Transcription factor shapes chromosomal conformation and regulates gene expression in bacterial adaptation.

Genomic mutations allow bacteria to adapt rapidly to adverse stress environments. The three-dimensional conformation of the genome may also play an important role in transcriptional regulation and environmental adaptation. Here, using chromosome conformation capture, we investigate the high-order architecture of the Zymomonas mobilis chromosome in response to genomic mutation and ambient stimuli (acetic acid and furfural, derived from lignocellulosic hydrolysate). We find that genomic mutation only influences the local chromosome contacts, whereas stress of acetic acid and furfural restrict the long-range contacts and significantly change the chromosome organization at domain scales. Further deciphering the domain feature unveils the important transcription factors, Ferric uptake regulator (Fur) proteins, which act as nucleoid-associated proteins to promote long-range (>200 kb) chromosomal communications and regulate the expression of genes involved in stress response. Our work suggests that ubiquitous transcription factors in prokaryotes mediate chromosome organization and regulate stress-resistance genes in bacterial adaptation.

Rhizosphere metabolic cross-talk from plant-soil-microbe tapping into agricultural sustainability: Current advance and perspectives.

Rhizosphere interactions from plant-soil-microbiome occur dynamically all the time in the "black microzone" underground, where we can't see intuitively. Rhizosphere metabolites including root exudates and microbial metabolites act as various chemical signalings involving in rhizosphere interactions, and play vital roles on plant growth, development, disease suppression and resistance to stress conditions as well as proper soil health. Although rhizosphere metabolites are a mixture from plant roots and soil microbes, they often are discussed alone. As a rapid appearance of various omics platforms and analytical methods, it offers possibilities and opportunities for exploring rhizosphere interactions in unprecedented breadth and depth. However, our comprehensive understanding about the fine-tuning mechanisms of rhizosphere interactions mediated by these chemical compounds still remain clear. Thus, this review summarizes recent advances systemically including the features of rhizosphere metabolites and their effects on rhizosphere ecosystem, and looks forward to the future research perspectives, which contributes to facilitating better understanding of biochemical communications belowground and helping identify novel rhizosphere metabolites. We also address challenges for promoting the understanding about the roles of rhizosphere metabolites in different environmental stresses.

Simultaneous production of algal biomass and lipid by heterotrophic cultivation of linoleic acid-rich oleaginous microalga Chlorella sorokiniana using high acetate dosage.

The low-cost carbon source, acetate, was utilized to feed a linoleic acid-rich Chlorella sorokiniana for microalgal biomass and lipid accumulation. Remarkably high tolerance capability to high acetate dosage up to 30 g/L was observed, with heterotrophy being the preferred trophic mode for algal growth and lipogenesis when supplemented 20 g/L acetate. Transcriptome analysis revealed a marked activation of pathways involved in acetate bioconversion and lipogenesis upon exposure to high-level of acetate. However, the enhancement of photorespiration inhibited photosynthesis, which ultimately led to a decrease in biomass and lipid under mixotrophy. Heterotrophic acetate-feeding generated more superior amino acid profiling of algal biomass and a predominant linoleic acid content (50 %). Heterotrophic repeat fed-batch strategy in 5 L fermenter significantly increased the growth performance and lipid titer, with the highest levels achieved being 23.4 g/L and 7.0 g/L, respectively. This work provides a viable approach for bio-products production through acetate-based heterotrophic algal cultivation.

Economic co-production of cellulosic ethanol and microalgal biomass through efficient fixation of fermentation carbon dioxide.

An integrated process for the co-production of cellulosic ethanol and microalgal biomass by fixing CO2 generated from bioethanol fermentation is proposed. Specifically, over one-fifth of the fermentative carbon was converted into high-purity CO2 during ethanol production. The optimal concentration of 4 % CO2 was identified for the growth and metabolism of Chlorella sp. BWY-1. A multiple short-term intermittent CO2 supply system was established to efficiently fix and recycle the waste CO2. Using this system, economical co-production of cellulosic ethanol by Zymomonas mobilis and microalgal biomass in biogas slurry wastewater was achieved, resulting in the production of ethanol at a rate of 0.4 g/L/h and a fixed fermentation CO2 of 3.1 g/L/d. Moreover, the amounts of algal biomass and chlorophyll a increased by over 50 % and two-fold, respectively. Through techno-economic analysis, the integrated process demonstrated its cost-effectiveness for cellulosic ethanol production. This study presents an innovative approach to a low-carbon circular bioeconomy.

