High methionine intake alters gut microbiota and lipid profile and leads to liver steatosis in mice.

Methionine is an important sulfur-containing amino acid. Health effects of both methionine restriction (MR) and methionine supplementation (MS) have been studied. This study aimed to investigate the impact of a high-methionine diet (HMD) (1.64% methionine) on both the gut and liver functions in mice through multi-omic analyses. Hepatic steatosis and compromised gut barrier function were observed in mice fed the HMD. RNA-sequencing (RNA-seq) analysis of liver gene expression patterns revealed the upregulation of lipid synthesis and degradation pathways, cholesterol metabolism and inflammation-related nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway. Metagenomic sequencing of cecal content demonstrated a shift in gut microbial composition with an increased abundance of opportunistic pathogens and gut microbial functions with up-regulated lipopolysaccharide (LPS) biosynthesis in mice fed HMD. Metabolomic study of cecal content showed an altered gut lipid profile and the level of bioactive lipids, including docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), palmitoylethanolamide (PEA), linoleoyl ethanolamide (LEA) and arachidonoyl ethanolamide (AEA), that carry anti-inflammatory effects significantly reduced in the gut of mice fed the HMD. Correlation analysis demonstrated that gut microbiota was highly associated with liver and gut functions and gut bioactive lipid content. In conclusion, this study suggested that the HMD exerted negative impacts on both the gut and liver, and an adequate amount of methionine intake should be carefully determined to ensure normal physiological function without causing adverse effects.

Restricted intake of sulfur-containing amino acids reversed the hepatic injury induced by excess Desulfovibrio through gut-liver axis.

Diet is a key player in gut-liver axis. However, the effect of different dietary patterns on gut microbiota and liver functions remains unclear. Here, we used rodent standard chow and purified diet to mimic two common human dietary patterns: grain and plant-based diet and refined-food-based diet, respectively and explored their impacts on gut microbiota and liver. Gut microbiota experienced a great shift with notable increase in Desulfovibrio, gut bile acid (BA) levels elevated significantly, and liver inflammation was observed in mice fed with the purified diet. Liver inflammation and elevated gut BA levels also occurred in mice fed with the chow diet after receiving Desulfovibrio desulfuricans ATCC 29,577 (DSV). Restriction of sulfur-containing amino acids (SAAs) prevented liver injury mainly through higher hepatic antioxidant and detoxifying ability and reversed the elevated BA levels due to excess Desulfovibrio. Ex vivo fermentation of human fecal microbiota with primary BAs demonstrated that DSV enhanced production of secondary BAs. Higher concentration of both primary and secondary BAs were found in the gut of germ-free mice after receiving DSV. In conclusion, Restriction of SAAs in diet may become an effective dietary intervention to prevent liver injury associated with excess Desulfovibrio in the gut.

Decoding the Qu-aroma of medium-temperature Daqu starter by volatilomics, aroma recombination, omission studies and sensory analysis.

Qu-aroma is of great significance for evaluation the quality of Daqu starter. This study aimed to decode the Qu-aroma of medium-temperature Daqu (MT-Daqu) via "top-down" and "bottom-up" approaches. Firstly, 52 aroma descriptors were defined to describe the MT-Daqu aroma by quantitative descriptive analysis. Secondly, 193 volatile organic compounds (VOCs) were identified from 42 MT-Daqu samples by HS-SPME-GC-MS, and 43 dominant VOCs were screened out by frequence of occurrence or abundance. By Thin Film (TF)-SPME-GC-O-MS, 27 odors and 90 VOCs were detected in MT-Daqu mixture, and 14 odor-active VOCs were screened out by odor intensity. Thirdly, a five-level MT-Daqu aroma wheel was constructed by matching 52 aroma descriptors and 37 aroma-active VOCs. Finally, Qu-aroma of MT-Daqu was reconstructed with 37 aroma-active VOCs and evaluated by omission experiments. Hereinto, 26 key aroma-active VOCs were determined by OAV value ≥1, including isovaleric acid, 1-hexanol, isovaleraldehyde, 2-octanone, trimethylpyrazine, γ-nonalactone, 4-vinylguaiacol, etc.

