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.

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.

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.

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.

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.

Bio-solar cell factories for photosynthetic isoprenoids production.

MAIN CONCLUSION: Photosynthetic production of isoprenoids in cyanobacteria is considered in terms of metabolic engineering and biological importance. Metabolic engineering of photosynthetic bacteria (cyanobacteria) has been performed to construct bio-solar cell factories that convert carbon dioxide to various value-added chemicals. Isoprenoids, which are found in nature and range from essential cell components to defensive molecules, have great value in cosmetics, pharmaceutics, and biofuels. In this review, we summarize the recent engineering of cyanobacteria for photosynthetic isoprenoids production as well as carbon molar basis comparisons with heterotrophic isoprenoids production in engineered Escherichia coli.