Elimination of editing plasmid mediated by theophylline riboswitch in Zymomonas mobilis.

Zymomonas mobilis is regarded as a potential chassis for the production of platform chemicals. Genome editing using the CRISPR-Cas system could meet the need for gene modification in metabolic engineering. However, the low curing efficiency of CRISPR editing plasmid is a common bottleneck in Z. mobilis. In this study, we utilized a theophylline-dependent riboswitch to regulate the expression of the replicase gene of the editing plasmid, thereby promoting the elimination of exogeneous plasmid. The riboswitch D (RSD) with rigorous regulatory ability was identified as the optimal candidate by comparing the transformation efficiency of four theophylline riboswitch-based backbone editing plasmids, and the optimal theophylline concentration for inducing RSD was determined to be 2 mM. A highly effective method for eliminating the editing plasmid, cells with RSD-based editing plasmid which were cultured in liquid and solid RM media in alternating passages at 37 °C without shaking, was established by testing the curing efficiency of backbone editing plasmids pMini and pMini-RSD in RM medium with or without theophylline at 30 °C or 37 °C. Finally, the RSD-based editing plasmid was applied to genome editing, resulting in an increase of more than 10% in plasmid elimination efficiency compared to that of pMini-based editing plasmid. KEY POINTS: • An effective strategy for curing CRISPR editing plasmid has been established in Z. mobilis. • Elimination efficiency of the CRISPR editing plasmid was enhanced by 10% to 20% under the regulation of theophylline-dependent riboswitch RSD.

Valorization of waste streams and C1 gases for sustainable food nutrients and value-added compounds production: Acetate as a promising intermediate.

Resource recovery from waste streams and C1 gaseous substrates (CO2, CO and CH4) are of extensive interest due to the insufficient utilization and threats to the environment. From a perspective of sustainability, valorization of waste streams and C1 gases into target energy-rich value-added products in a sustainable way offers tempting approaches for simultaneously alleviating the environmental problems and achieving a circular carbon economy, while it still suffers from the complicated compositions of feedstocks or the low solubility of gaseous feeds. Recently, a C2 feedstock-based biomanufacturing serving acetate as potential next-generation platform has received much attention, where different gaseous or cellulosic wastes are recycling into acetate and then be further processed into a wide range of valuable long-chain compounds. The different alternative waste-processing technologies that are being developed to generate acetate from various wastes or gaseous substrates are summarized, in which gas fermentation and electrochemical reduction from CO2 represent the most promising routes for achieving high acetate yield. The recent advances and innovations in metabolic engineering for acetate bioconversion into various bioproducts ranging from food nutrients to value-added compounds were then highlighted. The challenges and promising strategies to reinforce microbial acetate conversion were also proposed, which conferred a new horizon for future food and chemical manufacturing with reduced carbon footprint.

Molecular mechanism of engineered Zymomonas mobilis to furfural and acetic acid stress.

Acetic acid and furfural (AF) are two major inhibitors of microorganisms during lignocellulosic ethanol production. In our previous study, we successfully engineered Zymomonas mobilis 532 (ZM532) strain by genome shuffling, but the molecular mechanisms of tolerance to inhibitors were still unknown. Therefore, this study investigated the responses of ZM532 and its wild-type Z. mobilis (ZM4) to AF using multi-omics approaches (transcriptomics, genomics, and label free quantitative proteomics). Based on RNA-Seq data, two differentially expressed genes, ZMO_RS02740 (up-regulated) and ZMO_RS06525 (down-regulated) were knocked out and over-expressed through CRISPR-Cas technology to investigate their roles in AF tolerance. Overall, we identified 1865 and 14 novel DEGs in ZM532 and wild-type ZM4. In contrast, 1532 proteins were identified in ZM532 and wild-type ZM4. Among these, we found 96 important genes in ZM532 involving acid resistance mechanisms and survival rates against stressors. Furthermore, our knockout results demonstrated that growth activity and glucose consumption of mutant strains ZM532∆ZMO_RS02740 and ZM4∆ZMO_RS02740 decreased with increased fermentation time from 42 to 55 h and ethanol production up to 58% in ZM532 than that in ZM532∆ZMO_RS02740. Hence, these findings suggest ZMO_RS02740 as a protective strategy for ZM ethanol production under stressful conditions.

