Targeted mutagenesis and high-throughput screening of diversified gene and promoter libraries for isolating gain-of-function mutations.

Targeted mutagenesis of a promoter or gene is essential for attaining new functions in microbial and protein engineering efforts. In the burgeoning field of synthetic biology, heterologous genes are expressed in new host organisms. Similarly, natural or designed proteins are mutagenized at targeted positions and screened for gain-of-function mutations. Here, we describe methods to attain complete randomization or controlled mutations in promoters or genes. Combinatorial libraries of one hundred thousands to tens of millions of variants can be created using commercially synthesized oligonucleotides, simply by performing two rounds of polymerase chain reactions. With a suitably engineered reporter in a whole cell, these libraries can be screened rapidly by performing fluorescence-activated cell sorting (FACS). Within a few rounds of positive and negative sorting based on the response from the reporter, the library can rapidly converge to a few optimal or extremely rare variants with desired phenotypes. Library construction, transformation and sequence verification takes 6-9 days and requires only basic molecular biology lab experience. Screening the library by FACS takes 3-5 days and requires training for the specific cytometer used. Further steps after sorting, including colony picking, sequencing, verification, and characterization of individual clones may take longer, depending on number of clones and required experiments.

Tackling the Catch-22 Situation of Optimizing a Sensor and a Transporter System in a Whole-Cell Microbial Biosensor Design for an Anthropogenic Small Molecule.

Whole-cell biosensors provide a convenient detection tool for the high-throughput screening of genetically engineered biocatalytic activity. But establishing a biosensor for an anthropogenic molecule requires both a custom transporter and a transcription factor. This results in an unavoidable "Catch-22" situation in which transporter activity cannot be easily confirmed without a biosensor and a biosensor cannot be established without a functional transporter in a host organism. We overcame this type of circular problem while developing an adipic acid (ADA) sensor. First, leveraging an established cis,cis-muconic acid (ccMA) sensor, an annotated ccMA transporter MucK, which is expected to be broadly responsive to dicarboxylates, was stably expressed in the genome of Pseudomonas putida to function as a transporter for ADA, and then a PcaR transcription factor (endogenous to the strain and naturally induced by ß-ketoadipic acid, BKA) was diversified and selected to detect the ADA molecule. While MucK expression is otherwise very unstable in P. putida under strong promoter expression, our optimized mucK expression was functional for over 70 generations without loss of function, and we selected an ADA sensor that showed a specificity switch of over 35-fold from BKA at low concentrations (typically <0.1 mM of inducers). Our ADA and BKA biosensors show high sensitivity (low detection concentration <10 µM) and dynamic range (∼50-fold) in an industrially relevant organism and will open new avenues for high throughput discovery and optimization of enzymes and metabolic pathways for the biomanufacture of these molecules. In particular, the novel ADA sensor will aid the discovery and evolution of efficient biocatalysts for the biological recycling of ADA from the degradation of nylon-6,6 waste.

Precise Genomic Riboregulator Control of Metabolic Flux in Microbial Systems.

Engineered microbes can be used for producing value-added chemicals from renewable feedstocks, relieving the dependency on nonrenewable resources such as petroleum. These microbes often are composed of synthetic metabolic pathways; however, one major problem in establishing a synthetic pathway is the challenge of precisely controlling competing metabolic routes, some of which could be crucial for fitness and survival. While traditional gene deletion and/or coarse overexpression approaches do not provide precise regulation, cis-repressors (CRs) are RNA-based regulatory elements that can control the production levels of a particular protein in a tunable manner. Here, we describe a protocol for a generally applicable fluorescence-activated cell sorting technique used to isolate eight subpopulations of CRs from a semidegenerate library in Escherichia coli, followed by deep sequencing that permitted the identification of 15 individual CRs with a broad range of protein production profiles. Using these new CRs, we demonstrated a change in production levels of a fluorescent reporter by over two orders of magnitude and further showed that these CRs are easily ported from E. coli to Pseudomonas putida. We next used four CRs to tune the production of the enzyme PpsA, involved in pyruvate to phosphoenolpyruvate (PEP) conversion, to alter the pool of PEP that feeds into the shikimate pathway. In an engineered P. putida strain, where carbon flux in the shikimate pathway is diverted to the synthesis of the commodity chemical cis,cis-muconate, we found that tuning PpsA translation levels increased the overall titer of muconate. Therefore, CRs provide an approach to precisely tune protein levels in metabolic pathways and will be an important tool for other metabolic engineering efforts.

High-Quality Genome Assembly of Nannochloris desiccata 2437 and Its Associated Bacterial Community.

