Transcriptomics and metabolomics analysis of L-phenylalanine overproduction in Escherichia coli.

BACKGROUND: Highly efficient production of L-phenylalanine (L-Phe) in E. coli has been achieved by multiple rounds of random mutagenesis and modification of key genes of the shikimate (SHIK) and L-Phe branch pathways. In this study, we performed transcriptomic (16, 24 and 48 h) and metabolomic analyses (8, 16, 24, 32,40, and 48 h) based on time sequences in an engineered E. coli strain producing L-Phe, aiming to reveal the overall changes of metabolic activities during the fermentation process. RESULTS: The largest biomass increase rate and the highest production rate were seen at 16 h and 24 h of fermentation, respectively reaching 5.9 h-1 and 2.76 g/L/h, while the maximal L-Phe titer of 60 g/L was accumulated after 48 h of fermentation. The DEGs and metabolites involved in the EMP, PP, TCA, SHIIK and L-Phe-branch pathways showed significant differences at different stages of fermentation. Specifically, the significant upregulation of genes encoding rate-limiting enzymes (aroD and yidB) and key genes (aroF, pheA and aspC) pushed more carbon flux toward the L-Phe synthesis. The RIA changes of a number of important metabolites (DAHP, DHS, DHQ, Glu and PPN) enabled the adequate supply of precursors for high-yield L-Phe production. In addition, other genes related to Glc transport and phosphate metabolism increased the absorption of Glc and contributed to rerouting the carbon flux into the L-Phe-branch. CONCLUSIONS: Transcriptomic and metabolomic analyses of an L-Phe overproducing strain of E. coli confirmed that precursor supply was not a major limiting factor in this strain, whereas the rational distribution of metabolic fluxes was achieved by redistributing the carbon flux (for example, the expression intensity of the genes tyrB, aspC, aroL and aroF/G/H or the activity of these enzymes is increased to some extent), which is the optimal strategy for enhancing L-Phe production.

Analyzing the genetic characteristics of a tryptophan-overproducing Escherichia coli.

L-tryptophan (L-trp) production in Escherichia coli has been developed by employing random mutagenesis and selection for a long time, but this approach produces an unclear genetic background. Here, we generated the L-trp overproducer TPD5 by combining an intracellular L-trp biosensor and fluorescence-activated cell sorting (FACS) in E. coli, and succeeded in elucidating the genetic basis for L-trp overproduction. The most significant identified positive mutations affected TnaA (deletion), AroG (S211F), TrpE (A63V), and RpoS (nonsense mutation Q33*). The underlying structure-function relationships of the feedback-resistant AroG (S211F) and TrpE (A63V) mutants were uncovered based on protein structure modeling and molecular dynamics simulations, respectively. According to transcriptomic analysis, the global regulator RpoS not only has a great influence on cell growth and morphology, but also on carbon utilization and the direction of carbon flow. Finally, by balancing the concentrations of the L-trp precursors' serine and glutamine based on the above analysis, we further increased the titer of L-trp to 3.18 g/L with a yield of 0.18 g/g. The analysis of the genetic characteristics of an L-trp overproducing E. coli provides valuable information on L-trp synthesis and elucidates the phenotype and complex cellular properties in a high-yielding strain, which opens the possibility to transfer beneficial mutations and reconstruct an overproducer with a clean genetic background.

Metabolic engineering of Escherichia coli for production of chemicals derived from the shikimate pathway.

The shikimate pathway is indispensable for the biosynthesis of natural products with aromatic moieties. These products have wide current and potential applications in food, cosmetics and medicine, and consequently have great commercial value. However, compounds extracted from various plants or synthesized from petrochemicals no longer satisfy the requirements of contemporary industries. As a result, an increasing number of studies has focused on this pathway to enable the biotechnological manufacture of natural products, especially in E. coli. Furthermore, the development of synthetic biology, systems metabolic engineering and high flux screening techniques has also contributed to improving the biosynthesis of high-value compounds based on the shikimate pathway. Here, we review approaches based on a combination of traditional and new metabolic engineering strategies to increase the metabolic flux of the shikimate pathway. In addition, applications of this optimized pathway to produce aromatic amino acids and a range of natural products is also elaborated. Finally, this review sums up the opportunities and challenges facing this field.

