Measuring the economic efficiency of laboratory automation in biotechnology.

Laboratory automation with robot-assisted processes enhances synthetic biology, but its economic impact on projects is uncertain. We have proposed an experiment price index (EPI) for a quantitative comparison of factors in time, cost, and sample numbers, helping measure the efficiency of laboratory automation in synthetic biology and biomolecular engineering.

CRISPR-dCas13a system for programmable small RNAs and polycistronic mRNA repression in bacteria.

Bacterial small RNAs (sRNAs) function in post-transcriptional regulatory responses to environmental changes. However, the lack of eukaryotic RNA interference-like machinery in bacteria has limited the systematic engineering of RNA repression. Here, we report the development of clustered regularly interspaced short palindromic repeats (CRISPR)-guided dead CRIPSR-associated protein 13a (dCas13a) ribonucleoprotein that utilizes programmable CRISPR RNAs (crRNAs) to repress trans-acting and cis-acting sRNA as the target, altering regulatory mechanisms and stress-related phenotypes. In addition, we implemented a modular loop engineering of the crRNA to promote modular repression of the target gene with 92% knockdown efficiency and a single base-pair mismatch specificity. With the engineered crRNAs, we achieved targetable single-gene repression in the polycistronic operon. For metabolic application, 102 crRNAs were constructed in the biofoundry and used for screening novel knockdown sRNA targets to improve lycopene (colored antioxidant) production in Escherichia coli. The CRISPR-dCas13a system will assist as a valuable systematic tool for the discovery of novel sRNAs and the fine-tuning of bacterial RNA repression in both scientific and industrial applications.

Cytosine base editing in cyanobacteria by repressing archaic Type IV uracil-DNA glycosylase.

Base editing enables precise gene editing without requiring donor DNA or double-stranded breaks. To facilitate base editing tools, a uracil DNA glycosylase inhibitor (UGI) was fused to cytidine deaminase-Cas nickase to inhibit uracil DNA glycosylase (UDG). Herein, we revealed that the bacteriophage PBS2-derived UGI of the cytosine base editor (CBE) could not inhibit archaic Type IV UDG in oligoploid cyanobacteria. To overcome the limitation of the CBE, dCas12a-assisted gene repression of the udg allowed base editing at the desired targets with up to 100% mutation frequencies, and yielded correct phenotypes of desired mutants in cyanobacteria. Compared with the original CBE (BE3), base editing was analyzed within a broader C4-C16 window with a strong TC-motif preference. Using multiplexed CyanoCBE, while udg was repressed, simultaneous base editing at two different sites was achieved with lower mutation frequencies than single CBE. Our discovery of a Type IV UDG that is not inhibited by the UGI of the CBE in cyanobacteria and the development of dCas12a-mediated base editing should facilitate the application of base editing not only in cyanobacteria, but also in archaea and green algae that possess Type IV UDGs. We revealed the bacteriophage-derived UGI of the base editor did not repress Type IV UDG in cyanobacteria. To overcome the limitation, orthogonal dCas12a interference was successfully applied to repress the UDG gene expression in cyanobacteria during base editing occurred, yielding a premature translational termination at desired targets. This study will open a new opportunity to perform base editing with Type IV UDGs in archaea and green algae.

High-Fidelity Cytosine Base Editing in a GC-Rich Corynebacterium glutamicum with Reduced DNA Off-Target Editing Effects.

Genome editing technology is a powerful tool for programming microbial cell factories. However, rat APOBEC1-derived cytosine base editor (CBE) that converts C•G to T•A at target genes induced DNA off-targets, regardless of single-guide RNA (sgRNA) sequences. Although the high efficiencies of the bacterial CBEs have been developed, a risk of unidentified off-targets impeded genome editing for microbial cell factories. To address the issues, we demonstrate the genome engineering of Corynebacterium glutamicum as a GC-rich model industrial bacterium by generating premature termination codons (PTCs) in desired genes using high-fidelity cytosine base editors (CBEs). Through this CBE-STOP approach of introducing specific cytosine conversions, we constructed several single-gene-inactivated strains for three genes (ldh, idsA, and pyc) with high base editing efficiencies of average 95.6% (n = 45, C6 position) and the highest success rate of up to 100% for PTCs and ultimately developed a strain with five genes (ldh, actA, ackA, pqo, and pta) that were inactivated sequentially for enhancing succinate production. Although these mutant strains showed the desired phenotypes, whole-genome sequencing (WGS) data revealed that genome-wide point mutations occurred in each strain and further accumulated according to the duration of CBE plasmids. To lower the undesirable mutations, high-fidelity CBEs (pCoryne-YE1-BE3 and pCoryne-BE3-R132E) was employed for single or multiplexed genome editing in C. glutamicum, resulting in drastically reduced sgRNA-independent off-targets. Thus, we provide a CRISPR-assisted bacterial genome engineering tool with an average high efficiency of 90.5% (n = 76, C5 or C6 position) at the desired targets. IMPORTANCE Rat APOBEC1-derived cytosine base editor (CBE) that converts C•G to T•A at target genes induced DNA off-targets, regardless of single-guide RNA (sgRNA) sequences. Although the high efficiencies of bacterial CBEs have been developed, a risk of unidentified off-targets impeded genome editing for microbial cell factories. To address the issues, we identified the DNA off-targets for single and multiple genome engineering of the industrial bacterium Corynebacterium glutamicum using whole-genome sequencing. Further, we developed the high-fidelity (HF)-CBE with significantly reduced off-targets with comparable efficiency and precision. We believe that our DNA off-target analysis and the HF-CBE can promote CRISPR-assisted genome engineering over conventional gene manipulation tools by providing a markerless genetic tool without need for a foreign DNA donor.

