorescence of the cells correlated with the substrate concentration added and with LbAdh enzyme activity, supporting the NADPH responsiveness of the sensor. These properties enabled sorting of single cells harboring wild-type LbAdh from those with lowered or without LbAdh activity by fluorescence-activated cell sorting (FACS). In a proof-of-principle application, the system was used successfully to screen a mutant LbAdh library for variants showing improved activity with the substrate 4-methyl-2-pentanone.

Transcription factor-based biosensors are used to identify producer strains, a critical bottleneck in cell factory engineering. Here, we address two challenges with this methodology: transplantation of heterologous transcriptional regulators into new hosts to generate functional biosensors and biosensing of the extracellular product concentration that accurately reflects the effective cell factory production capacity. We describe the effects of different translation initiation rates on the dynamic range of a p-coumaric acid biosensor based on the Bacillus subtilis transcriptional repressor PadR by varying its ribosomal binding site. Furthermore, we demonstrate the functionality of this p-coumaric acid biosensor in Escherichia coli and Corynebacterium glutamicum. Finally, we encapsulate yeast p-coumaric acid-producing cells with E. coli-biosensing cells in picoliter droplets and, in a microfluidic device, rapidly sort droplets containing yeast cells producing high amounts of extracellular p-coumaric acid using the fluorescent E. coli biosensor signal. As additional biosensors become available, such approaches will find broad applications for screening of an extracellular product.

Only 25% of bacterial membrane transporters have functional annotation owing to the difficulty of experimental study and of accurate prediction of their function. Here we report a sequence-independent method for high-throughput mining of novel transporters. The method is based on ligand-responsive biosensor systems that enable selective growth of cells only if they encode a ligand-specific importer. We developed such a synthetic selection system for thiamine pyrophosphate and mined soil and gut metagenomes for thiamine-uptake functions. We identified several members of a novel class of thiamine transporters, PnuT, which is widely distributed across multiple bacterial phyla. We demonstrate that with modular replacement of the biosensor, we could expand our method to xanthine and identify xanthine permeases from gut and soil metagenomes. Our results demonstrate how synthetic-biology approaches can effectively be deployed to functionally mine metagenomes and elucidate sequence-function relationships of small-molecule transport systems in bacteria.