Differences in the Ability of Lactic Acid Bacteria To Prevent Acute Alcohol-Induced Liver Injury via the Gut Microbiota-Bile Acid-Liver Axis.

Probiotics can regulate gut microbiota and protect against acute alcohol-induced liver injury through the gut-liver axis. However, efficacy is strain-dependent, and their mechanism remains unclear. This study investigated the effect of lactic acid bacteria (LAB), including Lacticaseibacillus paracasei E10 (E10), Lactiplantibacillus plantarum M (M), Lacticaseibacillus rhamnosus LGG (LGG), Lacticaseibacillus paracasei JN-1 (JN-1), and Lacticaseibacillus paracasei JN-8 (JN-8), on the prevention of acute alcoholic liver injury in mice. We found that LAB pretreatment reduced serum alanine transaminase (ALT) and aspartate transaminase (AST) and reduced hepatic total cholesterol (TC) and triglyceride (TG). JN-8 pretreatment exhibited superior efficacy in improving hepatic antioxidation. LGG and JN-8 pretreatment significantly attenuated hepatic and colonic inflammation by decreasing the expression of interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) and increasing the expression of interleukin 10 (IL-10). JN-1 and JN-8 pretreatments have better preventive effects than other LAB pretreatment on intestinal barrier dysfunction. In addition, the LAB pretreatment improved gut microbial dysbiosis and bile acid (BA) metabolic abnormality. All of the strains were confirmed to have bile salt deconjugation capacities in vitro, where M and JN-8 displayed higher activities. This study provides new insights into the prevention and mechanism of LAB strains in preventing acute alcoholic liver injury.

EHHADH deficiency regulates pexophagy and accelerates tubulointerstitial injury in diabetic kidney disease.

Peroxisomal L-bifunctional enzyme (EHHADH) plays a role in the classic peroxisomal fatty acid ß-oxidation pathway; however, the relationship between EHHADH expression and diabetic kidney disease has not been well understood. Here, we found that endogenous EHHADH levels were strongly correlated with the progression and severity of diabetic nephropathy in T2D patients. EHHADH knockout mice exhibited worsened renal tubular injury in diabetic mice. Furthermore, EHHADH is a modulator of pexophagy. In renal tubular epithelial cells (RTECs) in vitro, the knockdown of EHHADH induced a dramatic loss of peroxisomes. The loss of peroxisomes in EHHADH-deficient RTECs was restored by either an autophagic inhibitor 3-methyladenine or bafilomycin A1 both in vitro and in vivo. NBR1 was required for pexophagy in EHHADH-knockdown cells, where the level of reactive oxygen species (ROS) was increased, while inhibition of ROS blocked pexophagy. In summary, our findings revealed EHHADH deficiency accelerated renal injury in DKD as a modulator of pexophagy.

Cordycepin alleviates hepatic fibrosis in association with the inhibition of glutaminolysis to promote hepatic stellate cell senescence.

Cordycepin (CRD) is an active component derived from Cordyceps militaris, which possesses multiple biological activities and uses in liver disease. However, whether CRD improves liver fibrosis by regulating hepatic stellate cell (HSC) activation has remained unknown. The study aims further to clarify the activities of CRD on liver fibrosis and elucidate the possible mechanism. Our results demonstrated that CRD significantly relieved hepatocyte injury and inhibited HSC activation, alleviating hepatic fibrogenesis in the Diethyl 1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC)-induced mice model. In vitro, CRD exhibited dose-dependent repress effects on HSC proliferation, migration, and pro-fibrotic function in TGF-ß1-activated LX-2 and JS-1 cells. The functional enrichment analysis of RNA-seq data indicated that the pathway through which CRD alleviates HSC activation involves cellular senescence and cell cycle-related pathways. Furthermore, it was observed that CRD accumulated the number of senescence-associated a-galactosidase positive cells and the levels of senescencemarker p21, and provoked S phasearrestof activated HSC. Remarkably, CRD treatment abolished TGF-ß-induced yes-associated protein (YAP) nuclear translocation that acts upstream of glutaminolysis in activated HSC. On the whole, CRD significantly inhibited glutaminolysis of activated-HSC and induced cell senescence through the YAP signaling pathway, consequently alleviating liver fibrosis, which may be a valuable supplement for treating liver fibrosis.

