Cervicovaginal microbiota long-term dynamics and prediction of different outcomes in persistent human papillomavirus infection.

Persistent human papillomavirus (HPV) infection can lead to cervical intraepithelial neoplasia (CIN) and cervical cancer, posing serious threats to the health of women. Although the cervicovaginal microbiota is strongly associated with CIN, the dynamics of the microbiota during CIN development are unknown. In this retrospective cohort study, we analyzed 3-year longitudinal data from 72 patients diagnosed with a persistent HPV infection almost all caused by high-risk HPV types. Patients were categorized into groups with HPV persistent infection (n = 37), progression to CIN (n = 16), and CIN regression (n = 19) based on infection outcome during the follow-up period. Furthermore, 16S rRNA gene sequencing was performed on consecutively collected cervical samples to explore the composition and dynamics of the cervicovaginal microbiota during the development and regression of CIN. Our results showed that the composition of the cervicovaginal microbiota varied among women with different HPV infection outcomes and remained relatively stable during the follow-up period. Notably, the serial follow-up data showed that these microbial alterations were present for at least 1-2 years and occurred before pathologic changes. In addition, microbial markers that were highly discriminatory for CIN progression or regression were identified. This study provides evidence for a temporal relationship between changes in the cervicovaginal microbiota and the development of CIN, and our findings provide support for future microbial intervention strategies for CIN.

Metabolic engineering of fast-growing Vibrio natriegens for efficient pyruvate production.

BACKGROUND: Pyruvate is a widely used value-added chemical which also serves as a hub of various metabolic pathways. The fastest-growing bacterium Vibrio natriegens is a promising chassis for synthetic biology applications with high substrate uptake rates. The aim of this study was to investigate if the high substrate uptake rates of V. natriegens enable pyruvate production at high productivities. RESULTS: Two prophage gene clusters and several essential genes for the biosynthesis of byproducts were first deleted. In order to promote pyruvate accumulation, the key gene aceE encoding pyruvate dehydrogenase complex E1 component was down-regulated to reduce the carbon flux into the tricarboxylic acid cycle. Afterwards, the expression of ppc gene encoding phosphoenolpyruvate carboxylase was fine-tuned to balance the cell growth and pyruvate synthesis. The resulting strain PYR32 was able to produce 54.22 g/L pyruvate from glucose within 16 h, with a yield of 1.17 mol/mol and an average productivity of 3.39 g/L/h. In addition, this strain was also able to efficiently convert sucrose or gluconate into pyruvate at high titers. CONCLUSION: A novel strain of V. natriegens was engineered which was capable to provide higher productivity in pyruvate synthesis. This study lays the foundation for the biosynthesis of pyruvate and its derivatives in fast-growing V. natriegens.

The Partial Reconstruction Symplectic Geometry Mode Decomposition and Its Application in Rolling Bearing Fault Diagnosis.

Extracting the fault characteristic information of rolling bearings from intense noise disturbance has been a heated research issue. Symplectic geometry mode decomposition (SGMD) has already been adopted for bearing fault diagnosis due to its advantages of no subjective customization of parameters and the ability to reconstruct existing modes. However, SGMD suffers from rapidly decreasing calculation efficiency as the amount of data increases, in addition to invalid symplectic geometry components affecting decomposition accuracy. The regularized composite multiscale fuzzy entropy (RCMFE) operator is constructed to evaluate the complexity of each initial single component and minimize the residual energy. Combined with the partial reconstruction threshold indicator to filter out specific significant initial single components, the raw signal can be decomposed into multiple physically meaningful symplectic geometric mode components. Therefore, the decomposition efficiency and accuracy can be enhanced. Thus, a rolling bearing fault diagnosis method is proposed based on partial reconstruction symplectic geometry mode decomposition (PRSGMD). Both simulated and experimental analysis results show that PRSGMD can improve the speed of SGMD analysis while increasing the decomposition accuracy, thereby augmenting the robustness and effectiveness of the algorithm.

