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Abstract l-Threonine is an important feed additive with the third largest market size among the amino acids produced by microbial fermentation. The GRAS (generally regarded as safe) industrial workhorse Corynebacterium glutamicum is an attractive chassis for l-threonine production. However, the present l-threonine production in C. glutamicum cannot meet the requirement of industrialization due to the relatively low production level of l-threonine and the accumulation of large amounts of by-products (such as l-lysine, l-isoleucine, and glycine). Herein, to enhance the l-threonine biosynthesis in C. glutamicum, releasing the aspartate kinase (LysC) and homoserine dehydrogenase (Hom) from feedback inhibition by l-lysine and l-threonine, respectively, and overexpressing four flux-control genes were performed. Next, to reduce the formation of by-products l-lysine and l-isoleucine without the cause of an auxotrophic phenotype, the feedback regulation of dihydrodipicolinate synthase (DapA) and threonine dehydratase (IlvA) was strengthened by replacing the native enzymes with heterologous analogues with more sensitive feedback inhibition by l-lysine and l-isoleucine, respectively. The resulting strain maintained the capability of synthesizing enough amounts of l-lysine and l-isoleucine for cell biomass formation but exhibited almost no extracellular accumulation of these two amino acids. To further enhance l-threonine production and reduce the by-product glycine, l-threonine exporter and homoserine kinase were overexpressed. Finally, the rationally engineered non-auxotrophic strain ZcglT9 produced 67.63 g/L (17.2% higher) l-threonine with a productivity of 1.20 g/L/h (108.0% higher) in fed-batch fermentation, along with significantly reduced by-product accumulation, representing the record for l-threonine production in C. glutamicum. In this study, we developed a strategy of reconstructing the feedback regulation of amino acid metabolism and successfully applied this strategy to de novo construct a non-auxotrophic l-threonine producing C. glutamicum. The main end by-products including l-lysine, l-isoleucine, and glycine were almost eliminated in fed-batch fermentation of the engineered C. glutamicum strain. This strategy can also be used for engineering producing strains for other amino acids and derivatives.

AimsCitruses often occur with imbalance in iron nutrition in coastal saline-alkali lands, which severely limits the yield and quality of the fruit. In the rhizosphere, the salt content plays a crucial role in reducing uptake of iron, as well as the activity and abundance of bacteria. However, few studies have explored how salt content affects the effectiveness of iron and the community structure of bacteria across different vertical spatial scales.MethodsWe investigated the citrus rhizosphere (0–30 cm) and bulk (0–60 cm) soil microenvironments of the coastal saline soil were analyzed using the 16S rRNA amplicon and inductively coupled plasma-optical emission spectroscopy.ResultsWe found that the nutrient-related elements in the rhizosphere and bulk soil decreased with increasing soil depth, while the salinity-related elements showed the opposite trend. The nutrient-related element content in the rhizosphere was higher than that in the bulk, whereas the salinity-alkaline-related element content was lower than that in the bulk. The structure and diversity of bacterial communities are affected by the rhizosphere and soil depth. In the bulk, there are enriched bacteria such as WB1-A12, Nitrospiraceae and Anaerolineae that are tolerant to salt-alkali stress. In the rhizosphere, bacteria that promote plant nutrient absorption and secretion of iron carriers, such as Pseudomonas, Streptomyces, and Duganella, are prominent.ConclusionsThe soil depth and rhizosphere affect soil nutrients and saline alkali-related factors. Changes in soil depth and rhizosphere determine the structure and diversity of bacterial communities. Rhizosphere enhances iron absorption promoting bacteria to alleviate iron deficiency stress in saline-alkali soils. Our results indicate that citrus roots maybe can resist the stress of iron deficiency in saline-alkali soils by enhancing iron absorption promoting bacteria.