Co-production of l-Lysine and Heterologous Squalene in CRISPR/dCas9-Assisted Corynebacterium glutamicum.

Corynebacterium glutamicum is widely used for a large-scale industrial producer of feed additive amino acids, such as l-lysine. Moreover, C. glutamicum has been engineered for producing various non-native chemicals, including terpenes. For the first time, C. glutamicum was engineered for co-production of l-lysine and heterologous squalene. To control metabolic fluxes for either the l-lysine biosynthesis pathway or the squalene biosynthesis pathway, pyruvate, an intermediate in the central metabolism, a node was regulated by a clustered regularly interspaced short palindromic repeat (CRISPR) interference system. Repressing pyc encoding for pyruvate carboxylase in the l-lysine producer (DM1919) and its derivatives resulted in 99.24 ± 7.63 mg/L total squalene and 6.25 ± 0.20 g/L extracellular lysine at 120 h. Furthermore, various oil overlays were tested for efficient co-productions. In situ extraction with corn oil (10%, v/v) exhibited a separation of 99.75% (w/v) of total squalene (intra- and extracellular squalene), while l-lysine can be secreted in the medium. This co-production strategy will help a potential bioprocess of amino acid production with various terpenes.

Biofoundry Palette: Planning-Assistant Software for Liquid Handler-Based Experimentation and Operation in the Biofoundry Workflow.

Lab automation has facilitated synthetic biology applications in an automated workflow, and biofoundry facilities have enabled automated high-throughput experiments of gene cloning and genome engineering to be conducted following a precise experimental design and protocol. However, before-experiment procedures in biofoundry applications have been underdetermined. We aimed to develop a Python-based planning-assistant software, namely Biofoundry Palette, for liquid handler-based experimentation and operation in the biofoundry workflow. Depending on the synthetic biology project, variable information and content information may vary; the Biofoundry Palette provides precise information for the before-experiment units for each process module in the biofoundry workflow. As a demonstration, more than 200 unique information sets, generated by Biofoundry Palette, were used in automated gene cloning or pathway construction. The information on planning and management can potentially help the operator faithfully execute the biofoundry workflow after securing the before-experiment unit, thereby lowering the risk of human errors and performing successful biofoundry operations for synthetic biology applications.

DeepTESR: A Deep Learning Framework to Predict the Degree of Translational Elongation Short Ramp for Gene Expression Control.

Controlling translational elongation is essential for efficient protein synthesis. Ribosome profiling has revealed that the speed of ribosome movement is correlated with translational efficiency in the translational elongation ramp. In this work, we present a new deep learning model, called DeepTESR, to predict the degree of translational elongation short ramp (TESR) from mRNA sequence. The proposed deep learning model exhibited superior performance in predicting the TESR scores for 226 981 TESR sequences, resulting in the mean absolute error (MAE) of 0.285 and a coefficient of determination R2 of 0.627, superior to the conventional machine learning models (e.g., MAE of 0.335 and R2 of 0.571 for LightGBM). We experimentally validated that heterologous fluorescence expression of proteins with randomly selected TESR was moderately correlated with the predictions. Furthermore, a genome-wide analysis of TESR prediction in the 4305 coding sequences of Escherichia coli showed conserved TESRs over the clusters of orthologous groups. In this sense, DeepTESR can be used to predict the degree of TESR for gene expression control and to decipher the mechanism of translational control with ribosome profiling. DeepTESR is available at https://github.com/fmblab/DeepTESR.