[Metabolic engineering of Escherichia coli for production of salicylate 2-O-ß-d-glucoside].

Salicylate 2-O-ß-d-glucoside (SAG) is a derivative of salicylate in plants. Recent reports showed that SAG could be considered as a potential anti-inflammatory substance due to its anti-inflammatory and analgesic effects, and less irritation compared with salicylic acid and aspirin. The biological method uses renewable resources to produce salicylic acid compounds, which is more environmentally friendly than traditional industry methods. In this study, Escherichia coli Tyr002 was used as the starting strain, and a salicylic acid producing strain of E. coli was constructed by introducing the isochorismate pyruvate lyase gene pchB from Pseudomonas aeruginosa. By regulating the expression of the key genes in the downstream aromatic amino acid metabolic pathways, the titer of salicylic acid reached 1.05 g/L in shake flask fermentation. Subsequently, an exogenous salicylic acid glycosyltransferase was introduced into the salicylic acid producing strain to glycosylate the salicylic acid. The newly engineered strain produced 5.7 g/L SAG in shake flask fermentation. In the subsequent batch fed fermentation in a 5 L fermentation tank, the titer of SAG reached 36.5 g/L, which is the highest titer reported to date. This work provides a new route for biosynthesis of salicylate and its derivatives.

Multi-step biosynthesis of the biodegradable polyester monomer 2-pyrone-4,6-dicarboxylic acid from glucose.

BACKGROUND: 2-Pyrone-4,6-dicarboxylic acid (PDC), a chemically stable pseudoaromatic dicarboxylic acid, represents a promising building block for the manufacture of biodegradable polyesters. Microbial production of PDC has been extensively investigated, but low titers and yields have limited industrial applications. RESULTS: In this study, a multi-step biosynthesis strategy for the microbial production of PDC was demonstrated using engineered Escherichia coli whole-cell biocatalysts. The PDC biosynthetic pathway was first divided into three synthetic modules, namely the 3-dehydroshikimic acid (DHS) module, the protocatechuic acid (PCA) module and the PDC module. Several effective enzymes, including 3-dehydroshikimate dehydratase for the PCA module as well as protocatechuate 4,5-dioxygenase and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase for the PDC module were isolated and characterized. Then, the highly efficient whole-cell bioconversion systems for producing PCA and PDC were constructed and optimized, respectively. Finally, the efficient multi-step biosynthesis of PDC from glucose was achieved by smoothly integrating the above three biosynthetic modules, resulting in a final titer of 49.18 g/L with an overall 27.2% molar yield, which represented the highest titer for PDC production from glucose reported to date. CONCLUSIONS: This study lays the foundation for the microbial production of PDC, including one-step de novo biosynthesis from glucose as well as the microbial transformation of monoaromatics.

Characterization of glycosylation regulator-mediated glycosylation modification patterns and tumor microenvironment infiltration in hepatocellular carcinoma.

Background: Previous studies have shown that glycosylation of proteins ofen plays an important role in HCC. However, the potential mechanism of glycosylation in HCC has not been described systematically. Methods: We comprehensively evaluated the glycosylation patterns in HCC samples based on 43 glycosylation regulators, and annotated the modification patterns with the enrichment of immune cells and stromal cells. Considering the heterogeneity of HCC patients, the glycosylation score was constructed using single-sample gene set enrichment analysis (ssGSEA). We also explored the drugs that different HCC patients were sensitive to based on glycosylation mode and score. Results: We identified three glycosylation-regulated gene subtypes. By annotating the subtypes, it was found that the glycosylation regulated gene subtypes was highly matched with three immunophenotypes of HCC (immune-inflamed, immune-excluded, and immune-desert), regardless of the characteristics of immune cell infiltration or prognosis. Based on the characteristic genes of glycosylation-regulated gene subtypes, we constructed a glycosylation-related model, and found that glycosylation-related model was highly consistent with the glycosylation regulated gene subtypes. The glycosylation score that evaluates the glycosylation characteristics of a single HCC sample has high prognostic value, and the prognosis of patients with high glycosylation score is significantly worse. Interestingly, we found that the glycosylation score was closely related to tumor node metastasis (TNM) staging. By applying glycosylation-regulated gene subtypes and glycosylation score to explore the sensitivity of different patients to anticancer drugs, it was found that the sensitivity of Thapsigargin, Shikonin, Embelin and Epothilone. B was closely related to the glycosylation mode. Conclusion: This study reveals that the diversity of glycosylation patterns plays an important role in HCC. Therefore, evaluating the glycosylation patterns of patients with HCC will be helpful in identifying the characteristics of immune cell infiltration and selecting accurate treatment methods.