High-quality genome sequences were generated for the nonaxenic marine microalga Nannochloris desiccata UTEX 2437 and eight of its associated environmental bacterial species. N. desiccata UTEX 2437 is diploid, and its 20.738-Mbp nuclear genome sequence is assembled in 29 contigs.

Phylogenetic analyses and reclassification of the oleaginous marine species Nannochloris sp. "desiccata" (Trebouxiophyceae, Chlorophyta), formerly Chlorella desiccata, supported by a high-quality genome assembly.

Microalgae are diverse, with many gaps remaining in phylogenetic and physiological understanding. Thus, studying new microalgae species increases our broader comprehension of biological diversity, and evaluation of new candidates as algal production platforms can lead to improved productivity under a variety of cultivation conditions. Chlorella is a genus of fast-growing species often isolated from freshwater habitats and cultivated as a source of nutritional supplements. However, the use of freshwater increases competition with other freshwater needs. We identified Chlorella desiccata to be worthy of further investigation as a potential algae production strain, due to its isolation from a marine environment and its promising growth and biochemical composition properties. Long-read genomic sequencing was conducted for C. desiccata UTEX 2526, resulting in a high-quality, near chromosome level, diploid genome with an assembly length of 21.55 Mbp in only 18 contigs. We also report complete circular mitochondrial and chloroplast genomes. Phylogenomic and phylogenetic analyses using nuclear, chloroplast, 18S rRNA, and actin sequences revealed that this species clades within strains currently identified as Nannochloris (Trebouxiophyceae, Chlorophyta), leading to its reclassification as Nannochloris sp. "desiccata" UTEX 2526. The mode of cell division for this species is autosporulation, differing from the type species N. bacillaris. As has occurred across multiple microalgae genera, there are repeated examples of Nannochloris species reclassification in the literature. This high-quality genome assembly and phylogenetic analysis of the potential algal production strain Nannochloris sp. "desiccata" UTEX 2526 provides an important reference and useful tool for further studying this region of the phylogenetic tree.

Gene amplification, laboratory evolution, and biosensor screening reveal MucK as a terephthalic acid transporter in Acinetobacter baylyi ADP1.

Microbial terephthalic acid (TPA) catabolic pathways are conserved among the few bacteria known to turnover this xenobiotic aromatic compound. However, to date there are few reported cases in which this pathway has been successfully expressed in heterologous hosts to impart efficient utilization of TPA as a sole carbon source. In this work, we aimed to engineer TPA conversion in Acinetobacter baylyi ADP1 via the heterologous expression of catabolic and transporter genes from a native TPA-utilizing bacterium. Specifically, we obtained ADP1-derived strains capable of growing on TPA as the sole carbon source using chromosomal insertion and targeted amplification of the tph catabolic operon from Comamonas sp. E6. Adaptive laboratory evolution was then used to improve growth on this substrate. TPA consumption rates of the evolved strains, which retained multiple copies of the tph genes, were ~0.2 g/L/h (or ~1 g TPA/g cells/h), similar to that of Comamonas sp. E6 and almost 2-fold higher than that of Rhodococcus jostii RHA1, another native TPA-utilizing strain. To evaluate TPA transport in the evolved ADP1 strains, we engineered a TPA biosensor consisting of the transcription factor TphR and a fluorescent reporter. In combination with whole-genome sequencing, the TPA biosensor revealed that transport of TPA was not mediated by the heterologous proteins from Comamonas sp. E6. Instead, the endogenous ADP1 muconate transporter MucK, a member of the major facilitator superfamily, was responsible for TPA transport in several evolved strains in which MucK variants were found to enhance TPA uptake. Furthermore, the IclR-type transcriptional regulator DcaS was identified as a repressor of mucK expression. Overall, this work presents an unexpected function of a native protein identified through gene amplification, adaptive laboratory evolution, and a combination of screening methods. This study also provides a TPA biosensor for application in ADP1 and identifies transporter variants for use in metabolic engineering applications focused on plastic upcycling of polyesters.

Engineering glucose metabolism for enhanced muconic acid production in Pseudomonas putida KT2440.

