CRISPR-COPIES: an in silico platform for discovery of neutral integration sites for CRISPR/Cas-facilitated gene integration.

The CRISPR/Cas system has emerged as a powerful tool for genome editing in metabolic engineering and human gene therapy. However, locating the optimal site on the chromosome to integrate heterologous genes using the CRISPR/Cas system remains an open question. Selecting a suitable site for gene integration involves considering multiple complex criteria, including factors related to CRISPR/Cas-mediated integration, genetic stability, and gene expression. Consequently, identifying such sites on specific or different chromosomal locations typically requires extensive characterization efforts. To address these challenges, we have developed CRISPR-COPIES, a COmputational Pipeline for the Identification of CRISPR/Cas-facilitated intEgration Sites. This tool leverages ScaNN, a state-of-the-art model on the embedding-based nearest neighbor search for fast and accurate off-target search, and can identify genome-wide intergenic sites for most bacterial and fungal genomes within minutes. As a proof of concept, we utilized CRISPR-COPIES to characterize neutral integration sites in three diverse species: Saccharomyces cerevisiae, Cupriavidus necator, and HEK293T cells. In addition, we developed a user-friendly web interface for CRISPR-COPIES (https://biofoundry.web.illinois.edu/copies/). We anticipate that CRISPR-COPIES will serve as a valuable tool for targeted DNA integration and aid in the characterization of synthetic biology toolkits, enable rapid strain construction to produce valuable biochemicals, and support human gene and cell therapy applications.

An end-to-end pipeline for succinic acid production at an industrially relevant scale using Issatchenkia orientalis.

Microbial production of succinic acid (SA) at an industrially relevant scale has been hindered by high downstream processing costs arising from neutral pH fermentation for over three decades. Here, we metabolically engineer the acid-tolerant yeast Issatchenkia orientalis for SA production, attaining the highest titers in sugar-based media at low pH (pH 3) in fed-batch fermentations, i.e. 109.5 g/L in minimal medium and 104.6 g/L in sugarcane juice medium. We further perform batch fermentation using sugarcane juice medium in a pilot-scale fermenter (300×) and achieve 63.1 g/L of SA, which can be directly crystallized with a yield of 64.0%. Finally, we simulate an end-to-end low-pH SA production pipeline, and techno-economic analysis and life cycle assessment indicate our process is financially viable and can reduce greenhouse gas emissions by 34-90% relative to fossil-based production processes. We expect I. orientalis can serve as a general industrial platform for production of organic acids.

A landing pad system for multicopy gene integration in Issatchenkia orientalis.

The robust nature of the non-conventional yeast Issatchenkia orientalis allows it to grow under highly acidic conditions and therefore, has gained increasing interest in producing organic acids using a variety of carbon sources. Recently, the development of a genetic toolbox for I. orientalis, including an episomal plasmid, characterization of multiple promoters and terminators, and CRISPR-Cas9 tools, has eased the metabolic engineering efforts in I. orientalis. However, multiplex engineering is still hampered by the lack of efficient multicopy integration tools. To facilitate the construction of large, complex metabolic pathways by multiplex CRISPR-Cas9-mediated genome editing, we developed a bioinformatics pipeline to identify and prioritize genome-wide intergenic loci and characterized 47 gRNAs located in 21 intergenic regions. These loci are screened for guide RNA cutting efficiency, integration efficiency of a gene cassette, the resulting cellular fitness, and GFP expression level. We further developed a landing pad system using components from these well-characterized loci, which can aid in the integration of multiple genes using single guide RNA and multiple repair templates of the user's choice. We have demonstrated the use of the landing pad for simultaneous integrations of 2, 3, 4, or 5 genes to the target loci with efficiencies greater than 80%. As a proof of concept, we showed how the production of 5-aminolevulinic acid can be improved by integrating five copies of genes at multiple sites in one step. We have further demonstrated the efficiency of this tool by constructing a metabolic pathway for succinic acid production by integrating five gene expression cassettes using a single guide RNA along with five different repair templates, leading to the production of 9 g/L of succinic acid in batch fermentations. This study demonstrates the effectiveness of a single gRNA-mediated CRISPR platform to build complex metabolic pathways in a non-conventional yeast. This landing pad system will be a valuable tool for the metabolic engineering of I. orientalis.

Metabolic Engineering: Methodologies and Applications.

