Recombinant Strains Research Articles

Mycobacterium tuberculosis Rv2617c is involved in stress response and phage infection resistance

Mycobacterium tuberculosis (M. tuberculosis) is the pathogen of human tuberculosis (TB). Resistance to numerous in vivo stresses, including oxidative stress, is determinant for M. tuberculosis intracellular survival, and understanding associated mechanisms is crucial for developing new therapeutic strategies. M. tuberculosis Rv2617c has been associated with oxidative stress response when interacting with other proteins in M. tuberculosis; however, its functional promiscuity and underlying molecular mechanisms remain elusive. In this study, we investigated the phenotypic changes of Mycobacterium smegmatis (M. smegmatis) expressing Rv2617c (Ms_Rv2617c) and its behavior in the presence of various in vitro stresses and phage infections. We found that Rv2617c conferred resistance to SDS and diamide while sensitizing M. smegmatis to oxidative stress (H2O2) and altered mycobacterial phenotypic properties (single-cell clone and motility), suggestive of reprogrammed mycobacterial cell wall lipid contents exemplified by increased cell wall permeability. Interestingly, we also found that Rv2617c promoted M. smegmatis resistance to infection by phages (SWU1, SWU2, D29, and TM4) and kept phage TM4 from destroying mycobacterial biofilms. Our findings provide new insights into the role of Rv2617c in resistance to oxide and acid stresses and report for the first time on its role in phage resistance in Mycobacterium.

Effect of modification of penicillin-binding protein 3 on susceptibility to ceftazidime-avibactam, imipenem-relebactam, meropenem-vaborbactam, aztreonam-avibactam, cefepime-taniborbactam, and cefiderocol of Escherichia coli strains producing broad-spectrum β-lactamases.

The impact of penicillin-binding protein 3 (PBP3) modifications that may be identified in Escherichia coli was evaluated with respect to susceptibility to β-lactam/β-lactamase inhibitor combinations including ceftazidime-avibactam, imipenem-relebactam, meropenem-vaborbactam, aztreonam-avibactam, cefepime-taniborbactam, and to cefiderocol. A large series of E. coli recombinant strains producing broad-spectrum β-lactamases was evaluated. While imipenem-relebactam showed a similar activity regardless of the PBP3 background, susceptibility to other molecules tested was affected at various levels. This was particularly the case for ceftazidime-avibactam, aztreonam-avibactam, and cefepime-taniborbactam.

Comparative Study of the Convertibility of Pretreated Miscanthus Straw Using Enzyme Preparations Produced by Different Recombinant Strains of Penicillium verruculosum

Non-edible cellulosic biomass from perennial herbaceous plants is a promising and abundant feedstock for replacing slow-growing woody plants used in biotechnological applications. Herbaceous plant biomass, as other types of plant biomass, requires pretreatment before biochemical conversion. In this study, miscanthus straw was pretreated using different methods and subjected to enzymatic hydrolysis with Penicillium verruculosum enzyme complexes under laboratory conditions. The convertibility after enzymatic hydrolysis varied from 15% to 66%, depending on the pretreatment method. Dilute alkaline pretreatment showed the highest convertibility compared to other methods, reaching up to 66%. The efficiency of dilute acid pretreatment was relatively low compared to other methods. The maximum convertibility was 37% for sulfuric acid pretreatment (the least efficient) and 51% for nitric acid. Convertibility was almost equal with 43% for white liquor and 46% for hot water. The glucose-to-xylose ratio was 4.7:1 for dilute alkaline pretreatment and 11–13:1 for white liquor. Both sulfuric and nitric acid resulted in a low xylose content in the enzymatic hydrolysates. Low-xylose hydrolysates with less than 2% of the glucose amount can be produced by hot water pretreatment. Preparation C, enriched with endoglucanase I from T. reesei and endoglucanase II from P. verruculosum, was found to be the most effective of the different enzyme preparations (EPs) tested.

