Establishment and application of a CRISPR-Cas12a assisted genome-editing system in Zymomonas mobilis.

BACKGROUND: Efficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking. RESULTS: Using Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR-Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR-Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR-Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed. CONCLUSIONS: This study applied CRISPR-Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR-Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.

Biosensor-assisted CRISPRi high-throughput screening to identify genetic targets in Zymomonas mobilis for high d-lactate production.

Lactate is an important monomer for the synthesis of poly-lactate (PLA), which is a substitute for the petrochemical plastics. To achieve the goal of high lactate titer, rate, and yield for commercial production, efficient lactate production pathway is needed as well as genetic targets that affect high lactate production and tolerance. In this study, an LldR-based d-lactate biosensor with a broad dynamic range was first applied into Zymomonas mobilis to select mutant strains with strong GFP fluorescence, which could be the mutant strains with increased d-lactate production. Then, LldR-based d-lactate biosensor was combined with a genome-wide CRISPR interference (CRISPRi) library targeting the entire genome to generate thousands of mutants with gRNA targeting different genetic targets across the whole genome. Specifically, two mutant libraries were selected containing 105 and 104 mutants with different interference sites from two rounds of fluorescence-activated cell sorting (FACS), respectively. Two genetic targets of ZMO1323 and ZMO1530 were characterized and confirmed to be associated with the increased d-lactate production, further knockout of ZMO1323 and ZMO1530 resulted in a 15% and 21% increase of d-lactate production, respectively. This work thus not only established a high-throughput approach that combines genome-scale CRISPRi and biosensor-assisted screening to identify genetic targets associated with d-lactate production in Z. mobilis, but also provided a feasible high-throughput screening approach for rapid identification of genetic targets associated with strain performance for other industrial microorganisms.

Identification and Characterization of Genes Related to Ampicillin Antibiotic Resistance in Zymomonas mobilis.

Antibiotics can inhibit or kill microorganisms, while microorganisms have evolved antibiotic resistance strategies to survive antibiotics. Zymomonas mobilis is an ideal industrial microbial chassis and can tolerate multiple antibiotics. However, the mechanisms of antibiotic resistance and genes associated with antibiotic resistance have not been fully analyzed and characterized. In this study, we investigated genes associated with antibiotic resistance using bioinformatic approaches and examined genes associated with ampicillin resistance using CRISPR/Cas12a-based genome-editing technology. Six ampicillin-resistant genes (ZMO0103, ZMO0893, ZMO1094, ZMO1650, ZMO1866, and ZMO1967) were identified, and five mutant strains ZM4∆0103, ZM4∆0893, ZM4∆1094, ZM4∆1650, and ZM4∆1866 were constructed. Additionally, a four-gene mutant ZM4∆ARs was constructed by knocking out ZMO0103, ZMO0893, ZMO1094, and ZMO1650 continuously. Cell growth, morphology, and transformation efficiency of mutant strains were examined. Our results show that the cell growth of ZM4∆0103 and ZM4∆ARs was significantly inhibited with 150 µg/mL ampicillin, and cells changed to a long filament shape from a short rod shape. Moreover, the transformation efficiencies of ZM4∆0103 and ZM4∆ARs were decreased. Our results indicate that ZMO0103 is the key to ampicillin resistance in Z. mobilis, and other ampicillin-resistant genes may have a synergetic effect with it. In summary, this study identified and characterized genes related to ampicillin resistance in Z. mobilis and laid a foundation for further study of other antibiotic resistance mechanisms.

A plasmid-free Zymomonas mobilis mutant strain reducing reactive oxygen species for efficient bioethanol production using industrial effluent of xylose mother liquor.

