Artificial Biosynthetic Pathway for Efficient Synthesis of Vanillin, a Feruloyl-CoA-Derived Natural Product from Eugenol.

Eugenol, the main component of essential oil from the Syzygium aromaticum clove tree, has great potential as an alternative bioresource feedstock for biosynthesis purposes. Although eugenol degradation to ferulic acid was investigated, an efficient method for directly converting eugenol to targeted natural products has not been established. Herein we identified the inherent inhibitions by simply combining the previously reported ferulic acid biosynthetic pathway and vanillin biosynthetic pathway. To overcome this, we developed a novel biosynthetic pathway for converting eugenol into vanillin, by introducing cinnamoyl-CoA reductase (CCR), which catalyzes conversion of coniferyl aldehyde to feruloyl-CoA. This approach bypasses the need for two catalysts, namely coniferyl aldehyde dehydrogenase and feruloyl-CoA synthetase, thereby eliminating inhibition while simplifying the pathway. To further improve efficiency, we enhanced CCR catalytic efficiency via directed evolution and leveraged an artificialvanillin biosensor for high-throughput screening. Switching the cofactor preference of CCR from NADP+ to NAD+ significantly improved pathway efficiency. This newly designed pathway provides an alternative strategy for efficiently biosynthesizing feruloyl-CoA-derived natural products using eugenol.

Engineering and application of LacI mutants with stringent expressions.

Optimal transcriptional regulatory circuits are expected to exhibit stringent control, maintaining silence in the absence of inducers while exhibiting a broad induction dynamic range upon the addition of effectors. In the Plac /LacI pair, the promoter of the lac operon in Escherichia coli is characterized by its leakiness, attributed to the moderate affinity of LacI for its operator target. In response to this limitation, the LacI regulatory protein underwent engineering to enhance its regulatory properties. The M7 mutant, carrying I79T and N246S mutations, resulted in the lac promoter displaying approximately 95% less leaky expression and a broader induction dynamic range compared to the wild-type LacI. An in-depth analysis of each mutation revealed distinct regulatory profiles. In contrast to the wild-type LacI, the M7 mutant exhibited a tighter binding to the operator sequence, as evidenced by surface plasmon resonance studies. Leveraging the capabilities of the M7 mutant, a high-value sugar biosensor was constructed. This biosensor facilitated the selection of mutant galactosidases with approximately a seven-fold improvement in specific activity for transgalactosylation. Consequently, this advancement enabled enhanced biosynthesis of galacto-oligosaccharides (GOS).

Designing glucose utilization "highway" for recombinant biosynthesis.

cAMP receptor protein (CRP) is known as a global regulatory factor mainly mediating carbon source catabolism. Herein, we successfully engineered CRP to develop microbial chassis cells with improved recombinant biosynthetic capability in minimal medium with glucose as single carbon source. The obtained best-performing cAMP-independent CRPmu9 mutant conferred both faster cell growth and a 133-fold improvement in expression level of lac promoter in presence of 2% glucose, compared with strain under regulation of CRPwild-type. Promoters free from "glucose repression" are advantageous for recombinant expression, as glucose is a frequently used inexpensive carbon source in high-cell-density fermentations. Transcriptome analysis demonstrated that the CRP mutant globally rewired cell metabolism, displaying elevated tricarboxylic acid cycle activity; reduced acetate formation; increased nucleotide biosynthesis; and improved ATP synthesis, tolerance, and stress-resistance activity. Metabolites analysis confirmed the enhancement of glucose utilization with the upregulation of glycolysis and glyoxylate-tricarboxylic acid cycle. As expected, an elevated biosynthetic capability was demonstrated with vanillin, naringenin and caffeic acid biosynthesis in strains regulated by CRPmu9. This study has expanded the significance of CRP optimization into glucose utilization and recombinant biosynthesis, beyond the conventionally designated carbon source utilization other than glucose. The Escherichiacoli cell regulated by CRPmu9 can be potentially used as a beneficial chassis for recombinant biosynthesis.

