Antifungal activity and possible mechanism of Streptomyces nojiriensis 9-13 against Mycogone sp., causing wet bubble disease on Agaricus bisporus.

Wet bubble disease (WBD) in Agaricus bisporus caused by Mycogone species imposes a substantial economic loss to mushroom production in China. Currently, fungicide application is the main method to control WBD. However, excessive use of fungicide is challenged by the appearance of resistance and food safety. Therefore, it is necessary to explore safe and efficient strategies to control WBD. Strain 9-13, isolated from the rhizosphere soil of Taxus chinensis, showed strong inhibitory activity against three Mycogone species. According to morphological and biochemical characteristics, and multilocus phylogenetic analysis, the strain was identified as Streptomyces nojiriensis. In addition, strain 9-13 extracts significantly inhibited mycelial growth and spore germination of M. perniciosa, M. rosea and M. xinjiangensis in vitro. Strain 9-13 and its extracts also exhibited broad-spectrum antifungal activities against 12 selected plant pathogenic fungi. Scanning electron microscopic observations showed that extracts destroyed mycelial structure, inducing mycelia to twist and shrink. Moreover, transmission electron microscopy revealed that extracts resulted in severe plasmolysis, rupture of cell membrane and a decrease in cell inclusions, and the cell wall appeared a rough and uneven surface. Notably, the extracts obviously reduced disease severity and incidence of WBD by from 83.85% to 87.32% in fruiting bodies and 77.36% in mushroom beds, and maintained fruiting time and color on harvested mushroom. Collectively, these results clearly indicate that S. nojiriensis 9-13 is a promising biocontrol agent to control WBD on A. bisporus.

Construction of prokaryotic nanocompartment in Yarrowia lipolytica to assist phloroglucinol production.

Phloroglucinol is an important chemical intermediate which has been tentatively produced by engineered bacteria. However, its biosynthesis in industry is limited due to its natural antibacterial activity. Our study firstly selected Yarrowia lipolytica as the chassis strain, which was verified to be tolerable to phloroglucinol. Then the gene of type III polyketone synthase PhlD (the key biosynthetic gene) was overexpressed to facilitate phloroglucinol production with a concentration of 107.4 mg/L. Furthermore, we introduced the prokaryotic nanocompartment to assist the intracellular catalytic activity. The results showed that the concentration of phloroglucinol was increased by about 2.5 times, indicating this multifunctional nanocompartment is orthogonal to the physiological activities of Y. lipolytica. Additionally, fermentations with xylose and lignocellulosic hydrolysates as the carbon source were performed with the engineered Y. lipolytica, resulting in a total concentration of 580.2 mg/L and 328.9 mg/L, respectively. These findings revealed the potential of Y. lipolytica in phloroglucinol production and provided an effective nanocompartment strategy to improve the catalytic activity of the enzyme for boosting phloroglucinol production. KEY POINTS: • The first time to select and use Y. lipolytica to produce phloroglucinol. • Successful construction of prokaryotic nanocompartment in Y. lipolytica to increase production of phloroglucinol. • Lignocellulose hydrolysate is used as a substrate in fermentation.

First report of cobweb disease of Auricularia heimuer caused by Hypomyces mycophilus in Fujian Province, China.