An Efficient Probe for Bacterial Nitroreductase Imaging and Detection Based on NanoLuc‐Furimazine Bioluminescent Pair

Comprehensive SummaryThe detection of critical endogenous species, such as bacteria in microenvironments in the body, requires better imaging tools for visualization and monitoring of biological events. Bioluminescence imaging is the most popular strategy for obtaining real‐time in living cells and organisms. Herein, we introduced a nitroaryl group on the C‐3 position and a hydroxy group at the C‐6 phenyl ring on furimazine to report the first bioluminescent probe (7) based on NanoLuc‐furimazine bioluminescent pair for the detection of nitroreductase in bacteria. The probe, which possessed up to 560‐fold intensity increase with a low detection limit of 16 ng/mL of nitroreductase, has the most efficient uncage efficiency in comparison with other bioluminescent congeners, thus enabling highly selective and sensitive visualization of NTR activity in a panel of clinical priority pathogens. Additionally, imaging of the recombinant strain as well as the NTR from mouse feces indicated the potential of this probe in the application of different mouse disease models.

Insights into the complementation potential of the extreme acidophile's orthologue in replacing Escherichia coli hfq gene-particularly in bacterial resistance to environmental stress.

Acidithiobacillus caldus is a typical extreme acidophile widely used in the biohydrometallurgical industry, which often experiences extreme environmental stress in its natural habitat. Hfq, an RNA-binding protein, typically functions as a global regulator involved in various cellular physiological processes. Yet, the biological functions of Hfq derived from such extreme acidophile have not been extensively investigated. In this study, the recombinant strain Δhfq/Achfq, constructed by CRISPR/Cas9-mediated chromosome integration, fully or partially restored the phenotypic defects caused by hfq deletion in Escherichia coli, including impaired growth performance, abnormal cell morphology, impaired swarming motility, decreased stress resistance, decreased intracellular ATP and free amino acid levels, and attenuated biofilm formation. Particularly noteworthy, the intracellular ATP level and biofilm production of the recombinant strain were increased by 12.2% and 7.0%, respectively, compared to the Δhfq mutant. Transcriptomic analysis revealed that even under heterologous expression, AcHfq exerted global regulatory effects on multiple cellular processes, including metabolism, environmental signal processing, and motility. Finally, we established a potential working model to illustrate the regulatory mechanism of AcHfq in bacterial resistance to environmental stress.

Isolation, identification, recombination analysis and pathogenicity experiment of a PRRSV recombinant strain in Sichuan Province, China.

Since 2013, the porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2), lineage 1.8 (NADC30-like PRRSV) has emerged and become widely prevalent in China. The NADC30-like PRRSV poses significant challenges for disease control, primarily because of its propensity for frequent mutations and recombinations. We successfully isolated and identified a NADC30-like strain, designated SCCD22, in Chengdu, Sichuan Province, China. We meticulously examined the genetic recombination properties and evaluated its pathogenicity in 28-day-old piglets. SCCD22 showed 93.02% nucleotide homology with the NADC30 PRRSV strain, and its non-structural protein 2 coding region showed the same 131 amino acid deletion pattern as that seen in NADC30. Furthermore, we identified two recombination events in SCCD22: one in the NSP2 region (1,028-3,290 nt), where it was highly similar to the JXA1-like strain GZ106; and another in the NSP10 ~ 12 region (9,985-12,279 nt), closely resembling the NADC30-like strain CY2-1604. Piglets infected with SCCD22 exhibited clinical symptoms such as elevated body temperature, prolonged fever, reduced appetite, and roughened fur. Postmortem examinations underscored the typical lung pathology associated with PRRSV, indicating that the lungs were the primary affected organs. Furthermore, extended viral shedding accompanied by progressive viremia was observed in the serum and nasal excretions of infected piglets. In summary, this study reports a domestic PRRSV recombination strain in the Sichuan Province that can provide critical insights into preventing and controlling PRRSV in this region.

Combinatorial Biosynthesis of 3-O-Carbamoylmaytansinol by Rational Engineering of the Tailoring Steps of Ansamitocins.

