Effects of different replicons in conjugative plasmids on transformation efficiency,plasmid stability,gene expression and n-butanol biosynthesis in <Emphasis Type="Italic">Clostridium tyrobutyricum</Emphasis>

Clostridium tyrobutyricum ATCC 25755 can produce butyric acid, acetic acid, and hydrogen as the main products from various carbon sources. In this study, C. tyrobutyricum was used as a host to produce n-butanol by expressing adhE2 gene under the control of a native thiolase promoter using four different conjugative plasmids (pMTL82151, 83151, 84151, and 85151) each with a different replicon (pBP1 from C. botulinum NCTC2916, pCB102 from C. butyricum, pCD6 from Clostridium difficile, and pIM13 from Bacillus subtilis). The effects of different replicons on transformation efficiency, plasmid stability, adhE2 expression and aldehyde/alcohol dehydrogenase activities, and butanol production by different mutants of C. tyrobutyricum were investigated. Among the four plasmids and replicons studied, pMTL82151 with pBP1 gave the highest transformation efficiency, plasmid stability, gene expression, and butanol biosynthesis. Butanol production from various substrates, including glucose, xylose, mannose, and mannitol were then investigated with the best mutant strain harboring adhE2 in pMTL82151. A high butanol titer of 20.5 g/L with 0.33 g/g yield and 0.32 g/L h productivity was obtained with mannitol as the substrate in batch fermentation with pH controlled at ~6.0.     

Enhancement of the yield of long helical structure of recombinant nucleocapsid protein of Newcastle disease virus

A deletion mutant of the nucleocapsid protein (NP_Δc375) of Newcastle disease virus self-assembles into a long helical structure when expressed in Escherichia coli. However, the NP_Δc375 subjects to proteolytic activity of host cell endogenous proteases during the protein recovery process. Image analysis of Western blots using the Quantity One software was performed to identify the size of the degraded bands and hence the potential proteases cleavage sites were predicted. The data obtained from this image analysis were compared to those identified with the PeptideCutter program; the potential proteases that degrade the NP_Δc375 were identified to be mainly the metallo and serine proteases. Combination of ethylenediaminetetraacetic acid and phenylmethylsulfonyl fluoride at their optimal concentration gave a synergistic effect and increased the NP_Δc375 yield by 2.1-fold. The antigenicity and self-assembled long helical structure long helical structure of NP_Δc375 were preserved after treatment with the protease inhibitors.     

Catecholate siderophore esterases Fes,IroD and IroE are required for salmochelins secretion following utilization,but only IroD contributes to virulence of extra‐intestinal pathogenic Escherichia coli

Salmochelins are glucosylated forms of enterobactin (enterochelin) and contribute to the virulence of Salmonella enterica and some extra‐intestinal pathogenic Escherichia coli (ExPEC). Fes, IroD and IroE esterases degrade salmochelins and enterobactin to release iron. We investigated the apparently redundant role of these esterases in virulence and in salmochelin production and utilization of the ExPEC strain χ7122. The ΔiroD, ΔfesΔiroD and ΔfesΔiroDΔiroE mutants displayed attenuated virulence phenotypes in an avian systemic infection model. Growth of ΔfesΔiroD and ΔfesΔiroDΔiroE mutants was severely reduced in the presence of conalbumin, and although enterobactin was produced, no salmochelins were detected in the culture supernatants of these mutants. Elimination of catecholate synthesis via an entA deletion in a ΔfesΔiroDΔiroE restored growth in the presence of conalbumin, but only partially restored the virulence of the strain. Salmochelin production was reestablished by reintroducing active esterases. Intracellular accumulation of cyclic mono‐glucosylated enterobactin was observed in the triple mutant ΔfesΔiroDΔiroE, and deletion of fepC, required for catecholate import into the cytoplasm, restored salmochelin detection in supernatants. These results suggest that in the absence of esterases, cyclic salmochelins are synthesized and secreted, but remain cell‐bound after internalization indicating that esterase‐mediated degradation is required for re‐secretion of catecholate siderophore molecules following their utilization.     

