ASIA: An automated stress-inducible adaptor for enhanced stress protein expression in engineered Escherichia coli.

Orthogonal T7 RNA polymerase (T7RNAP) and T7 promoter is a potent technique for protein expression in broad cells, but the energy requirements associated with this method impede the growth, leading to cell lysis when dealing with toxic and stress proteins. A Lemo21(DE3) strain denoted as L21 offers a solution by fine-tuning T7RNAP levels under rhamnose to induce T7 lysozyme (LysY) and enhance the protein production, but it requires optimization of inducer concentration, cultural temperature, and condition, even the types of carbon sources. Herein, we construct an automated stress-inducible adaptor (ASIA) employing different stress-inducible promoters from Escherichia coli. The ASIA system is designed to automatically regulate LysY expression in response to stress signals, thereby suppressing T7RNAP and amplifying the overexpression of stress protein cutinase ICCM. This approach fine-tunes T7RNAP levels and outperforms L21 in various temperatures and carbon source conditions. The ASIAhtp strain maintains ICCM yield at 91.6 mg/g-DCW even in the limiting carbon source at 1 g/L, which is 12-fold higher in protein productivity compared to using L21. ASIA as a versatile and robust tool for enhancing overexpression of stress proteins in E. coli is expected to address more difficult proteins in the future.

Polyhydroxyalkanoates bioproduction from bench to industry: Thirty years of development towards sustainability.

The pursuit of carbon neutrality goals has sparked considerable interest in expanding bioplastics production from microbial cell factories. One prominent class of bioplastics, polyhydroxyalkanoates (PHA), is generated by specific microorganisms, serving as carbon and energy storage materials. To begin with, a native PHA producer, Cupriavidus necator (formerly Ralstonia eutropha) is extensively studied, covering essential topics such as carbon source selection, cultivation techniques, and accumulation enhancement strategies. Recently, various hosts including archaea, bacteria, cyanobacteria, yeast, and plants have been explored, stretching the limit of microbial PHA production. This review provides a comprehensive overview of current advancements in PHA bioproduction, spanning from the native to diversified cell factories. Recovery and purification techniques are discussed, and the current status of industrial applications is assessed as a critical milestone for startups. Ultimately, it concludes by addressing contemporary challenges and future prospects, offering insights into the path towards reduced carbon emissions and sustainable development goals.

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.

Tunable T7 Promoter Orthogonality on T7RNAP for cis-Aconitate Decarboxylase Evolution via Base Editor and Screening from Itaconic Acid Biosensor.

The deaminase-fused T7 RNA polymerase (T7RNAP) presents a promising toolkit for in vivo target-specific enzyme evolution, offering the unique advantage of simultaneous DNA modification and screening. Previous studies have reported the mutation efficiency of base editors relying on different resources. In contrast, the mechanism underlying the T7RNAP/T7 system is well-understood. Therefore, this study aimed to establish a new platform, termed dT7-Muta, by tuning the binding efficiency between T7RNAP and the T7 promoter for gene mutagenesis. The strategy for proof-of-concept involves alterations in the fluorescence distribution through dT7-Muta and screening of the mutants via flow cytometry. The cis-aconitate decarboxylase from Aspergillus terreus (AtCadA) was evolved and screened via an itaconate-induced biosensor as proof-of-function of enzyme evolution. First, the degenerated codons were designed within the binding and initial region of T7 promoters (dT7s), including upstream (U), central (C), and downstream (D) regions. Three strength variants of dT7 promoter from each design, i.e., strong (S), medium (M), and weak (W), were used for evaluation. Mutation using dT7s of varying strength resulted in a broader fluorescence distribution in sfGFP mutants from the promoters CW and DS. On the other hand, broader fluorescence distribution was observed in the AtCadA mutants from the original promoter T7, UW, and DS, with the highest fluorescence and itaconic acid titer at 860 a.u. and 0.51 g/L, respectively. The present platform introduces a novel aspect of the deaminase-based mutagenesis, emphasizing the potential of altering the binding efficiency between T7RNAP and the T7 promoter for further efforts in enzyme evolution.

Optimization of 4-aminobutyric acid feeding strategy and clustered regularly interspaced short palindromic repeats activation for enhanced value-added chemicals in halophilic Chlorella sorokiniana.

Chlorella sorokiniana (CS) is a prominent microalga with vast potential as a biocarrier for carbon mitigation toward a green process. However, challenges remain in achieving high biomass levels and production rates. Therefore, a systematic feeding strategy using 4-aminobutyric acid (GABA) and CRISPR technology was applied to improve microalgal productivity. At first, GABA increased protein content by 1.4-fold, while intermittent supplementation during cultivation resulted in a 1.58-fold and 2.13-fold increase in biomass and pigment content, respectively. Under halophilic conditions, the optimal approach involved repeated feeding of 5 mM GABA at the initial and mid-log phases of growth, resulting in biomass, protein, and pigment levels of 6.74 g/L, 3.24 g/L, and 49.87 mg/L. CRISPRa mediated glutamate synthase and using monosodium glutamate (MSG) as a cheap precursor for GABA has effectively enhanced the biomass, protein, and lutein content, thus offers a cost-effective approach to commercialize high-valued chemical using algae towards a low-carbon paradigm.