Currently, most maytansine-containing antibody-drug conjugates (ADCs) in clinical trials are prepared with DM1 or DM4, which in turn is synthesized mainly from ansamitocin P-3 (AP-3), a bacterial maytansinoid, isolated from Actinosynnema pretiosum. However, due to the high self-toxicity of AP-3 to A.pretiosum, the yield of AP-3 has been difficult to improve. Herein, a new maytansinoid with much lower self-toxicity to A.pretiosum, 3-O-carbamoylmaytansinol (CAM, 3), was designed and generated by introducing the 3-O-carbamoyltransferase gene asc21b together with the N-methyltransferase genes from exogenous maytansinoid gene clusters into the 3-O-acyltransferase gene (asm19) deleted mutant HGF052. Meanwhile, two new shunt products, 20-O-demethyl-19-dechloro-N-demethyl-4,5-desepoxy-CAM (4) and 20-O-demethyl-N-demethyl-4,5-desepoxy-CAM (5) were identified from the recombinant strain. Furthermore, by screening of liquid fermentation media, overexpression of bottleneck tailoring enzymes and the pathway-specific activator, the titer of CAM reached 498 mg/L in the engineered strain. Since the 3-O-carbamoyl group of CAM can be removed by chemical cleavage as AP-3 to produce maytansinol, our work suggests that CAM may be a promising alternative to AP-3 in the future development of ADCs.

Metabolic Engineering of Escherichia coli for Production of 2,5-Dimethylpyrazine.

2,5-Dimethylpyrazine (2,5-DMP) is a high-value-added alkylpyrazine compound with important applications in both the food and pharmaceutical fields. In response to the increasing consumer preference for natural products over chemically synthesized ones, efforts have been made to develop efficient microbial cell factories for the production of 2,5-DMP. However, the previously reported recombinant strains have exhibited low yields and relied on expensive antibiotics and inducers. In this study, we employed metabolic engineering strategies to develop an Escherichia coli strain capable of producing 2,5-DMP at high levels without the need for inducers or antibiotics. Initially, the biosynthesis pathway of 2,5-DMP was constructed that realized 2,5-DMP production from glucose. Subsequently, efforts focused on enhancing 2,5-DMP production by improving the availability of the cofactor NAD+ and precursor l-threonine. Additionally, the supply and conversion of l-threonine were balanced by optimizing the copy number of the key gene tdh on the chromosome and by modifying the l-threonine transport system. The final engineering strain D19 produced 3.1 g/L of 2,5-DMP, which is the highest titer for fermentative production of 2,5-DMP using glucose as the carbon source up to date. The strategies used in this study lay a good foundation for the production of 2,5-DMP on a large scale.

The VP1/2 Protein of a New Recombinant PRV Strain Promotes the Infectivity and Pathogenicity of PRV in Northeastern China

Pseudorabies virus (PRV) is an acute infectious disease characterized by neurological and respiratory symptoms. In order to have a better understanding of the current prevalence of PRV in northeastern China, a strain of PRV was isolated by us. Then, protein structure analysis and pathogenicity testing of the virus were performed to give insight into the characterization of the isolated PRV strains. In this study, the PRV strain named CH/HLJPRVJ/2023 was isolated and identified. Genome-wide phylogenetic analysis shows CH/HLJPRVJ/2023 and HeN1 have higher homology. The CH/HLJPRVJ/2023 strain had the highest homology with HeN1 strain (97.3%) and the lowest homology with Bartha-K61 (89.2%). Recombinant evolution analysis shows CH/HLJPRVJ/2023 shows many variants in OBP, AN, UL21, UL17, VP11/12, and VP1/2 fragments, which predict its unique genetically. VP1/2, an effector protein of capsid transport and neuroinvasion, has mutations and deletions in its amino acids, which cause changes in the protein conformation of CH/HLJPRVJ/2023. Besides the typical neurologic and respiratory lesions, infection with highly pathogenic CH/HLJPRVJ/2023 can lead to damage to the colonic villi and colonic barrier in piglets. This study will provide a basis for knowledge about the prevalence, genetic evolution, and vaccine optimization of endemic PRV strains in northeastern China.

Corynebacterium glutamicum cell factory design for the efficient production of cis, cis-muconic acid