The escape of CRISPR-mediated gene editing in Zymomonas mobilis.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems have been widely applied for gene or genome editing. Adequate checking is important to screen mutants after CRISPR-mediated editing events. Here, we report gene escape cases after the knockout by Type I-F native CRISPR system in Zymomonas mobilis. Through amplifying both the gene of interest and its flanking homologous arms, followed by curing the editing plasmid, we found different destinies for gene-editing events. Some genes were readily knocked out and followed by the easy plasmid curing. In some other cases, however, the editing plasmid was difficult to remove from the cell, or the deleted genes were transferred into the editing plasmid. For example, the targeted region of fur can be integrated into the editing plasmid after the knockout, resulting in a spurious editing event. We supposed that the transfer of the gene may be attributed to bacterial insertion sequences. Searching for literatures on the gene knockout using CRISPR in bacteria reveals that the escape event is likely underestimated due to inadequate validation in other microbes. Hence, several strategies are proposed to enhance gene knockout and plasmid curing.

Highly efficient reduction of ammonia emissions from livestock waste by the synergy of novel manure acidification and inhibition of ureolytic bacteria.

The global livestock system is one of the largest sources of ammonia emissions and there is an urgent need for ammonia mitigation. Here, we designed and constructed a novel strategy to abate ammonia emissions via livestock manure acidification based on a synthetic lactic acid bacteria community (LAB SynCom). The LAB SynCom possessed a wide carbon source spectrum and pH profile, high adaptability to the manure environment, and a high capability of generating lactic acid. The mitigation strategy was optimized based on the test and performance by adjusting the LAB SynCom inoculation ratio and the adding frequency of carbon source, which contributed to a total ammonia reduction efficiency of 95.5 %. Furthermore, 16S rDNA amplicon sequencing analysis revealed that the LAB SynCom treatment reshaped the manure microbial community structure. Importantly, 22 manure ureolytic microbial genera and urea hydrolysis were notably inhibited by the LAB SynCom treatment during the treatment process. These findings provide new insight into manure acidification that the conversion from ammonia to ammonium ions and the inhibition of ureolytic bacteria exerted a synergistic effect on ammonia mitigation. This work systematically developed a novel strategy to mitigate ammonia emissions from livestock waste, which is a crucial step forward from traditional manure acidification to novel and environmental-friendly acidification.

The potential use of Zymomonas mobilis for the food industry.

Zymomonas mobilis is a gram-negative facultative anaerobic spore, which is generally recognized as a safe. As a promising ethanologenic organism for large-scale bio-ethanol production, Z. mobilis has also shown a good application prospect in food processing and food additive synthesis for its unique physiological characteristics and excellent industrial characteristics. It not only has obvious advantages in food processing and becomes the biorefinery chassis cell for food additives, but also has a certain healthcare effect on human health. Until to now, most of the research is still in theory and laboratory scale, and further research is also needed to achieve industrial production. This review summarized the physiological characteristics and advantages of Z. mobilis in food industry for the first time and further expounds its research status in food industry from three aspects of food additive synthesis, fermentation applications, and prebiotic efficacy, it will provide a theoretical basis for its development and applications in food industry. This review also discussed the shortcomings of its practical applications in the current food industry, and explored other ways to broaden the applications of Z. mobilis in the food industry, to promote its applications in food processing.