Engineering an SspB-mediated degron for novel controllable protein degradation.

Elegant controllable protein degradation tools have great applications in metabolic engineering and synthetic biology designs. SspB-mediated ClpXP proteolysis system is well characterized, and SspB acts as an adaptor tethering ssrA-tagged substrates to the ClpXP protease. This degron was applied in metabolism optimization, but the efficiency was barely satisfactory. Limited high-quality tools are available for controllable protein degradation. By coupling structure-guided modeling and directed evolution, we establish state-of-the-art high-throughput screening strategies for engineering both degradation efficiency and SspB-ssrA binding specificity of this degron. The reliability of our approach is confirmed by functional validation of both SspB and ssrA mutants using fluorescence assays and metabolic engineering of itaconic acid or ferulic acid biosynthesis. Isothermal titration calorimetry analysis and molecular modeling revealed that an appropriate instead of excessively strong interaction between SspB and ssrA benefited degradation efficiency. Mutated SspB-ssrA pairs with 7-22-fold higher binding KD than the wild-type pair led to higher degradation efficiency, revealing the advantage of directed evolution over rational design in degradation efficiency optimization. Furthermore, an artificial SspB-ssrA pair exhibiting low crosstalk of interactions with the wild-type SspB-ssrA pair was also developed. Efforts in this study have demonstrated the plasticity of SspB-ssrA binding pocket for designing high-quality controllable protein degradation tools. The obtained mutated degrons enriched the tool box of metabolic engineering designs.

Whole-Cell Biosensors Aid Exploration of Vanillin Transmembrane Transport.

Transcriptional regulatory protein (TRP)-based whole-cell biosensors are widely used nowadays. Here, they were demonstrated to have great potential application in screening cell efflux and influx pumps for small molecules. First, a vanillin whole-cell biosensor was developed by altering the specificity of a TRP, VanR, and strains with improved vanillin productions that were selected from a random genome mutagenesis library by using this biosensor as a high-throughput screening tool. A high intracellular vanillin concentration was found to accumulate due to the inactivation of the AcrA protein, indicating the involvement of this protein in vanillin efflux. Then, the application of this biosensor was extended to explore efflux and influx pumps, combined with directed genome evolution. Elevated intracellular vanillin levels resulting from efflux pump inactivation or influx pump overexpression could be rapidly detected by the whole-cell biosensor, markedly facilitating the identification of genome targets related to small-molecule transmembrane transport, which is of great importance in metabolic engineering.

De Novo Biosynthesis of Chlorogenic Acid Using an Artificial Microbial Community.

Engineering an artificial microbial community for natural product production is a promising strategy. As mono- and dual-culture systems only gave non-detectable or minimal chlorogenic acid (CGA) biosynthesis, here, a polyculture of three recombinant Escherichia coli strains, acting as biosynthetic modules of caffeic acid (CA), quinic acid (QA), and CGA, was designed and used for de novo CGA biosynthesis. An influx transporter of 3-dehydroshikimic acid (DHS)/shikimic acid (SA), ShiA, was introduced into the QA module-a DHS auxotroph. The QA module proportion in the polyculture and CGA production were found to be dependent on ShiA expression, providing an alternative approach for controlling microbial community composition. The polyculture strategy avoids metabolic flux competition in the biosynthesis of two CGA precursors, CA and QA, and allows production improvement by balancing module proportions. The performance of this polyculture approach was superior to that of previously reported approaches of de novo CGA production.

Metabolic Engineering for Improved Curcumin Biosynthesis in Escherichia coli.