The low‐molecular‐mass,penicillin‐binding proteins DacB and DacC combine to modify peptidoglycan cross‐linking and allow stable Type IV pilus expression in Neisseria gonorrhoeae

Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection gonorrhea and is adapted to survive in humans, its only host. The N. gonorrhoeae cell wall is critical for maintaining envelope integrity, resisting immune cell killing and production of cytotoxic peptidoglycan (PG) fragments. Deletion of the N. gonorrhoeae strain FA1090 genes encoding two predicted low‐molecular‐mass, penicillin‐binding proteins (LMM PBPs), DacB and DacC, substantially altered the PG cross‐linking. Loss of the DacB peptidase resulted in global alterations to the PG composition, while loss of the DacC protein affected a much narrower subset of PG peptide components. A double ΔdacB/ΔdacC mutant resembled the ΔdacB single mutant, but had an even greater level of cross‐linked PG. While single ΔdacB or ΔdacC mutants did not show any major phenotypes, the ΔdacB/ΔdacC mutant displayed an altered cellular morphology, decreased resistance to antibiotics and increased sensitivity to detergent‐mediated death. Loss of the two proteins also drastically reduced the number of Type IV pili (Tfp), a critical virulence factor. The decreased piliation reduced transformation efficiency and correlated with increased growth rate. While these two LMM PBPs differentially alter the PG composition, their overlapping effects are essential to proper envelope function and expression of factors critical for pathogenesis.     

Search for live attenuated vaccine candidate against edwardsiellosis by mutating virulence-related genes of fish pathogen Edwardsiella tarda

Aims: The aims of this study were to construct and evaluate the live attenuated vaccine against edwardsiellosis on zebra fish model. Methods and Results: In this study, the deletion mutant of aroC gene for the biosynthesis of chorismic acid in Edwardsiella tarda EIB202 was firstly constructed by allelic exchange strategy. According to the genome information, 19 double mutants and one multiple mutant were successively constructed by deleting virulence‐associated genes based on the ΔaroC mutant. Zebra fish model was used to assay the virulence of the mutants by intramuscular (i.m.) injection. Fourteen mutants were significantly attenuated with accumulated mortality ranged from 0 to 63% (P < 0·05). The zebra fish vaccinated with ΔaroC, ΔaroCΔesrC, ΔaroCΔslyA and ΔaroCΔeseBCDΔesaC via i.m. injection showed ideal protection, resulting in relative per cent survival (RPS) of 68·3, 71·3, 80·1 and 81% against subsequent challenge with the wild‐type Edw. tarda EIB202. Conclusions: ΔaroCΔeseBCDΔesaC behaved a low virulence and the highest RPS on zebra fish model. When the zebra fish were vaccinated with ΔaroCΔeseBCDΔesaC via injection, the expression of immune‐related factors including IgM and MHC II was up‐regulated. Significance and Impact: The mutant ΔaroCΔeseBCDΔesaC might serve as an effective live attenuated vaccine against edwardsiellosis.     

Heterologous production of epothilones B and D in Streptomyces venezuelae

Epothilones, produced from the myxobacterium Sorangium cellulosum, are potential anticancer agents that stabilize microtubules in a similar manner to paclitaxel. The entire epothilone biosynthetic gene cluster was heterologously expressed in an engineered strain of Streptomyces venezuelae bearing a deletion of pikromycin polyketide synthase gene cluster. The resulting strains produced approximately 0.1 μg/l of epothilone B as a sole product after 4 days cultivation. Deletion of an epoF encoding the cytochrome P450 epoxidase gave rise to a mutant that selectively produces 0.4 μg/l of epothilone D. To increase the production level of epothilones B and D, an additional copy of the positive regulatory gene pikD was introduced into the chromosome of both S. venezuleae mutant strains. The resulting strains showed enhanced production of corresponding compounds (approximately 2-fold). However, deletion of putative transport genes, orf3 and orf14 in the epothilone D producing S. venezuelae mutant strain, led to an approximately 3-fold reduction in epothilone D production. These results introduce S. venezuelae as an alternative heterologous host for the production of these valuable anticancer agents and demonstrate the possibility of engineering this strain as a generic heterologous host for the production of polyketides and hybrid polyketide-nonribosomal peptides.     