Pseudomonas putida KT2440 has received increasing attention as an important biocatalyst for the conversion of diverse carbon sources to multiple products, including the olefinic diacid, cis,cis-muconic acid (muconate). P. putida has been previously engineered to produce muconate from glucose; however, periplasmic oxidation of glucose causes substantial 2-ketogluconate accumulation, reducing product yield and selectivity. Deletion of the glucose dehydrogenase gene (gcd) prevents 2-ketogluconate accumulation, but dramatically slows growth and muconate production. In this work, we employed adaptive laboratory evolution to improve muconate production in strains incapable of producing 2-ketogluconate. Growth-based selection improved growth, but reduced muconate titer. A new muconate-responsive biosensor was therefore developed to enable muconate-based screening using fluorescence activated cell sorting. Sorted clones demonstrated both improved growth and muconate production. Mutations identified by whole genome resequencing of these isolates indicated that glucose metabolism may be dysregulated in strains lacking gcd. Using this information, we used targeted engineering to recapitulate improvements achieved by evolution. Deletion of the transcriptional repressor gene hexR improved strain growth and increased the muconate production rate, and the impact of this deletion was investigated using transcriptomics. The genes gntZ and gacS were also disrupted in several evolved clones, and deletion of these genes further improved strain growth and muconate production. Together, these targets provide a suite of modifications that improve glucose conversion to muconate by P. putida in the context of gcd deletion. Prior to this work, our engineered strain lacking gcd generated 7.0 g/L muconate at a productivity of 0.07 g/L/h and a 38% yield (mol/mol) in a fed-batch bioreactor. Here, the resulting strain with the deletion of hexR, gntZ, and gacS achieved 22.0 g/L at 0.21 g/L/h and a 35.6% yield (mol/mol) from glucose in similar conditions. These strategies enabled enhanced muconic acid production and may also improve production of other target molecules from glucose in P. putida.

Sensor-Enabled Alleviation of Product Inhibition in Chorismate Pyruvate-Lyase.

Product inhibition is a frequent bottleneck in industrial enzymes, and testing mutations to alleviate product inhibition via traditional methods remains challenging as many variants need to be tested against multiple substrate and product concentrations. Further, traditional screening methods are conducted in vitro, and resulting enzyme variants may perform differently in vivo in the context of whole-cell metabolism and regulation. In this study, we address these two problems by establishing a high-throughput screening method to alleviate product inhibition in an industrially relevant enzyme, chorismate pyruvate-lyase (UbiC). First, we engineered a highly specific, genetically encoded biosensor for 4-hydroxybenzoate (4HB) in an industrially relevant host, Pseudomonas putida KT2440. We subsequently applied the biosensor to detect the activity of a heterologously expressed UbiC that converts chorismate into 4HB and pyruvate. By using benzoate as a product surrogate that inhibits UbiC without activating the biosensor, we were able to efficiently create and screen a diversified library for UbiC variants with reduced product inhibition. Introduction of the improved UbiC enzyme variant into an experimental production strain for the industrial precursor cis,cis-muconic acid (muconate), enabled a >2-fold yield improvement for glucose to muconate conversion when the new UbiC variant was expressed from a plasmid and a 60% yield increase when the same UbiC variant was genomically integrated into the strain. Overall, this work demonstrates that by coupling a library of enzyme variants to whole-cell catalysis and biosensing, variants with reduced product inhibition can be identified, and that this improved enzyme can result in increased titers of a downstream molecule of interest.

A protocatechuate biosensor for Pseudomonas putida KT2440 via promoter and protein evolution.

Robust fluorescence-based biosensors are emerging as critical tools for high-throughput strain improvement in synthetic biology. Many biosensors are developed in model organisms where sophisticated synthetic biology tools are also well established. However, industrial biochemical production often employs microbes with phenotypes that are advantageous for a target process, and biosensors may fail to directly transition outside the host in which they are developed. In particular, losses in sensitivity and dynamic range of sensing often occur, limiting the application of a biosensor across hosts. Here we demonstrate the optimization of an Escherichia coli-based biosensor in a robust microbial strain for the catabolism of aromatic compounds, Pseudomonas putida KT2440, through a generalizable approach of modulating interactions at the protein-DNA interface in the promoter and the protein-protein dimer interface. The high-throughput biosensor optimization approach demonstrated here is readily applicable towards other allosteric regulators.

Discovery of Bioactive Metabolites in Biofuel Microalgae That Offer Protection against Predatory Bacteria.