The biosynthetic efficiency of curcumin, a highly bioactive compound from the plant Curcuma longa, needs to be improved. In this study, we performed host cell and biosynthetic pathway engineering to improve curcumin biosynthesis. Using in vivo-directed evolution, the expression level of curcuminoid synthase (CUS), the rate-limiting enzyme in the curcumin biosynthetic pathway, was significantly improved. Furthermore, as curcumin is a highly hydrophobic compound, two cell membrane engineering strategies were applied to optimize the biosynthetic efficiency. Curcumin storage was increased by overexpression of monoglucosyldiacylglycerol synthase from Acholeplasma laidlawii, which optimized the cell membrane morphology. Furthermore, unsaturated fatty acid supplementation was used to enhance membrane fluidity, which greatly ameliorated the damaging effect of curcumin on the cell membrane. These two strategies enhanced curcumin biosynthesis and demonstrated an additive effect.

Developing a highly efficient hydroxytyrosol whole-cell catalyst by de-bottlenecking rate-limiting steps.

Hydroxytyrosol is an antioxidant free radical scavenger that is biosynthesized from tyrosine. In metabolic engineering efforts, the use of the mouse tyrosine hydroxylase limits its production. Here, we design an efficient whole-cell catalyst of hydroxytyrosol in Escherichia coli by de-bottlenecking two rate-limiting enzymatic steps. First, we replace the mouse tyrosine hydroxylase by an engineered two-component flavin-dependent monooxygenase HpaBC of E. coli through structure-guided modeling and directed evolution. Next, we elucidate the structure of the Corynebacterium glutamicum VanR regulatory protein complexed with its inducer vanillic acid. By switching its induction specificity from vanillic acid to hydroxytyrosol, VanR is engineered into a hydroxytyrosol biosensor. Then, with this biosensor, we use in vivo-directed evolution to optimize the activity of tyramine oxidase (TYO), the second rate-limiting enzyme in hydroxytyrosol biosynthesis. The final strain reaches a 95% conversion rate of tyrosine. This study demonstrates the effectiveness of sequentially de-bottlenecking rate-limiting steps for whole-cell catalyst development.

CRISPRi/dCpf1-mediated dynamic metabolic switch to enhance butenoic acid production in Escherichia coli.

Butenoic acid is a short-chain unsaturated fatty acid and important precursor for pharmaceutical and other applications. Heterologous thioesterases are able to convert a fatty acid biosynthesis intermediate in Escherichia coli to butenoic acid. In order to acquire high titer and yield of the product, dynamically switching the metabolic flux from fatty acid biosynthesis pathway to butenoic acid is critical after achieving enough cell mass of the host. A previous developed switch for butenoic acid fermentation is based on triclosan molecule as the FabI inhibitor in the fatty acid biosynthesis cycle. However, triclosan is toxic to human, which may limit its pharmaceutical application. Alternatively, we here purposed a nontoxic switch of carbon flux by harnessing recently developed CRISPR interference (CRISPRi) approach. In our work, we constructed a CRISPRi/dCpf1-mediated dynamic metabolic switch to separate the host growth and production phase via switching the expression of the fabI gene in fatty acid biosynthesis pathway. After optimizing the programmable targets, the CRISPRi-based switch boosted the titer of butenoic acid by 6-fold (1.41 g/L) in fed-batch fermentation. Our work supported that the CRISPRi/dCpf1 switch could replace triclosan-based switch as a nontoxic switch for butenoic acid production, and outcompeted the later switch in the biomass accumulation of the host cell. Moreover, the CRISPRi/dCpf1 system was integrated into the chromosome of the host to improve its genetic stability for long-term fermentation and other applications.Key Points• A programmable metabolic switch was developed to replace the toxic chemical switch to separate the growth phase and production phase of the butenoic acid.• The programmable CRISPRi/dCpf1 switch was efficiently and stably integrated into the host genome to increase their genetic stability during fermentation.• The optimized metabolic switch simultaneously increased the host biomass and butenoic acid titer, and solved the paradox of the competition between growth and production.

Development of a highly efficient and specific L-theanine synthase.