Semiautomated design and soluble expression of a chimeric antigen TbpAB01 from Glaesserella parasuis.

Pathogenic bacterial membrane proteins (MPs) are a class of vaccine and antibiotic development targets with widespread clinical application. However, the inherent hydrophobicity of MPs poses a challenge to fold correctly in living cells. Herein, we present a comprehensive method to improve the soluble form of MP antigen by rationally designing multi-epitope chimeric antigen (ChA) and screening two classes of protein-assisting folding element. The study uses a homologous protein antigen as a functional scaffold to generate a ChA possessing four epitopes from transferrin-binding protein A of Glaesserella parasuis. Our engineered strain, which co-expresses P17 tagged-ChA and endogenous chaperones groEL-ES, yields a 0.346 g/L highly soluble ChA with the property of HPS-positive serum reaction. Moreover, the protein titer of ChA reaches 4.27 g/L with >90% soluble proportion in 5-L bioreactor, which is the highest titer reported so far. The results highlight a timely approach to design and improve the soluble expression of MP antigen in industrially viable applications.

Enhanced synthesis of S-adenosyl-L-methionine through combinatorial metabolic engineering and Bayesian optimization in Saccharomyces cerevisiae.

S-Adenosyl-L-methionine (SAM) is a substrate for many enzyme-catalyzed reactions and provides methyl groups in numerous biological methylations, and thus has vast applications in the agriculture and medical field. Saccharomyces cerevisiae has been engineered as a platform with significant potential for producing SAM, but the current production has room for improvement. Thus, a method that consists of a series of metabolic engineering strategies was established in this study. These strategies included enhancing SAM synthesis, increasing ATP supply, down-regulating SAM metabolism, and down-regulating competing pathway. After combinatorial metabolic engineering, Bayesian optimization was conducted on the obtained strain C262P6S to optimize the fermentation medium. A final yield of 2972.8 mg·L-1 at 36 h with 29.7% of the L-Met conversion rate in the shake flask was achieved, which was 26.3 times higher than that of its parent strain and the highest reported production in the shake flask to date. This paper establishes a feasible foundation for the construction of SAM-producing strains using metabolic engineering strategies and demonstrates the effectiveness of Bayesian optimization in optimizing fermentation medium to enhance the generation of SAM.

[Metagenomics unveils the differences in the functions of microbial community of medium-temperature Daqu before and after maturation].