Cis, cis-muconic acid (MA) is widely used as a key starting material in the synthesis of diverse polymers. The growing demand in these industries has led to an increased need for MA. Here, we constructed recombinant Corynebacterium glutamicum by systems metabolic engineering, which exhibit high efficiency in the production of MA. Firstly, the three major degradation pathways were disrupted in the MA production process. Subsequently, metabolic optimization strategies were predicted by computational design and the shikimate pathway was reconstructed, significantly enhancing its metabolic flux. Finally, through optimization and integration of key genes involved in MA production, the recombinant strain produced 88.2 g/L of MA with the yield of 0.30 mol/mol glucose in the 5 L bioreactor. This titer represents the highest reported titer achieved using glucose as the carbon source in current studies, and the yield is the highest reported for MA production from glucose in Corynebacterium glutamicum. Furthermore, to enable the utilization of more cost-effective glucose derived from corn straw hydrolysate, we subjected the strain to adaptive laboratory evolution in corn straw hydrolysate. Ultimately, we successfully achieved MA production in a high solid loading of corn straw hydrolysate (with the glucose concentration of 83.56 g/L), resulting in a titer of 19.9 g/L for MA, which is 4.1 times higher than that of the original strain. Additionally, the glucose yield was improved to 0.33 mol/mol. These provide possibilities for a greener and more sustainable production of MA.

Microbial Synthesis of Lacto-N-fucopentaose I with High Titer and Purity by Screening of Specific Glycosyltransferase and Elimination of Residual Lacto-N-triose II and Lacto-N-tetraose.

Lacto-N-fucopentaose I (LNFP I) has recently been approved as generally recognized as safe, demonstrating its great commercial potential in the food industry. Microbial synthesis through metabolic engineering strategies is an effective approach for large-scale production of LNFP I. Biosynthesis of LNFP I requires consideration of two key points: high titer with low byproduct 2'-fucosyllactose (2'-FL) generation and high purity with low lacto-N-triose II (LNTri II) and lacto-N-tetraose (LNT) residues. Herein, α1,2-fucosyltransferase from Thermoanaerobacterium sp. RBIITD was screened from 16 selected LNFP I-producing glycosyltransferase candidates, showing the highest in vivo LNFP I productivity. Chromosomal integration of wbgO enhanced the LNFP I production by improving the precursor conversion from LNTri II to LNT. The best engineered strain produced 4.42 and 35.1 g/L LNFP I in shake-flask and fed-batch cultivation, respectively. The residual LNTri II and LNT were eliminated by further cultivation with a recombinant strain coexpressing Bifidobacterium bifidum β-N-acetylhexosaminidase and lacto-N-biosidase. A strategy for LNFP I biosynthesis with high yield and purity was finally realized, providing support for its practical application in large-scale production.

Biocontrol of black rot of sweetpotato by Pichia pastoris recombinant strain expressing chitinase IbChiA

Black rot disease, caused by Ceratocystis fimbriata, is highly destructive to sweetpotato (Ipomoea batatas). IbChiA is a chitinase identified from sweetpotato with strong antifungal activity against C. fimbriata. The recombinant yeast X33/pGAPZαA-IbChiA was obtained by transforming IbChiA into Pichia pastoris using the constitutive secretory expression vector pGAPZαA. IbChiA was stably expressed and secreted into the extracellular space without an inducer. The recombinant yeast X33/pGAPZαA-IbChiA could not only distinctly inhibit the conidium germination and mycelial growth of C. fimbriata, but also exhibit excellent colonization ability on the tuberous root wounds of sweetpotato. Biocontrol experiments further revealed that recombinant yeast X33/pGAPZαA-IbChiA could effectively limit the expansion of lesions and reduce the severity of black rot in sweetpotato. The findings present a novel approach for the biological control of black rot disease in postharvest sweetpotato.

Nitric oxide synthase expression in Pseudomonas koreensis MME3 improves plant growth promotion traits

The development of novel biotechnologies that promote a better use of N to optimize crop yield is a central goal for sustainable agriculture. Phytostimulation, biofertilization, and bioprotection through the use of bio-inputs are promising technologies for this purpose. In this study, the plant growth–promoting rhizobacteria Pseudomonas koreensis MME3 was genetically modified to express a nitric oxide synthase of Synechococcus SyNOS, an atypical enzyme with a globin domain that converts nitric oxide to nitrate. A cassette for constitutive expression of synos was introduced as a single insertion into the genome of P. koreensis MME3 using a miniTn7 system. The resulting recombinant strain MME3:SyNOS showed improved growth, motility, and biofilm formation. The impact of MME3:SyNOS inoculation on Brachypodium distachyon growth and N uptake and use efficiencies under different N availability situations was analyzed, in comparison to the control strain MME3:c. After 35 days of inoculation, plants treated with MME3:SyNOS had a higher root dry weight, both under semi-hydroponic and greenhouse conditions. At harvest, both MME3:SyNOS and MME3:c increased N uptake and use efficiency of plants grown under low N soil. Our results indicate that synos expression is a valid strategy to boost the phytostimulatory capacity of plant-associated bacteria and improve the adaptability of plants to N deficiency.Key points• synos expression improves P. koreensis MME3 traits important for rhizospheric colonization• B. distachyon inoculated with MME3:SyNOS shows improved root growth• MME3 inoculation improves plant N uptake and use efficiencies in N-deficient soil