Enhanced carbon capture, lipid and lutein production in Chlamydomonas reinhardtii under meso-thermophilic conditions using chaperone and CRISPRi system.

Microalgae are widely recognized as a promising bioresource for producing renewable fuels and chemicals. Microalgal biorefinery has tremendous potential for incorporation into circular bioeconomy, including sustainability, cascading use, and waste reduction. In this study, genetic engineering was used to enhance the growth, lipid and lutein productivity of Chlamydomonas reinhardtii including strains of CC400, PY9, pCHS, and PG. Notably, CRISPRi mediated on phosphoenolpyruvate carboxylase (PEPC1) gene to down-regulate the branch pathway from glycolysis to partitioning more carbon flux to lipid was explored under meso-thermophilic condition. The best chassis PGi, which has overexpressed chaperone GroELS and applied CRISPRi resulting in the highest biomass of 2.56 g/L and also boosted the lipids and lutein with 893 and 23.5 mg/L, respectively at 35°C. Finally, all strains with CRISPRi exhibited higher transcriptional levels of the crucial genes from photosynthesis, starch, lipid and lutein metabolism, thus reaching a CO2 assimilation of 1.087 g-CO2/g-DCW in mixotrophic condition.

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.

Microbial itaconic acid bioproduction towards sustainable development: Insights, challenges, and prospects.

Microbial biomanufacturing is a promising approach to produce high-value compounds with low-carbon footprint and significant economic benefits. Among twelve "Top Value-Added Chemicals from Biomass", itaconic acid (IA) stands out as a versatile platform chemical with numerous applications. IA is naturally produced by Aspergillus and Ustilago species through a cascade enzymatic reaction between aconitase (EC 4.2.1.3) and cis-aconitic acid decarboxylase (EC 4.1.1.6). Recently, non-native hosts such as Escherichia coli, Corynebacterium glutamicum, Saccharomyces cerevisiae, and Yarrowia lipolytica have been genetically engineered to produce IA through the introduction of key enzymes. This review provides an up-to-date summary of the progress made in IA bioproduction, from native to engineered hosts, covers in vivo and in vitro approaches, and highlights the prospects of combination tactics. Current challenges and recent endeavors are also addressed to envision comprehensive strategies for renewable IA production in the future towards sustainable development goals (SDGs).

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.

Investigation of enzymatic quality and quantity using pyridoxal 5'-phosphate (PLP) regeneration system as a decoy in Escherichia coli.

Pyridoxal 5'-phosphate (PLP), an essential cofactor for multiple enzymes, was used as a protein decoy to prompt enzyme expression and activity for the first time. The best chassis, denoted as WJK, was developed using a pyridoxal kinase (PdxK) and integrated at the HK022 phage attack site of Escherichia coli W3110. When compared with the original strain, the amount and activity of lysine decarboxylase (CadA) in WJK were significantly increased by 100 % and 120 %, respectively. When supplementary nineteen amino acids as second carbon source, cell growth and protein trade-off were observed. The transcriptional levels of genes from glycolysis to TCA cycle, adhE, argH and gdhA were dominating and redirected more flux into α-ketoglutarate, thus facilitated cell growth. Stepwise improvement was conducted with pyridoxal and nitrogen-rich medium; hence, CadA activity was increased to 60 g-cadaverine/g-dry cell weight/h. By reutilizing the whole-cell biocatalysts in two repeated reactions with the supplementation of fresh cells, a total cadaverine of 576 g/L was obtained even without additional PLP. Notably, PLP decoy augment the enzymatic activities of 5-aminolevulinic acid synthase and glutamate/lysine/arginine decarboxylases by over 100 %. Finally, a conserved PLP-binding pocket, Ser-His-Lys, was identified as a vital PLP sponge site that simultaneously improved protein quality and quantity.

Heterologous Expression of Toxic White Spot Syndrome Virus (WSSV) Protein in Eengineered Escherichia coli Strains.