γ-Glutamylcysteine synthetase (γ-GCS) from Escherichia coli, which catalyzes the formation of L-glutamylcysteine from L-glutamic acid and L-cysteine, was engineered into an L-theanine synthase using L-glutamic acid and ethylamine as substrates. A high-throughput screening method using a 96-well plate was developed to evaluate the L-theanine synthesis reaction. Both site-saturation mutagenesis and random mutagenesis were applied. After three rounds of directed evolution, 13B6, the best-performing mutant enzyme, exhibited 14.6- and 17.0-fold improvements in L-theanine production and catalytic efficiency for ethylamine, respectively, compared with the wild-type enzyme. In addition, the specific activity of 13B6 for the original substrate, L-cysteine, decreased to approximately 14.6% of that of the wild-type enzyme. Thus, the γ-GCS enzyme was successfully switched to a specific L-theanine synthase by directed evolution. Furthermore, an ATP-regeneration system was introduced based on polyphosphate kinases catalyzing the transfer of phosphates from polyphosphate to ADP, thus lowering the level of ATP consumption and the cost of L-theanine synthesis. The final L-theanine production by mutant 13B6 reached 30.4 ± 0.3 g/L in 2 h, with a conversion rate of 87.1%, which has great potential for industrial applications.

Dynamic control of toxic natural product biosynthesis by an artificial regulatory circuit.

To mimic the delicately regulated metabolism in nature for improved efficiency, artificial and customized regulatory components for dynamically controlling metabolic networks in multiple layers are essential in laboratory engineering. For this purpose, a novel regulatory component for controlling vanillin biosynthetic pathway was developed through directed evolution, which was responsive to both the product vanillin and substrate ferulic acid, with different capacities. This regulatory component facilitated pathway expression via dynamic control of the intracellular substrate and product concentrations. As vanillin is an antimicrobial compound, low pathway expression and vanillin formation levels enabled better cell growth at an early stage, and the product feedback-activated pathway expression at later stages significantly improved biosynthesis efficiency. This novel multiple-layer dynamic control was demonstrated effective in managing the trade-off between cell growth and production, leading to improved cell growth and vanillin production compared to the conventional or quorum-sensing promoter-controlled pathway. The multiple-layer dynamic control enabled by designed regulatory components responsive to multiple signals shows potential for wide applications in addition to the dynamic controls based on biosynthetic intermediate sensing and quorum sensing reported to date.

Promiscuous enzymatic activity-aided multiple-pathway network design for metabolic flux rearrangement in hydroxytyrosol biosynthesis.

Genetic diversity is a result of evolution, enabling multiple ways for one particular physiological activity. Here, we introduce this strategy into bioengineering. We design two hydroxytyrosol biosynthetic pathways using tyrosine as substrate. We show that the synthetic capacity is significantly improved when two pathways work simultaneously comparing to each individual pathway. Next, we engineer flavin-dependent monooxygenase HpaBC for tyrosol hydroxylase, tyramine hydroxylase, and promiscuous hydroxylase active on both tyrosol and tyramine using directed divergent evolution strategy. Then, the mutant HpaBCs are employed to catalyze two missing steps in the hydroxytyrosol biosynthetic pathways designed above. Our results demonstrate that the promiscuous tyrosol/tyramine hydroxylase can minimize the cell metabolic burden induced by protein overexpression and allow the biosynthetic carbon flow to be divided between two pathways. Thus, the efficiency of the hydroxytyrosol biosynthesis is significantly improved by rearranging the metabolic flux among multiple pathways.

Engineering the effector specificity of regulatory proteins for the in vitro detection of biomarkers and pesticide residues.