Potential applications of Zymomonas mobilis in food industry summarized for the first time.Research status of Z. mobilis in food additive synthesis, fermentation applications, and probiotics are discussed in details.Future research perspectives of Z. mobilis in food industry further proposed.

Enhanced biomass and lipid production by light exposure with mixed culture of Rhodotorula glutinis and Chlorella vulgaris using acetate as sole carbon source.

Microbial biomass and lipid production with mixed-culture of Rhodotorula glutinis and Chlorella vulgaris using acetate as sole carbon source was investigated. Synergistic effect of mixed-culture using 20 g/L acetate significantly promoted cell growth and acetate utilization efficiency. Increasing the proportion of algae in co-culture was beneficial for biomass and lipid accumulation and the optimal ratio of yeast/algae was 1:2. Light exposure further enhanced biomass and lipid titer with 6.9 g/L biomass and 2.6 g/L lipid (38.3 % lipid content) obtained in a 5L bioreactor. The results of lipid classes and fatty acid profiles moreover indicated that more neutral lipids and linolenic acid were synthesized in mixed-culture under light exposure condition, suggesting the great potential in applications of biofuels production. This study provided new insight and strategy for economical microbial biomass and lipid production by light-exposed mixed-culture using inexpensive acetate as carbon source.

D-Lactic acid production from agricultural residues by membrane integrated continuous fermentation coupled with B vitamin supplementation.

BACKGROUND: D-Lactic acid played an important role in the establishment of PLA as a substitute for petrochemical plastics. But, so far, the D-lactic acid production was limited in only pilot scale, which was definitely unable to meet the fast growing market demand. To achieve industrial scale D-lactic acid production, the cost-associated problems such as high-cost feedstock, expensive nutrient sources and fermentation technology need to be resolved to establish an economical fermentation process. RESULTS: In the present study, the combined effect of B vitamin supplementation and membrane integrated continuous fermentation on D-lactic acid production from agricultural lignocellulosic biomass by Lactobacillus delbrueckii was investigated. The results indicated the specific addition of vitamins B1, B2, B3 and B5 (VB1, VB2, VB3 and VB5) could reduce the yeast extract (YE) addition from 10 to 3 g/l without obvious influence on fermentation efficiency. By employing cell recycling system in 350 h continuous fermentation with B vitamin supplementation, YE addition was further reduced to 0.5 g/l, which resulted in nutrient source cost reduction of 86%. A maximum D-lactate productivity of 18.56 g/l/h and optical purity of 99.5% were achieved and higher than most recent reports. CONCLUSION: These findings suggested the novel fermentation strategy proposed could effectively reduce the production cost and improve fermentation efficiency, thus exhibiting great potential in promoting industrial scale D-lactic acid production from lignocellulosic biomass.

Bioenergy production in Pakistan: Potential, progress, and prospect.

Pakistan is a developing country with a rapidly growing population. It is currently facing serious economic and energy challenges. Pakistan's energy demand is increasing by the day, and it now stands at 84 MTOE. Currently, the use of fossil fuels dominates Pakistan's energy sector. Conversely, indigenous fossil fuel resources are rapidly depleting and will be unable to meet rising energy demands in the future. Therefore, to withstand its energy needs, the country will need to explore alternative energy production methods. Biomass is one of the alternatives that has enormous potential to help Pakistan combat its growing energy crisis. In this review, we first present an overview of bioenergy, biomass resources, and biomass conversion technologies. We then discuss in detail the current state of the energy mix of Pakistan. Subsequently, we show that annual production of about 121 MT of agricultural residues, 427 MT of animal manure, and 7.5 MT of MSW in Pakistan offer a variety of bioenergy options ranging from biofuels to bio-electricity production. Overall, these biomass resources in Pakistan have the potential to generate 20,709 MW of bio-electricity and 12,615 million m3 of biogas annually in Pakistan. Though these resources hold promising potential for bioenergy production in the country, however, there are some critical challenges that need to be considered, and some of which are extremely difficult to overcome for a developing country like Pakistan. This work is expected to provide a useful basis for biomass management and utilization in Pakistan to harvest eco-friendly and sustainable green energy locally.