Microalgae could become an important resource for addressing increasing global demand for food, energy, and commodities while helping to reduce atmospheric greenhouse gasses. Even though Chlorophytes are generally regarded safe for human consumption, there is still much we do not understand about the metabolic and biochemical potential of microscopic algae. The aim of this study was to evaluate biofuel candidate strains of Chlorella and Scenedesmus for the potential to produce bioactive metabolites when grown under nutrient depletion regimes intended to stimulate production of triacylglycerides. Strain specific combinations of macro- and micro-nutrient restricted growth media did stimulate neutral lipid accumulation by microalgal cultures. However, cultures that were restricted for iron consistently and reliably tested positive for cytotoxicity by in vivo bioassays. The addition of iron back to these cultures resulted in the disappearance of the bioactive components by LC/MS fingerprinting and loss of cytotoxicity by in vivo bioassay. Incomplete NMR characterization of the most abundant cytotoxic fractions suggested that small molecular weight peptides and glycosides could be responsible for Chlorella cytotoxicity. Experiments were conducted to determine if the bioactive metabolites induced by Fe-limitation in Chlorella sp. cultures would elicit protection against Vampirovibrio chlorellavorus, an obligate predator of Chlorella. Introduction of V. chlorellavorus resulted in a 72% decrease in algal biomass in the experimental controls after 7 days. Conversely, only slight losses of algal biomass were measured for the iron limited Chlorella cultures (0-9%). This study demonstrates a causal linkage between iron bioavailability and bioactive metabolite production in strains of Chlorella and Scenedesmus. Further study of this phenomenon could contribute to the development of new strategies to extend algal production cycles in open, outdoor systems while ensuring the protection of biomass from predatory losses.

Tunable Riboregulator Switches for Post-transcriptional Control of Gene Expression.

Until recently, engineering strategies for altering gene expression have focused on transcription control using strong inducible promoters or one of several methods to knock down wasteful genes. Recently, synthetic riboregulators have been developed for translational regulation of gene expression. Here, we report a new modular synthetic riboregulator class that has the potential to finely tune protein expression and independently control the concentration of each enzyme in an engineered metabolic pathway. This development is important because the most straightforward approach to altering the flux through a particular metabolic step is to increase or decrease the concentration of the enzyme. Our design includes a cis-repressor at the 5' end of the mRNA that forms a stem-loop helix, occluding the ribosomal binding sequence and blocking translation. A trans-expressed activating-RNA frees the ribosomal-binding sequence, which turns on translation. The overall architecture of the riboregulators is designed using Watson-Crick base-pairing stability. We describe here a cis-repressor that can completely shut off translation of antibiotic-resistance reporters and a trans-activator that restores translation. We have established that it is possible to use these riboregulators to achieve translational control of gene expression over a wide dynamic range. We have also found that a targeting sequence can be modified to develop riboregulators that can, in principle, independently regulate translation of many genes. In a selection experiment, we demonstrated that by subtly altering the sequence of the trans-activator it is possible to alter the ratio of the repressed and activated states and to achieve intermediate translational control.

Specificity of the ribosomal A site for aminoacyl-tRNAs.

Although some experiments suggest that the ribosome displays specificity for the identity of the esterified amino acid of its aminoacyl-tRNA substrate, a study measuring dissociation rates of several misacylated tRNAs containing the GAC anticodon from the A site showed little indication for such specificity. In this article, an expanded set of misacylated tRNAs and two 2'-deoxynucleotide-substituted mRNAs are used to demonstrate the presence of a lower threshold in k(off) values for aa-tRNA binding to the A site. When a tRNA binds sufficiently well to reach this threshold, additional stabilizing effects due to the esterified amino acid or changes in tRNA sequence are not observed. However, specificity for different amino acid side chains and the tRNA body is observed when tRNA binding is sufficiently weaker than this threshold. We propose that uniform aa-tRNA binding to the A site may be a consequence of a conformational change in the ribosome, induced by the presence of the appropriate combination of contributions from the anticodon, amino acid and tRNA body.

miRNA editing--we should have inosine this coming.

In a recent issue of Science, Nishikura and colleagues provide the first evidence that editing of a microRNA (miRNA) precursor by ADARs can modulate the target specificity of the mature miRNA (Kawahara et al., 2007).

Amino acid specificity in translation.

Recent structural and biochemical experiments indicate that bacterial elongation factor Tu and the ribosomal A-site show specificity for both the amino acid and the tRNA portions of their aminoacyl-tRNA (aa-tRNA) substrates. These data are inconsistent with the traditional view that tRNAs are generic adaptors in translation. We hypothesize that each tRNA sequence has co-evolved with its cognate amino acid, such that all aa-tRNAs are translated uniformly.

Binding of misacylated tRNAs to the ribosomal A site.

To test whether the ribosome displays specificity for the esterified amino acid and the tRNA body of an aminoacyl-tRNA (aa-tRNA), the stabilities of 4 correctly acylated and 12 misacylated tRNAs in the ribosomal A site were determined. By introducing the GAC (valine) anticodon into each tRNA, a constant anticodon.codon interaction was maintained, thus removing concern that different anticodon.codon strengths might affect the binding of the different aa-tRNAs to the A site. Surprisingly, all 16 aa-tRNAs displayed similar dissociation rate constants from the A site. These results suggest that either the ribosome is not specific for different amino acids and tRNA bodies when intact aa-tRNAs are used or the specificity for the amino acid side chain and tRNA body is masked by a conformational change upon aa-tRNA release.