Construction of hypervesiculation Escherichia coli strains and application for secretory protein production

Outer membrane vesicles (OMVs) are extracellular vesicles released from the surface of Gram-negative bacteria, including Escherichia coli. Several gene-deficient mutants relating to envelope stress (nlpI and degP) and phospholipid accumulation in the outer leaflet of the outer membrane (mlaA and mlaE) increase OMV production. This study examined the combinatorial deletion of these genes in E. coli and its effect on OMV production. The nlpI and mlaE double-gene-knockout mutant (ΔmlaEΔnlpI) showed the highest OMV production. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis-based quantitative analysis showed that OMV production by strain ΔmlaEΔnlpI was ~30 times that by the wild-type (WT). In addition, to evaluate the protein secretion capacity of OMVs, a green fluorescent protein (GFP) fused with outer membrane protein W (OmpW) was expressed in OMVs. Western blot analysis showed that GFP secretion through OMVs reached 3.3 mg/L in the culture medium of strain ΔmlaEΔnlpI/gfp, 500 times that for the WT. Our approach using OMVs for extracellular protein secretion in E. coli is an entirely new concept compared with existing secretion systems.     

Disruption of genes involved in CORVET complex leads to enhanced secretion of heterologous carboxylesterase only in protease deficient Pichia pastoris

The methylotrophic yeast Pichia pastoris (Komagataella spp.) is a popular microbial host for the production of recombinant proteins. Previous studies have shown that mis‐sorting to the vacuole can be a bottleneck during production of recombinant secretory proteins in yeast, however, no information was available for P. pastoris. In this work the authors have therefore generated vps (vacuolar protein sorting) mutant strains disrupted in genes involved in the CORVET (class C core vacuole/endosome tethering) complex at the early stages of endosomal sorting. Both Δvps8 and Δvps21 strains contained lower extracellular amounts of heterologous carboxylesterase (CES) compared to the control strain, which could be attributed to a high proteolytic activity present in the supernatants of CORVET engineered strains due to rerouting of vacuolar proteases. Serine proteases were identified to be responsible for this proteolytic degradation by liquid chromatography‐mass spectrometry and protease inhibitor assays. Deletion of the major cellular serine protease Prb1 in Δvps8 and Δvps21 strains did not only rescue the extracellular CES levels, but even outperformed the parental CES strain (56 and 80% higher yields, respectively). Further deletion of Ybr139W, another serine protease, did not show a further increase in secretion levels. Higher extracellular CES activity and low proteolytic activity were detected also in fed batch cultivation of Δvps21Δprb1 strains, thus confirming that modifying early steps in the vacuolar pathway has a positive impact on heterologous protein secretion.     

High ethanol titers from cellulose by using metabolically engineered thermophilic, anaerobic microbes

This work describes novel genetic tools for use in Clostridium thermocellum that allow creation of unmarked mutations while using a replicating plasmid. The strategy employed counter-selections developed from the native C. thermocellum hpt gene and the Thermoanaerobacterium saccharolyticum tdk gene and was used to delete the genes for both lactate dehydrogenase (Ldh) and phosphotransacetylase (Pta). The Δldh Δpta mutant was evolved for 2,000 h, resulting in a stable strain with 40:1 ethanol selectivity and a 4.2-fold increase in ethanol yield over the wild-type strain. Ethanol production from cellulose was investigated with an engineered coculture of organic acid-deficient engineered strains of both C. thermocellum and T. saccharolyticum. Fermentation of 92 g/liter Avicel by this coculture resulted in 38 g/liter ethanol, with acetic and lactic acids below detection limits, in 146 h. These results demonstrate that ethanol production by thermophilic, cellulolytic microbes is amenable to substantial improvement by metabolic engineering.     

Enhanced synthesis of poly gamma glutamic acid by increasing the intracellular reactive oxygen species in the Bacillus licheniformis Δ1-pyrroline-5-carboxylate dehydrogenase gene ycgN-deficient strain

Poly gamma glutamic acid (γ-PGA) is an anionic polyamide with numerous applications. Previous studies revealed that L-proline metabolism is implicated in a wide range of cellular processes by increasing intercellular reactive oxygen species (ROS) generation. However, the relationship between L-proline metabolism and γ-PGA synthesis has not yet been analyzed. In this study, our results confirmed that deletion of Δ1-pyrroline-5-carboxylate dehydrogenase gene ycgN in Bacillus licheniformis WX-02 increased γ-PGA yield to 13.91 g L⁻¹, 85.22% higher than that of the wild type (7.51 g L⁻¹). However, deletion of proline dehydrogenase gene ycgM had no effect on γ-PGA synthesis. Furthermore, a 2.92-fold higher P5C content (19.24 μmol gDCW⁻¹) was detected in the ycgN deficient strain WXΔycgN, while the P5C levels of WXΔycgM and the double mutant strain WXΔycgMN showed no difference, compared to WX-02. Moreover, the ROS level of WXΔycgN was increased by 1.18-fold, and addition of n-acetylcysteine (antioxidant) decreased its ROS level, which further reduced γ-PGA synthesis capability of WXΔycgN. Collectively, our results demonstrated that proline catabolism played an important role in maintaining ROS homeostasis, and deletion of ycgN-enhanced P5C accumulation, which induced a transient ROS signal to promote γ-PGA synthesis in B. licheniformis.