RoboMoClo: A Robotics-Assisted Modular Cloning Framework for Multiple Gene Assembly in Biofoundry.

Efficient and versatile DNA assembly frameworks have had an impact on promoting synthetic biology to build complex biological systems. To accelerate system development, laboratory automation (or biofoundry) provides an opportunity to construct organisms and DNA assemblies via computer-aided design. However, a modular cloning (MoClo) system for multiple DNA assemblies limits the biofoundry workflow in terms of simplicity and feasibility by preparing the number of cloning materials such as destination vectors prior to the automation process. Herein, we propose robot-assisted MoClo (RoboMoClo) to accelerate a synthetic biology project with multiple gene expressions at the biofoundry. The architecture of the RoboMoClo framework provides a hybrid strategy of hierarchical gene assembly and iterative gene assembly, and fewer destination vectors compared with other MoClo systems. An industrial bacterium, Corynebacterium glutamicum, was used as a model host for RoboMoClo. After building a biopart library (promoter and terminator; level 0) and evaluating its features (level 1), various transcriptional directions in multiple gene assemblies (level 2) were studied using the RoboMoClo vectors. Among the constructs, the convergent construct exhibited potential transcriptional interference through the collision of RNA polymerases. To study design of experiment-guided lycopene biosynthesis in C. glutamicum (levels 1, 2, and 3), the biofoundry-assisted multiple gene assembly was demonstrated as a proof-of-concept by constructing various sub-pathway units (level 2) and pathway units (level 3) for C. glutamicum. The RoboMoClo framework provides an improved MoClo toolkit for laboratory automation in a synthetic biology application.

Improved CO2-derived polyhydroxybutyrate (PHB) production by engineering fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 for potential utilization of flue gas.

Industrial application of cyanobacterial poly-ß-hydroxybutyrate (PHB) production from CO2 is currently challenged by slow growth rate and low photoautotrophic PHB productivity of existing cyanobacteria species. Herein, a novel PHB-producing cyanobacterial strain was developed by harnessing fast-growing cyanobacteria Synechococcus elongatus UTEX 2973 with introduction of heterologous phaCAB genes. Under photoautotrophic condition, the engineered strain produced 420 mg L-1 (16.7% of dry cell weight) with the highest specific productivity of 75.2 mg L-1 d-1. When compared with a native PHB producer Synechocystis PCC 6803 under nitrogen deprivation, the engineered strain exhibited 2.4-fold higher PHB productivity. The performance of the engineered strain was further demonstrated in large scale cultivation using photobioreactor and outdoor cultivation employing industrial flue gas as the sole carbon source. This study can provide a promising solution to address petroleum-based plastic waste and contribute to CO2 mitigation.

Biocontainment of Engineered Synechococcus elongatus PCC 7942 for Photosynthetic Production of α-Farnesene from CO2.

Biocontainment systems have been developed to mitigate the concerns regarding biosafety and environmental risk because of the possible escape of genetically modified organisms into the environment following large-scale outdoor cultivation. Here, we present a biocontainment system entailing genetically modified Synechococcus elongatus PCC 7942, also engineered for α-farnesene production using a de-evolutionary strategy. In this approach, the gene cluster encoding the ß-carboxysome and the associated carbon concentrating mechanism (CCM) were deleted in the α-farnesene-producing cyanobacteria, resulting in no cell growth and no α-farnesene production at ambient CO2 concentrations (100% air bubbling). However, cell growth and α-farnesene production were detected in the CCM-deficient strains at high CO2 concentrations (5% CO2 [v/v], 10% CO2 [v/v]), albeit at levels lower than those of the parental control. To overcome this limitation, the overexpression of carbonic anhydrase and bicarbonate transporter genes in the CCM-deficient strains restored cell growth and the production level of α-farnesene (5.0 ± 0.6 mg/L) to that of the parental control. The production of α-farnesene in the later strains strictly depended on CO2 concentration in the photobioreactor and did not rely on a chemical induction process. Thus, next generation bio-solar cell factories could be promoted with the suggested biocontainment system.

A Logic NAND Gate for Controlling Gene Expression in a Circadian Rhythm in Cyanobacteria.