Metabolic engineering aims to improve the production of economically valuable molecules through the genetic manipulation of microbial metabolism. While the discipline is a little over 30 years old, advancements in metabolic engineering have given way to industrial-level molecule production benefitting multiple industries such as chemical, agriculture, food, pharmaceutical, and energy industries. This review describes the design, build, test, and learn steps necessary for leading a successful metabolic engineering campaign. Moreover, we highlight major applications of metabolic engineering, including synthesizing chemicals and fuels, broadening substrate utilization, and improving host robustness with a focus on specific case studies. Finally, we conclude with a discussion on perspectives and future challenges related to metabolic engineering.

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.

CRISPRi-Mediated NIMPLY Logic Gate for Fine-Tuning the Whole-Cell Sensing toward Simple Urine Glucose Detection.

Whole-cell biosensors have been regarded as a prominent alternative to chemical and physical biosensors due to their renewability, environmental friendliness, and biocompatibility. However, there is still a lack of noninvasive measurements of urine glucose, which plays a vital role in monitoring the risk of diabetes in the healthcare system, via whole-cell biosensors. In this study, we characterized a glucose-inducible promoter and further enhanced the sensing performance using three genetic effectors, which encompassed ribozyme regulator (RiboJ), clustered regularly interspaced short palindromic repeat interference (CRISPRi), and plasmid-based T7RNA polymerase (PDT7), to develop the noninvasive glucose biosensor by fluorescent signal. As a result, RiboJ increased dynamic range to 2989 au, but declined signal-to-noise (S/N) to 1.59, while CRISPRi-mediated NIMPLY gate intensified both dynamic range to 5720 au and S/N to 4.58. The use of single PDT7 orthogonal with T7 promoter in cells (i.e., P strain) achieved a 44 180 au of dynamic range with S/N at 3.08. By coupling the PDT7 and NIMPLY-mediated CRISPRi, we constructed an optimum PIGAS strain with the highest S/N value of 4.95. Finally, we adopted the synthetic bacteria into a microdevice to afford an integrative and portable system for daily urine glucose inspection, which would be an alternative approach for medical diagnosis in the future.

Pyridoxal kinase PdxY mediated carbon dioxide assimilation to enhance the biomass in Chlamydomonas reinhardtii CC-400.

Microalga served as the promising bioresources due to the high efficiency of carbon dioxide conversion. However, the application of microalga is still restricted by low biomass, easier contamination, and high cost of production. To overcome the challenge, engineered Chlamydomonas reinhardtii CC-400 with pyridoxal kinase gene (pdxY) has demonstrated in this study. The results indicated CC-400 with pdxY reached enhanced algal biomass in three different systems, including flask, Two-layer Photo-Reactor (TPR) and airlift Photo-Bioreactor (PBR). The genetic strain PY9 cultured with 1% CO2 in the PBR showed a significant enhancement of biomass up to 1.442 g/L, a 2-times of that of the wild type. We also found the transcriptional levels of carbonic anhydrase (CA) dropped down in PY9 while higher levels of RuBisCo and pdxY occurred, thus the carbon dioxide assimilation under mixotrophic culture dramatically increased. We proofed that pdxY successfully mediated carbon dioxide utilization in CC-400.

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.

Enhanced recombinant Sulfurihydrogenibium yellowstonense carbonic anhydrase activity and thermostability by chaperone GroELS for carbon dioxide biomineralization.

Biological carbon fixation is a feasible strategy to reduce atmospheric carbon dioxide levels (CO2). In this platform, carbonic anhydrase (CA) enzyme is employed to accelerate the sequestration of CO2. The present work explored the effect of chaperone GroELS and TrxA-tag on improving soluble expression of the recombinant Sulfurihydrogenibium yellowstonense CA which activity and biomineralization capability were taken into consideration. At first, the expression of GroELS using the inducible T7 promoter and constitutive J23100 promoter were investigated. The results indicated that 1.4 folds increment of soluble protein and 100% of CA activity enhancement were achieved with GroELS co-expression driven by J23100 promoter. Furthermore, the involvement of TrxA fusion tag displayed a significant enhancement of soluble protein production which was about 2.67 times higher than that of original SyCA. Besides, co-expression with GroELS intensified the thermostability of SyCA at 60 °C owing to changes in the structural conformation of the protein, which was proved by an in vitro assay. The SyCA was further entrapped and immobilized into polyacrylamide gel (i.e., PAGE-SyCA). The biomineralization capability of the PAGE-SyCA and whole-cell (WC) was compared in a two-column system. After 5 cycles of reuse, PAGE-SyCA maintained 29.8% activity and formed 774 mg of CaCO3 solids in the B::JG strain. This study presents the recombinant engineering strategies to improve SyCA productivity, activity, thermostability, and effective carbon dioxide conversion.