Daqu is the saccharifying, fermenting, and aroma-producing agent used in Baijiu brewing, and its maturation is crucial for obtaining high-quality Daqu. Previous studies have explored the microbial community composition and diversity before and after maturation. However, little is known about the changes in the functions of microbial community. In this study, based on the analyses of enzyme activities and volatile compounds of medium-temperature Daqu before and after maturation, metagenomics was used to analyze the differences in the composition of microbial community and the potential functions, with the aim to explore the microorganisms involved in changes in enzyme activities and important volatiles. The results showed that the moisture (P≤0.05), starch content, liquefying activity, saccharifying activity (P≤0.05), and fermentative activity decreased, while the acidity and esterifying activity (P≤0.05) increased after Daqu maturation. In the meantime, the composition of volatile compounds changed significantly (P=0.001), with significant decreases in the contents of aromatic alcohols and esters as well as significant increases in the contents of pyrazines, ketones, and higher fatty alcohols. The relative abundances of Mucorales (34.8%-23.0%) and Eurotiales (34.3%-20.1%) decreased in matured Daqu, and functional predictions showed these changes decreased the gene abundances of α-amylase, α-glucosidase, alcohol dehydrogenase, and alcohol dehydrogenase (NADP+) (P > 0.05), resulting in lower levels of liquefying activity (P > 0.05), saccharifying activity (P≤0.05), fermentative activity (P > 0.05), as well as aromatic alcohols such as phenylethyl alcohol (P≤0.05). In addition, higher relative abundances of Saccharomycetales (2.9%-16.6%), Lactobacillales (14.9%-23.6%), and Bacillales (0.8%-3.8%) were observed after maturation, and they were conducive to improving the gene abundances of alcohol O-acetyltransferase, carboxylesterase, acetolactate decarboxylase, (R)-acetoin dehydrogenase, and (S)-acetoin dehydrogenase (P≤0.05), resulting in significantly higher levels of esterifying activity and pyrazines (P≤0.05). The microorganisms involved in the changes in enzyme activities and important volatiles before and after Daqu maturation were studied at the gene level in this work, which may facilitate further rational regulation for Daqu production.

High-Level Extracellular Expression of Hyaluronate Lyase HylP in Bacillus subtilis for Hyaluronan Degradation.

Hyaluronate lyase (HA lyase) has potential in the industrial processing of hyaluronan. In this study, HylP, an HA lyase from Streptococcus pyogenes phage (SPB) was successfully expressed in Bacillus subtilis. To improve the extracellular enzyme activity of HylP in B. subtilis, signal peptide engineering systematic optimization was carried out, and cultured it from shake flasks and fermenters, followed by purification, characterization, and analysis of degradation products. The results showed that the replacement of the signal peptide increased the extracellular enzyme activity of HylP from 1.0 × 104 U/mL to 1.86 × 104 U/mL in the shake flask assay, and using a 20 L fermenter in a batch fermentation process, the extracellular enzyme activity achieved the level of 1.07 × 105 U/mL. HylP exhibited significant thermal and pH stability in the temperature range of 40 °C and pH range of 4-8, respectively. The enzyme showed optimum activity at 40 °C and pH 6, with significant activity in the presence of Na+, Mg2+, and Co2+ ions. Degradation analysis showed that HylP efficiently degraded hyaluronan as an endonuclease, releasing unsaturated disaccharides. These comprehensive findings underscore the substantial industrial potential of HylP for hyaluronan processing applications, offering valuable insights into enzyme characterization and optimization of expression for potential industrial utilization.

Rapid detection of six Oceanobacillus species in Daqu starter using single-cell Raman spectroscopy combined with machine learning.

Many traditional fermented foods and beverages industries around the world request the addition of multi-species starter cultures. However, the microbial community in starter cultures is subject to fluctuations due to their exposure to an open environment during fermentation. A rapid detection approach to identify the microbial composition of starter culture is essential to ensure the quality of the final products. Here, we applied single-cell Raman spectroscopy (SCRS) combined with machine learning to monitor Oceanobacillus species in Daqu starter, which plays crucial roles in the process of Chinese baijiu. First, a total of six Oceanobacillus species (O. caeni, O. kimchii, O. iheyensis, O. sojae, O. oncorhynchi subsp. Oncorhynchi and O. profundus) were detected in 44 Daqu samples by amplicon sequencing and isolated by pure culture. Then, we created a reference database of these Oceanobacillus strains which correlated their taxonomic data and single-cell Raman spectra (SCRS). Based on the SCRS dataset, five machine-learning algorithms were used to classify Oceanobacillus strains, among which support vector machine (SVM) showed the highest rate of accuracy. For validation of SVM-based model, we employed a synthetic microbial community composed of varying proportions of Oceanobacillus species and demonstrated a remarkable accuracy, with a mean error was less than 1% between the predicted result and the expected value. The relative abundance of six different Oceanobacillus species during Daqu fermentation was predicted within 60 min using this method, and the reliability of the method was proved by correlating the Raman spectrum with the amplicon sequencing profiles by partial least squares regression. Our study provides a rapid, non-destructive and label-free approach for rapid identification of Oceanobacillus species in Daqu starter culture, contributing to real-time monitoring of fermentation process and ensuring high-quality products.