Auricularia heimuer is a gelatinous fungus with great edible and medicinal values. In September 2021, a suspected cobweb disease was found in some A. heimuer farms in Fujian Province, China. The disease caused white cottony mycelium to grow on the basal surface of the A. heimuer at the beginning of infection and gradually spread along the outer edge of the fruiting body, and eventually the white pathogen mycelium covered the entire fruiting body, which eventually led to the wilting and death of about 35% of A. heimuer . Two heavily infected tissues of A. heimuer were collected and two isolates were obtained by single spore isolation and purification technique. The pathogen colonies grew 10 to 12 mm per day on potato dextrose agar (PDA), and the colonies were initially white in color and gradually changed to yellowish brown with neat margins. Well-developed mycelium with septum, Conidiophores are bottle-stemmed and whorl-shaped branches, Conidia solitary, as ovoid, colorless singletons or doublets. The chlamydospores are yellowish, smooth surface, with 2-3 septa, size 9-22 µm × 30-38 µm. These morphological features are consistent with the Hypomyces mycophilus (Gea et al. 2019; Wang et al. 2021). For molecular identification, genomic DNA of the two isolates was obtained using an extraction kit (Biocolor, Shanghai, China), internal transcribed spacers (ITS) regions and 5SrRNA were amplified using ITS1 and ITS4 primers (White et al. 1990). A 590 bp DNA fragment was obtained and the sequences were deposited in GenBank (Accession Nos. OP715875 and OP782039), A BLAST search in GenBank revealed the highest similarity (≥99%) to H. mycophilus (GenBank Accession Nos. MH857567 and KU937111) . To fulfill Koch's postulates, the isolates cultured on PDA plates for 10 days were made into a spore suspension with sterile water at a concentration of 5 × 106 conidia/ml and sprayed onto twenty healthy fruiting bodies grown to about 2 cm in diameter, and another ten healthy fruiting bodies sprayed with sterile water as control, and incubated in an artificial climate chamber at 25℃ and relative humidity of 90%-95% (Back et al. 2012). After 4 days of inoculation, the pathogen started to germinate and slowly grew white mycelium, then the white mycelium multiplied at the base of the fruiting bodies and spread from the base to the periphery, and finally the fruiting bodies were completely covered by the pathogenic mycelium and gradually wilted. The symptoms were consistent with the natural disease symptoms under cultivation conditions, while the control group had normal growth of the seeds and no disease symptoms. H. mycophilus was reisolated and purified from symptomatic cotyledons and identified by the above method, and the results of the two experiments were consistent. To our knowledge, this is the first report of H. mycophilus causing cobweb disease in A. heimuer.

Bacterial mandelic acid degradation pathway and its application in biotechnology.

Mandelic acid and its derivatives are an important class of chemical synthetic blocks, which is widely used in drug synthesis and stereochemistry research. In nature, mandelic acid degradation pathway has been widely identified and analysed as a representative pathway of aromatic compounds degradation. The most studied mandelic acid degradation pathway from Pseudomonas putida consists of mandelate racemase, S-mandelate dehydrogenase, benzoylformate decarboxylase, benzaldehyde dehydrogenase and downstream benzoic acid degradation pathways. Because of the ability to catalyse various reactions of aromatic substrates, pathway enzymes have been widely used in biocatalysis, kinetic resolution, chiral compounds synthesis or construction of new metabolic pathways. In this paper, the physiological significance and the existing range of the mandelic acid degradation pathway were introduced first. Then each of the enzymes in the pathway is reviewed one by one, including the researches on enzymatic properties and the applications in biotechnology as well as efforts that have been made to modify the substrate specificity or improving catalytic activity by enzyme engineering to adapt different applications. The composition of the important metabolic pathway of bacterial mandelic acid degradation pathway as well as the researches and applications of pathway enzymes is summarized in this review for the first time.

Recent advances in genetic technology development of oleaginous yeasts.

As important chemical raw materials and potential nutritional supplements, microbial lipids play an important role in ensuring economic development, food security, and energy security. Compared with non-natural hosts, oleaginous yeasts exhibit obvious advantages in lipid yield and productivity and have great potential to be genetically engineered into an oil cell factory. The main bottleneck in the current oleaginous yeasts engineering is the lack of genetic manipulation tools. Fortunately, the rapid development of synthetic biology has provided numerous new approaches, resources, and ideas for the field. Most importantly, gene editing technology mediated by CRISPR/Cas systems has been successfully applied to some oleaginous yeasts, almost completely rewriting the development pattern of genetic manipulation technology applicable. This paper reviews recent progress in genetic technology with regard to oleaginous yeasts, with a special focus on transformation methods and genome editing tools, discussing the effects of some important genetic parts. KEY POINTS: •Contribution of microbiotechnology in food safety and biofuel by oleaginous yeasts. •Advancement of genetic manipulation and transformation for oleaginous yeasts.