Idiosyncratic tuning of tRNAs to achieve uniform ribosome binding.

The binding of seven tRNA anticodons to their complementary codons on Escherichia coli ribosomes was substantially impaired, as compared with the binding of their natural tRNAs, when they were transplanted into tRNA(2)(Ala). An analysis of chimeras composed of tRNA(2)(Ala) and various amounts of either tRNA(3)(Gly) or tRNA(2)(Arg) indicates that the presence of the parental 32-38 nucleotide pair is sufficient to restore ribosome binding of the transplanted anticodons. Furthermore, mutagenesis of tRNA(2)(Ala) showed that its highly conserved A32-U38 pair serves to weaken ribosome affinity. We propose that this negative binding determinant is used to offset the very tight codon-anticodon interaction of tRNA(2)(Ala). This suggests that each tRNA sequence has coevolved with its anticodon to tune ribosome affinity to a value that is the same for all tRNAs.

Uniform binding of aminoacylated transfer RNAs to the ribosomal A and P sites.

The association and dissociation rate constants of eight different E. coli aminoacyl-tRNAs (aa-tRNAs) for E. coli ribosomes programmed with mRNAs of defined sequences were determined. Identical association and dissociation rate constants were observed for all eight aa-tRNAs in both the ribosomal A and P sites despite substantial differences in tRNA sequence, the type of esterified amino acid, and posttranscriptional modifications. These results indicate that the overall binding of all aa-tRNAs to the ribosome is uniform. However, when either the esterified amino acid or the tRNA modifications were removed, binding was no longer uniform. These results suggest that differences in tRNA sequences and tRNA modifications have evolved to offset differential thermodynamic contributions of the esterified amino acid and the codon-anticodon interaction so that ribosomal binding of all aa-tRNAs remains uniform.

The affinity of elongation factor Tu for an aminoacyl-tRNA is modulated by the esterified amino acid.

When different mutations were introduced into the anticodon loop and at position 73 of YFA2, a derivative of yeast tRNA(Phe), a single tRNA body was misacylated with 13 different amino acids. The affinities of these misacylated tRNAs for Thermus thermophilus elongation factor Tu (EF-Tu).GTP were determined using a ribonuclease protection assay. A range of 2.5 kcal/mol in the binding energies was observed, clearly demonstrating that EF-Tu specifically recognizes the side chain of the esterified amino acid. Furthermore, this specificity can be altered by introducing a mutation in the amino acid binding pocket on the surface of EF-Tu. Also, when discussed in conjunction with the previously determined specificity of EF-Tu for the tRNA body, these experiments further demonstrate that EF-Tu uses thermodynamic compensation to bind cognate aminoacyl-tRNAs similarly.

Effects of magnesium ions on the stabilization of RNA oligomers of defined structures.

Optical melting was used to determine the stabilities of 11 small RNA oligomers of defined secondary structure as a function of magnesium ion concentration. The oligomers included helices composed of Watson-Crick base pairs, GA tandem base pairs, GU tandem base pairs, and loop E motifs (both eubacterial and eukaryotic). The effect of magnesium ion concentration on stability was interpreted in terms of two simple models. The first assumes an uptake of metal ion upon duplex formation. The second assumes nonspecific electrostatic attraction of metal ions to the RNA oligomer. For all oligomers, except the eubacterial loop E, the data could best be interpreted as nonspecific binding of metal ions to the RNAs. The effect of magnesium ions on the stability of the eubacterial loop E was distinct from that seen with the other oligomers in two ways. First, the extent of stabilization by magnesium ions (as measured by either change in melting temperature or free energy) was three times greater than that observed for the other helical oligomers. Second, the presence of magnesium ions produces a doubling of the enthalpy for the melting transition. These results indicate that magnesium ion stabilizes the eubacterial loop E sequence by chelating the RNA specifically. Further, these results on a rather small system shed light on the large enthalpy changes observed upon thermal unfolding of large RNAs like group I introns. It is suggested that parts of those large enthalpy changes observed in the folding of RNAs may be assigned to variations in the hydration states and types of coordinating atoms in some specifically bound magnesium ions and to an increase in the observed cooperativity of the folding transition due to the binding of those magnesium ions coupling the two stems together. Brownian dynamic simulations, carried out to visualize the metal ion binding sites, reveal rather delocalized ionic densities in all oligomers, except for the eubacterial loop E, in which precisely located ion densities were previously calculated.