Non-native Pathway Engineering with CRISPRi for Carbon Dioxide Assimilation and Valued 5-Aminolevulinic Acid Synthesis in Escherichia coli Nissle.

Carbon dioxide emission and acidification during chemical biosynthesis are critical challenges toward microbial cell factories' sustainability and efficiency. Due to its acidophilic traits among workhorse lineages, the probiotic Escherichia coli Nissle (EcN) has emerged as a promising chemical bioproducer. However, EcN lacks a CO2-fixing system. Herein, EcN was equipped with a simultaneous CO2 fixation system and subsequently utilized to produce low-emission 5-aminolevulinic acid (5-ALA). Two different artificial CO2-assimilating pathways were reconstructed: the novel ribose-1,5-bisphosphate (R15P) route and the conventional ribulose-5-phosphate (Ru5P) route. CRISPRi was employed to target the pfkAB and zwf genes in order to redirect the carbon flux. As expected, the CRISPRi design successfully strengthened the CO2 fixation. The CO2-fixing route via R15P resulted in high biomass, while the engineered Ru5P route acquired the highest 5-ALA and suppressed the CO2 release by 77%. CO2 fixation during 5-ALA production in EcN was successfully synchronized through fine-tuning the non-native pathways with CRISPRi.

Challenges and opportunities for engineered Escherichia coli as a pivotal chassis toward versatile tyrosine-derived chemicals production.

Growing concerns over limited fossil resources and associated environmental problems are motivating the development of sustainable processes for the production of high-volume fuels and high-value-added compounds. The shikimate pathway, an imperative pathway in most microorganisms, is branched with tyrosine as the rate-limiting step precursor of valuable aromatic substances. Such occurrence suggests the shikimate pathway as a promising route in developing microbial cell factories with multiple applications in the nutraceutical, pharmaceutical, and chemical industries. Therefore, an increasing number of studies have focused on this pathway to enable the biotechnological manufacture of pivotal and versatile aromatic products. With advances in genome databases and synthetic biology tools, genetically programmed Escherichia coli strains are gaining immense interest in the sustainable synthesis of chemicals. Engineered E. coli is expected to be the next bio-successor of fossil fuels and plants in commercial aromatics synthesis. This review summarizes successful and applicable genetic and metabolic engineering strategies to generate new chassis and engineer the iterative pathway of the tyrosine route in E. coli, thus addressing the opportunities and current challenges toward the realization of sustainable tyrosine-derived aromatics.

High value ferulic acid biosynthesis using modular design and spent coffee ground in engineered Escherichia coli chassis.

Sophisticated genetic engineering enables microbial hosts to derive high-value aromatics in a green manner. Ferulic acid (FA) is one of the noteworthy aromatics due to its potent pharmacokinetic properties. However, the current approaches to FA biosynthesis still decamp from time- and cost-effectiveness. Herein, FA pathway was artificially reconstructed in Escherichia coli using modular designs. Comprehensive screening of E. coli lineages was reckoned for efficient synthesis of p-coumaric acid (pCA) as a precursor and FA eventually. The modular design was further advanced by harboring tyrosine transporter, adapting the heterologous codon, utilizing pCA symporter, and enriching FADH2 cofactor pools via in vivo regeneration. Taken together with simultaneous optimization of culture condition, a remarkable FA yield of 972.6 mg/L with 89.4 % conversion was achieved in 48 h, circumventing the time-consuming issue. Moreover, this study successfully exported inexpensive precursor from spent coffee ground for the first time, paving the economical way of FA biosynthesis.

High-level itaconic acid (IA) production using engineered Escherichia coli Lemo21(DE3) toward sustainable biorefinery.

Itaconic acid (IA) serves as a prominent building block for polyamides as sustainable material. In vivo IA production is facing the competing side reactions, byproducts accumulation, and long cultivation time. Therefore, the utilization of whole-cell biocatalysts to carry out production from citrate is an alternative approach to sidestep the current limitations. In this study, in vitro reaction of IA was obtained 72.44 g/L by using engineered E. coli Lemo21(DE3) harboring the aconitase (Acn, EC 4.2.1.3) and cis-aconitate decarboxylase (CadA, EC 4.1.1.6) which was cultured in glycerol-based minimal medium. IA productivity enhancement was observed after cold-treating the biocatalysts in - 80 °C for 24 h prior to the reaction, reaching 81.6 g/L. On the other hand, a new seeding strategy in Terrific Broth (TB) as a nutritionally rich medium was employed to maintain the biocatalysts stability up to 30 days. Finally, the highest IA titer of 98.17 g/L was attained using L21::7G chassis, that has a pLemo plasmid and integration of GroELS to the chromosome. The high-level of IA production along with the biocatalyst reutilization enables the economic viability toward a sustainable biorefinery.

Prospective and challenges of live bacterial therapeutics from a superhero Escherichia coli Nissle 1917.

Escherichia coli Nissle 1917 (EcN), the active component of Mutaflor(R), is a notable probiotic from Gram-negative to treat Crohn's disease and irritable bowel syndrome. Therefore, a comprehensive genomic database maximizes the systemic probiotic assessment to discover EcN's role in human health. Recently, advanced synthetic and genetic tools have opened up a rich area to execute EcN as "living medicines" with controllable functions. Incorporating unique biomarkers allows the engineered EcN to switch genes on and off in response to environmental cues. Since EcN holds promise as a safe nature vehicle, more studies are desired to fully realize a wide range of probiotic potential for disease treatment. This review aims to deliver a historical origin of EcN, discuss the recent promising genetic toolbox in the rational design of probiotics, and pinpoint the clinical translation and evaluation of engineered EcN in vitro and in vivo. The summary of safety concerns, strategies of biotherapeutics development, and the challenges and prospects of engineered EcN is also concluded.