Risk factors for venoarterial-extracorporeal membrane oxygenation related nosocomial infection in children after cardiac surgery. / 儿童心脏术后静脉-åŠ¨è„‰ä½“å¤–è†œè‚ºæ°§åˆç›¸å ³åŒ»é™¢å† æ„ŸæŸ“çš„å±é™©å› ç´ .

OBJECTIVES: Extracorporeal membrane oxygenation (ECMO) is an extracorporeal life support strategy for the treatment of critically ill children with reversible heart and lung failure, increasingly being used in patients with low cardiac output after cardiac surgery. However, the mortality of patients is closely related to the complications of ECMO, especially bleeding, thrombosis, and infection, ECMO-related nosocomial infection has become a challenge to the success of ECMO. This study aims to analyze the incidence and risk factors for venoarterial-ECMO (VA-ECMO)-related nosocomial infections in children after cardiac surgery. METHODS: We retrospectively collected the data of patients who underwent VA-ECMO treatment after pediatric cardiac surgery in the Second Xiangya Hospital of Central South University from July 2015 to March 2021, and divided them into an infected group and a non-infected group. The clinical characteristics of the 2 groups of patients, VA-ECMO-related nosocomial infection factors, pathogenic microorganisms, and patient mortality were compared. Logistic regression was used to analyze the risk factors for nosocomial infection related to VA-ECMO after cardiac surgery. RESULTS: Of the 38 pediatric patients, 18 patients (47.37%) had VA-ECMO related nosocomial infection, served as the infected group, including 7 patients with blood infections and 11 respiratory tract infections. Gram-negative pathogens (16 strains, 88.9%) were the main bacteria, such as Acinetobacter baumannii (6 strains), Klebsiella pneumoniae (3 strains), and Stenotrophomonas maltophilia (3 strains). Compared with the non-infected group (n=20), the infection group had longer time of cardiopulmonary bypass, time of myocardial block, and time of VA-ECMO assistance (All P<0.05). Multivariate logistic regression analysis showed that time of cardiopulmonary bypass (OR=1.012, 95% CI 1.002 to 1.022; P=0.021) was an independent risk factor for ECMO-related nosocomial infection. The number of surviving discharges in the infected group was less than that in the non-infected group (1 vs 11, P<0.05). CONCLUSIONS: Cardiopulmonary bypass time is an independent risk factor for VA-ECMO-related nosocomial infection in children after cardiac surgery. Shortening the duration of extracorporeal circulation may reduce the incidence of VA-EMCO-related nosocomial infections in children after cardic surgery. The occurrence of VA-ECMO-related nosocomial infections affects the number of patient's discharge alive.

Steroidobacter gossypii sp. nov., isolated from cotton field soil.

A Gram-negative bacterium, designated S1-65T, was isolated from soil samples collected from a cotton field located in the Xinjiang region of PR China. Results of 16S rRNA gene sequence analysis revealed that strain S1-65T was affiliated to the genus Steroidobacter with its closest phylogenetic relatives being 'Steroidobacter cummioxidans' 35Y (98.4 %), 'Steroidobacter agaridevorans' SA29-B (98.3 %) and Steroidobacter agariperforans KA5-BT (98.3 %). 16S rRNA-directed phylogenetic analysis showed that strain S1-65T formed a unique phylogenetic subclade next to 'S. agaridevorans' SA29-B and S. agariperforans KA5-BT, suggesting that strain S1-65T should be identified as a member of the genus Steroidobacter. Further, substantial differences between the genotypic properties of strain S1-65T and the members of the genus Steroidobacter, including average nucleotide identity and digital DNA-DNA hybridization, resolved the taxonomic position of strain S1-65T and suggested its positioning as representing a novel species of the genus Steroidobacter. The DNA G+C content of strain S1-65T was 62.5 mol%, based on its draft genome sequence. The predominant respiratory quinone was ubiquinone-8. The main fatty acids were identified as summed feature 3 (C16:1ω6c/C16:1ω7c), C16 : 0 and iso-C15 : 0. In addition, its polar lipid profile was composed of aminophospholipid, diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. Here, we propose a novel species of the genus Steroidobacter: Steroidobacter gossypii sp. nov. with the type strain S1-65T (=JCM 34287T=CGMCC 1.18736T).