Pinpointing the L-phenylalanine binding sites of TyrR using biosensors and computer-aided simulation.

OBJECTIVES: To determine the binding sites for L-phenylalanine in TyrR protein via a rational mutation analysis combining biosensors and computer-aided simulation. RESULTS: TyrR protein of Escherichia coli is the chief transcriptional regulator of several genes essential for the biosynthesis and transport of aromatic amino acids. The identification of ligand-binding sites is often the starting point for protein function annotation and structure-based protein design. Here we combined computer-aided prediction methods and biosensors to identify the ligand-binding sites for L-Phe in TyrR protein. CONCLUSIONS: Residues at positions 160, 173 and 184 of TyrR protein are important for transcriptional activation of target genes tyrP induced by L-Phe, which indicates that they are the bona fide L-Phe binding sites of TyrR protein.

Genetic engineering of Escherichia coli to improve L-phenylalanine production.

BACKGROUND: L-phenylalanine (L-Phe) is an essential amino acid for mammals and applications expand into human health and nutritional products. In this study, a system level engineering was conducted to enhance L-Phe biosynthesis in Escherichia coli. RESULTS: We inactivated the PTS system and recruited glucose uptake via combinatorial modulation of galP and glk to increase PEP supply in the Xllp01 strain. In addition, the HTH domain of the transcription factor TyrR was engineered to decrease the repression on the transcriptional levels of L-Phe pathway enzymes. Finally, proteomics analysis demonstrated the third step of the SHIK pathway (catalyzed via AroD) as the rate-limiting step for L-Phe production. After optimization of the aroD promoter strength, the titer of L-Phe increased by 13.3%. Analysis of the transcriptional level of genes involved in the central metabolic pathways and L-Phe biosynthesis via RT-PCR showed that the recombinant L-Phe producer exhibited a great capability in the glucose utilization and precursor (PEP and E4P) generation. Via systems level engineering, the L-Phe titer of Xllp21 strain reached 72.9 g/L in a 5 L fermenter under the non-optimized fermentation conditions, which was 1.62-times that of the original strain Xllp01. CONCLUSION: The metabolic engineering strategy reported here can be broadly employed for developing genetically defined organisms for the efficient production of other aromatic amino acids and derived compounds.

Rational design and analysis of an Escherichia coli strain for high-efficiency tryptophan production.

L-tryptophan (L-trp) is a precursor of various bioactive components and has great pharmaceutical interest. However, due to the requirement of several precursors and complex regulation of the pathways involved, the development of an efficient L-trp production strain is challenging. In this study, Escherichia coli (E. coli) strain KW001 was designed to overexpress the L-trp operator sequences (trpEDCBA) and 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (aroG fbr ). To further improve the production of L-trp, pyruvate kinase (pykF) and the phosphotransferase system HPr (ptsH) were deleted after inactivation of repression (trpR) and attenuation (attenuator) to produce strain KW006. To overcome the relatively slow growth and to increase the transport rate of glucose, strain KW018 was generated by combinatorial regulation of glucokinase (galP) and galactose permease (glk) expression. To reduce the production of acetic acid, strain KW023 was created by repressive regulation of phosphate acetyltransferase (pta) expression. In conclusion, strain KW023 efficiently produced 39.7 g/L of L-trp with a conversion rate of 16.7% and a productivity of 1.6 g/L/h in a 5 L fed-batch fermentation system.

A hybrid RNA-protein biosensor for high-throughput screening of adenosylcobalamin biosynthesis.

Genetically encoded circuits have been successfully utilized to assess and characterize target variants with desirable traits from large mutant libraries. Adenosylcobalamin is an essential coenzyme that is required in many intracellular physiological reactions and is widely used in the pharmaceutical and food industries. High-throughput screening techniques capable of detecting adenosylcobalamin productivity and selecting superior adenosylcobalamin biosynthesis strains are critical for the creation of an effective microbial cell factory for the production of adenosylcobalamin at an industrial level. In this study, we developed an RNA-protein hybrid biosensor whose input part was an endogenous RNA riboswitch to specifically respond to adenosylcobalamin, the inverter part was an orthogonal transcriptional repressor to obtain signal inversion, and the output part was a fluorescent protein to be easily detected. The hybrid biosensor could specifically and positively correlate adenosylcobalamin concentrations to green fluorescent protein expression levels in vivo. This study also improved the operating concentration and dynamic range of the hybrid biosensor by systematic optimization. An individual cell harboring the hybrid biosensor presented over 20-fold higher fluorescence intensity than the negative control. Then, using such a biosensor combined with fluorescence-activated cell sorting, we established a high-throughput screening platform for screening adenosylcobalamin overproducers. This study demonstrates that this platform has significant potential to quickly isolate high-productive strains to meet industrial demand and that the framework is acceptable for various metabolites.

