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.

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.

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.

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.

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.

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.

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.

Improving the thermoactivity and thermostability of pectate lyase from Bacillus pumilus for ramie degumming.

Thermostable alkaline pectate lyases can be potentially used for enzymatically degumming ramie in an environmentally sustainable manner and as an alternative to the currently used chemical-based ramie degumming processes. To assess its potential applications, pectate lyase from Bacillus pumilus (ATCC 7061) was cloned and expressed in Escherichia coli. Evolutionary strategies were applied to generate efficient ramie degumming enzymes. Obtained from site-saturation mutagenesis and random mutagenesis, the best performing mutant enzyme M3 exhibited a 3.4-fold higher specific activity on substrate polygalacturonic acid, compared with the wild-type enzyme. Furthermore, the half-life of inactivation at 50 °C for M3 mutant extended to over 13 h. In contrast, the wild-type enzyme was completely inactivated in less than 10 min under the same conditions. An upward shift in the optimal reaction temperature of M3 mutant, to 75 °C, was observed, which was 10 °C higher than that of the wild-type enzyme. Kinetic parameter data revealed that the catalysis efficiency of M3 mutant was higher than that of the wild-type enzyme. Ramie degumming with M3 mutant was also demonstrated to be more efficient than that with the wild-type enzyme. Collectively, our results suggest that the M3 mutant, with remarkable improvements in thermoactivity and thermostability, has potential applications for ramie degumming in the textile industry.

Development of a novel uric-acid-responsive regulatory system in Escherichia coli.

A novel uric-acid-responsive regulatory system was developed in Escherichia coli by adapting the HucR-related regulatory elements from Deinococcus radiodurans into E. coli. The induction performance of this system was compared to the performance of both the pBAD and pET systems. Our novel regulatory system was induced in a dose-dependent manner in the presence of uric acid and exhibited low basal expression in its absence. The system was characterized by a wide dynamic range of induction, being compatible with various E. coli strains and not requiring genomic modifications of the bacterial host. E. coli DH5α and DH10B were the most suitable host strains for optimal performance of this system. In conclusion, we developed a regulatory system with potential for applications in both recombinant protein expression and metabolic optimization.

[Design and application of high-throughput screening tools: a review].

As an efficient and promising protein engineering strategy, directed evolution includes the construction of mutant libraries and screening of desirable mutants. A rapid and high-throughput screening method has played a critical role in the successful application of directed evolution strategy. We reviewed several high-throughput screening tools which have great potential to be applied in directed evolution. The development of powerful high-throughput screening tools will make great contributions to the advancement of protein engineering.

Directed evolution of a thermophilic endoglucanase (Cel5A) into highly active Cel5A variants with an expanded temperature profile.

Cel5A is a highly active endoglucanase from Thermoanaerobacter tengcongensis MB4, displaying an optimal temperature range between 75 and 80°C. After three rounds of error-prone PCR and screening of 4700 mutants, five variants of Cel5A with improved activities were identified by Congo Red based screening method. Compared with the wild type, the best variants 3F6 and C3-13 display 135±6% and 193±8% of the wild type specific activity for the substrate carboxymethyl cellulose (CMC), besides improvements in the relative expression level in Escherichia coli system. Remarkable are especially the improvements in activities at reduced temperatures (50% of maximum activity at 50°C and about 45°C respectively, while 65°C for the wild type). Molecular Dynamics simulations performed on the 3F6 and C3-13 variants show a decreased number of intra-Cel5A hydrogen bonds compared to the wild type, implying a more flexible protein skeleton which correlates well to the higher catalytic activity at lower temperatures. To investigate functions of each individual amino acid position site-directed (saturation) mutagenesis were generated and screened. Amino acid positions Val249 and Ile321 were found to be crucial for improving activity and residue Ile13 (encoded by rare codon AUA) yields an improved expression level in E. coli.

Cloning and characterization of a thermostable and halo-tolerant endoglucanase from Thermoanaerobacter tengcongensis MB4.

A ß-1,4-endoglucanase (Cel5A) was cloned from the genomic DNA of saccharolytic thermophilic eubacterium Thermoanaerobacter tengcongensis MB4 and functionally expressed in Escherichia coli. Substrate specificity analysis revealed that Cel5A cleaves specifically the ß-1,4-glycosidic linkage in cellulose with high activity (294 U mg(-1); carboxymethyl cellulose sodium (CMC)). On CMC, kinetics of Cel5A was determined (K (m) 1.39 ± 0.12 g l(-1); k (cat)/K (m) 1.41 ± 0.13 g(-1) s(-1)). Cel5A displays an activity optimum between 75 and 80 °C. Residues Glu187 and Glu289 were identified as key catalytic amino acids by sequence alignment. Interestingly, derived from a non-halophilic bacterium, Cel5A exhibits high residual activities in molar concentration of NaCl (3 M, 49.3%) and KCl (4 M, 48.6%). In 1 M NaCl, 82% of Cel5A activity is retained after 24 h incubation. Molecular Dynamics studies performed at 0 and 3 M NaCl, correlate the Cel5A stability to the formation of R-COO(-)···Na(+) ···(-)OOC-R salt bridges within the Cel5A tertiary structure, while activity possibly relates to the number of Na(+) ions trapped into the negatively charged active site, involving a competition mechanism between substrate and Na(+). Additionally, Cel5A is remarkably resistant in ionic liquids 1-butyl-3-methyllimidazolium chloride (1 M, 54.4%) and 1-allyl-3-methylimidazolium chloride (1 M, 65.1%) which are promising solvents for cellulose degradation and making Cel5A an attractive candidate for industrial applications.

[Synergistic systems for biodegradations of lignocellulose in microorganisms: a review].

Lignocellulose is the most abundant natural biomass. Bioconversion of lignocelluloses becomes a bottleneck for biorefinery, because of its complex structures and heterogeneous composition. Besides screening or engineering approach for single free enzymes with improved properties, an alternative approach is to study synergistic pattern with hydrolysis systems or mimic natural cellulosome for better performance in cellulolytic substrate degradation. Besides, bacterial co-cultures provide another synergistic cellulolytic system. Engineered strains with modified metabolic network could facilitate consolidated bioprocess by increasing yields as well as reducing costs.