Precision strategies for cancer treatment by modifying the tumor-related bacteria.

Research on the roles of the bacteria in tumor development and progression is a rapidly emerging field. Increasing evidence links bacteria with the modification of the tumor immune microenvironment, which greatly influences the antitumor response. In view of the individual immune effects of various bacteria in various tumors, developing personalized bacteria-modulating therapy may be a key to successful antitumor treatment. This review emphasizes the critical role of the bacteria in immune regulation, including both the tumor bacteria and gut bacteria. Aiming at tumor-related bacteria, we focus on various precise modulation strategies and discuss their impact and potential for tumor suppression. Finally, engineered bacteria with tumor-targeting ability could achieve precise delivery of various payloads into tumors, acting as a precision tool. Therefore, a precise tumor-related bacteria therapy may be a promising approach to suppress the development of tumors, as well as an adjuvant therapy to improve the antitumor efficacy of other approaches. KEY POINTS: • The mini-review updates the knowledge on complex effect of bacteria in TME. • Insight into the interaction and adjustment of bacteria in gut for TME. • Prospects and limitations of bacteria-related personalized therapy in the clinical anticancer therapy.

Adaptive laboratory evolution of Yarrowia lipolytica improves ferulic acid tolerance.

Yarrowia lipolytica strain is a promising cell factory for the conversion of lignocellulose to biofuels and bioproducts. Despite the inherent robustness of this strain, further improvements to lignocellulose-derived inhibitors toxicity tolerance of Y. lipolytica are also required to achieve industrial application. Here, adaptive laboratory evolution was employed with increasing concentrations of ferulic acid. The adaptive laboratory evolution experiments led to evolve Y. lipolytica strain yl-XYL + *FA*4 with increased tolerance to ferulic acid as compared to the parental strain. Specifically, the evolved strain could tolerate 1.5 g/L ferulic acid, whereas 0.5 g/L ferulic acid could cause about 90% lethality of the parental strain. Transcriptome analysis of the evolved strain revealed several targets underlying toxicity tolerance enhancements. YALI0_E25201g, YALI0_F05984g, YALI0_B18854g, and YALI0_F16731g were among the highest upregulated genes, and the beneficial contributions of these genes were verified via reverse engineering. Recombinant strains with overexpressing each of these four genes obtained enhanced tolerance to ferulic acid as compared to the control strain. Fortunately, recombinant strains with overexpression of YALI0_E25201g, YALI0_B18854g, and YALI0_F16731g individually also obtained enhanced tolerance to vanillic acid. Overall, this work demonstrated a whole strain improvement cycle by "non-rational" metabolic engineering and presented new targets to modify Y. lipolytica for microbial lignocellulose valorization. KEY POINTS: • Adaptive evolution improved the ferulic acid tolerance of Yarrowia lipolytica • Transcriptome sequence was applied to analyze the ferulic acid tolerate strain • Three genes were demonstrated for both ferulic acid and vanillic acid tolerance.

Genomic molecular signatures determined characterization of Mycolicibacterium gossypii sp. nov., a fast-growing mycobacterial species isolated from cotton field soil.