7-Hydroxyflavone Alleviates Myocardial Ischemia/Reperfusion Injury in Rats by Regulating Inflammation.

Inflammation is the primary pathological process of myocardial ischemia/reperfusion injury (MI/RI). 7-Hydroxyflavone (HF), a natural flavonoid with a variety of bioactivities, plays a crucial role in various biological processes. However, its cardioprotective effects and the underlying mechanisms of MI/RI have not been investigated. This study aimed to explore whether pretreatment with HF could attenuate MI/RI-induced inflammation in rats and investigate its potential mechanisms. The results showed that pretreatment with HF could significantly improve the anatomic data and electrocardiograph parameters, reduce the myocardial infarct size, decrease markers of myocardial injury (aspartate transaminase, creatine kinase, lactate dehydrogenase, and cardiac troponin I), inhibit inflammatory cytokines (IL-1ß, IL-6, and TNF-α), suppress oxidative stress, and recover the architecture of the cardiomyocytes. The cardioprotective effect of HF was connected with the regulation of the MAPK/NF-κB signaling pathway. What is more, molecular docking was carried out to prove that HF could be stably combined with p38, ERK1/2, JNK, and NF-κB. In summary, this is a novel study demonstrating the cardioprotective effects of HF against MI/RI in vivo. Consequently, these results demonstrate that HF can be considered a promising potential therapy for MI/RI.

Anti-Fatigue Effects of Lycium barbarum Polysaccharide and Effervescent Tablets by Regulating Oxidative Stress and Energy Metabolism in Rats.

The purpose of this study was to investigate the anti-fatigue effect of natural Lycium barbarum polysaccharide (LBP) during exercise, develop a functional anti-fatigue effervescent tablet by applying LBP to practical products, and help patients who have difficulty swallowing conventional tablets or capsules. LBP was extracted with water, and DEAE-52 cellulose was used for purification. The chemical structure and monosaccharide composition of LBP by Fourier transform infrared spectroscopy (FI-IR) and ion chromatography (IC). Lycium barbarum polysaccharide effervescent tablets (LBPT) were prepared by mixing LBP and an excipient. Animal experiments showed that LBP and LBPT significantly increased the exhaustive swimming time in rats. LBP and LBPT improved biochemical markers in rat serum, such as lactic acid and creatine kinase, enhanced the antioxidant capacity of rat muscle, and reversed the decrease in serum glucose, ATP and glycogen content caused by exercise. Transmission electron microscopy showed that LBP and LBPT increased the density of mitochondria in rat liver. In addition, molecular experiments showed that LBP and LBPT could improve oxidative stress caused by exercise by regulating the Nrf2/HO-1 signaling pathway and regulating energy metabolism via the AMPK/PGC-1α signaling pathway.

Identification of hub biomarkers of myocardial infarction by single-cell sequencing, bioinformatics, and machine learning.