Genomic surveillance and evolutionary dynamics of type 2 porcine reproductive and respiratory syndrome virus in China spanning the African swine fever outbreak.

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a serious threat to the pig industry in China. Our previous study demonstrated that PRRSV persists with local circulations and overseas imports in China and has formed a relatively stable epidemic pattern. However, the sudden African swine fever (ASF) outbreak in 2018 caused serious damage to China's pig industry structure, which resulted in about 40 per cent of pigs being slaughtered. The pig yields recovered by the end of 2019. Thus, whether the ASF outbreak reframed PRRSV evolution with changes in pig populations and further posed new threats to the pig industry becomes a matter of concern. For this purpose, we conducted genomic surveillance and recombination, NSP2 polymorphism, population dynamics, and geographical spread analysis of PRRSV-2, which is dominant in China. The results showed that the prevalence of ASF had no significant effects on genetic diversities like lineage composition, recombination patterns, and NSP2 insertion and deletion patterns but was likely to lead to changes in PRRSV-2 recombination frequency. As for circulation of the two major sub-lineages of Lineage 1, there was no apparent transmission of NADC30-like among provinces, while NADC34-like had obvious signs of inter-provincial transmission and foreign importation during the ASF epidemic. In addition, two suspected vaccine recombinant epidemic strains suggest a slight safety issue of vaccine use. Herein, the interference of ASF to the PRRSV-2 evolutionary pattern was evaluated and vaccine safety was analyzed, in order to monitor the potential threat of PRRSV-2 to China's pig industry in the post-epidemic era of ASF.

Reduced susceptibility to aztreonam-avibactam conferred by acquired AmpC-type β-lactamases in PBP3-modified Escherichia coli.

Carbapenemase-producingEnterobacterales are a growing threat, and very few therapeutic options remainactive against those multidrug resistant bacteria. Aztreonam is the molecule of choice against metallo-beta-lactamases (MBL)producers since it is not hydrolyzed by those enzymes, but the co-production of acquired plasmidic cephalosporinases orextended-spectrum β-lactamases leading to aztreonam resistance may reduce the efficacy of this molecule. Hence, thedevelopment of the aztreonam-avibactam (AZA) combination provides an interesting therapeutic alternative since avibactaminhibits the activity of both cephalosporinases and extended-spectrum β-lactamases. However, structural modifications ofpenicillin binding protein PBP3, the target of aztreonam, may lead to reduced susceptibility to aztreonam-avibactam. Here the impact of various plasmid-encoded AmpC-type β-lactamases (ACC-1, ACT-7, ACT-17, CMY-2, CMY-42,DHA-1, FOX-1, and FOX-5) on susceptibility to aztreonam-avibactam was evaluated using isogenic E. coli MG1655 strainsharboring insertions in PBP3 (YRIN and YRIK). The inhibitory activity of various β-lactamase inhibitors (clavulanic acid,tazobactam, avibactam, relebactam, and vaborbactam) were also compared against these enzymes. Hence, we showedthat reduced susceptibility to AZA was due to the combined effect of both AmpC production and amino acid insertions in PBP3.The highest resistance level was achieved in strains possessing the insertions in PBP3 in association with the production ofACT-7, ACC-1, or CMY-42. Although none of the recombinant strains tested displayed clinical resistance toaztreonam-avibactam, our data emphasize that the occurrence of such profile might be of clinical relevance for MBL-producingstrains.