A Gram-positive, acid-fast and rapidly growing rod, designated S2-37 T, that could form yellowish colonies was isolated from one soil sample collected from cotton cropping field located in the Xinjiang region of China. Genomic analyses indicated that strain S2-37 T harbored T7SS secretion system and was very likely able to produce mycolic acid, which were typical features of pathogenetic mycobacterial species. 16S rRNA-directed phylogenetic analysis referred that strain S2-37 T was closely related to bacterial species belonging to the genus Mycolicibacterium, which was further confirmed by pan-genome phylogenetic analysis. Digital DNA-DNA hybridization and the average nucleotide identity presented that strain S2-37 T displayed the highest values of 39.1% (35.7-42.6%) and 81.28% with M. litorale CGMCC 4.5724 T, respectively. And characterization of conserved molecular signatures further supported the taxonomic position of strain S2-37 T belonging to the genus Mycolicibacterium. The main fatty acids were identified as C16:0, C18:0, C20:3ω3 and C22:6ω3. In addition, polar lipids profile was mainly composed of diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. Phylogenetic analyses, distinct fatty aids and antimicrobial resistance profiles indicated that strain S2-37 T represented genetically and phenotypically distinct from its closest phylogenetic neighbour, M. litorale CGMCC 4.5724 T. Here, we propose a novel species of the genus Mycolicibacterium: Mycolicibacterium gossypii sp. nov. with the type strain S2-37 T (= JCM 34327 T = CGMCC 1.18817 T).

Intraspecific Mitochondrial DNA Comparison of Mycopathogen Mycogone perniciosa Provides Insight Into Mitochondrial Transfer RNA Introns.

Mycogone perniciosa is the main causative agent of wet bubble disease, which causes severe damage to the production of the cultivated mushroom Agaricus bisporus around the world. Whole-genome sequencing of 12 isolates of M. perniciosa was performed using the Illumina sequencing platform, and the obtained paired-end reads were used to assemble complete mitochondrial genomes. Intraspecific comparisons of conserved protein-coding genes, transfer RNA (tRNA) and ribosomal RNA (rRNA) genes, introns, and intergenic regions were conducted. Five different mitochondrial DNA (mtDNA) haplotypes were detected among the tested isolates, ranging from 89,080 to 93,199 bp in length. All of the mtDNAs contained the same set of 14 protein-coding genes and 2 rRNA and 27 tRNA genes, which shared high sequence similarity. In contrast, the number, insertion sites, and sequences of introns varied greatly among the mtDNAs. Eighteen of 43 intergenic regions differed among the isolates, reflecting 65 single nucleotide polymorphisms, 76 indels, and the gain/loss of nine long fragments. Intraspecific comparison revealed that two introns were located within tRNA genes, which is the first detailed description of mitochondrial tRNA introns. Intronic sequence comparison within the same insertion sites revealed the formation process of two introns, which also illustrated a fast evolutionary rate of introns among M. perniciosa isolates. Based on the intron distribution pattern, a pair of universal primers and four pairs of isolate-specific primers were designed and were used to identify the five mtDNA types. In summary, the rapid gain or loss of mitochondrial introns could be an ideal marker for population genetics analysis.

Rhodosporidium toruloides - A potential red yeast chassis for lipids and beyond.

The red yeast Rhodosporidium toruloides naturally produces microbial lipids and carotenoids. In the past decade or so, many studies demonstrated R. toruloides as a promising platform for lipid production owing to its diverse substrate appetites, robust stress resistance and other favorable features. Also, significant progresses have been made in genome sequencing, multi-omic analysis and genome-scale modeling, thus illuminating the molecular basis behind its physiology, metabolism and response to environmental stresses. At the same time, genetic parts and tools are continuously being developed to manipulate this distinctive organism. Engineered R. toruloides strains are emerging for enhanced production of conventional lipids, functional lipids as well as other interesting metabolites. This review updates those progresses and highlights future directions for advanced biotechnological applications.

Combined evolutionary engineering and genetic manipulation improve low pH tolerance and butanol production in a synthetic microbial Clostridium community.