Background: Myocardial infarction (MI) is one of the first cardiovascular diseases endangering human health. Inflammatory response plays a significant role in the pathophysiological process of MI. Messenger RNA (mRNA) has been proven to play a key role in cardiovascular diseases. Single-cell sequencing (SCS) technology is a new technology for high-throughput sequencing analysis of genome, transcriptome, and epigenome at the single-cell level, and it also plays an important role in the diagnosis and treatment of cardiovascular diseases. Machine learning algorithms have a wide scope of utilization in biomedicine and have demonstrated superior efficiency in clinical trials. However, few studies integrate these three methods to investigate the role of mRNA in MI. The aim of this study was to screen the expression of mRNA, investigate the function of mRNA, and provide an underlying scientific basis for the diagnosis of MI. Methods: In total, four RNA microarray datasets of MI, namely, GSE66360, GSE97320, GSE60993, and GSE48060, were downloaded from the Gene Expression Omnibus database. The function analysis was carried out by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Disease Ontology (DO) enrichment analysis. At the same time, inflammation-related genes (IRGs) were acquired from the GeneCards database. Then, 52 co-DEGs were acquired from differentially expressed genes (DEGs) in differential analysis, IRGs, and genes from SCS, and they were used to construct a protein-protein interaction (PPI) network. Two machine learning algorithms, namely, (1) least absolute shrinkage and selection operator and (2) support vector machine recursive feature elimination, were used to filter the co-DEGs. Gene set enrichment analysis (GSEA) was performed to screen the hub-modulating signaling pathways associated with the hub genes. The results were validated in GSE97320, GSE60993, and GSE48060 datasets. The CIBERSORT algorithm was used to analyze 22 infiltrating immune cells in the MI and healthy control (CON) groups and to analyze the correlation between these immune cells. The Pymol software was used for molecular docking of hub DEGs and for potential treatment of MI drugs acquired from the COREMINE. Results: A total of 126 DEGs were in the MI and CON groups. After screening two machine learning algorithms and key co-DEGs from a PPI network, two hub DEGs (i.e., IL1B and TLR2) were obtained. The diagnostic efficiency of IL1B, TLR2, and IL1B + TLR2 showed good discrimination in the four cohorts. GSEA showed that KEGG enriched by DEGs were mainly related to inflammation-mediated signaling pathways, and GO biological processes enriched by DEGs were linked to biological effects of various inflammatory cells. Immune analysis indicated that IL1B and TLR2 were correlated with various immune cells. Dan shen, san qi, feng mi, yuan can e, can sha, san qi ye, san qi hua, and cha shu gen were identified as the potential traditional Chinese medicine (TCM) for the treatment of MI. 7-hydroxyflavone (HF) had stable combinations with IL1B and TLR2, respectively. Conclusion: This study identified two hub DEGs (IL1B and TLR2) and illustrated its potential role in the diagnosis of MI to enhance our knowledge of the underlying molecular mechanism. Infiltrating immune cells played an important role in MI. TCM, especially HF, was a potential drug for the treatment of MI.

Mechanism of Action of Flavonoids of Oxytropis falcata on the Alleviation of Myocardial Ischemia-Reperfusion Injury.

Oxytropis falcata Bunge is a plant used in traditional Tibetan medicine, with reported anti-inflammatory and antioxidants effects and alleviation of myocardial ischemia reperfusion injury (MIRI). However, the underlying mechanism against MIRI and the phytochemical composition of O. falcata are vague. One fraction named OFF1 with anti-MIRI activity was obtained from O. falcata, and the chemical constituents were identified by ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS). The potential targets and signaling pathways involved in the action of O. falcata against MIRI were predicted by network pharmacology analysis, and its molecular mechanism on MIRI was determined by in vitro assays. The results revealed that flavonoids are the dominant constituents of OFF1. A total of 92 flavonoids reported in O. falcata targeted 213 potential MIRI-associated factors, including tumor necrosis factor (TNF), prostaglandin-endoperoxide synthase 2 (PTGS2), and the NF-κB signaling pathway. The in vitro assay on H9c2 cardiomyocytes subjected to hypoxia/reoxygenation injury confirmed that the flavonoids in OFF1 reduced myocardial marker levels, apoptotic rate, and the inflammatory response triggered by oxidative stress. Moreover, OFF1 attenuated MIRI by downregulating the ROS-mediated JNK/p38MAPK/NF-κB pathway. Collectively, these findings provide novel insights into the molecular mechanism of O. falcata in alleviating MIRI, being a potential therapeutic candidate.