     

A metabolic engineering strategy for producing free fatty acids by the Yarrowia lipolytica yeast based on impairment of glycerol metabolism

In recent years, bio‐based production of free fatty acids from renewable resources has attracted attention for their potential as precursors for the production of biofuels and biochemicals. In this study, the oleaginous yeast Yarrowia lipolytica was engineered to produce free fatty acids by eliminating glycerol metabolism. Free fatty acid production was monitored under lipogenic conditions with glycerol as a limiting factor. Firstly, the strain W29 (Δgpd1), which is deficient in glycerol synthesis, was obtained. However, W29 (Δgpd1) showed decreased biomass accumulation and glucose consumption in lipogenic medium containing a limiting supply of glycerol. Analysis of substrate utilization from a mixture of glucose and glycerol by the parental strain W29 revealed that glycerol was metabolized first and glucose utilization was suppressed. Thus, the Δgpd1Δgut2 double mutant, which is deficient also in glycerol catabolism, was constructed. In this genetic background, growth was repressed by glycerol. Oleate toxicity was observed in the Δgpd1Δgut2Δpex10 triple mutant strain which is deficient additionally in peroxisome biogenesis. Consequently, two consecutive rounds of selection of spontaneous mutants were performed. A mutant released from growth repression by glycerol was able to produce 136.8 mg L^?1 of free fatty acids in a test tube, whereas the wild type accumulated only 30.2 mg L^?1. Next, an isolated oleate‐resistant strain produced 382.8 mg L^?1 of free fatty acids. Finely, acyl‐CoA carboxylase gene (ACC1) over‐expression resulted to production of 1436.7 mg L^?1 of free fatty acids. The addition of dodecane promoted free fatty acid secretion and enhanced the level of free fatty acids up to 2033.8 mg L^?1 during test tube cultivation.

     

The autophagy genes atg8 and atg1 affect morphogenesis and pathogenicity in Ustilago maydis

Autophagy is a complex degradative process in which cytosolic material, including organelles, is randomly sequestered within double‐membrane vesicles termed autophagosomes. In Saccharomyces cerevisiae, the autophagy genes ATG1 and ATG8 are crucial for autophagy induction and autophagosome assembly, respectively, and their deletion has an impact on the autophagic potential of the corresponding mutant strains. We were interested in the role of autophagy in the development and virulence of U. maydis. Using a reverse genetic approach, we showed that the U. maydis ATG8 orthologue, atg8, is associated with autophagy‐dependent processes. Deletion of atg8 abolished autophagosome accumulation in the vacuoles of carbon‐starved cells and drastically reduced the survival of U. maydisΔatg8 mutant strains during these conditions. In addition, atg8 deletion had an impact on the budding process during saprobic haploid growth. The infection of maize with compatible Δatg8 strains resulted in fewer galled plants, and fungal gall colonization was strongly reduced, as reflected by the very low hyphal density in these tissues. Δatg8 infections resulted in the formation of very few teliospores. To corroborate the role of autophagy in U. maydis development, we also deleted the ATG1 orthologue, atg1. Deletion of atg1 yielded phenotypes similar to the Δatg8 strains during saprobic growth, but of lower magnitude. The Δatg1 strains were only slightly less pathogenic than the wild‐type and teliospore production was not affected. Surprisingly, atg1 deletion in the Δatg8 background exacerbated those phenotypes already observed in the Δatg8 and Δatg1 single‐mutant strains, strongly suggesting an additive phenotype. In particular, the double mutant was completely suppressed for plant gall induction.     

Extracellular Production of Human Tumor Necrosis Factor-α by Escherichia coli Using a Chemically-synthesized Gene

A DNA fragment of approximately 490 base pairs encoding human TNF was chemically synthesized and expressed within Escherichia coli cells. Furthermore, extracellular production of human TNF and several N-terminal deletion mutants of TNF was attempted using the excretion vector pEAP8. The TNF mutant with two N-terminal amino acids deleted (NΔ2-TNF) was efficiently excreted into the culture medium by E. coli carrying the plasmid pEXTNF3. In this clone, the signal peptide was correctly processed during the excretion. The E. coli-excreted NΔ2-TNF had higher antitumor activity than wild-type TNF or NΔ2-TNF produced intracellularly by E. coli.     