A new HaCV-EBHSV recombinant lagovirus circulating in European brown hares (Lepus europaeus) from Catalonia, Spain

In 2020/2021, several European brown hare syndrome virus (EBHSV) outbreaks were recorded in European hares (Lepus europaeus) from Catalonia, Spain. Recombination analysis combined with phylogenetic reconstruction and estimation of genetic distances of the complete coding sequences revealed that 5 strains were recombinants. The recombination breakpoint is located within the non-structural protein 2C-like RNA helicase (nucleotide position ~ 1889). For the genomic fragment upstream of the breakpoint, a non-pathogenic EBHSV-related strain (hare calicivirus, HaCV; GII.2) was the most closely related sequence; for the rest of the genome, the most similar strains were the European brown hare syndrome virus (EBHSV) strains recovered from the same 2020/2021 outbreaks, suggesting a recent origin. While the functional impact of the atypical recombination breakpoint remains undetermined, the novel recombinant strain was detected in different European brown hare populations from Catalonia, located 20–100 km apart, and seems to have caused a fatal disease both in juvenile and adult animals, confirming its viability and ability to spread and establish infection. This is the first report of a recombination event involving HaCV and EBHSV and, despite the recombination with a non-pathogenic strain, it appears to be associated with mortality in European brown hares, which warrants close monitoring.

Equilibrium, kinetic, and thermodynamic studies on the biosorption of lead by human metallothionein gene-cloned bacteria as a novel biosorbent.

Heavy metals are the main pollutants in water and are an important global problem that threatens human health and ecosystems. In recent years, there has been an increasing interest in the use of genetically modified bacteria as an eco-friendly method to solve heavy metal pollution problems. The goal of this study was to generate genetically modified Escherichia coli expressing human metallothioneins (hMT2A and hMT3) and to determine their tolerance, bioaccumulation, and biosorption capacity to lead (Pb2+ ). Recombinant MT2A and MT3 strains expressing MT were successfully generated. Minimum inhibition concentrations (MIC) of Pb for MT2A and MT3 were found to be 1750 and 2000 mg L-1 , respectively. Pb2+ resistance and bioaccumulation capacity of MT3 were higher than MT2A. Therefore, only MT3 biosorbent was used in Pb2+ biosorption, and its efficiency was examined by performing experiments in a batch system. Pb2+ biosorption by MT3 was evaluated in terms of isotherms, kinetics, and thermodynamics. The results showed that Pb biosorption fits to the Langmuir isotherm model and the pseudo-first-order kinetic model, and the reaction is exothermic. The maximum Pb2+ capacity of the biosorbent was 50 mg Pb2+ g-1 . The potential of MT3 in Pb biosorption was characterized by Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and scanning transmission electron microscopy (STEM) analyses. The desorption study showed that the sorbent had up to 74% recovery and could be effectively used four times. These findings imply that this biosorbent can be applied as a promising, precise, and effective means of removing Pb2+ from contaminated waters. PRACTITIONER POINTS: In this study, the tolerance levels, bioaccumulation, and biosorption capacities of Pb in aqueous solutions were determined for the first time in recombinant MT2A and MT3 strains in which human MT2A and MT3 genes were cloned. The biosorbent of MT3, which was determined to be more effective in Pb bioaccumulation, was synthesized and used in Pb biosorption. The Pb biosorption mechanism of MT3 biosorbent was identified using isotherm modeling, kinetic modeling, and thermodynamic studies. The maximum Pb removal percentage capacity of the biosorbent was 90%, whereas the maximum biosorption capacity was up to 50 mg Pb2+ g-1 . These results indicated that MT3 biosorbent has a higher Pb biosorption capacity than existing recombinant biosorbents. MT3 biosorbent can be used as a promising and effective biosorbent for removing Pb from wastewater.

Efficient production of 2′-fucosyllactose from fructose through metabolically engineered recombinant Escherichia coli