Abstract Background Plant hemoglobin shows great potential as a food additive to circumvent the controversy of using animal materials. Microbial fermentation with engineered microorganisms is considered as a promising strategy for sustainable production of hemoglobin. As an endotoxin-free and GRAS (generally regarded as safe) bacterium, Corynebacterium glutamicum is an attractive host for hemoglobin biosynthesis. Results Herein, C. glutamicum was engineered to efficiently produce plant hemoglobin. Hemoglobin genes from different sources including soybean and maize were selected and subjected to codon optimization. Interestingly, some candidates optimized for the codon usage bias of Escherichia coli outperformed those for C. glutamicum regarding the heterologous expression in C. glutamicum. Then, saturated synonymous mutation of the N-terminal coding sequences of hemoglobin genes and fluorescence-based high-throughput screening produced variants with 1.66- to 3.45-fold increase in hemoglobin expression level. To avoid the use of toxic inducers, such as isopropyl-β-d-thiogalactopyranoside, two native inducible expression systems based on food additives propionate and gluconate were developed. Promoter engineering improved the hemoglobin expression level by 2.2- to 12.2-fold. Combination of these strategies and plasmid copy number modification allowed intracellular production of hemoglobin up to approximately 20% of total protein. Transcriptome and proteome analyses of the hemoglobin-producing strain revealed the cellular response to excess hemoglobin accumulation. Several genes were identified as potential targets for further enhancing hemoglobin production. Conclusions In this study, production of plant hemoglobin in C. glutamicum was systematically engineered by combining codon optimization, promoter engineering, plasmid copy number modification, and multi-omics-guided novel target discovery. This study offers useful design principles to genetically engineer C. glutamicum for the production of hemoglobin and other recombinant proteins.

Allosteric regulation by pathway products plays a vital role in amino acid metabolism. Homoserine dehydrogenase (HSD), the key enzyme for the biosynthesis of various aspartate family amino acids, is subject to feedback inhibition by l-threonine and l-isoleucine. The desensitized mutants with the potential for amino acid production remain limited. Herein, a semi-rational approach was proposed to relieve the feedback inhibition. HSD from Corynebacterium glutamicum (CgHSD) was first characterized as a homotetramer, and nine conservative sites at the tetramer interface were selected for saturation mutagenesis by structural simulations and sequence analysis. Then, we established a high-throughput screening (HTS) method based on resistance to l-threonine analog and successfully acquired two dominant mutants (I397V and A384D). Compared with the best-ever reported desensitized mutant G378E, both new mutants qualified the engineered strains with higher production of CgHSD-dependent amino acids. The mutant and wild-type enzymes were purified and assessed in the presence or absence of inhibitors. Both purified mutants maintained >90% activity with 10 mM l-threonine or 25 mM l-isoleucine. Moreover, they showed >50% higher specific activities than G378E without inhibitors. This work provides two competitive alternatives for constructing cell factories of CgHSD-related amino acids and derivatives. Moreover, the proposed approach can be applied to engineering other allosteric enzymes in the amino acid synthesis pathway.

Abstract Background 5-Aminolevulinic acid (5-ALA) is a promising biostimulant, feed nutrient, and photodynamic drug with wide applications in modern agriculture and therapy. Although microbial production of 5-ALA has been improved realized by using metabolic engineering strategies during the past few years, there is still a gap between the present production level and the requirement of industrialization. Results In this study, pathway, protein, and cellular engineering strategies were systematically employed to construct an industrially competitive 5-ALA producing Escherichia coli. Pathways involved in precursor supply and product degradation were regulated by gene overexpression and synthetic sRNA-based repression to channel metabolic flux to 5-ALA biosynthesis. 5-ALA synthase was rationally engineered to release the inhibition of heme and improve the catalytic activity. 5-ALA transport and antioxidant defense systems were targeted to enhance cellular tolerance to intra- and extra-cellular 5-ALA. The final engineered strain produced 30.7 g/L of 5-ALA in bioreactors with a productivity of 1.02 g/L/h and a yield of 0.532 mol/mol glucose, represent a new record of 5-ALA bioproduction. Conclusions An industrially competitive 5-ALA producing E. coli strain was constructed with the metabolic engineering strategies at multiple layers (protein, pathway, and cellular engineering), and the strategies here can be useful for developing industrial-strength strains for biomanufacturing.

Heme is an important tetrapyrrole compound, and has been widely applied in food and medicine industries. Although microbial production of heme has been developed with metabolic engineering strategies during the past 20 years, the production levels are relatively low due to the multistep enzymatic processes and complicated regulatory mechanisms of microbes. Previous studies mainly adopted the strategies of strengthening precursor supply and product transportation to engineer microbes for improving heme biosynthesis. Few studies focused on the engineering and screening of efficient enzymes involved in heme biosynthesis. Herein, a growth-coupled, high-throughput selection platform based on the detoxification of Zinc-protoporphyrin IX (an analogue of heme) was developed and applied to directed evolution of coproporphyrin ferrochelatase, catalyzing the insertion of metal ions into porphyrin ring to generate heme or other tetrapyrrole compounds. A mutant with 3.03-fold increase in kcat/KM was selected. Finally, growth-coupled directed evolution of another three key enzymes involved in heme biosynthesis was tested by using this selection platform. The growth-coupled selection platform developed here can be a simple and effective strategy for directed evolution of the enzymes involved in the biosynthesis of heme or other tetrapyrrole compounds.