[Advances in metabolic engineering for the production of aromatic chemicals].

Metabolic engineering has been developed for nearly 30 years since the early 1990s, and it has given a great impetus to microbial strain breeding and improvement. Aromatic chemicals are a variety of important chemicals that can be produced by microbial fermentation and are widely used in the pharmaceutical, food, feed, and material industry. Microbial cells can be engineered to accumulate a variety of useful aromatic chemicals in a targeted manner through rational engineering of the biosynthetic pathways of shikimate and the derived aromatic amino acids. This review summarizes the metabolic engineering strategies and biosynthetic pathways for the production of aromatic chemicals developed in the past 30 years, with the aim to provide a valuable reference and promote the research in this field.

Design and Reconstruction of Regulatory Parts for Fast-frowing Vibrio natriegens Synthetic Biology.

The fast-growing Vibrio natriegens is an attractive robust chassis for diverse synthetic biology applications. However, V. natriegens lacks the suitable constitutive regulatory parts for precisely tuning the gene expression and, thus, recapitulating physiologically relevant changes in gene expression levels. In this study, we designed, constructed, and screened the synthetic regulatory parts by varying the promoter region and ribosome binding site element for V. natriegens with different transcriptional or translational strengths, respectively. The fluorescence intensities of the cells with different synthetic regulatory parts could distribute evenly over a wide range of 5 orders of magnitude. The selected synthetic regulatory parts had good stability in both nutrient-rich and minimal media. The precise combinatorial modulation of galP (GalP = galactose permease) and glk (Glk = glucokinase) from Escherichia coli by using three synthetic regulatory parts with different strengths was confirmed in a phosphoenolpyruvate:carbohydrate phosphotransferase system with inactive V. natriegens strain to alter the glucose transport. This work provides the simple, efficient, and standardized constitutive regulatory parts for V. natriegens synthetic biology.

Noise Reduction of Welding Crack AE Signal Based on EMD and Wavelet Packet.

The acoustic emission (AE) signal collected by a sensor in the welding process has an overlapping frequency band and weak characteristics under a complex noise background. It is difficult for the wavelet noise reduction method, with single basis function, to effectively match the different characteristic information of the welding crack AE signal. Taking into account the adaptive decomposition characteristics of Empirical Mode Decomposition (EMD), a novel wavelet packet noise reduction method for welding AE signal was proposed. The welding AE signal was adaptively decomposed into several Intrinsic Mode Functions (IMFs) by the EMD. The effective IMFs were selected by the frequency distribution characteristics of the welding crack AE signal. A wavelet packet, with a specific basis function, was subsequently performed on the effective IMFs, which were reconstructed to be the welding crack AE signal. The simulated and experimental results indicated that the proposed method can effectively achieve noise reduction of the welding crack AE signal, which provided a mean for structure crack detection in the welding process.

Nucleocytoplasmic shuttling of SAMHD1 is important for LINE-1 suppression.