Aquacultural shrimps suffer economic lost due to the white spot syndrome virus (WSSV) that is the most notorious virus for its fatality and contagion, leading to a 100% death rate on infected shrimps within 7 days. However, the infection of mechanism remains a mystery and crucial problem. To elucidate the pathogenesis of WSSV, a high abundance of protein is required to identify and characterize its functions. Therefore, the optimal WSSV355 overexpression was explored in engineered Escherichia coli strains, in particular C43(DE3) as a toxic tolerance strain remedied 40% of cell growth from BL21(DE3). Meanwhile, a trace amount of WSSV355 was observed in both strains. To optimize the codon of WSSV355 using codon adaption index (CAI), an overexpression was observed with 1.32 mg/mL in C43(DE3), while the biomass was decreased by 35%. Subsequently, the co-expression with pRARE boosted the target protein up to 1.93 mg/mL. Finally, by scaling up production of WSSV355 in the fermenter with sufficient oxygen supplied, the biomass and total and soluble protein were enhanced 67.6%, 44.9%, and 7.8% compared with that in flask condition. Herein, the current approach provides efficacious solutions to produce toxic proteins via codon usage, strain selection, and processing optimization by alleviating the burden and boosting protein production in E. coli.

A direct enzymatic evaluation platform (DEEP) to fine-tuning pyridoxal 5'-phosphate-dependent proteins for cadaverine production.

Pyridoxal 5'-phosphate (pyridoxal phosphate, PLP) is an essential cofactor for multiple enzymatic reactions in industry. However, cofactor engineering based on PLP regeneration and related to the performance of enzymes in chemical production has rarely been discussed. First, we found that MG1655 strain was sensitive to nitrogen source and relied on different amino acids, thus the biomass was significantly reduced when PLP excess in the medium. Then, the six KEIO collection strains were applied to find out the prominent gene in deoxyxylulose-5-phosphate (DXP) pathway, where pdxB was superior in controlling cell growth. Therefore, the clustered regularly interspaced short palindromic repeats interference (CRISPRi) targeted on pdxB in MG1655 was employed to establish a novel direct enzymatic evaluation platform (DEEP) as a high-throughput tool and obtained the optimal modules for incorporating of PLP to enhance the biomass and activity of PLP-dependent enzymes simultaneously. As a result, the biomass has increased by 55% using PlacI promoter driven pyridoxine 5'-phosphate oxidase (PdxH) with a trace amount of precursor. When the strains incorporated DEEP and lysine decarboxylase (CadA), the cadaverine productivity was increased 32% due to the higher expression of CadA. DEEP is not only feasible for high-throughput screening of the best chassis for PLP engineering but also practical in fine-tuning the quantity and quality of enzymes.

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.

Pathogenesis-related protein 1 suppresses oomycete pathogen by targeting against AMPK kinase complex.

INTRODUCTION: During the arms race between plants and pathogens, pathogenesis-related proteins (PR) in host plants play a crucial role in disease resistance, especially PR1. PR1 constitute a secretory peptide family, and their role in plant defense has been widely demonstrated in both hosts and in vitro. However, the mechanisms by which they control host-pathogen interactions and the nature of their targets within the pathogen remain poorly understood. OBJECTIVES: The present study was aimed to investigate the anti-oomycete activity of secretory PR1 proteins and elaborate their underlying mechanisms. METHODS: This study was conducted in the potato-Phytophthora infestans pathosystem. After being induced by the pathogen infection, the cross-kingdom translocation of secretory PR1 was demonstrated by histochemical assays and western blot, and their targets in P. infestans were identified by yeast-two-hybrid assays, bimolecular fluorescence complementation assays, and co-immunoprecipitation assay. RESULTS: The results showed that the expression of secretory PR1-encoding genes was induced during pathogen infection, and the host could deliver PR1 into P. infestans to inhibit its vegetative growth and pathogenicity. The translocated secretory PR1 targeted the subunits of the AMPK kinase complex in P. infestans, thus affecting the AMPK-driven phosphorylation of downstream target proteins, preventing ROS homeostasis, and down-regulating the expression of RxLR effectors. CONCLUSION: The results provide novel insights into the molecular function of PR1 in protecting plants against pathogen infection, and uncover a potential target for preventing pre- and post-harvest late blight.

Clustered regularly interspaced short palindromic repeats (CRISPR) technology and genetic engineering strategies for microalgae towards carbon neutrality: A critical review.

Carbon dioxide is the major greenhouse gas and regards as the critical issue of global warming and climate changes. The inconspicuous microalgae are responsible for 40% of carbon fixation among all photosynthetic plants along with a higher photosynthetic efficiency and convert the carbon into lipids, protein, pigments, and bioactive compounds. Genetic approach and metabolic engineering are applied to accelerate the growth rate and biomass of microalgae, hence achieve the mission of carbon neutrality. Meanwhile, CRISPR/Cas9 is efficiently to enhance the productivity of high-value compounds in microalgae for it is easier operation, more affordable and is able to regulate multiple genes simultaneously. The genetic engineering strategies provide the multidisciplinary concept to evolute and increase the CO2 fixation rate through Calvin-Benson-Bassham cycle. Therefore, the technologies, bioinformatics tools, systematic engineering approaches for carbon neutrality and circular economy are summarized and leading one step closer to the decarbonization society in this review.