Abstract The development trend of Fischer–Tropsch (F–T) technology is to develop high value-added products. The separation of linear α-olefins with low cost is an effective method. Nevertheless, the lack of thermodynamic data and the huge energy consumption are the two main problems restricting the development of the separation process. The thermodynamic data of the key components (1-dodecene and n-dodecane) in the F–T product were measured. The Wilson binary interaction parameters of the key components were obtained. Next, one traditional distillation column sequence and two dividing wall column (DWC) sequences were designed to separate the F–T heavy oil to obtain narrow fractions with different carbon numbers. Then, the obtained fractions of C10 and C12 were simulated to obtain 1-decene and 1-dodecene, respectively. There was a traditional distillation and a differential pressure thermal coupling distillation process. When separating 95.0% purity 1-decene and 1-octene, the direct DWC process and differential pressure thermal coupled distillation are an excellent combination, which can reduce the energy by 33.1% (i.e., 11,286 kW) and total annual cost by 15.9% (i.e., 3.96 × 106 $) compared with traditional distillation.

Understanding the influence of the heavy metal cadmium (Cd) on the phyllosphere microbiome of hyperaccumulator plants is crucial for enhancing phytoremediation. The characteristics of the phyllosphere of Sedum alfredii Hance, a hyperaccumulator plant, were investigated using 16S rRNA and internal transcribed spacer amplicon sequencing of powdery mildew-infected leaves treated or untreated with Cd. The results showed that the colonization of powdery mildew caused severe chlorosis and necrosis in S. alfredii leaves, and the relative abundance of Leotiomycetes in infected leaves increased dramatically and significantly decreased phyllosphere microbiome diversity. However, S. alfredii preferentially accumulated higher concentrations of Cd in the leaves of infected plants than in uninfected plants by powdery mildew, which in turn significantly inhibited powdery mildew colonization in leaves; the relative abundance of the fungal class Leotiomycetes in infected leaves decreased, and alpha and beta diversities of the phyllosphere microbiome significantly increased with Cd treatment in the infected plants. In addition, the inter-kingdom networks in the microbiota of the infected leaves treated with Cd presented many nodes and edges, and the highest inter-kingdom modularity compared to the untreated infected leaves, indicating a highly connected microbial community. These results suggest that Cd significantly inhibits powdery mildew colonization by altering the composition of the phyllosphere microbiome in S. alfredii leaves, paving the way for efficient heavy metal phytoremediation and providing a new perspective on defense strategies against heavy metals.

BackgroundThere is increasing evidence that neoadjuvant chemoradiotherapy is superior to neoadjuvant chemotherapy for patients with locally advanced gastric cancer. However, a number of studies have come to the opposite conclusion. Therefore, our meta-analysis is to evaluate the efficacy and safety of neoadjuvant chemoradiotherapy versus neoadjuvant chemotherapy in the treatment of locally advanced gastric cancer.MethodsWe searched Wanfang Database, China National Knowledge Network database, VIP database, China Biomedical Literature Database, PubMed, Embase and Cochrane Library. The searched terms included’Stomach Neoplasms’, ‘Neoadjuvant Therapy’ and ‘Chemoradiotherapy’. The retrieval time was from the establishment of the corresponding database to September 2022, and our meta-analysis was performed using RevMan (version 5.3) and Stata (version 17) software.ResultsA total of 17 literatures were included, which involved 7 randomized controlled trials and 10 retrospective studies, with a total of 6831 patients. The results of meta-analysis showed that compared with NACT group, the complete response rate(RR=1.95, 95%CI 1.39-2.73, p=0.0001), the partial response rate(RR=1.44, 95%CI 1.22-1.71, p=0.0001), the objective response rate(RR=1.37, 95%CI 1.27-1.54, p=0.00001), the pathologic complete response rate(RR=3.39, 95%CI 2.17-5.30, p=0.00001), the R0 resection rate(RR=1.18, 95%CI 1.09-1.29, p=0.0001) and 3-year overall survival rate(HR=0.89, 95%CI 0.82-0.96, p=0.002) of neoadjuvant chemoradiotherapy group were significantly improved. The results of subgroup analyses of gastric cancer subgroup and gastroesophageal junction cancer subgroup were consistent with the overall results. Meanwhile, the stable disease(RR=0.59, 95%CI:0.44-0.81, P=0.0010) of neoadjuvant chemoradiotherapy group was lower than that of neoadjuvant chemotherapy group, and there were no statistical significance in the progressive disease rate(RR=0.57, 95%CI:0.31-1.03, P=0.06), five-year overall survival rate(HR=1.03, 95%CI:0.99-1.07, P=0.839), postoperative complications and adverse reactions between the neoadjuvant chemoradiotherapy group and neoadjuvant chemotherapy group.ConclusionCompared with neoadjuvant chemotherapy, neoadjuvant chemoradiotherapy might bring more survival benefits without significantly increasing adverse reactions. neoadjuvant chemoradiotherapy may be a recommended treatment for patients with locally advanced gastric cancer.Systematic Review Registrationhttps://inplasy.com/inplasy-2022-12-0068/, identifier INPLASY202212068.