Synthetic microbial communities have become a focus of biotechnological research since they can overcome several of the limitations of single-specie cultures. A paradigmatic example is Clostridium cellulovorans DSM 743B, which can decompose lignocellulose but cannot produce butanol. Clostridium beijerinckii NCIMB 8052 however, is unable to use lignocellulose but can produce high amounts of butanol from simple sugars. In our previous studies, both organisms were cocultured to produce butanol by consolidated bioprocessing. However, such consolidated bioprocessing implementation strongly depends on pH regulation. Since low pH (pH 4.5-5.5) is required for butanol fermentation, C. cellulovorans cannot grow well and saccharify sufficient lignocellulose to feed both strains at a pH below 6.4. To overcome this bottleneck, this study engineered C. cellulovorans by adaptive laboratory evolution, inactivating cell wall lyases genes (Clocel_0798 and Clocel_2169), and overexpressing agmatine deiminase genes (augA, encoded by Cbei_1922) from C. beijerinckii NCIMB 8052. The generated strain WZQ36: 743B*6.0*3△lyt0798△lyt2169-(pXY1-Pthl -augA) can tolerate a pH of 5.5. Finally, the alcohol aldehyde dehydrogenase gene adhE1 from Clostridium acetobutylicum ATCC 824 was introduced into the strain to enable butanol production at low pH, in coordination with solvent fermentation of C. beijerinckii in consortium. The engineered consortium produced 3.94 g/L butanol without pH control within 83 hr, which is more than 5-fold of the level achieved by wild consortia under the same conditions. This exploration represents a proof of concept on how to combine metabolic and evolutionary engineering to coordinate coculture of a synthetic microbial community.

Optimization of n-butanol synthesis in Lactobacillus brevis via the functional expression of thl, hbd, crt and ter.

N-butanol is an important chemical and can be naturally synthesized by Clostridium species; however, the poor n-butanol tolerance of Clostridium impedes the further improvement in titer. In this study, Lactobacillus brevis, which possesses a higher butanol tolerance, was selected as host for heterologous butanol production. The Clostridium acetobutylicum genes thl, hbd, and crt which encode thiolase, ß-hydroxybutyryl-CoA dehydrogenase, and crotonase, and the Treponema denticola gene ter, which encodes trans-enoyl-CoA reductase were cloned into a single plasmid to express the butanol synthesis pathway in L. brevis. A titer of 40 mg/L n-butanol was initially achieved with plasmid pLY15-opt, in which all pathway genes are codon-optimized. A titer of 450 mg/L of n-butanol was then synthesized when ter was further overexpressed in this pathway. The role of metabolic flux was reinforced with pLY15, in which only the ter gene was codon-optimized, which greatly increased the n-butanol titer to 817 mg/L. Our strategy significantly improved n-butanol synthesis in L. brevis and the final titer is the highest achieved amongst butanol-tolerant lactic acid bacteria.

Improved n-Butanol Production from Clostridium cellulovorans by Integrated Metabolic and Evolutionary Engineering.

Clostridium cellulovorans DSM 743B offers potential as a chassis strain for biomass refining by consolidated bioprocessing (CBP). However, its n-butanol production from lignocellulosic biomass has yet to be demonstrated. This study demonstrates the construction of a coenzyme A (CoA)-dependent acetone-butanol-ethanol (ABE) pathway in C. cellulovorans by introducing adhE1 and ctfA-ctfB-adc genes from Clostridium acetobutylicum ATCC 824, which enabled it to produce n-butanol using the abundant and low-cost agricultural waste of alkali-extracted, deshelled corn cobs (AECC) as the sole carbon source. Then, a novel adaptive laboratory evolution (ALE) approach was adapted to strengthen the n-butanol tolerance of C. cellulovorans to fully utilize its n-butanol output potential. To further improve n-butanol production, both metabolic engineering and evolutionary engineering were combined, using the evolved strain as a host for metabolic engineering. The n-butanol production from AECC of the engineered C. cellulovorans was increased 138-fold, from less than 0.025 g/liter to 3.47 g/liter. This method represents a milestone toward n-butanol production by CBP, using a single recombinant clostridium strain. The engineered strain offers a promising CBP-enabling microbial chassis for n-butanol fermentation from lignocellulose.IMPORTANCE Due to a lack of genetic tools, Clostridium cellulovorans DSM 743B has not been comprehensively explored as a putative strain platform for n-butanol production by consolidated bioprocessing (CBP). Based on the previous study of genetic tools, strain engineering of C. cellulovorans for the development of a CBP-enabling microbial chassis was demonstrated in this study. Metabolic engineering and evolutionary engineering were integrated to improve the n-butanol production of C. cellulovorans from the low-cost renewable agricultural waste of alkali-extracted, deshelled corn cobs (AECC). The n-butanol production from AECC was increased 138-fold, from less than 0.025 g/liter to 3.47 g/liter, which represents the highest titer of n-butanol produced using a single recombinant clostridium strain by CBP reported to date. This engineered strain serves as a promising chassis for n-butanol production from lignocellulose by CBP.