Genome minimization is an effective way for industrial chassis development. In this study, Zymomonas mobilis ZMNP, a plasmid-free mutant strain of Z. mobilis ZM4 with four native plasmids deleted, was constructed using native type I-F CRISPR-Cas system. Cell growth of ZMNP under different temperatures and industrial effluent of xylose mother liquor were examined to investigate the impact of native plasmid removal. Despite ZMNP grew similarly as ZM4 under different temperatures, ZMNP had better xylose mother liquor utilization than ZM4. In addition, genomic, transcriptomic, and proteomic analyses were applied to unravel the molecular changes between ZM4 and ZMNP. Whole-genome resequencing result indicated that an S267P mutation in the C-terminal of OxyR, a peroxide-sensing transcriptional regulator, probably alters the transcription initiation of antioxidant genes for stress responses. Transcriptomic and proteomic studies illustrated that the reason that ZMNP utilized the toxic xylose mother liquor better than ZM4 was probably due to the upregulation of genes in ZMNP involving in stress responses as well as cysteine biosynthesis to accelerate the intracellular ROS detoxification and nucleic acid damage repair. This was further confirmed by lower ROS levels in ZMNP compared to ZM4 in different media supplemented with furfural or ethanol. The upregulation of stress response genes due to the OxyR mutation to accelerate ROS detoxification and DNA/RNA repair not only illustrates the underlying mechanism of the robustness of ZMNP in the toxic xylose mother liquor, but also provides an idea for the rational design of synthetic inhibitor-tolerant microorganisms for economic lignocellulosic biochemical production.

Synthetic refactor of essential genes decodes functionally constrained sequences in yeast genome.

The relationship between gene sequence and function matters for fundamental and practical reasons. Here, yeast essential genes were systematically refactored to identify invariable sequences in the coding and regulatory regions. The coding sequences were synonymously recoded with all optimal codons to explore the importance of codon choice. The promoters and terminators were swapped with well-characterized CYC1 promoter and terminator to examine whether a specialized expression is required for the function of a specific gene. Among the 10 essential genes from Chr.XIIL, this scheme successfully generated 7 refactored genes that can effectively support wild-type-like fitness under various conditions, thereby revealing amazing sequence plasticity of yeast genes. Moreover, different invariable elements were identified from the remaining 3 genes, exampling the logics for genetic information encoding and regulation. Further refactoring of all essential genes using this strategy will generate comprehensive understanding of gene sequence choice, thereby guiding its design in various applications.

[Progress and perspective on development of non-model industrial bacteria as chassis cells for biochemical production in the synthetic biology era].

The development and implement of microbial chassis cells can provide excellent cell factories for diverse industrial applications, which help achieve the goal of environmental protection and sustainable bioeconomy. The synthetic biology strategy of Design-Build-Test-Learn (DBTL) plays a crucial role on rational and/or semi-rational construction or modification of chassis cells to achieve the goals of "Building to Understand" and "Building for Applications". In this review, we briefly comment on the technical development of the DBTL cycle and the research progress of a few model microorganisms. We mainly focuse on non-model bacterial cell factories with potential industrial applications, which possess unique physiological and biochemical characteristics, capabilities of utilizing one-carbon compounds or of producing platform compounds efficiently. We also propose strategies for the efficient and effective construction and application of synthetic microbial cell factories securely in the synthetic biology era, which are to discover and integrate the advantages of model and non-model industrial microorganisms, to develop and deploy intelligent automated equipment for cost-effective high-throughput screening and characterization of chassis cells as well as big-data platforms for storing, retrieving, analyzing, simulating, integrating, and visualizing omics datasets at both molecular and phenotypic levels, so that we can build both high-quality digital cell models and optimized chassis cells to guide the rational design and construction of microbial cell factories for diverse industrial applications.

Whole genome engineering by synthesis.

Whole genome engineering is now feasible with the aid of genome editing and synthesis tools. Synthesizing a genome from scratch allows modifications of the genomic structure and function to an extent that was hitherto not possible, which will finally lead to new insights into the basic principles of life and enable valuable applications. With several recent genome synthesis projects as examples, the technical details to synthesize a genome and applications of synthetic genome are addressed in this perspective. A series of ongoing or future synthetic genomics projects, including the different genomes to be synthesized in GP-write, synthetic minimal genome, massively recoded genome, chimeric genome and synthetic genome with expanded genetic alphabet, are also discussed here with a special focus on theoretical and technical impediments in the design and synthesis process. Synthetic genomics will become a commonplace to engineer pathways and genomes according to arbitrary sets of design principles with the development of high-efficient, low-cost genome synthesis and assembly technologies.