Design and optimization of bioreactor to boost carbon dioxide assimilation in RuBisCo-equipped Escherichia coli.

Global warming is a surging issue that has provoked the demand of green process to mitigate carbon dioxide. In this context, RuBisCo-equipped Escherichia coli has first developed and evaluated the CO2-assimiliable capability based on the mass balance in three devices: Flask-based in CO2 incubator (FIC), two-layered device (TLD) and CO2 bubbling device (CBD) systematically. With the forced diffusion of 5% CO2 in CBD, which confers an efficient attack of CO2 to RuBisCo, the CO2 assimilation increased from -5.03 to -2.63 g-CO2/g-DCW. Furthermore, boosted CO2 assimilation ability was observed by co-expression of GroELS chaperone with 71% reduction on CO2 release. By DNA sequencing and tandem MS/MS analysis, the toxicity of RuBisCo and PRK was identified to interfere the sugar metabolism and energy producing, while the cell morphology was changed and observed in RuBisCo-equipped E. coli. Our study provides a new perspective of higher CO2 assimilation for sustainable to eco-friendly green bioprocess.

CRISPRi-mediated programming essential gene can as a Direct Enzymatic Performance Evaluation & Determination (DEPEND) system.

Harnessing enzyme expression for production of target chemicals is a critical and multifarious process, where screening of different genes by inspection of enzymatic activity plays an imperative role. Here, we conceived an idea to improve the time-consuming and labor-intensive process of enzyme screening. Controlling cell growth was achieved by the Cluster Regularly Interspaced Short Palindromic Repeat (CRISPRi) system with different single guide RNA targeting the essential gene can (CRISPRi::CA) that encodes a carbonic anhydrase for CO2 uptake. CRISPRi::CA comprises a whole-cell biosensor to monitor CO2 concentration, ranging from 1% to 5%. On the basis of CRISPRi::CA, an effective and simple Direct Enzymatic Performance Evaluation & Determination (DEPEND) system was developed by a single step of plasmid transformation for targeted enzymes. As a result, the activity of different carbonic anhydrases corresponded to the colony-forming units. Furthermore, the enzymatic performance of 5-aminolevulinic acid synthetase (ALAS), which converts glycine and succinate-CoA to release a molecule of CO2 , has also been distinguished, and the effect of the chaperone GroELS on ALAS enzyme folding was successfully identified in the DEPEND system. We provide a highly feasible, time-saving, and flexible technology for the screening and inspection of high-performance enzymes, which may accelerate protein engineering in the future.

New Insight into Plasmid-Driven T7 RNA Polymerase in Escherichia coli and Use as a Genetic Amplifier for a Biosensor.

T7 RNA polymerase (T7RNAP) and T7 promoter are powerful genetic components, thus a plasmid-driven T7 (PDT7) genetic circuit could be broadly applied for synthetic biology. However, the limited knowledge of the toxicity and instability of such a system still restricts its application. Herein, we constructed 16 constitutive genetic circuts of PDT7 and investigated the orthogonal effects in toxicity and instability. The T7 toxicity was elucidated from the construction processes and cell growth characterization, showing the importance of optimal orthogonality for PDT7. Besides, a protein analysis was performed to validate how the T7 system affected cell metabolism and led to the instability. The application of constitutive PDT7 in functional protein expressions, including carbonic anhydrase, lysine decarboxylase, and 5-ALA synthetase was demonstrated. Furthermore, PDT7 working as a genetic amplifier had been designed for E. coli cell-based biosensors, which illustrated the opportunities in the future of PDT7 used in synthetic biology.

A Critical Review of Genome Editing and Synthetic Biology Applications in Metabolic Engineering of Microalgae and Cyanobacteria.

Being the green gold of the future, microalgae and cyanobacteria have recently attracted considerable interest worldwide, for their metabolites such as lipids, protein, pigments, and bioactive compounds have immense potential for sustainable energy and pharmaceutical production capabilities. In the last decades, the efforts attended to enhance the usage of microalgae and cyanobacteria by genetic manipulation, synthetic and metabolic engineering. However, the development of photoautotrophic cell factories have rarely compared to the heterotrophic counterparts due to limited tools, bioinformatics, and multi-omics database. Therefore, recent advances of their genome editing techniques by clustered regularly interspaced short palindromic repeats (CRISPR) technology, and potential applications of their metabolic engineering and regulation approaches are examined in this review. Moreover, the contemporary achievements of synthetic biology approaches of microalgae and cyanobacteria in carbon fixation and sequestration, lipid and triacylglycerol (TAG), and sustainable production of high value-added chemicals, such as carotenoids and docosahexaenoic acid (DHA), have been also discussed. From recent genomic study to trends in metabolic regulation of microalgae and cyanobacteria and a comprehensive assessment of the current challenges and opportunities for microalgae and cyanobacteria is also conducted.