Corynebacterium glutamicum is a major workhorse in industrial biomanufacturing of amino acids. Here, the authors employ CRISPR-assisted rational flux-tuning and CRISPRi screening of a L-proline exporter to covert a wild-type C. glutamicum to a hyperproducer of L-proline.

ObjectivesAndrogen deprivation therapy combined with radiotherapy for intermediate-risk prostate cancer is still a matter of debate. We conducted a meta-analysis to evaluate the necessity of androgen deprivation therapy combined with radiotherapy for intermediate-risk prostate cancer patients.MethodsA comprehensive literature search of articles was performed in PubMed, Embase, Cochrane library, Web of Science, Chinese National Knowledge Infrastructure, Chinese Biological Medicine, Wanfang, and VIP Databases published between February 1988 and April 2022. Studies comparing the survival of patients diagnosed with intermediate-risk prostate cancer who were treated with androgen deprivation therapy combined with radiotherapy or radiotherapy alone were included. Data were extracted and analyzed with the RevMan software (version 5.3) and the Stata software (version 17).ResultsSix randomized controlled trials and nine retrospective studies, including 6853 patients (2948 in androgen deprivation therapy combined with radiotherapy group and 3905 in radiotherapy alone group) were enrolled. Androgen deprivation therapy combined with radiotherapy did not provide an overall survival (HR 1.12, 95% CI 1.01-1.12, p=0.04) or biochemical recurrence-free survival (HR 1.23, 95% CI 1.09-1.39, P=0.001) advantage to intermediate-risk prostate cancer patients.ConclusionAndrogen deprivation therapy combined with radiotherapy did not show some advantages in terms of overall survival and biochemical recurrence-free survival and radiotherapy alone may be the effective therapy for intermediate-risk prostate cancer patients.Systematic review registrationhttps://inplasy.com/inplasy-2022-8-0095/, identifier 202280095.

ObjectivesTo assess the clinical efficacy and toxicity of nimotuzumab in combination with chemoradiotherapy or chemoradiotherapy alone in the treatment of cervical cancer.MethodsThe PubMed, Embase, Cochrane Library, Web of Science, China National Knowledge Infrastructure, China Biomedical Medicine, Wanfang, and VIP databases were systematically searched for relevant literature. Ultimately, six randomised controlled trials (n=393) were included in our meta-analysis.ResultsA total of 393 patients were included, of which 197 were in the nimotuzumab combined with chemoradiotherapy group and 196 were in the chemoradiotherapy group. The results of our meta-analysis showed that the complete remission rate (risk ratio [RR] = 1.34, 95% confidence interval [CI]: 1.08-1.65, P = 0.007), objective response rate (RR = 1.30, 95% CI: 1.16-1.44, P < 0.05), and three-year survival rate (RR = 1.27, 95% CI: 1.06-1.51, P = 0.008) in the nimotuzumab combined with chemoradiotherapy group were significantly improved compared with the chemoradiotherapy group. This difference was not statistically significant when comparing the incidence of adverse reactions (such as leukocytopenia, gastrointestinal reaction, radiocystitis, and radioproctitis) between the two groups.ConclusionsNimotuzumab in combination with chemoradiotherapy has some advantages over chemoradiotherapy alone in the treatment of cervical cancer and does not increase toxicity. Therefore, nimotuzumab has the potential to be an effective treatment for cervical cancer; however, further evidence from large-scale randomised controlled trials is needed.