BackgroundThe biosynthesis of human milk oligosaccharides (HMOs) using several microbial systems has garnered considerable interest for their value in pharmaceutics and food industries. 2′-Fucosyllactose (2′-FL), the most abundant oligosaccharide in HMOs, is usually produced using chemical synthesis with a complex and toxic process. Recombinant E. coli strains have been constructed by metabolic engineering strategies to produce 2′-FL, but the low stoichiometric yields (2′-FL/glucose or glycerol) are still far from meeting the requirements of industrial production. The sufficient carbon flux for 2′-FL biosynthesis is a major challenge. As such, it is of great significance for the construction of recombinant strains with a high stoichiometric yield.ResultsIn the present study, we designed a 2′-FL biosynthesis pathway from fructose with a theoretical stoichiometric yield of 0.5 mol 2′-FL/mol fructose. The biosynthesis of 2′-FL involves five key enzymes: phosphomannomutase (ManB), mannose-1-phosphate guanylytransferase (ManC), GDP-d-mannose 4,6-dehydratase (Gmd), and GDP-l-fucose synthase (WcaG), and α-1,2-fucosyltransferase (FucT). Based on starting strain SG104, we constructed a series of metabolically engineered E. coli strains by deleting the key genes pfkA, pfkB and pgi, and replacing the original promoter of lacY. The co-expression systems for ManB, ManC, Gmd, WcaG, and FucT were optimized, and nine FucT enzymes were screened to improve the stoichiometric yields of 2′-FL. Furthermore, the gene gapA was regulated to further enhance 2′-FL production, and the highest stoichiometric yield (0.498 mol 2′-FL/mol fructose) was achieved by using recombinant strain RFL38 (SG104ΔpfkAΔpfkBΔpgi119-lacYΔwcaF::119-gmd-wcaG-manC-manB, 119-AGGAGGAGG-gapA, harboring plasmid P30). In the scaled-up reaction, 41.6 g/L (85.2 mM) 2′-FL was produced by a fed-batch bioconversion, corresponding to a stoichiometric yield of 0.482 mol 2′-FL/mol fructose and 0.986 mol 2′-FL/mol lactose.ConclusionsThe biosynthesis of 2′-FL using recombinant E. coli from fructose was optimized by metabolic engineering strategies. This is the first time to realize the biological production of 2′-FL production from fructose with high stoichiometric yields. This study also provides an important reference to obtain a suitable distribution of carbon flux between 2′-FL synthesis and glycolysis.

Baculovirus entry into the central nervous system of Spodoptera exigua caterpillars is independent of the viral protein tyrosine phosphatase.

Neuroparasitism concerns the hostile take-over of a host's nervous system by a foreign invader, in order to alter the behaviour of the host in favour of the parasite. One of the most remarkable cases of parasite-induced host behavioural manipulation comprises the changes baculoviruses induce in their caterpillar hosts. Baculoviruses may manipulate caterpillar behaviour in two ways: hyperactivity (increased movement in the horizontal plane) and/or tree-top disease (movement to elevated levels in the vertical plane). Those behavioural changes are followed by liquefaction and death of the caterpillar. In Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected Spodoptera exigua caterpillars, an enzymatic active form of the virally encoded protein tyrosine phosphatase (PTP) is needed for the expression of hyperactivity from 3 days post infection (dpi). Using eGFP-expressing recombinant AcMNPV strains, we show that infection of the caterpillar's central nervous system (CNS) can be observed primarily from 3 dpi onwards. In addition, we demonstrate that the structural and enzymatic function of PTP does not play a role in infection of the CNS. Instead we show that the virus entered the CNS via the trachea, progressing caudally to frontally through the CNS and that the infection progressed from the outermost cell layers towards the inner cell layers of the CNS, in a PTP independent manner. These findings help to further understand parasitic manipulation and the mechanisms by which neuroparasites infect the host nervous system to manipulate host behaviour.

Tubercidin inhibits PRRSV replication via RIG-I/NF-κB pathways and interrupting viral nsp2 synthesis.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important swine diseases, which causes huge economic loss worldwide. However, there is no effective therapeutic method for PRRS prevention and control. Here, we found that tubercidin, a naturally extracted adenosine analog, exhibited strong anti-porcine reproductive and respiratory syndrome virus (PRRSV) activity. Mechanically, tubercidin inhibited viral binding, replication, and release. Tubercidin suppressed PRRSV non-structural protein 2 expression, which is important for the formation of replication and transcription complex, leading to the block of viral RNA synthesis and PRRSV replication. Moreover, tubercidin could activate retinoic acid-inducible gene I/nuclear factor kappa-light-chain-enhancer of activated B cell innate immune signaling pathway and increased the expression of interferons and proinflammatory cytokines, which was the other way to inhibit PRRSV replication. Our work evaluated the potential value of tubercidin as an antiviral agent on PRRSV replication and provided a new way to prevent PRRSV replication in vitro.