Effective 5-aminolevulinic acid production via T7 RNA polymerase and RuBisCO equipped Escherichia coli W3110.

Chromosome-based engineering is a superior approach for gene integration generating a stable and robust chassis. Therefore, an effective amplifier, T7 RNA polymerase (T7RNAP) from bacteriophage, has been incorporated into Escherichia coli W3110 by site-specific integration. Herein, we performed the 5-aminolevulinic acid (5-ALA) production in four T7RNAP-equipped W3110 strains using recombinant 5-aminolevulinic synthase and further explored the metabolic difference in best strain. The fastest glucose consumption resulted in the highest biomass and the 5-ALA production reached to 5.5 g/L; thus, the least by-product of acetate was shown in RH strain in which T7RNAP was inserted at HK022 phage attack site. Overexpression of phosphoenolpyruvate (PEP) carboxylase would pull PEP to oxaloacetic acid in tricarboxylic acid cycle, leading to energy conservation and even no acetate production, thus, 6.53 g/L of 5-ALA was achieved. Amino acid utilization in RH deciphered the major metabolic flux in α-ketoglutaric acid dominating 5-ALA production. Finally, the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase were expressed for carbon dioxide recycling; a robust and efficient chassis toward low-carbon assimilation and high-level of 5-ALA production up to 11.2 g/L in fed-batch fermentation was established.

Microalgae-based biotechnological sequestration of carbon dioxide for net zero emissions.

Excessive carbon dioxide (CO2) emissions into the atmosphere have become a dire threat to the human race and environmental sustainability. The ultimate goal of net zero emissions requires combined efforts on CO2 sequestration (natural sinks, biomass fixation, engineered approaches) and reduction in CO2 emissions while delivering economic growth (CO2 valorization for a circular carbon bioeconomy, CCE). We discuss microalgae-based CO2 biosequestration, including flue gas cultivation, biotechnological approaches for enhanced CO2 biosequestration, technological innovations for microalgal cultivation, and CO2 valorization/biofuel productions. We highlight challenges to current practices and future perspectives with the goal of contributing to environmental sustainability, net zero emissions, and the CCE.

Simultaneous carbon dioxide sequestration and utilization for cadaverine production using dual promoters in engineered Escherichia coli strains.

Human carbonic anhydrase II (hCAII) is a rapid-acting zinc-metalloenzyme that catalyzes CO2 hydration reversibly, with encouraging applications in carbon capture, sequestration, and utilization (CCSU). However, biocatalyst durability is a major challenge. Herein, hCAII is emphasized in 4 different Escherichia coli strains and designated under dual promoters from sigma factor 70 (σ70) and heat shock protein (HSP70A) to suppress the usage of inducer and stimulate activity in heat environments. As a result, hCAII under high-efficient dual promoters regulation retained high residual activity in CO2 biomineralization of 68.8 % after 4 cycles at 40 °C. Moreover, co-expression of CAC9 with lysine decarboxylase (CadA) simultaneously sequestered CO2 release up to 95.7 % and increased cadaverine titer from 18.0 to 36.7 g/L by using E. coli MG1655. The remnant biomass from cadaverine synthesis sustained converting CO2 to 57.9 mg-CaCO3. Thus, the dual promoters design demonstrated the promising potential for CCSU through simultaneous CO2 utilization and cadaverine synthesis.

Enhanced recombinant carbonic anhydrase in T7RNAP-equipped Escherichia coli W3110 for carbon capture storage and utilization (CCSU).

Sulfurihydrogenibium yellowstonense carbonic anhydrase (SyCA) is a well-known thermophilic CA for carbon mineralization. To broaden the applications of SyCA, the activity of SyCA was improved through stepwise engineering and in different cultural conditions, as well as extended to co-expression with other enzymes. The engineered W3110 strains with 4 different T7 RNA polymerase levels were employed for SyCA production. As a result, the best strain WT7L cultured in modified M9 medium with temperature shifted from 37 to 30 °C after induction increased SyCA activity to 9122 U/mL. The SyCA whole-cell biocatalyst was successfully applied for carbon capture and storage (CCS) of CaCO3. Furthermore, SyCA was applied for low-carbon footprint synthesis of 5-aminolevulinic acid (5-ALA) and cadaverine (DAP) by coupling with ALA synthetase (ALAS) and lysine decarboxylase (CadA), suppressing CO2 release to -6.1 g-CO2/g-ALA and -2.53 g-CO2/g-DAP, respectively. Harnessing a highly active SyCA offers a complete strategy for CCSU in a green process.

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.