ObjectivesTo evaluate the clinical curative effects and toxicity of neoadjuvant chemoradiotherapy for resectable gastric cancer compared to those of neoadjuvant chemotherapy.MethodsA systematic review and meta-analysis of the randomized controlled trials (RCTs) of neoadjuvant chemoradiotherapy versus neoadjuvant chemotherapy were performed in patients with resectable gastric cancer.ResultsSeven RCTs were included (601 patients; 302 in the neoadjuvant chemoradiotherapy group and 299 in the neoadjuvant chemotherapy group). The neoadjuvant chemoradiotherapy group had an increased number of patients with a complete response [odds ratio (OR) = 3.79, 95% confidence interval (CI): 1.68–8.54, p = 0.001] and improved objective response rate (OR = 2.78, 95% CI: 1.69–4.57, p < 0.0001), 1-year (OR = 3.51, 95% CI: 1.40–8.81, p = 0.007) and 3-year (OR = 2.14, 95% CI: 1.30–3.50, p = 0.003) survival rates, R0 resection rate (OR = 2.21, 95% CI: 1.39–3.50, p = 0.0008), and complete pathologic response (OR = 4.39, 95% CI: 1.59–12.14, p = 0.004). Regarding the incidence of adverse effects after neoadjuvant therapy, only the occurrence rate of gastrointestinal reaction in the neoadjuvant chemoradiotherapy group was higher than that in the neoadjuvant chemotherapy group (OR = 1.76, 95% CI: 1.09–2.85, p = 0.02), and there was no significant difference in other adverse effects. There was no difference in the incidence of postoperative complications between the two groups.ConclusionNeoadjuvant chemoradiotherapy for resectable gastric cancer has several advantages in terms of efficacy and safety compared to neoadjuvant chemotherapy. Therefore, neoadjuvant chemoradiotherapy has great potential as an effective therapy for resectable gastric cancers.Systematic Review Registrationhttps://inplasy.com/inplasy-2022-3-0164, registration number INPLASY202230164.

Citrus are one of the most important fruit crops in the world, and the citrus hybrid cv. Beni-Madonna (BM) is widely cultivated in Xiangshan County in China because of its profitability. However, due to the lack of technical guidance and management, nutritional unbalances of trees occur frequently and are a major constraint for fruit quality and yield. In this study, the soil and fruit nutritional status of 62 citrus orchards in three diverse landforms (mountain, flat, and coastal saline–alkali land) located in Xiangshan County has been investigated. Higher pH, calcium, and magnesium content but lower soil organic matter, nitrogen, iron, and boron content were observed in orchards of the coastal saline–alkali land compared with the mountain and flat lands. Compared to Citrus x unshiu, another major hybrid citrus cultivar in Xiangshan county, the Fe deficiency in fruits of BM in coastal saline–alkali lands was more severe, leading to chlorosis symptoms. In addition, long-term cultivation under plastic housing increased soil salinization and affected the absorption of Fe and other nutrients by BM. In conclusion, organic fertilizers, acidic fertilizers, and micronutrient fertilizers should be applied in citrus orchards located in coastal saline–alkali lands. Especially, BM orchards should be supplemented with iron fertilizer and not rely excessively on protection with plastic films.

To obtain optimal yield and productivity in bioproduction, expression of pathway genes must be appropriately coordinated. Here, the authors report repurposing of base editors for simultaneous regulation of multiple gene expression and demonstrate its application in industrially important and model microorganisms.

Abstract Background 5-Aminolevulinic acid (5-ALA) is a promising biostimulant, feed nutrient, and photodynamic drug with wide applications in modern agriculture and therapy. Considering the complexity and low yield of chemical synthesis methods, bioproduction of 5-ALA has drawn intensive attention recently. However, the present bioproduction processes use refined glucose as the main carbon source and the production level still needs further enhancement. Results To lay a solid technological foundation for large-scale commercialized bioproduction of 5-ALA, an industrial workhorse Corynebacterium glutamicum was metabolically engineered for high-level 5-ALA biosynthesis from cheap renewable bioresources. After evaluation of 5-ALA synthetases from different sources, the 5-ALA biosynthetic pathway and anaplerotic pathway were rebalanced by regulating intracellular activities of 5-ALA synthetase and phosphoenolpyruvate carboxylase. The engineered biocatalyst produced 5.5 g/L 5-ALA in shake flasks and 16.3 g/L in 5-L bioreactors with a one-step fermentation process from glucose. To lower the cost of feedstock, cheap raw materials were used to replace glucose. Enzymatically hydrolyzed cassava bagasse was proven to be a perfect alternative to refined sugars since the final 5-ALA titer further increased to 18.5 g/L. Use of corn starch hydrolysate resulted in a similar 5-ALA production level (16.0 g/L) with glucose, whereas use of beet molasses caused seriously inhibition. The results obtained here represent a new record of 5-ALA bioproduction. It is estimated that replacing glucose with cassava bagasse will reduce the carbon source cost by 90.1%. Conclusions The high-level biosynthesis of 5-ALA from cheap bioresources will brighten the prospects for industrialization of this sustainable and environment-friendly process. The strategy for balancing metabolic flux developed in this study can also be used for improving the bioproduction of other value-added chemicals.