Molecular mechanism of enhanced ethanol tolerance associated with hfq overexpression in Zymomonas mobilis.

Zymomonas mobilis is a promising microorganism for industrial bioethanol production. However, ethanol produced during fermentation is toxic to Z. mobilis and affects its growth and bioethanol production. Although several reports demonstrated that the RNA-binding protein Hfq in Z. mobilis contributes to the tolerance against multiple lignocellulosic hydrolysate inhibitors, the role of Hfq on ethanol tolerance has not been investigated. In this study, hfq in Z. mobilis was either deleted or overexpressed and their effects on cell growth and ethanol tolerance were examined. Our results demonstrated that hfq overexpression improved ethanol tolerance of Z. mobilis, which is probably due to energy saving by downregulating flagellar biosynthesis and heat stress response proteins, as well as reducing the reactive oxygen species induced by ethanol stress via upregulating the sulfate assimilation and cysteine biosynthesis. To explore proteins potentially interacted with Hfq, the TEV protease mediated Yeast Endoplasmic Reticulum Sequestration Screening system (YESS) was established in Z. mobilis. YESS results suggested that Hfq may modulate the cytoplasmic heat shock response by interacting with the heat shock proteins DnaK and DnaJ to deal with the ethanol inhibition. This study thus not only revealed the underlying mechanism of enhanced ethanol tolerance by hfq overexpression, but also provided an alternative approach to investigate protein-protein interactions in Z. mobilis.

Enhancing Secretion of Endoglucanase in Zymomonas mobilis by Disturbing Peptidoglycan Synthesis.

Zymomonas mobilis (Z. mobilis) is a potential candidate strain for consolidated bioprocessing (CBP) in lignocellulosic biorefinery. However, the low-level secretion of cellulases limits this CBP process, and the mechanism of protein secretion that is affected by cell wall peptidoglycan is also not well understood. Here, we constructed several penicillin-binding protein (PBP)-deficient strains derived from Z. mobilis S192 to perturb the cell wall peptidoglycan network and then investigated the effects of peptidoglycan on the endoglucanase secretion. The results showed that extracellular recombinant endoglucanase production was significantly enhanced in PBP mutant strains, notably, Δ1089/0959 (4.09-fold) and Δ0959 (5.76-fold) in comparison to parent strains. For PBP-deficient strains, the growth performance was not significantly inhibited, but cell morphology was altered. In addition, enhanced antibiotic sensitivity and reduced inhibitor tolerance were also detected in our study. The concentration of intracellular soluble peptidoglycan was increased, especially for single-gene deletion. Outer membrane permeability of PBP-deficient strains was also improved, notably, Δ1089/0959 (1.14-fold) and Δ0959 (1.07-fold), which might explain the increased endoglucanase extracellular secretion. Our findings indicated that PBP-deficient Z. mobilis was capable of increasing endoglucanase extracellular secretion via cell wall peptidoglycan disturbance, and it will provide a foundation for the development of CBP technology in Z. mobilis in the future. IMPORTANCE Cell wall peptidoglycan has the function to maintain cell robustness and acts as the barrier to secret recombinant proteins from the cytoplasm to extracellular space in Z. mobilis and other bacteria. Herein, we perturbed the peptidoglycan synthesis network via knocking out PBPs (ZMO0197, ZMO0959, ZMO1089) to enhance recombinant endoglycanase extracellular secretion in Z. mobilis S912. This study could lay the foundation for understanding the regulatory network of cell wall synthesis and guide the construction of CBP strains in Z. mobilis.

Functional Gene Identification and Corresponding Tolerant Mechanism of High Furfural-Tolerant Zymomonas mobilis Strain F211.