Efficient production and characterization of a newly identified trehalase for inhibiting the formation of bacterial biofilms.

Trehalase has attracted widespread attention in medicine, agriculture, food, and ethanol industry due to its ability to specifically degrade trehalose. Efficient expression of trehalase remains a challenge. In this study, a putative trehalase-encoding gene (Tre-zm) from Zunongwangia mangrovi was explored using gene-mining strategy and heterologously expressed in E. coli. Trehalase activity reached 3374 U·mL-1 after fermentation optimization. The scale-up fermentation in a 15 L fermenter was achieved with a trehalase production of 15,068 U·mL-1. The recombinant trehalase TreZM was purified and characterized. It displayed optimal activity at 35 °C and pH 8.5, with Mn2+, Sn2+, Na+, and Fe2+ promoting the activity. Notably, TreZM showed significant inhibition effect on biofilm forming of Staphylococcus epidermidis. The combination of TreZM with a low concentration of antibiotics could inhibit 70 % biofilm formation of Staphylococcus epidermidis and 28 % of Pseudomonas aeruginosa. Hence, this study provides a promising candidate for industrial production of trehalase and highlights its potential application to control harmful biofilms.

Selective synthesis of rebaudioside M2 through structure-guided engineering of glycosyltransferase UGT94D1.

Rebaudioside M2 (Reb M2), a novel steviol glycoside derivative, has limited industrial applications due to its low synthetic yield and selectivity. Herein, we identify UGT94D1 as a selective glycosyltransferase for rebaudioside D (Reb D), leading to the production of a mono ß-1,6-glycosylated derivative, Reb M2. A variant UGT94D1-F119I/D188P was developed through protein engineering. This mutant exhibited a 6.33-fold improvement in catalytic efficiency, and produced Reb M2 with 92% yield. Moreover, molecular dynamics simulations demonstrated that UGT94D1-F119I/D188P exhibited a shorter distance between the nucleophilic oxygen (OH6) of the substrate Reb D and uridine diphosphate glucose, along with an increased Ophosphate-C1-Oacceptor angle, thus improving the catalytic activity of the enzyme. Therefore, this study provides an efficient method for the selective synthesis of Reb M2 and paves the way for its applications in various fields.

Enhanced Production of Sisomicin in Micromonospora inyoensis by Protoplast Mutagenesis and Fermentation Optimization.

Sisomicin is a broad-spectrum aminoglycoside antibiotic and is the precursor of netilmicin and plazomicin. However, the fermentation level of sisomicin is still low compared with other antibiotics, which restricts the application of sisomicin and its derivatives. In this study, to improve sisomicin production, breeding of high-yielding sisomicin strains was conducted with chemical mutagenesis using Micromonospora inyoensis OG-1 (titer, 1042 U·mL-1) as the starting strain. Protoplast preparation was conducted under optimal conditions, and protoplast mutagenesis was performed with a suitable concentration of diethyl sulfate. Subsequently, a high-yielding and genetically stable strain (H6-32) was obtained by screening, with a sisomicin titer of 1486 U·mL-1 (an increase of 42.6%). Finally, carbon and nitrogen sources were optimized to further improve sisomicin production, and a sisomicin titer of 1780 U·mL-1 was ultimately obtained by controlling the dissolved oxygen level at 30% in a 5-L fermenter, which to the best of our knowledge is the highest reported titer ever achieved by fermentation. Comparative genome analysis showed that a total of 13 genes in the genome of the mutant strain H6-32 were mutated compared to the original strain. This study not only provides a reference for further breeding of high-yielding strains and fermentation optimization, but also enhances our understanding of sisomicin production.

Lactic acid fermentation of goji berries (Lycium barbarum) prevents acute alcohol liver injury and modulates gut microbiota and metabolites in mice.