To enable circadian control of gene expression in cyanobacteria, we constructed a genetic logic gate (NAND) using orthogonal promoters via modular CRISPR interference. The NAND gates were tested in Synechococcus elongatus PCC 7942 using a fluorescent reporter. The NAND gate dynamics were characterized based on the affinity of the dCas9 complex to the output element. Upon connecting tight gene repressions with the circadian promoter (the purF gene; peak expression at dawn), inversed peak expressions were obtained as an output of the NAND gate although the retroactivities were shown in the ON and OFF states. A dark-responsive genetic element of the NAND gate was also expanded to an AND gate in S. elongatus PCC 7942. These cyanobacterial NAND and AND gates could facilitate the control of gene expressions in dynamic metabolic engineering technologies, thereby enabling the cyanobacteria to serve as biosolar cell factories.

Biosynthesis of the Calorie-Free Sweetener Precursor ent-Kaurenoic Acid from CO2 Using Engineered Cyanobacteria.

To supply the sustainable calorie-free sweetener stevioside, synthetic photosynthetic bacteria were developed to produce ent-kaurenoic acid as a precursor of stevioside directly from CO2. By the use of a combinatorial and modular approach for gene expression, including a cytochrome P450 and the corresponding reductase, engineered Synechoccous elongatus PCC 7942 as a model cyanobacterium enabled the biosynthesis of ent-kaurenoic acid at 2.9 ± 0.01 mg L-1 from CO2. We found that the order of genes for expression was critical, producing ent-kaurenoic acid by balancing gene expressions and accumulation of the toxic intermediate in a cell. The engineered bacteria allowed the complete biosynthesis of ent-kaurenoic acid, and it will be used for stevioside biosynthesis from CO2 as a controlled fermentation.

Current understanding of the cyanobacterial CRISPR-Cas systems and development of the synthetic CRISPR-Cas systems for cyanobacteria.

Cyanobacteria are photosynthetic microorganisms that are capable of converting CO2 to value-added chemicals. Engineering of cyanobacteria with synthetic biology tools, including the CRISPR-Cas system, has allowed an opportunity for biological CO2 utilization. Here, we described natural CRISPR-Cas systems for understanding cyanobacterial genomics and synthetic CRISPR-Cas systems for metabolic engineering applications. The natural CRISPR-Cas systems in cyanobacteria have been identified as Class 1, with type I and III, and some Class 2, with type V, as an adaptive immune system against viral invasion. As synthetic tools, CRISPR-Cas9 and -Cas12a have been successfully established in cyanobacteria to delete a target gene without a selection marker. Deactivated Cas9 and Cas12a have also been used to repress genes for metabolic engineering. In addition, a perspective on how advanced CRISPR-Cas systems and a pool of the guide RNAs can be advantageous for precise genome engineering and understanding of unknown functions was discussed for advanced engineering of cyanobacteria.

Scalable Cultivation of Engineered Cyanobacteria for Squalene Production from Industrial Flue Gas in a Closed Photobioreactor.

Economically feasible photosynthetic cultivation of microalgal and cyanobacterial strains is crucial for the biological conversion of CO2 and potential CO2 mitigation to challenge global warming. To overcome the economic barriers, the production of value-added chemicals was desired by compensating for the overall processing cost. Here, we engineered cyanobacteria for photosynthetic squalene production and cultivated them in a scalable photobioreactor using industrial flue gas. First, an inducer-free gene expression system was developed for the cyanobacteria to lower production const. Then, the recombinant cyanobacteria were cultivated in a closed photobioreactor (100 L) using flue gas (5% CO2) as the sole carbon source under natural sunlight as the only energy source. Seasonal light intensities and temperatures were analyzed along with cyanobacterial cell growth and squalene production in August and October 2019. As a result, the effective irradiation hours were the most critical factor for the large-scale cultivation of cyanobacteria. Thus, an automated photobioprocess system will be developed based on the regional light sources.

Overexpression of the Key Enzymes in the Methylerythritol 4-phosphate Pathway in Corynebacterium glutamicum for Improving Farnesyl Diphosphate-Derived Terpene Production.

A systematic and combinatorial optimization has been employed to metabolically engineer microbes for identifying key gene targets for overexpression to increase the intermediate pools for terpenoid production. Herein, the methylerythritol 4-phosphate (MEP) pathway in Corynebacterium glutamicum, an industrial host, was investigated to identify the key genes whose overexpression would improve the production of farnesyl diphosphate (FPP)-derived terpenoids (squalene and α-farnesene). Using a combinatorial approach with the single, double, and triple expression of genes in the MEP pathway in a high-throughput fermentation, overexpression of the ispDF genes, along with the known dxs and idi genes, was most effective at increasing the squalene contents, i.e., by 14-fold. The dxr gene was identified as the key target enzyme for α-farnesene production. This result could provide fundamental information for improving the metabolic engineering of C. glutamicum for terpene production via an optimized MEP pathway.