URA3基因影响青蒿酸酿酒酵母工程菌中试发酵产量

CRISPR/Cas9基因编辑技术已经被广泛应用于工程酿酒酵母的基因插入、基因替换和基因敲除,通过使用选择标记进行基因编辑具有简单高效的特点。前期利用CRISPR/Cas9系统敲除青蒿酸生产菌株酿酒酵母(Saccharomyces cerevisiae) 1211半乳糖代谢负调控基因GAL80,获得菌株S. cerevisiae 1211-2,在不添加半乳糖诱导的情况下,青蒿酸摇瓶发酵产量达到了740 mg/L。但在50 L中试发酵实验中,S. cerevisiae 1211-2很难利用对青蒿酸积累起到决定性作用的碳源-乙醇,青蒿酸的产量仅为亲本菌株S.cerevisiae 1211的20%–25%。我们推测因遗传操作所需的筛选标记URA3突变,影响了其生长及青蒿酸产量。随后我们使用重组质粒pML104-KanMx4-u连同90 bp供体DNA成功恢复了URA3基因,获得了工程菌株S. cerevisiae 1211-3。S. cerevisiae 1211-3能够在葡萄糖和乙醇分批补料的发酵罐中正常生长,其青蒿酸产量超过20g/L,与亲本菌株产量相当。研究不但获得了不加半乳糖诱导的青...     

Dihydroxyacetone production in an engineered Escherichia coli through expression of Corynebacterium glutamicum dihydroxyacetone phosphate dephosphorylase

Dihydroxyacetone (DHA) has several industrial applications such as a tanning agent in tanning lotions in the cosmetic industry; its production via microbial fermentation would present a more sustainable option for the future. Here we genetically engineered Escherichia coli (E. coli) for DHA production from glucose. Deletion of E. coli triose phosphate isomerase (tpiA) gene was carried out to accumulate dihydroxyacetone phosphate (DHAP), for use as the main intermediate or precursor for DHA production. The accumulated DHAP was then converted to DHA through the heterologous expression of Corynebacterium glutamicum DHAP dephosphorylase (cghdpA) gene. To conserve DHAP exclusively for DHA production we removed methylglyoxal synthase (mgsA) gene in the ΔtpiA strain. This drastically improved DHA production from 0.83 g/l (0.06 g DHA/g glucose) in the ΔtpiA strain bearing cghdpA to 5.84 g/l (0.41 g DHA/g glucose) in the ΔtpiAΔmgsA double mutant containing the same gene. To limit the conversion of intracellular DHA to glycerol, glycerol dehydrogenase (gldA) gene was further knocked out resulting in a ΔtpiAΔmgsAΔgldA triple mutant. This triple mutant expressing the cghdpA gene produced 6.60 g/l of DHA at 87% of the maximum theoretical yield. In summary, we demonstrated an efficient system for DHA production in genetically engineered E. coli strain.     

Heterologous expression of tylosin polyketide synthase and production of a hybrid bioactive macrolide in Streptomyces venezuelae

Tylosin polyketide synthase (Tyl PKS) was heterologously expressed in an engineered strain of Streptomyces venezuelae bearing a deletion of pikromycin PKS gene cluster using two compatible low-copy plasmids, each under the control of a pikAI promoter. The mutant strain produced 0.5 mg/l of the 16-membered ring macrolactone, tylactone, after a 4-day culture, which is a considerably reduced culture period to reach the maximum production level compared to other Streptomyces hosts. To improve the production level of tylactone, several precursors for ethylmalonyl-CoA were fed to the growing medium, leading to a 2.8-fold improvement (1.4 mg/ml); however, switching the pikAI promoter to an actI promoter had no observable effect. In addition, a small amount of desosamine-glycosylated tylactone was detected from the extract of the mutant strain, revealing that the native glycosyltransferase DesVII displayed relaxed substrate specificity in accepting the 16-membered ring macrolactone to produce the glycosylated tylactone. These results demonstrate a successful attempt for a heterologous expression of Tyl PKS in S. venezuelae and introduce S. venezuelae as a rapid heterologous expression system for the production of secondary metabolites.