Currently, SAMHD1 is the only known dNTPase in human cells. It also suppresses the replication of both retroviruses and retroelements. SAMHD1 contains a classic nuclear localization sequence (NLS) and resides in the nucleus in live or fixed cells. It has been reported that alteration or removal of NLS does not affect the dNTPase or the antiviral activity of SAMHD1. However, it was unclear whether the nuclear localization was involved in SAMHD1-mediated suppression against retroelements such as long interspersed element type 1 (LINE-1 or L1). In this study, we reported that SAMHD1 is a nucleocytoplasmic shuttling protein. Digitonin-based cytoplasm/nucleus fractionation tests suggested that SAMHD1 is capable of being exported from the nucleus, which was confirmed by introducing exogenous exportin Xpo1 in live cells. Interestingly, altering the protein's subcellular localization by mutating or removing NLS significantly enhances SAMHD1's potency in L1 suppression. Further tests with SAMHD1 mutants indicated that nucleocytoplasmic shuttling is important for SAMHD1-mediated L1 suppression. Finally, we demonstrated that the cytoplasmic distribution of SAMHD1 leads to an enhanced depletion of L1 ORF2p. Taken together, our data have revealed SAMHD1 as a nucleocytoplasmic shuttling protein, and associated such a new feature of SAMHD1 with its potency against L1 retrotransposition, which provides more insights to the understanding of SAMHD1 and its role in L1 suppression.

LINE1 contributes to autoimmunity through both RIG-I- and MDA5-mediated RNA sensing pathways.

Improper host immune activation leads to the development of the autoimmune disease Aicardi-Goutières syndrome (AGS), which is attributed to defined genetic mutations in such proteins as TREX1 and ADAR1. The mechanism of immune activation in AGS patients has not been thoroughly elucidated to date. In this study, we report that endogenous LINE1 components trigger IFNß production in multiple human cell types, including those defective for cGAS/STING-mediated DNA sensing. In these cells, LINE1 DNA synthesis and retrotransposition were not required for LINE1-triggered immune activation, but RNA sensing pathways were essential. LINE1-triggered immune activation could be suppressed by diverse LINE1 inhibitors, including AGS-associated proteins targeting LINE1 RNA or proteins. However, AGS-associated ADAR1 or TREX1 mutants were defective in suppressing LINE1 retrotransposition or LINE1-triggered immune activation. Therefore, we have revealed a new function for LINE1 as an endogenous trigger of innate immune activation, which is important for understanding the molecular basis of IFN-based autoimmune diseases and may offer new intervention strategies.

[Improvement of pyruvate production by Escherichia coli via pathway engineering and Tn5 transposon mediated mutagenesis].

To develop a high-yield pyruvate strain, we first engineered a pyruvate-producing Escherichia coli KLPP from wild-type E. coli MG1655 by blocking the pathways for byproduct formation via gene knockout. Then, we built a library of mutant containing 7 197 monoclones by using the pUT Mini-Tn5 transposon vector for random mutagenesis with E. coli KLPP. We developed a high-throughput method for pyruvate detection based on dinitrophenylhydrazine reaction using 96-well microplate reader. After two-round screening we successfully obtained six mutants with increased pyruvate titer using this method, the titer of pyruvate was increased by 38%, 31%, 19%, 28%, 44% and 14%, respectively. The position of transposon insertion was determined by whole genome re-sequencing, and the gene locus possibly influencing pyruvate production was analyzed, which laid the foundation for subsequent strain improvement by metabolic engineering.

Designing and Creating a Synthetic Omega Oxidation Pathway in Saccharomyces cerevisiae Enables Production of Medium-Chain α, ω-Dicarboxylic Acids.

Medium-chain (C8-C14) α, ω-dicarboxylic acids (α, ω-DCAs), which have numerous applications as raw materials for producing various commodities and polymers in chemical industry, are mainly produced from chemical or microbial conversion of petroleum-derived alkanes or plant-derived fatty acids at present. Recently, significant attention has been gained to microbial production of medium-chain α, ω-DCAs from simple renewable sugars. Here, we designed and created a synthetic omega oxidation pathway in Saccharomyces cerevisiae to produce C10 and C12 α, ω-DCAs from renewable sugars and fatty acids by introducing a heterogeneous cytochrome P450 CYP94C1 and cytochrome reductase ATR1. Furthermore, the deletion of fatty acyl-CoA synthetase genes FAA1 and FAA4 increased the production of medium-chain α, ω-DCAs from 4.690 ± 0.088 mg/L to 12.177 ± 0.420 mg/L and enabled the production of C14 and C16 α, ω-DCAs at low percentage. But blocking ß-oxidation pathway by deleting fatty-acyl coenzyme A oxidase gene POX1 and overexpressing different thioesterase genes had no significant impact on the production and the composition of α, ω-dicarboxylic acids. Overall, our study indicated the potential of microbial production of medium-chain α, ω-DCAs from renewable feedstocks using engineered yeast.