Furfural is a major inhibitor in lignocellulose hydrolysate for Zymomonas mobilis. A mutant F211 strain with high furfural tolerance was obtained from our previous study. Thus, its key tolerance mechanism was studied in the present study. The function of mutated genes in F211 was identified by functional complementation experiments, revealing that the improved furfural tolerance was resulted from the C493T mutation of the ZCP4_0270 gene promoting cell flocculation and the mutation (G1075A)/downregulation of ZCP4_0970. Comparative transcriptome analysis revealed 139 differentially expressed genes between F211 and the control, CP4, in response to furfural stress. In addition, the reliability of the RNA-Seq data was also confirmed. The potential tolerance mechanism was further demonstrated by functional identification of tolerance genes as follows: (I) some upregulated or downregulated genes increase the levels of NAD(P)H, which is involved in the reduction of furfural to less toxic furfuryl alcohol, thus accelerating the detoxification of furfural; (II) the mutated ZCP4_0270 and upregulated cellulose synthetase gene (ZCP4_0241 and ZCP4_0242) increased flocculation to resist furfural stress; (III) upregulated molecular chaperone genes promote protein synthesis and repair stress-damaged proteins; and (IV) transporter genes ZCP4_1623-1,625 and ZCP4_1702-1703 were downregulated, saving energy for cell growth. The furfural-tolerant mechanism and corresponding functional genes were revealed, which provides a theoretical basis for developing robust chassis strains for synthetic biology efforts.

Genome-Wide Analyses of Proteome and Acetylome in Zymomonas mobilis Under N2-Fixing Condition.

Zymomonas mobilis, a promising candidate for industrial biofuel production, is capable of nitrogen fixation naturally without hindering ethanol production. However, little is known about the regulation of nitrogen fixation in Z. mobilis. We herein conducted a high throughput analysis of proteome and protein acetylation in Z. mobilis under N2-fixing conditions and established its first acetylome. The upregulated proteins mainly belong to processes of nitrogen fixation, motility, chemotaxis, flagellar assembly, energy production, transportation, and oxidation-reduction. Whereas, downregulated proteins are mainly related to energy-consuming and biosynthetic processes. Our acetylome analyses revealed 197 uniquely acetylated proteins, belonging to major pathways such as nitrogen fixation, central carbon metabolism, ammonia assimilation pathway, protein biosynthesis, and amino acid metabolism. Further, we observed acetylation in glycolytic enzymes of central carbon metabolism, the nitrogenase complex, the master regulator NifA, and the enzyme in GS/GOGAT cycle. These findings suggest that protein acetylation may play an important role in regulating various aspects of N2-metabolism in Z. mobilis. This study provides new knowledge of specific proteins and their associated cellular processes and pathways that may be regulated by protein acetylation in Z. mobilis.

Cellulosic ethanol production by consortia of Scheffersomyces stipitis and engineered Zymomonas mobilis.

BACKGROUND: As one of the clean and sustainable energies, lignocellulosic ethanol has achieved much attention around the world. The production of lignocellulosic ethanol does not compete with people for food, while the consumption of ethanol could contribute to the carbon dioxide emission reduction. However, the simultaneous transformation of glucose and xylose to ethanol is one of the key technologies for attaining cost-efficient lignocellulosic ethanol production at an industrial scale. Genetic modification of strains and constructing consortia were two approaches to resolve this issue. Compared with strain improvement, the synergistic interaction of consortia in metabolic pathways should be more useful than using each one separately. RESULTS: In this study, the consortia consisting of suspended Scheffersomyces stipitis CICC1960 and Zymomonas mobilis 8b were cultivated to successfully depress carbon catabolite repression (CCR) in artificially simulated 80G40XRM. With this strategy, a 5.52% more xylose consumption and a 6.52% higher ethanol titer were achieved by the consortium, in which the inoculation ratio between S. stipitis and Z. mobilis was 1:3, compared with the Z. mobilis 8b mono-fermentation. Subsequently, one copy of the xylose metabolic genes was inserted into the Z. mobilis 8b genome to construct Z. mobilis FR2, leading to the xylose final-consumption amount and ethanol titer improvement by 15.36% and 6.81%, respectively. Finally, various corn stover hydrolysates with different sugar concentrations (glucose and xylose 60, 90, 120 g/L), were used to evaluate the fermentation performance of the consortium consisting of S. stipitis CICC1960 and Z. mobilis FR2. Fermentation results showed that a 1.56-4.59% higher ethanol titer was achieved by the consortium compared with the Z. mobilis FR2 mono-fermentation, and a 46.12-102.14% higher ethanol titer was observed in the consortium fermentation when compared with the S. stipitis CICC1960 mono-fermentation. Furthermore, qRT-PCR analysis of xylose/glucose transporter and other genes responsible for CCR explained the reason why the initial ratio inoculation of 1:3 in artificially simulated 80G40XRM had the best fermentation performance in the consortium. CONCLUSIONS: The fermentation strategy used in this study, i.e., using a genetically modified consortium, had a superior performance in ethanol production, as compared with the S. stipitis CICC1960 mono-fermentation and the Z. mobilis FR2 mono-fermentation alone. This result showed that this strategy has potential for future lignocellulosic ethanol production.