Genome editing and transcriptional repression in Pseudomonas putida KT2440 via the type II CRISPR system.

BACKGROUND: The soil bacterium Pseudomonas putida KT2440 is a "generally recognized as safe"-certified strain with robust property and versatile metabolism. Thus, it is an ideal candidate for synthetic biology, biodegradation, and other biotechnology applications. The known genome editing approaches of Pseudomonas are suboptimal; thus, it is necessary to develop a high efficiency genome editing tool. RESULTS: In this study, we established a fast and convenient CRISPR-Cas9 method in P. putida KT2440. Gene deletion, gene insertion and gene replacement could be achieved within 5 days, and the mutation efficiency reached > 70%. Single nucleotide replacement could be realized, overcoming the limitations of protospacer adjacent motif sequences. We also applied nuclease-deficient Cas9 binding at three locations upstream of enhanced green fluorescent protein (eGFP) for transcriptional inhibition, and the expression intensity of eGFP reduced to 28.5, 29.4, and 72.1% of the control level, respectively. Furthermore, based on this CRISPR-Cas9 system, we also constructed a CRISPR-Cpf1 system, which we validated for genome editing in P. putida KT2440. CONCLUSIONS: In this research, we established CRISPR based genome editing and regulation control systems in P. putida KT2440. These fast and efficient approaches will greatly facilitate the application of P. putida KT2440.

Enhanced solvent production by metabolic engineering of a twin-clostridial consortium.

The efficient fermentative production of solvents (acetone, n-butanol, and ethanol) from a lignocellulosic feedstock using a single process microorganism has yet to be demonstrated. Herein, we developed a consolidated bioprocessing (CBP) based on a twin-clostridial consortium composed of Clostridium cellulovorans and Clostridium beijerinckii capable of producing cellulosic butanol from alkali-extracted, deshelled corn cobs (AECC). To accomplish this a genetic system was developed for C. cellulovorans and used to knock out the genes encoding acetate kinase (Clocel_1892) and lactate dehydrogenase (Clocel_1533), and to overexpress the gene encoding butyrate kinase (Clocel_3674), thereby pulling carbon flux towards butyrate production. In parallel, to enhance ethanol production, the expression of a putative hydrogenase gene (Clocel_2243) was down-regulated using CRISPR interference (CRISPRi). Simultaneously, genes involved in organic acids reassimilation (ctfAB, cbei_3833/3834) and pentose utilization (xylR, cbei_2385 and xylT, cbei_0109) were engineered in C. beijerinckii to enhance solvent production. The engineered twin-clostridia consortium was shown to decompose 83.2g/L of AECC and produce 22.1g/L of solvents (4.25g/L acetone, 11.5g/L butanol and 6.37g/L ethanol). This titer of acetone-butanol-ethanol (ABE) approximates to that achieved from a starchy feedstock. The developed twin-clostridial consortium serves as a promising platform for ABE fermentation from lignocellulose by CBP.