Reprogramming T7RNA Polymerase in Escherichia coli Nissle 1917 under Specific Lac Operon for Efficient p-Coumaric Acid Production.

Lac operon is the standard regulator used to control the orthogonality of T7RNA polymerase (T7RNAP) and T7 promoter inEscherichia coli BL21(DE3) strain for protein expression. However,E. coliNissle 1917 (EcN), the unique probiotic strain, has seldom been precisely adapted to the T7 system. Herein, we applied bioinformatics analysis on Lac operon from different strains, and it was observed that a weak promoter for LacI repressor existed in EcN. Furthermore, X-gal assay revealed a strong expression of lacZ in EcN. We demonstrated that Lac operon significantly affected the protein expression in the two T7-derived EcN, in which T7RNAP was integrated at lambda (ET7L) and HK022 (ET7H), respectively. Different combinations of replication origin, chaperonin GroELS, inducer, and medium were explored to fine-tune the best strain with tyrosine ammonia-lyase (TAL) for p-coumaric acid (pCA) production, which is one of the essential bioactive compounds for human health. Finally, the highest pCA conversion of 78.8% was achieved using RRtL (plasmid form) under the optimum condition, and a 51.5% conversion was obtained with L::Rt strain which has integrated T7-RtTAL at HK022 of ET7L in the simulated gut environment. The appropriate reprogramming of T7RNAP expedites EcN as an effective and promising cell factory for live bacterial therapeutics in the future.

Tailoring key enzymes for renewable and high-level itaconic acid production using genetic Escherichia coli via whole-cell bioconversion.

Renewable chemical productions through carbon-neutral design are widely concerned in recent years. Among all, itaconic acid (IA) is one of the most important building block chemicals from biorefinery. However, IA fermentation by the eukaryotic Aspergillus terreus is time-consuming and less productive. The whole-cell (WC) bioconversion, proposed as an alternative approach by transforming citrate into IA via two key enzymes of aconitase (ACN, EC 4.2.1.3) and cis-aconitate decarboxylase (CAD, EC 4.1.1.6), is attractive. In this study, we screened the best genes from genes library, studied the kinetics parameters of ACN from Corynebacterium glutamicum (Cg) and CAD from Aspergillus terreus (At), thus achieving the maximum IA production. The catalytic activity of CgAcnA was 39-fold of AtCadA, indicating CAD was the rate-determining step. For metal ions effect, copper and ferric ions inhibited 95% and 59% enzyme activity when both enzymes co-worked together. Finally, the engineered Escherichia coli expressing dual genes and cultured in glycerol-included medium reached the highest IA titer of 67 g/L and productivity of 8.375 g/L/h, which demonstrates as a promising renewable process.

Genetic design of co-expressed Mesorhizobium loti carbonic anhydrase and chaperone GroELS to enhancing carbon dioxide sequestration.

Mesorhizobium loti carbonic anhydrase (MlCA), an intrinsically high catalytic enzyme, has been employed for carbon dioxide capture and sequestration. However, recombinant expression of MlCA in Escherichia coli often forms inclusion bodies. Hence, protein partners such as fusion-tags and molecular chaperones are involved in regarding reduce the harshness of protein folding. TrxA-tag and GroELS have been chosen to co-express with MlCA in E. coli under an inducible T7 promoter or a constitutive J23100 promoter to compare productivity and activity. The results possessed that coupling protein partners effectively increased soluble MlCA up to 2.9-folds under T7 promoter, thus enhancing the CA activity by 120% and achieving a 5.2-folds turnover rate. Besides, it has also shifted the optimum temperature from 40 °C to 50 °C, promoted stability in the broad pH range (4.5 to 9.5) and the presence of various metal ions. Based on the in vitro assay and isothermal titration calorimetry (ITC) analysis, GroELS enhancing CA activity was due to change the intrinsic thermodynamic properties of the enzyme from endothermic to exothermic reaction (i.e., ∆H = 89.8 to -121.8 kJ/mol). Therefore, the collaboration of TrxA-MlCA with GroELS successfully augmented CO2 biomineralization.

Crosslinked on novel nanofibers with thermophilic carbonic anhydrase for carbon dioxide sequestration.

The recombinant Sulfurihydrogenibium yellowstonense carbonic anhydrase (SyCA) was covalently bonded on novel polyacrylonitrile (PAN) and polyethylene terephthalate (PET) nanofibers (PAN-PET-PAN donated as AEA) that was first fabricated by electrospinning. The resulting composite materials further crosslinked by the glutaraldehyde, which significantly increased thermostability up to 89.8% and 18.0% after heating at 60 °C for 1 h for immobilized crude and pure SyCA, respectively. After four repetitive attempts in the demonstration of CO2 sequestration, immobilized crude and pure SyCA on AEA also effectively improved the total CaCO3 yields to be 5.8 folds and 2.2 folds compared to free enzyme. Furthermore, the endurance of immobilized crude was investigated on flue gasses, which was retained its activity up to 57% on 50 mM NOx and 61% on 50 mM SOx presence. This is the first report of immobilized thermophilic SyCA on a novel nanofiber at the reusability, durability, sequestration of carbon dioxide, tolerant to sulfur oxides (SOx) and nitrogen oxides (NOx) toxic gases and to prevent air pollution.