Soil cadmium (Cd) extraction for hyperaccumulators is one of the most important technologies for the remediation of Cd-contaminated farmland soil. However, a phytoremediation model using a single hyperaccumulator cannot guarantee normal agricultural production in contaminated areas. To solve this problem, a combination of efficient remediation and safe production has been developed. Based on two-period field experiments, this study explored the effect of biofortification on soil Cd remediation using the fruit tree Sedum alfredii Hance and oil sunflower crop rotation and relay cropping mode. BioA and BioB treatments could markedly improve the efficiency of Cd extraction and remediation, and the maximum increase in Cd accumulation was 243.29%. When BioB treatment was combined with papaya–S. alfredii and oil sunflower crop rotation and relay cropping mode, the highest soil Cd removal rate in the two periods was 40.84%, whereas the Cd concentration of papaya fruit was lower than safety production standards (0.05 mg/kg). These results demonstrate that biofortification measures can significantly improve the Cd extraction effect of S. alfredii crop rotation and relay cropping restoration modes, which has guiding significance for Cd pollution remediation and safe production in farmland.

ObjectivesTo evaluate the clinical curative effects and toxicity of recombinant human adenovirus-p53 injection (rAd-p53) plus chemotherapy (CT), radiotherapy (RT), or concurrent chemoradiotherapy (CRT) for the treatment of cervical cancer.MethodsWe identified 14 eligible studies in the PubMed, Web of Science, Cochrane Library, Embase, CNKI, Wangfangdate, CBM, and VIP databases from their inception to May 2021 and performed meta-analyses using RevMan version 5.3.ResultsThis analysis included 14 studies involving 737 patients. The results of the meta-analysis results showed significantly improved complete remission (odds ratio [OR] = 2.54, 95% confidence interval [CI]: 1.74–3.70, p < 0.00001), partial remission (OR = 1.56, 95% CI: 1.14–2.14, p = 0.006), and object response (OR = 4.47, 95% CI: 3.02–6.60, p < 0.00001) rates in the rAd-p53 combination therapy group compared to those in the CT/RT/CRT group. The results of subgroup analyses of CT/RT/CRT were consistent with the overall results. Regarding the incidence of adverse reactions, only the occurrence rate of fever (OR = 18.21, 95% CI: 10.54–31.47, p < 0.00001) in the rAd-p53 combination group was higher than that in the CT/RT/CRT group. No other significant differences were observed in other adverse reactions.ConclusionRAd-p53 combined with CT/RT/CRT for the treatment of cervical cancer showed significant advantages in efficacy and safety compared to those in the CT/RT/CRT group. Therefore, rAd-p53 has great potential as an effective therapy for cervical cancer.Systematic Review Registrationhttps://inplasy.com/inplasy-2021-5-0058/.

Chinese bayberry (Myrica rubra) is a popular, nutrient- and antioxidant-rich fruit in Asia. However, it is susceptible to Drosophila during ripening, which disrupts production and causes economic loss. This study compared the effects of insecticides, insect-proof nets (IPNs), and insect- and rain-proof nets (IRPNs) on Chinese bayberry production and quality. Drosophila was absent in fruits from IPN- or IRPN-treated trees but only significantly reduced by insecticides. IPNs and IRPNs significantly increased fruit diameter, weight, edible rate and the Brix/acid ratio, and IRPNs had the strongest effect. Analysis of 16S rDNA showed that fruits collected from differently treated trees had unique bacterial communities. In IRPN fruits, Acetobacter and Gluconobacter were significantly decreased, reducing sugar consumption and disease; in addition, PICRUSt analysis predicted imputed functional profiles related to carbohydrate and nitrogen metabolism and mineral transport for fruit growth and development. This study proposed the use of IRPNs for improving Chinese bayberry production and quality.