Quantification, regulation and production of 5-aminolevulinic acid by green fluorescent protein in recombinant Escherichia coli.

5-Aminolevulinic acid (5-ALA) is an unnatural amino acid and has been approved as a biodegradable, non-toxic pesticide and herbicide with applications in sustainable agriculture. 5-ALA can also be applied for cancer targeting via tumor localization and photodynamic therapy. Herein, we developed a feasible quantification, regulation and production method of 5-ALA in Escherichia coli is based on the chimera of 5-ALA synthetase from Rhodobacter sphaeroides (RshemA) and super-fold green fluorescent protein (sfGFP) under the control of dual promoters/double plasmids. 5-ALA production based on quantification with the reporter sfGFP was unsuccessfully for the RshemA-sfGFP fusion protein owing to a steric hindrance effect, but was effective using dual constitutive promoters (i.e., J23100 and PLacI) for RshemA and sfGFP independently. Moreover, a simple quantification method based on the linear relationship between 5-ALA concentration and the change in sfGFP intensity was calculated with the Hill equation according to the results of dual plasmids which composed of RshemA-threonine/homoserine exporter (RhtA) and the sensing plasmid pSU-T7-sfGFP. Compared with the conventional detection method for 5-ALA using Ehrlich's reagent, our proposed method is advantages in effectiveness, real-time detection, and outstanding sensitivity. Finally, the highest yield of 5-ALA was obtained in E. coli D2TT strain, reaching 2.46 g/L of 5-ALA produced in a 2.5-L baffle flask fermentation. Hence, this approach shows strong potential for improving 5-ALA production with appropriate regulation and detection based on the fluorescent signal.

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.

ARduino-pH Tracker and screening platform for characterization of recombinant carbonic anhydrase in Escherichia coli.

Carbonic anhydrase (CA, EC 4.2.1.1) is an ancient enzyme with zinc ion as its active site, which catalyzes the chemical reaction of carbon dioxide (CO2 ) to react with water and form bicarbonate ions. Due to its high catalytic efficiency on CO2 assimilation, CA is expected to use for carbon sequestration in industry. However, the protein expression level, thermostability and high-throughput screening of an active CA are still with difficulty. In this study, the CA from Sulfurihydrogenibium yellowstonense (denoted as SyCA) was selected for overexpressed in Escherichia coli by different pET vectors. The enzymatic properties including thermo-stability, pH tolerance, effect of metal ion, and kinetic parameters were characterized through a novel ARduino-pH Tracker (ART) for monitoring online effectively. The SyCA is thermophilic and acidophilic as it maintains 100% activity at 50°C, while the residual activity is 34.8% after heating at 80°C for 150 min and the optimal pH is 3-5. The kinetic analysis by ART system showed that the k cat /K m of free enzyme was 4.4-folds that that of whole cell. On the other hand, the screening platforms as Wilbur-Anderson unit, phenol red indicator and size of colony forming unit have been established to explore CA with higher activity. The high-throughput screening platform is support in direct evolution of CA and further used in the industry.

Recent Developments on Genetic Engineering of Microalgae for Biofuels and Bio-Based Chemicals.

Microalgae serve as a promising source for the production of biofuels and bio-based chemicals. They are superior to terrestrial plants as feedstock in many aspects and their biomass is naturally rich in lipids, carbohydrates, proteins, pigments, and other valuable compounds. Due to the relatively slow growth rate and high cultivation cost of microalgae, to screen efficient and robust microalgal strains as well as genetic modifications of the available strains for further improvement are of urgent demand in the development of microalgae-based biorefinery. In genetic engineering of microalgae, transformation and selection methods are the key steps to accomplish the target gene modification. However, determination of the preferable type and dosage of antibiotics used for transformant selection is usually time-consuming and microalgal-strain-dependent. Therefore, more powerful and efficient techniques should be developed to meet this need. In this review, the conventional and emerging genome-editing tools (e.g., CRISPR-Cas9, TALEN, and ZFN) used in editing the genomes of nuclear, mitochondria, and chloroplast of microalgae are thoroughly surveyed. Although all the techniques mentioned above demonstrate their abilities to perform gene editing and desired phenotype screening, there still need to overcome higher production cost and lower biomass productivity, to achieve efficient production of the desired products in microalgal biorefineries.