Melatonin biosynthesis pathways in nature and its production in engineered microorganisms.

Melatonin is a biogenic amine that can be found in plants, animals and microorganism. The metabolic pathway of melatonin is different in various organisms, and biosynthetic endogenous melatonin acts as a molecular signal and antioxidant protection against external stress. Microbial synthesis pathways of melatonin are similar to those of animals but different from those of plants. At present, the method of using microorganism fermentation to produce melatonin is gradually prevailing, and exploring the biosynthetic pathway of melatonin to modify microorganism is becoming the mainstream, which has more advantages than traditional chemical synthesis. Here, we review recent advances in the synthesis, optimization of melatonin pathway. l-tryptophan is one of the two crucial precursors for the synthesis of melatonin, which can be produced through a four-step reaction. Enzymes involved in melatonin synthesis have low specificity and catalytic efficiency. Site-directed mutation, directed evolution or promotion of cofactor synthesis can enhance enzyme activity and increase the metabolic flow to promote microbial melatonin production. On the whole, the status and bottleneck of melatonin biosynthesis can be improved to a higher level, providing an effective reference for future microbial modification.

Establishment of a Biosensor-based High-Throughput Screening Platform for Tryptophan Overproduction.

With the flexibility to fold into complex structures, RNA is well-suited to act as a cellular sensor to recognize environmental fluctuations and respond to changes by regulating the corresponding genes. In this study, we established a high-throughput screening platform to screen tryptophan high-producing strains from a large repertoire of candidate strains. This platform consists of a tryptophan-specific aptamer-based biosensor and fluorescence-activated droplet sorting technology. One mutant strain, with a 165.9% increase in Trp titer compared with the parental strain, was successfully screened from a random mutagenesis library. Sequencing results revealed that a total of 10 single-nucleotide polymorphisms were discovered in the genome of the mutant strain, among which CRP(T29K) was proven to significantly increase Trp production through improving the strain's tolerance of the harsh environment during the stationary phase of the fermentation process. Our results indicate that this strategy has great potential for improving the production of other amino acids in Escherichia coli.

Biosensor-based monitoring of the central metabolic pathway metabolites.

In vivo biosensors have a wide range of applications, from the detection of metabolites to the regulation of metabolic networks, providing a versatile tool for cell biology and metabolic engineering. However, compared with the vast array of small molecules present in nature, the existing range of biosensors is far from sufficient. Here we describe the use of human hypoxanthine guanine phosphoribosyltransferase (HGPRT) as a ligand binding domain (LBD) protein, that acts by ligand-dependent stabilization, to build a biosensor for detection of the pentose phosphate pathway metabolite 5-phospho-α-D-ribose 1-diphosphate (PRPP). Using this protein as a template, we computationally redesigned a new pocket de novo according to the pose of the ligand, creating a binding mode exclusive to recognize another pentose phosphate metabolite, D-erythrose 4-phosphate (E4P), and glycerate-3-phosphate (3PG), from the glycolysis pathway. Furthermore, E4P biosensor was developed by fluorescence-activated cell sorting (FACS) and application of it enabled successful screening for the highest phenylalanine-producing strain reported to date. This work provides a strategy for computational design and development of biosensors for a broad range of molecules.

Engineering Escherichia coli to improve tryptophan production via genetic manipulation of precursor and cofactor pathways.