[Construction and optimization of microbial cell factories for producing cis, cis-muconic acid].

cis, cis-muconic acid (MA) is an important platform chemical. Now, majority of reported engineered strains are genetically instable, the exogenous genes are expressed under the control of expensive inducer and the components of their fermentation medium are complex, thus large-scale microbial production of MA is limited due to the lack of suitable strains. Hence, it is still necessary to construct novel high-performance strain that is genetically stable, no induction and grows in simple inorganic fermentation medium. In this study, after 3 exogenous genes (aroZ, aroY, catA) for biosynthesis of MA were integrated into previously constructed 3-hydroshikimate producing Escherichia coli WJ060 strain and combinatorially regulated with 3 constitutive promoters with different strengths, 27 engineered strains were constructed. The best engineered strain, E. coli MA30 could produce 1.7 g/L MA in the simple inorganic fermentation medium without induction. To further enhance the production capacity of MA, the mutant library of E. coli MA30 was constructed by genome replication engineering and screened via high-throughput assay. After two-round screening, the new strain, E. coli MA30-G2 with improved production of MA was obtained, and the titer of MA increased more than 8%. Under the condition of 5 L fed-batch fermentation, E. coli MA30-G2 could produce about 11.5 g/L MA. Combinatorial regulation and high-throughput screening provide important reference to microbial production of other bio-based chemicals.

[Construction and optimization of Escherichia coli for producing rhamnolipid biosurfactant].

Rhamnolipid biosurfactant is mainly produced by Pseudomonas aeruginosa that is the opportunistic pathogenic strain and not suitable for future industrial development. In order to develop a relatively safe microbial strain for the production of rhamnolipid biosurfactant, we constructed engineered Escherichia coli strains for rhamnolipid production by expressing different copy numbers of rhamnosyltransferase (rhlAB) gene with the constitutive synthetic promoters of different strengths in E. coli ATCC 8739. We further studied the combinatorial regulation of rhlAB gene and rhaBDAC gene cluster for dTDP-1-rhamnose biosynthesis with different synthetic promoters, and obtained the best engineered strain-E. coli TIB-RAB226. Through the optimization of culture temperature, the titer of rhamnolipd reached 124.3 mg/L, 1.17 fold higher than that under the original condition. Fed-batch fermentation further improved the production of rhamnolipid and the titer reached the highest 209.2 mg/L within 12 h. High performance liquid chromatography-mass spectrometry (LC-MS) analysis showed that there are total 5 mono-rhamnolipid congeners with different nuclear mass ratio and relative abundance. This study laid foundation for heterologous biosynthesis of rhanomilipd.

Rhamnolipid production, characterization and fermentation scale-up by Pseudomonas aeruginosa with plant oils.

OBJECTIVES: To be competitive with common chemical surfactants, the cost of rhamnolipid production must be minimized by selecting suitable substrates and optimizing the fermentation process. RESULTS: With different plant oils as substrates, Pseudomonas aeruginosa TIB-R02 can produce rhamnolipids with different structural characteristics that were confirmed by HPLC/MS analysis. Different rhamnolipids had different performances in interfacial tension. The production of rhamnolipid was greatly enhanced by fermentation optimization with palm oil as substrate. A fermentation-defoaming tandem system was developed to resolve the problems of foaming and medium overflow during scale-up. Finally, the titer of rhamnolipid reached 60 g/l and the yield reached 80% in a 300 l fermentation-defoaming tandem system. CONCLUSIONS: The work reveals the potential for producing high-performance rhamnolipids from renewable resources on a large scale.