CRISPRi-dCas12a: A dCas12a-Mediated CRISPR Interference for Repression of Multiple Genes and Metabolic Engineering in Cyanobacteria.

In cyanobacteria, metabolic engineering using synthetic biology tools is limited to build a biosolar cell factory that converts CO2 to value-added chemicals, as repression of essential genes has not been achieved. In this study, we developed a dCas12a-mediated CRISPR interference system (CRISPRi-dCas12a) in cyanobacteria that effectively blocked the transcriptional initiation by means of a CRISPR-RNA (crRNA) and 19-nt direct repeat, resulting in 53-94% gene repression. The repression of multiple genes in a single crRNA array was also successfully achieved without a loss in repression strength. In addition, as a demonstration of the dCas12a-mediated CRISPRi for metabolic engineering, photosynthetic squalene production was improved by repressing the essential genes of either acnB encoding for aconitase or cpcB2 encoding for phycocyanin ß-subunit in Synechococcus elongatus PCC 7942. The ability to regulate gene repression will promote the construction of biosolar cell factories to produce value-added chemicals.

Metabolic rewiring of synthetic pyruvate dehydrogenase bypasses for acetone production in cyanobacteria.

Designing synthetic pathways for efficient CO2 fixation and conversion is essential for sustainable chemical production. Here we have designed a synthetic acetate-acetyl-CoA/malonyl-CoA (AAM) bypass to overcome an enzymatic activity of pyruvate dehydrogenase complex. This synthetic pathway utilizes acetate assimilation and carbon rearrangements using a methyl malonyl-CoA carboxyltransferase. We demonstrated direct conversion of CO2 into acetyl-CoA-derived acetone as an example in photosynthetic Synechococcus elongatus PCC 7942 by increasing the acetyl-CoA pools. The engineered cyanobacterial strain with the AAM-bypass produced 0.41 g/L of acetone at 0.71 m/day of molar productivity. This work clearly shows that the synthetic pyruvate dehydrogenase bypass (AAM-bypass) is a key factor for the high-level production of an acetyl-CoA-derived chemical in photosynthetic organisms.

Case study of xylose conversion to glycolate in Corynebacterium glutamicum: Current limitation and future perspective of the CRISPR-Cas systems.

RNA-guided genome engineering technologies have been developed for the advanced metabolic engineering of microbial cells to enhance the production of value-added chemicals in Corynebacterium glutamicum as an industrial host. Here, we described the biotransformation of xylose to glycolate using engineered Corynebacterium glutamicum, a well-known industrial amino acid producer. A synthetic pathway involving heterologous D-tagatose 3-epimerase and L-fuculose kinase/aldolase reactions was introduced in C. glutamicum, resulting in 9.9 ±â€¯0.01 g/L glycolate from 20 g/L xylose at a yield of 0.51 g/g (equal to 1.0 mol/mol). Additional glyoxylate reduction pathway developed by CRISPR-Cas12a recombineering has been introduced and attempted to increase the maximum theoretical molar yield of 2.0 (mol/mol). Due to the limitation of the CRISPR-Cas12a recombineering with TTTV PAM sites, advanced CRISPR-Cas systems were suggested for the next-round metabolic engineering for improving the glycolate yield to overcome the current genome-editing tool for metabolic engineering in C. glutamicum.

Bioconversion of Xylose to Ethylene Glycol and Glycolate in Engineered Corynebacterium glutamicum.

The biological production of two-carbon compounds (ethylene glycol (EG) and glycolate) has been studied for the sustainable supply of the compounds to the polymer, cosmetic, textile, and medical industries. Here, we demonstrated the bioconversion of xylose to either ethylene glycol (EG) or glycolate using engineered Corynebacterium glutamicum, a well-known industrial amino acid producer. A synthetic ribulose 1-phosphate (Ru1P) pathway involving heterologous d-tagatose 3-epimerase and l-fuculose kinase/aldolase reactions was introduced in C. glutamicum. Subsequently, heterologous expression of Escherichia coli YqhD reductase with the synthetic Ru1P pathway led to ethylene glycol production from xylose. Additional pathway engineering in C. glutamicum by mutating ald, which encodes an aldehyde dehydrogenase, abolished the by-product formation of glycolate during xylose conversion to EG at a yield of 0.75 mol per mol. In addition, the bioconversion of xylose to glycolate was achieved, and the almost maximum molar yield was 0.99 mol per mol xylose in C. glutamicum via the Ru1P pathway. Thus, the synthetic Ru1P pathway in C. glutamicum led bioconversion of xylose to either ethylene glycol or glycolate with high molar yields.