Optimizing the supply of biosynthetic precursors and cofactors is usually an effective metabolic strategy to improve the production of target compounds. Here, the combination of optimizing precursor synthesis and balancing cofactor metabolism was adopted to improve tryptophan production in Escherichia coli. First, glutamine synthesis was improved by expressing heterologous glutamine synthetase from Bacillus subtilis and Bacillus megaterium in the engineered Escherichia coli strain KW001, resulting in the best candidate strain TS-1. Then icd and gdhA were overexpressed in TS-1, which led to the accumulation of 1.060 g/L tryptophan. Subsequently, one more copy of prs was introduced on the chromosome to increase the flux of 5-phospho-α-d-ribose 1-diphosphate followed by the expression of mutated serA and thrA to increase the precursor supply of serine, resulting in the accumulation of 1.380 g/L tryptophan. Finally, to maintain cofactor balance, sthA and pntAB, encoding transhydrogenase, were overexpressed. With sufficient amounts of precursors and balanced cofactors, the engineered strain could produce 1.710 g/L tryptophan after 48 h of shake-flask fermentation, which was 2.76-times higher than the titer of the parent strain. Taken together, our results demonstrate that the combination of optimizing precursor supply and regulating cofactor metabolism is an effective approach for high-level production of tryptophan. Similar strategies could be applied to the production of other amino acids or related derivatives.

Biosensor-Based Evolution and Elucidation of a Biosynthetic Pathway in Escherichia coli.

The successful evolution of metabolite-producing microbes requires a high-throughput screening method to obtain the desired properties within a short time. In this study, we developed a transcription-factor-driven device that combines a metabolite-responsive element and a selection module. This device was able to specifically sense intracellular l-phenylalanine (l-Phe) and convert this signal into an observable phenotype. Applying this device, we successfully improved l-Phe production by screening hyperproducing phenotypes from a ribonucleotide binding site library and a random mutagenesis library. In addition, several site mutations introduced by random mutagenesis were identified and elucidated to facilitate the improvement of l-Phe production. Our results present a paradigm for screening of compounds that are not easily observable to raise the yield of targeted compounds from a large candidate library. This approach may guide further applications in rewiring metabolic circuits and facilitate the directed evolution of recombinant strains.

Improving the Production of L-Phenylalanine by Identifying Key Enzymes Through Multi-Enzyme Reaction System in Vitro.

L-Phenylalanine (L-Phe) is an important amino acid used in both food and medicinal applications. We developed an in vitro system that allowed a direct, quantitative investigation of phenylalanine biosynthesis in E. coli. Here, the absolute concentrations of six enzymes (AroK, AroL, AroA, AroC, PheA and TyrB) involved in the shikimate (SHIK) pathway were determined by a quantitative proteomics approach and in vitro enzyme titration experiments. The reconstitution of an in vitro reaction system for these six enzymes was established and their effects on the phenylalanine production were tested. The results showed that the yield of phenylalanine increased 3.0 and 2.1 times when the concentrations of shikimate kinase (AroL) and 5-enolpyruvoyl shikimate 3-phosphate (EPSP) synthase (AroA) were increased 2.5 times. Consistent results were obtained from in vivo via the overexpression of AroA in a phenylalanine-producing strain, and the titer of phenylalanine reached 62.47 g/l after 48 h cultivation in a 5-liter jar fermentor. Our quantitative findings provide a practical method to detect the potential bottleneck in a specific metabolic pathway to determine which gene products should be targeted to improve the yield of the desired product.

Partially overlapping primer-based PCR for genome walking.

Current genome walking methods are cumbersome to perform and can result in non-specific products. Here, we demonstrate the use of partially overlapping primer-based PCR (POP-PCR), a direct genome walking technique for the isolation of unknown flanking regions. This method exploits the partially overlapping characteristic at the 3' ends of a set of POP primers (walking primers), which guarantees that the POP primer only anneals to the POP site of the preceding PCR product at relatively low temperatures. POP primer adaptation priming at the genomic DNA/POP site occurs only once due to one low-/reduced-stringency cycle in each nested PCR, resulting in the synthesis of a pool of single-stranded DNA molecules. Of this pool, the target single-stranded DNA is replicated to the double-stranded form bound by the specific primer and the POP primer in the subsequent high-stringency cycle due to the presence of the specific primer-binding site. The non-target single stranded DNA does not become double stranded due to the absence of a binding site for any of the primers. Therefore, the POP-PCR enriches target DNA while suppressing non-target products. We successfully used POP-PCR to retrieve flanking regions bordering the gadA locus in Lactobacillus brevis NCL912, malQ in Pichia pastoris GS115, the human aldolase A gene, and hyg in rice.