Transcriptional regulatory proteins (TRPs)-based whole-cell biosensors are promising owing to their specificity and sensitivity, but their applications are currently limited. Herein, TRPs were adapted for the extracellular detection of a disease biomarker, uric acid, and a typical pesticide residue, carbaryl. A mutant regulatory protein that specifically recognizes carbaryl as its non-natural effector and activates transcription upon carbaryl binding was developed by engineering the regulatory protein TtgR from Pseudomonas putida. The TtgR mutant responsive to carbaryl and a regulatory protein responsive to uric acid were used for in vitro detection, based on their allosteric binding of operator DNA and inducer molecules. Based on the quantitative polymerase chain reactions (qPCRs) output, the minimum detectable concentration was between 1 nM-1 µM and 1-10 nM for uric acid and carbaryl, respectively. Our results demonstrated that engineering the effector specificity of regulatory proteins is a potential technique for generating molecular recognition elements for not only in vivo but also in vitro applications.

Towards the construction of high-quality mutagenesis libraries.

OBJECTIVES: To improve the quality of mutagenesis libraries in directed evolution strategy. RESULTS: In the process of library transformation, transformants which have been shown to take up more than one plasmid might constitute more than 20% of the constructed library, thereby extensively impairing the quality of the library. We propose a practical transformation method to prevent the occurrence of multiple-plasmid transformants while maintaining high transformation efficiency. A visual library model containing plasmids expressing different fluorescent proteins was used. Multiple-plasmid transformants can be reduced through optimizing plasmid DNA amount used for transformation based on the positive correlation between the occurrence frequency of multiple-plasmid transformants and the logarithmic ratio of plasmid molecules to competent cells. CONCLUSIONS: This method provides a simple solution for a seemingly common but often neglected problem, and should be valuable for improving the quality of mutagenesis libraries to enhance the efficiency of directed evolution strategies.

Biosensor-aided high-throughput screening of hyper-producing cells for malonyl-CoA-derived products.

BACKGROUND: Malonyl-coenzyme A (CoA) is an important biosynthetic precursor in vivo. Although Escherichia coli is a useful organism for biosynthetic applications, its malonyl-CoA level is too low. RESULTS: To identify strains with the best potential for enhanced malonyl-CoA production, we developed a whole-cell biosensor for rapidly reporting intracellular malonyl-CoA concentrations. The biosensor was successfully applied as a high-throughput screening tool for identifying targets at a genome-wide scale that could be critical for improving the malonyl-CoA biosynthesis in vivo. The mutant strains selected synthesized significantly higher titers of the type III polyketide triacetic acid lactone (TAL), phloroglucinol, and free fatty acids compared to the wild-type strain, using malonyl-CoA as a precursor. CONCLUSION: These results validated this novel whole-cell biosensor as a rapid and sensitive malonyl-CoA high-throughput screening tool. Further analysis of the mutant strains showed that the iron ion concentration is closely related to the intracellular malonyl-CoA biosynthesis.

Monitoring in vivo metabolic flux with a designed whole-cell metabolite biosensor of shikimic acid.

Knowledge of intracellular metabolite levels is important for the understanding of metabolic flux distributions. Whole-cell biosensors of key metabolites are ideal for the monitoring of carbon flow in important metabolic pathways, thus guiding metabolic engineering for microbial improvement. However, lack of biosensors for metabolites of interests has limited their applications. In this study, a genetically encoded whole-cell biosensor specifically responding to shikimic acid has been developed by screening a site-saturation mutagenesis library of the binding pocket of a uric acid-responsive regulatory protein. This biosensor has been successfully applied in analyzing and engineering metabolic flux in the shikimic acid pathway, through genome-wide screening of gene targets critical for the pathway flux, and by improving the specific activity of pathway key enzyme, AroG. This work demonstrates the feasibility of monitoring metabolic flux with the aid of whole-cell biosensors designed for key metabolites.

Design and application of a lactulose biosensor.

In this study the repressor of Escherichia coli lac operon, LacI, has been engineered for altered effector specificity. A LacI saturation mutagenesis library was subjected to Fluorescence Activated Cell Sorting (FACS) dual screening. Mutant LacI-L5 was selected and it is specifically induced by lactulose but not by other disaccharides tested (lactose, epilactose, maltose, sucrose, cellobiose and melibiose). LacI-L5 has been successfully used to construct a whole-cell lactulose biosensor which was then applied in directed evolution of cellobiose 2-epimerase (C2E) for elevated lactulose production. The mutant C2E enzyme with ~32-fold enhanced expression level was selected, demonstrating the high efficiency of the lactulose biosensor. LacI-L5 can also be used as a novel regulatory tool. This work explores the potential of engineering LacI for customized molecular biosensors which can be applied in practice.