Corynebacterium glutamicum is an important workhorse for industrial production of diversiform bioproducts. Precise regulation of gene expression is crucial for metabolic balance and enhancing production of target molecules. Auto-inducible promoters, which can be activated without expensive inducers, are ideal regulatory tools for industrial-scale application. However, few auto-inducible promoters have been identified and applied in C. glutamicum. Here, a hyperosmotic stress inducible gene expression system was developed and used for metabolic engineering of C. glutamicum. The promoter of NCgl1418 (PNCgl1418) that was activated by the two-component signal transduction system MtrA/MtrB was found to exhibit a high inducibility under hyperosmotic stress conditions. A synthetic promoter library was then constructed by randomizing the flanking and space regions of PNCgl1418, and mutant promoters exhibiting high strength were isolated via fluorescence activated cell sorting (FACS)-based high-throughput screening. The hyperosmotic stress inducible gene expression system was applied to regulate the expression of lysE encoding a lysine exporter and repress four genes involved in lysine biosynthesis (gltA, pck, pgi, and hom) by CRISPR interference, which increased the lysine titer by 64.7% (from 17.0 to 28.0 g/L) in bioreactors. The hyperosmotic stress inducible gene expression system developed here is a simple and effective tool for gene auto-regulation in C. glutamicum and holds promise for metabolic engineering of C. glutamicum to produce valuable chemicals and fuels.

Lead (Pb) contamination presents a widespread environmental plague. Sedum alfredii is widely used for soil phytoremediation owing to its capacity to extract heavy metals, such as Pb. Although efficient Pb extraction is mediated by complex interactions between the roots and rhizospheric bacteria, the mechanism by which S. alfredii recruits microorganisms under Pb stress remains unclear. The Pb-accumulating ecotype (AE) and non-accumulating ecotype (NAE) of S. alfredii recruited different rhizobacterial communities. Under Pb stress, AE rhizosphere-enriched bacteria assembled into stable-connected clusters with higher phylogenetic and functional diversity. These microbes, e.g., Flavobacterium, could release indoleacetic acid to promote plant growth and siderophores, thereby increasing Pb availability. The NAE rhizosphere-enriched functional bacteria “desperately” assembled into highly specialized functional clusters with extremely low phylogenetic diversity. These bacteria, e.g., Pseudomonas, could enhance phosphorus solubilization and Pb precipitation, thereby reducing Pb stress and plant Pb accumulation. High niche overlap level of the rhizo-enriched species raised challenges in soil resource utilization, whereas the NAE community assembly was markedly constrained by environmental “selection effect” than that of AE rhizobacterial community. These results indicate that different ecotypes of S. alfredii recruit distinct bacterial functional assemblies to drive specific plant-soil feedbacks for different survival in Pb-contaminated soils. To cope with heavy metal stress, NAE formed a highly functional and specialized but vulnerable community and efficiently blocked heavy metal absorption by plants. However, the AE community adopted a more stable and elegant strategy to promote plant growth and the accumulation of dry matter via multiple evolutionary strategies that ensured a high yield of heavy metal phytoextraction. This for the first time provides new insights into the roles of rhizosphere microbes in plant adaptations to abiotic stresses.

RNA-guided and RNA-targeting type IV-D CRISPR/Cas systems (CRISPR/Cas13d) have recently been identified and employed for efficient and specific RNA knockdown in mammalian and plant cells. Cas13d possesses dual RNase activities and is capable of processing CRISPR arrays and cleaving target RNAs in a protospacer flanking sequence (PFS)-independent manner. These properties make this system a promising tool for multiplex gene expression regulation in microbes. Herein, we aimed to establish a CRISPR/Cas13d-mediated RNA knockdown platform for bacterial chassis. CasRx, Cas13d from Ruminococcus flavefaciens XPD3002, was selected due to its high activity. However, CasRx was found to be highly toxic to both Escherichia coli and Corynebacterium glutamicum, especially when it cooperated with its guide and target RNAs. After employing a low copy number vector, a tightly controlled promoter, and a weakened ribosome binding site, we successfully constructed an inducible expression system for CasRx and applied it for repressing the expression of a green fluorescent protein (GFP) in E. coli. Despite our efforts to optimize inducer usage, guide RNA (gRNA) architecture and combination, and target gene expression level, the highest gene repression efficiency was 30–50% at the protein level and ∼70% at the mRNA level. The moderate RNA knockdown is possibly caused by the collateral cleavage activity toward bystander RNAs, which acts as a mechanism of type IV-D immunity and perturbs microbial metabolism. Further studies on cellular response to CRISPR/Cas13d and improvement in RNA knockdown efficiency are required prior to practical application of this system in microbes.

