Guzhen Cui

Targeted gene engineering in Clostridium cellulolyticum H10 without methylation

Genetic engineering of Clostridium cellulolyticum has been developed slowly compared with that of other clostridial species, and one of the major reasons might be the restriction and modification (RM) system which degrades foreign DNA. Here, a putative MspI endonuclease gene, ccel2866, was inactivated by a ClosTron-based gene disruption method. The resulting C. cellulolyticum mutant H10ΔmspI lost the MspI endonuclease activity and can accept unmethylated DNA efficiently. Following that, an oxygen-independent green fluorescence protein gene was introduced into H10ΔmspI without methylation, generating a convenient reporter system to evaluate the expression of heterologous protein in C. cellulolyticum by green fluorescence. To further demonstrate the efficiency of the H10ΔmspI, double mutants H10ΔmspIΔldh and H10ΔmspIΔack were constructed by disrupting lactate dehydrogenase gene ccel2485 and acetate kinase gene ccel2136 in H10ΔmspI, respectively, without DNA methylation, and the stability of the double mutation was confirmed after the 100th generation. The mutant H10ΔmspI constructed here can be used as a platform for further targeted gene manipulation conveniently and efficiently. It will greatly facilitate the metabolic engineering of C. cellulolyticum aiming at faster cellulose degradation and higher biofuel production at the molecular level.

Transcriptome and gene expression analysis of DHA producer Aurantiochytrium under low temperature conditions

Aurantiochytrium is a promising docosahexaenoic acid (DHA) production candidate due to its fast growth rate and high proportions of lipid and DHA content. In this study, high-throughput RNA sequencing technology was employed to explore the acclimatization of this DHA producer under cold stress at the transcriptional level. The overall de novo assembly of the cDNA sequence data generated 29,783 unigenes, with an average length of 1,200 bp. In total, 13,245 unigenes were annotated in at least one database. A comparative genomic analysis between normal conditions and cold stress revealed that 2,013 genes were differentially expressed during the growth stage, while 2,071 genes were differentially expressed during the lipid accumulation stage. Further functional categorization and analyses showed some differentially expressed genes were involved in processes crucial to cold acclimation, such as signal transduction, cellular component biogenesis, and carbohydrate and lipid metabolism. A brief survey of the transcripts obtained in response to cold stress underlines the survival strategy of Aurantiochytrium; of these transcripts, many directly or indirectly influence the lipid composition. This is the first study to perform a transcriptomic analysis of the Aurantiochytrium under low temperature conditions. Our results will help to enhance DHA production by Aurantiochytrium in the future.

Cellulosome stoichiometry in Clostridium cellulolyticum is regulated by selective RNA processing and stabilization

The mechanism, physiological relevance and evolutionary implication of selective RNA processing and stabilization (SRPS) remain elusive. Here we report the genome-wide maps of transcriptional start sites (TSs) and post-transcriptional processed sites (PSs) for Clostridium cellulolyticum. The PS-associated genes are preferably associated with subunits of heteromultimeric protein complexes, and the intergenic PSs (iPSs) are enriched in operons exhibiting highly skewed transcript-abundance landscape. Stem-loop structures associated with those iPSs located at 3′ termini of highly transcribed genes exhibit folding free energy negatively correlated with transcript-abundance ratio of flanking genes. In the cellulosome-encoding cip-cel operon, iPSs and stem-loops precisely regulate structure and abundance of the subunit-encoding transcripts processed from a primary polycistronic RNA, quantitatively specifying cellulosome stoichiometry. Moreover, cellulosome evolution is shaped by the number, position and biophysical nature of TSs, iPSs and stem-loops. Our findings unveil a genome-wide RNA-encoded strategy controlling in vivo stoichiometry of protein complexes.

A new strategy for strain improvement of Aurantiochytrium sp. based on heavy-ions mutagenesis and synergistic effects of cold stress and inhibitors of enoyl-ACP reductase

Developing a strain with high docosahexaenoic acid (DHA) yield and stable fermenting-performance is an imperative way to improve DHA production using Aurantiochytrium sp., a microorganism with two fatty acid synthesis pathways: polyketide synthase (PKS) pathway and Type I fatty acid synthase (FAS) pathway. This study investigated the growth and metabolism response of Aurantiochytrium sp. CGMCC 6208 to two inhibitors of enoyl-ACP reductase of Type II FAS pathway (isoniazid and triclosan), and proposed a method of screening high DHA yield Aurantiochytrium sp. strains with heavy ion mutagenesis and pre-selection by synergistic usage of cold stress (4°C) and FAS inhibitors (triclosan and isoniazid). Results showed that (1) isoniazid and triclosan have positive effects on improving DHA level of cells; (2) mutants from irradiation dosage of 120Gy yielded more DHA compared with cells from 40Gy, 80Gy treatment and wild type; (3) DHA contents of mutants pre-selected by inhibitors of enoyl-ACP reductase of Type II FAS pathway (isoniazid and triclosan)at 4°C, were significantly higher than that of wild type; (4) compared to the wild type, the DHA productivity and yield of a mutant (T-99) obtained from Aurantiochytrium sp. CGMCC 6208 by the proposed method increased by 50% from 0.18 to 0.27g/Lh and 30% from 21 to 27g/L, respectively. In conclusion, this study developed a feasible method to screen Aurantiochytrium sp. with high DHA yield by a combination of heavy-ion mutagenesis and mutant-preselection by FAS inhibitors and cold stress.

A novel arabinose-inducible genetic operation system developed for Clostridium cellulolyticum.

