Tailored expression of ICCM cutinase in engineered Escherichia coli for efficient polyethylene terephthalate hydrolysis.

Enzymatic depolymerization of PET waste emerges as a crucial and sustainable solution for combating environmental pollution. Over the past decade, PET hydrolytic enzymes, such as PETase from Ideonella sakaiensis (IsPETases), leaf compost cutinases (LCC), and lipases, have been subjected to rational mutation to enhance their enzymatic properties. ICCM, one of the best LCC mutants, was selected for overexpression in Escherichia coli BL21(DE3) for in vitro PET degradation. However, overexpressing ICCM presents challenges due to its low productivity. A new stress-inducible T7RNA polymerase-regulating E. coli strain, ASIAhsp, which significantly enhances ICCM production by 72.8 % and achieves higher enzyme solubility than other strains. The optimal cultural condition at 30 °C with high agitation, corresponding to high dissolved oxygen levels, has brought the maximum productivity of ICCM and high PET-hydrolytic activity. The most effective PET biodegradation using crude or pure ICCM occurred at pH 10 and 60 °C. Moreover, ICCM exhibited remarkable thermostability, retaining 60 % activity after a 5-day reaction at 60 °C. Notably, crude ICCM eliminates the need for purification and efficiently degrades PET films.

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.

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.

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.

High-level production and extraction of C-phycocyanin from cyanobacteria Synechococcus sp. PCC7002 for antioxidation, antibacterial and lead adsorption.

Global warming and climate change because carbon dioxide (CO2) release to atmosphere is the forecasting challenges to human being. We are facing how to overcome the dilemma on the balance between economic and environment, thus taking more efforts on green processes to meet agreement of sustainable society are urgent and crucial. The absorption of CO2 by microalgae reduces the impact of CO2 on the environment. In this study, the CO2 removal efficiency was the highest in the culture of Cyanobacterium Synechococcus sp. PCC7002 (also called blue-green algae), at 2% CO2 to reach a value of 0.86 g-CO2/g-DCW. The main product of PCC7002 is C-phycocyanin (C-PC) which regarding to phycobilisome complex in all cyanobacterial species. A 160% increasing C-PC was achieved in the cultivation under 100 µmol/m2/s light intensity, 12:12 light-period with 2% CO2 at 30 °C. The mix-culture of nitric and ammonia ions had positive effect on the cell growth and C-PC accumulation, thus realized the highest yield of 0.439 g-CPC/g-DCW. Additionally, the partial purified C-PC displayed 89% antioxidant activity of 2,2-diphenyl-1-picryhydrazyl (DPPH) and 11% of superoxide free radical scavenging activity, respectively. The production of C-PC from PCC7002 reduced the CO2 emission and exhibited antibacterial activity against Escherichia coli and lead ion adsorption at room temperature, which has the great potential for eco-friendly application.

Tailoring Genetic Elements of the Plasmid-Driven T7 System for Stable and Robust One-Step Cloning and Protein Expression in Broad Escherichia coli.

The plasmid-driven T7 system (PDT7) is a flexible approach to trigger protein overexpression; however, most of the reported PDT7 rely on many auxiliary elements or inducible systems to attenuate the toxicity from the orthogonality of the T7 system, which limits its application as the one-step cloning and protein expression system. In this study, we developed a stable and robust PDT7 via tailoring the genetic elements. By error-prone mutagenesis, a mutated T7RNAP with TTTT insertion conferred a trace but enough amount of T7RNAP for stable and efficient PDT7, denoted as PDT7m. The replication origin was kept at the same level, while the ribosome binding site (RBS) of the T7 promoter was the most contributing factor, thus enhancing the protein expression twofold using PDT7m. For application as a host-independent screening platform, both constitutive and IPTG-inducible PDT7m were constructed. It was found that each strain harnessed different IPTG inducibilities for tailor-made strain selection. Constitutive PDT7m was successfully used to express the homologous protein (i.e., lysine decarboxylase) or heterologous protein (i.e., carbonic anhydrase, CA) as a one-step cloning and protein expression tool to select the best strain for cadaverine (DAP) or CA production, respectively. Additionally, PDT7m is compatible with the pET system for coproduction of DAP and CA simultaneously. Finally, PDT7m was used for in vivo high-end chemical production of aminolevulinic acid (ALA), in which addition of the T7 terminator successfully enhanced 340% ALA titer, thus paving the way to rapidly and effectively screening the superior strain as a cell factory.

Challenges and opportunity of recent genome editing and multi-omics in cyanobacteria and microalgae for biorefinery.

Microalgae and cyanobacteria are easy to culture, with higher growth rates and photosynthetic efficiencies compared to terrestrial plants, and thus generating higher productivity. The concept of microalgal biorefinery is to assimilate carbon dioxide and convert it to chemical energy/value-added products, such as vitamins, carotenoids, fatty acids, proteins and nucleic acids, to be applied in bioenergy, health foods, aquaculture feed, pharmaceutical and medical fields. Therefore, microalgae are annotated as the third generation feedstock in bioenergy and biorefinery. In past decades, many studies thrived to improve the carbon sequestration efficiency as well as enhance value-added compounds from different algae, especially via genetic engineering, synthetic biology, metabolic design and regulation. From the traditional Agrobacterium-mediated transformation DNA to novel CRISPR (clustered regularly interspaced short palindromic repeats) technology applied in microalgae and cyanobacteria, this review has highlighted the genome editing technology for biorefinery that is a highly environmental friendly trend to sustainable and renewable development.