Corynebacterium glutamicum is an important workhorse for industrial production of diversiform bioproducts. Multiplex control of metabolic pathway genes is crucial for maximizing biosynthesis of desired products. However, few tools for simultaneously regulating multiple genes in C. glutamicum have been reported. Here, a CRISPR-dCpf1-based multiplex gene repression system was developed for C. glutamicum. This system successfully repressed two fluorescent reporter genes simultaneously by expressing a dCpf1 (E1006A, D917A) and a designed single crRNA array. To demonstrate applications of this CRISPR-dCpf1 system in metabolic engineering, we applied this system to repress four genes involved in lysine biosynthesis (gltA, pck, pgi, and hom) with a single array, which increased the lysine titer and yield for over 4.0-fold. Quantitative PCR demonstrated that transcription of all the four endogenous target genes were repressed by over 90%. Thus, the CRISPR-dCpf1 system is a simple and effective technique for multiplex gene repression in C. glutamicum and holds promise for metabolic engineering of C. glutamicum to produce valuable chemicals and fuels.

Lipid A, the hydrophobic anchor of lipopolysaccharide (LPS), is an essential component in the outer membrane of Gram-negative bacteria. It can stimulate the innate immune system via Toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD2), leading to the release of inflammatory cytokines. In this study, six Escherichia coli strains which can produce lipid A with different acylation patterns were constructed; the influence of lipid A acylation pattern on the membrane permeability and innate immune stimulation has been systematically investigated. The lipid A species were isolated and identified by matrix assisted laser ionization desorption-time of flight/tandem mass spectrometry. N-Phenyl naphthylamine uptake assay and antibiotic susceptibility test showed that membrane permeability of these strains were different. The lower the number of acyl chains in lipid A, the stronger the membrane permeability. LPS purified from these strains were used to stimulate human or mouse macrophage cells, and different levels of cytokines were induced. Compared with wild type hexa-acylated LPS, penta-acylated, tetra-acylated and tri-acylated LPS induced lower levels of cytokines. These results suggest that the lipid A acylation pattern influences both the bacterial membrane permeability and innate immune stimulation. The results would be useful for redesigning the bacterial membrane structure and for developing lipid A vaccine adjuvant.

The lipid A moiety of Escherichia coli lipopolysaccharide is a hexaacylated disaccharide of glucosamine phosphorylated at the 1- and 4′-positions. It can be recognized by the TLR4/MD-2 complex of mammalian immune cells, leading to release of proinflammatory cytokines. The toxicity of lipid A depends on its structure. In this study, two E. coli mutants, HW001 and HW002, were constructed by deleting or integrating key genes related to lipid A biosynthesis in the chromosome of E. coli W3110. HW001 was constructed by deleting lacI and replacing lacZ with the Francisella novicida lpxE gene in the chromosome and only synthesizes monophosphoryl lipid A. HW002 was constructed by deleting lpxM in HW001 and synthesizes only the pentaacylated monophosphoryl lipid A. The structures of lipid A made in HW001 and HW002 were confirmed by thin layer chromatography and electrospray ionization mass spectrometry. HW001 and HW002 grew as well as the wild-type W3110. LPS purified from HW001 or HW002 was used to stimulate murine macrophage RAW264.7 cells, and less TNF-α were released. This study provides a feasible way to produce interesting lipid A species in E. coli.

Objectives: Androgen deprivation therapy combined with radiotherapy for intermediate-risk prostate cancer is still a matter of debate. We conducted a meta-analysis to evaluate the necessity of androgen deprivation therapy combined with radiotherapy for intermediate-risk prostate cancer patients. Methods: A comprehensive literature search of articles was performed in PubMed, Embase, Cochrane library, Web of Science, Chinese National Knowledge Infrastructure, Chinese Biological Medicine, Wanfang, and VIP Databases published between February 1988 and April 2022. Studies comparing the survival of patients diagnosed with intermediate-risk prostate cancer who were treated with androgen deprivation therapy combined with radiotherapy or radiotherapy alone were included. Data were extracted and analyzed with the RevMan software (version 5.3) and the Stata software (version 17). Results: Six randomized controlled trials and nine retrospective studies, including 6853 patients (2948 in androgen deprivation therapy combined with radiotherapy group and 3905 in radiotherapy alone group) were enrolled. Androgen deprivation therapy combined with radiotherapy did not provide an overall survival (HR 1.12, 95% CI 1.01-1.12, p=0.04) or biochemical recurrence-free survival (HR 1.23, 95% CI 1.09-1.39, P=0.001) advantage to intermediate-risk prostate cancer patients. Conclusion: Androgen deprivation therapy combined with radiotherapy did not show some advantages in terms of overall survival and biochemical recurrencefree survival and radiotherapy alone may be the effective therapy for intermediaterisk prostate cancer patients. [ABSTRACT FROM AUTHOR]