Endogenous CRISPR-assisted microhomology-mediated end joining enables rapid genome editing in Zymomonas mobilis.

BACKGROUND: Zymomonas mobilis is a natural ethanologen with many desirable characteristics, making it an ideal platform for future biorefineries. Recently, an endogenous CRISPR-based genome editing tool has been developed for this species. However, a simple and high-efficient genome editing method is still required. RESULTS: We developed a novel gene deletion tool based on the endogenous subtype I-F CRISPR-Cas system and the microhomology-mediated end joining (MMEJ) pathway. This tool only requires a self-interference plasmid carrying the mini-CRISPR (Repeat-Spacer-Repeat) expression cassette, where the spacer matches the target DNA. Transformation of the self-interference plasmid leads to target DNA damage and subsequently triggers the endogenous MMEJ pathway to repair the damaged DNA, leaving deletions normally smaller than 500 bp. Importantly, the MMEJ repair efficiency was increased by introducing mutations at the second repeat of the mini-CRISPR cassette expressing the guide RNA. Several genes have been successfully deleted via this method, and the phenotype of a σ28 deletion mutant generated in this study was characterized. Moreover, large fragment deletions were obtained by transformation of the self-interference plasmids expressing two guide RNAs in tandem. CONCLUSIONS: Here, we report the establishment of an efficient gene deletion tool based on the endogenous subtype I-F CRISPR-Cas system and the MMEJ pathway in Zymomonas mobilis. We achieved single gene deletion and large-fragment knockout using this tool. In addition, we further promoted the editing efficiency by modifying the guide RNA expression cassette and selecting lower GC% target sites. Our study has provided an effective method for genetic manipulation in Z. mobilis.

High-Efficiency Genome Editing Based on Endogenous CRISPR-Cas System Enhances Cell Growth and Lactic Acid Production in Pediococcus acidilactici.

Pediococcus acidilactici is commonly used for pediocin production and lactic acid fermentation. However, a high-efficiency genome editing tool is unavailable for this species. In this study, we constructed endogenous subtype II-A CRISPR-Cas system-based genome interference plasmids which carried a "repeat-spacer-repeat" cassette in the pMG36e shuttle vector. These plasmids exhibited self-interference activities in P. acidilactici LA412. Then, the genome-editing plasmids were constructed by cloning the upstream/downstream donor DNA into the corresponding interference plasmids to exert high-efficiency markerless gene deletion, gene integration, and point mutation in P. acidilactici LA412. We found that endogenous CRISPR-mediated depletion of the native plasmids enhanced the cell growth and that integration of an l-lactate dehydrogenase gene into the chromosome enhanced both cell growth and lactic acid production. IMPORTANCE A rapid and precise genome editing tool will promote the practical application of Pediococcus acidilactici, one type of lactic acid bacterium with excellent stress tolerance and probiotic characteristics. This study established a high-efficiency endogenous CRISPR-Cas system-based genome editing tool for P. acidilactici and achieved different genetic manipulations, including gene deletion, gene insertion, mononucleotide mutation, and endogenous plasmid depletion. The engineered strain edited by this tool showed significant advantages in cell growth and lactic acid fermentation. Therefore, our tool can satisfy the requirements for genetic manipulations of P. acidilactici, thus making it a sophisticated chassis species for synthetic biology and bioindustry.