BackgroundClostridium cellulolyticum and other cellulolytic Clostridium strains are natural producers of lignocellulosic biofuels and chemicals via the consolidated bioprocessing (CBP) route, and systems metabolic engineering is indispensable to meet the cost-efficient demands of industry. Several genetic tools have been developed for Clostridium strains, and an efficient and stringent inducible genetic operation system is still required for the precise regulation of the target gene function.ResultsHere, we provide a stringent arabinose-inducible genetic operation (ARAi) system for C. cellulolyticum, including an effective gene expression platform with an oxygen-independent fluorescent reporter, a sensitive MazF-based counterselection genetic marker, and a precise gene knock-out method based on an inducible ClosTron system. A novel arabinose-inducible promoter derived from Clostridium acetobutylicum is employed in the ARAi system to control the expression of the target gene, and the gene expression can be up-regulated over 800-fold with highly induced stringency. The inducible ClosTron method of the ARAi system decreases the off-target frequency from 100% to 0, which shows the precise gene targeting in C. cellulolyticum. The inducible effect of the ARAi system is specific to a universal carbon source L-arabinose, implying that the system could be used widely for clostridial strains with various natural substrates.ConclusionsThe inducible genetic operation system ARAi developed in this study, containing both controllable gene expression and disruption tools, has the highest inducing activity and stringency in Clostridium by far. Thus, the ARAi system will greatly support the efficient metabolic engineering of C. cellulolyticum and other mesophilic Clostridium strains for lignocellulose bioconversion.

Multiplexed CRISPR-Cpf1-Mediated Genome Editing in Clostridium difficile toward the Understanding of Pathogenesis of C. difficile Infection

Clostridium difficile is often the primary cause of nosocomial diarrhea, leading to thousands of deaths annually worldwide. The availability of an efficient genome editing tool for C. difficile is essential to understanding its pathogenic mechanism and physiological behavior. Although CRISPR-Cas9 has been extensively employed for genome engineering in various organisms, large gene deletion and multiplex genome editing is still challenging in microorganisms with underdeveloped genetic engineering tools. Here, we describe a streamlined CRISPR-Cpf1-based toolkit to achieve precise deletions of fur, tetM, and ermB1/2 in C. difficile with high efficiencies. All of these genes are relevant to important phenotypes (including iron uptake, antibiotics resistance, and toxin production) as related to the pathogenesis of C. difficile infection (CDI). Furthermore, we were able to delete an extremely large locus of 49.2-kb comprising a phage genome ( phiCD630-2) and realized multiplex genome editing in a single conjugation with high efficiencies (simultaneous deletion of cwp66 and tcdA). Our work highlighted the first application of CRISPR-Cpf1 for multiplexed genome editing and extremely large gene deletion in C. difficile, which are both crucial for understanding the pathogenic mechanism of C. difficile and developing strategies to fight against CDI. In addition, for the DNA cloning, we developed a one-step-assembly protocol along with a Python-based algorithm for automatic primer design, shortening the time for plasmid construction to half that of conventional procedures. The approaches we developed herein are easily and broadly applicable to other microorganisms. Our results provide valuable guidance for establishing CRISPR-Cpf1 as a versatile genome engineering tool in prokaryotic cells.

Current progress of targetron technology: Development, improvement and application in metabolic engineering

Targetrons are mobile group II introns that can recognize their DNA target sites by base‐pairing RNA‐DNA interactions with the aid of site‐specific binding reverse transcriptases. Targetron technology stands out from recently developed gene targeting methods because of the flexibility, feasibility, and efficiency, and is particularly suitable for the genetic engineering of difficult microorganisms, including cellulolytic bacteria that are considered promising candidates for biomass conversion via consolidated bioprocessing. Along with the development of the thermotargetron method for thermophiles, targetron technology becomes increasingly important for the metabolic engineering of industrial microorganisms aiming at biofuel/chemical production. To summarize the current progress of targetron technology and provide new insights on the use of the technology, this paper reviews the retrohoming mechanisms of both mesophilic and thermophilic targetron methods based on various group II introns, investigates the improvement of targetron tools for high target efficiency and specificity, and discusses the current applications in the metabolic engineering for bacterial producers. Although there are still intellectual property and technical restrictions in targetron applications, we propose that targetron technology will contribute to both biochemistry research and the metabolic engineering for industrial productions.

High Yield Recombinant Expression, Characterization and Homology Modeling of Two Types of Cis-epoxysuccinic Acid Hydrolases

The cis-epoxysuccinate hydrolases (CESHs), members of epoxide hydrolase, catalyze cis-epoxysuccinic acid hydrolysis to form d(−)-tartaric acid or l(+)-tartaric acid which are important chemicals with broad scientific and industrial applications. Two types of CESHs (CESH[d] and CESH[l], producing d(−)- and l(+)-tartaric acids, respectively) have been reported with low yield and complicated purification procedure in previous studies. In this paper, the two CESHs were overexpressed in Escherichia coli using codon-optimized genes. High protein yields by one-step purifications were obtained for both recombinant enzymes. The optimal pH and temperature were measured for both recombinant CESHs, and the properties of recombinant enzymes were similar to native enzymes. Kinetics parameters measured by Lineweaver–Burk plot indicates both enzymes exhibited similar affinity to cis-epoxysuccinic acid, but CESH[l] showed much higher catalytic efficiency than CESH[d], suggesting that the two CESHs have different catalytic mechanisms. The structures of both CESHs constructed by homology modeling indicated that CESH[l] and CESH[d] have different structural folds and potential active site residues. CESH[l] adopted a typical α/β-hydrolase fold with a cap domain and a core domain, whereas CESH[d] possessed a unique TIM barrel fold composed of 8 α-helices and 8 β-strands, and 2 extra short α-helices exist on the top and bottom of the barrel, respectively. A divalent metal ion, preferred to be zinc, was found in CESH[d], and the ion was proved to be crucial to the enzymatic activity. These results provide structural insight into the different catalytic mechanisms of the two CESHs.