Streptomyces tremellae sp. nov., isolated from a culture of the mushroom Tremella fuciformis.

A novel actinomycete strain, designated Js-1T, was isolated from Tremella fuciformis collected from Gutian, Fujian Province, in southeastern China. The taxonomic status of this strain was determined by a polyphasic approach, which demonstrated that the novel strain was a member of the genus Streptomyces. The cell walls of this strain were found to contain ll-diaminopimelic acid, muramic acid and glycine. An analysis of whole-cell hydrolysates revealed that no characteristic sugar was present. The key identified menaquinones were MK-9 (H6) and MK-9 (H8), while the diagnostic polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylmethylethanolamine and phosphatidylglycerol. The main cellular fatty acids were anteiso-C15 : 0, iso-C15 : 0, C16 : 0 and iso-C16 : 0. An analysis of an almost complete 16S rRNA gene sequence showed that the strain shared the highest levels of sequence similarity with Streptomyces sannanensisKC-7038T (97.87 %), Streptomyces hebeiensis YIM 001T (97.84 %), Streptomyces pathocidini NBRC 13812T (97.80 %), Streptomyces cocklensis BK168T (97.25 %), Streptomyces coerulescens NBRC 12758T (97.12 %), Streptomyces aurantiogriseus NBRC 12842T (97.06 %) and Streptomyces rimosussubsp. rimosus ATCC 10970T (97.04 %). The DNA G+C content of the genomic DNA of strain Js-1T was 70.1 mol%. Furthermore, DNA-DNA hybridization tests revealed that the relatedness values between strain Js-1T and the most closely related species ranged from 15.10 to 47.20 %. Based on its phenotypic and genotypic characteristics, strain Js-1T (=CCTCC M 2011365T=JCM 30846T) is considered to represent a novel species within the genus Streptomyces, which we classified as Streptomycestremellae sp. nov.

Analysis of Genetic Diversity and Development of SCAR Markers in a Mycogone perniciosa Population.

The fungus Mycogone perniciosa is a major pathogen of the common button mushroom Agaricus bisporus. Analysis of genetic diversity in M. Perniciosa may assist in developing methods for prophylaxis and treatment of M. Perniciosa infections. For this, it is necessary to classify M. Perniciosa into relevant class groups quickly and efficiently. Random amplified polymorphic DNA (RAPD), inter-simple sequence repeats (ISSR), and sequence-related amplified polymorphism (SRAP) markers were used to obtain genetic fingerprints and assess the genetic variation among 49 strains of M. perniciosa collected from different areas of Fujian Province in China. Analysis of DNA sequence polymorphism revealed two major distinct groups (Group I and Group II). Specific DNA fragments that were identified through RAPD, ISSR, and SRAP markers were sequenced and used for the designing of stable sequence-characterized amplified region (SCAR) markers. The resulting SCAR markers were then validated against the classified groups of M. perniciosa.

Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system.

Yarrowia lipolytica is categorized as a generally recognized as safe (GRAS) organism and is a heavily documented, unconventional yeast that has been widely incorporated into multiple industrial fields to produce valuable biochemicals. This study describes the construction of a CRISPR-Cas9 system for genome editing in Y. lipolytica using a single plasmid (pCAS1yl or pCAS2yl) to transport Cas9 and relevant guide RNA expression cassettes, with or without donor DNA, to target genes. Two Cas9 target genes, TRP1 and PEX10, were repaired by non-homologous end-joining (NHEJ) or homologous recombination, with maximal efficiencies in Y. lipolytica of 85.6 % for the wild-type strain and 94.1 % for the ku70/ku80 double-deficient strain, within 4 days. Simultaneous double and triple multigene editing was achieved with pCAS1yl by NHEJ, with efficiencies of 36.7 or 19.3 %, respectively, and the pCASyl system was successfully expanded to different Y. lipolytica breeding strains. This timesaving method will enable and improve synthetic biology, metabolic engineering and functional genomic studies of Y. lipolytica.