Improving key enzyme activity in phenylpropanoid pathway with a designed biosensor.

Overexpressing key enzymes of biosynthetic pathways for overproduction of value-added products usually imposes metabolic burdens on cells, which can be circumvented by improving the key enzyme activities. p-Coumarate: CoA ligase (4CL) is a critical enzyme in the phenylpropanoid pathway that synthesizes various natural products. To screen for 4CL with improved activity, a biosensor of resveratrol whose biosynthetic pathway involves 4CL was designed by engineering the TtgR regulatory protein. The biosensor exhibited good specificity and robustness, allowing rapid and sensitive selection of resveratrol hyper-producers. A 4CL variant with improved activity was selected from a 4CL mutagenesis library constructed in the resveratrol biosynthetic pathway in Escherichia coli. This mutant led to increased production of not only resveratrol but also the flavonoid naringenin, when introduced in their corresponding biosynthetic pathways. These findings demonstrate the feasibility of improving key enzyme activities in important biosynthetic pathways with the aid of designed biosensors of pathway products.

Putative Nonribosomal Peptide Synthetase and Cytochrome P450 Genes Responsible for Tentoxin Biosynthesis in Alternaria alternata ZJ33.

Tentoxin, a cyclic tetrapeptide produced by several Alternaria species, inhibits the F1-ATPase activity of chloroplasts, resulting in chlorosis in sensitive plants. In this study, we report two clustered genes, encoding a putative non-ribosome peptide synthetase (NRPS) TES and a cytochrome P450 protein TES1, that are required for tentoxin biosynthesis in Alternaria alternata strain ZJ33, which was isolated from blighted leaves of Eupatorium adenophorum. Using a pair of primers designed according to the consensus sequences of the adenylation domain of NRPSs, two fragments containing putative adenylation domains were amplified from A. alternata ZJ33, and subsequent PCR analyses demonstrated that these fragments belonged to the same NRPS coding sequence. With no introns, TES consists of a single 15,486 base pair open reading frame encoding a predicted 5161 amino acid protein. Meanwhile, the TES1 gene is predicted to contain five introns and encode a 506 amino acid protein. The TES protein is predicted to be comprised of four peptide synthase modules with two additional N-methylation domains, and the number and arrangement of the modules in TES were consistent with the number and arrangement of the amino acid residues of tentoxin, respectively. Notably, both TES and TES1 null mutants generated via homologous recombination failed to produce tentoxin. This study provides the first evidence concerning the biosynthesis of tentoxin in A. alternata.

Engineering a Carbohydrate-processing Transglycosidase into Glycosyltransferase for Natural Product Glycodiversification.

Glycodiversification broadens the scope of natural product-derived drug discovery. The acceptor substrate promiscuity of glucosyltransferase-D (GTF-D), a carbohydrate-processing enzyme from Streptococcus mutans, was expanded by protein engineering. Mutants in a site-saturation mutagenesis library were screened on the fluorescent substrate 4-methylumbelliferone to identify derivatives with improved transglycosylation efficiency. In comparison to the wild-type GTF-D enzyme, mutant M4 exhibited increased transglycosylation capabilities on flavonoid substrates including catechin, genistein, daidzein and silybin, using the glucosyl donor sucrose. This study demonstrated the feasibility of developing natural product glycosyltransferases by engineering transglycosidases that use donor substrates cheaper than NDP-sugars, and gave rise to a series of α-glucosylated natural products that are novel to the natural product reservoir. The solubility of the α-glucoside of genistein and the anti-oxidant capability of the α-glucoside of catechin were also studied.