Juice fermented with lactic acid bacteria (LAB) has received attention due to its health benefits, such as antioxidant and anti-inflammatory. Previous research on LAB-fermented goji juice mainly focused on exploring the changes in the metabolite profile and antioxidant activity in vitro, whereas the liver protection properties of LAB-fermented goji juice in vivo are still unknown. This study aimed to investigate the effects of Lacticaseibacillus paracasei E10-fermented goji juice (E10F), Lactiplantibacillus plantarum M-fermented goji juice (MF), Lacticaseibacillus rhamnosus LGG-fermented goji juice (LGGF) on preventing acute alcoholic liver injury with physiology, gut microbial, and metabolic profiles in mice. Compared with goji juice, E10F, MF, and LGGF enhanced the protective effect against liver injury by reducing serum alanine transaminase (ALT) levels, improving the hepatic glutathione (GSH) antioxidant system, and attenuating inflammation by decreasing the levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-ß. Furthermore, E10F, MF, and LGGF increased intestinal integrity, restructured the gut microbiota including Bacteroides and Lactobacillus, and altered gut microbial metabolites including kyotorphin, indolelactic acid, and N-methylserotonin. Pretreatment of different LAB-fermented goji juice in mice showed significant differences in gut microbiota and metabolism. The correlation analysis demonstrated that the increase of Lactobacillus, indolelactic acid, and N-methylserotonin by E10F, MF, and LGGF was positively correlated with reduced inflammation and improved liver and gut function. Taken together, E10F, MF, and LGGF all have the potential to be converted into dietary interventions to combat acute alcoholic liver injury. It provided a reference for the study of the hepatoprotective effect of LAB-fermented goji juice.

Engineering protein translocation and unfolded protein response enhanced human PH-20 secretion in Pichia pastoris.

Hyaluronidases catalyze the degradation of hyaluronan (HA), which is finding rising applications in medicine, cosmetic, and food industries. Recombinant expression of hyaluronidases in microbial hosts has been given special attention as a sustainable way to substitute animal tissue-derived hyaluronidases. In this study, we focused on optimizing the secretion of hyaluronidase from Homo sapiens in Pichia pastoris by secretion pathway engineering. The recombinant hyaluronidase was first expressed under the control of a constitutive promoter PGCW14. Then, two endoplasmic reticulum-related secretory pathways were engineered to improve the secretion capability of the recombinant strain. Signal peptide optimization suggested redirecting the protein into co-translational translocation using the ost1-proα signal sequence improved the secretion level by 20%. Enhancing the co-translational translocation by overexpressing signal recognition particle components further enhanced the secretory capability by 48%. Then, activating the unfolded protein response by overexpressing a transcriptional factor ScHac1p led to a secreted hyaluronidase activity of 4.06 U/mL, which was 2.1-fold higher than the original strain. Finally, fed-batch fermentation elevated the production to 19.82 U/mL. The combined engineering strategy described here could be applied to enhance the secretion capability of other proteins in yeast hosts. KEY POINTS: • Improving protein secretion by enhancing co-translational translocation in P. pastoris was reported for the first time. • Overexpressing Hac1p homologous from different origins improved the rhPH-20 secretion. • A 4.9-fold increase in rhPH-20 secretion was achieved after fermentation optimization and fed-batch fermentation.

Enhanced L-serine production by Corynebacterium glutamicum based on novel insights into L-serine exporters.

The L-serine exporters ThrE and SerE play important roles in L-serine production by Corynebacterium glutamicum. Deletion of both thrE and serE decreased L-serine titer by 60%, suggesting the existence of other L-serine exporters. A comparative transcriptomics identified NCgl0254 and NCgl0255 as novel L-serine exporters. Further analysis of the contributions of ThrE, SerE, NCgl0254, and NCgl0255 found that SerE was the major L-serine exporter in C. glutamicum and these four L-serine exporters were responsible for 79.7% of L-serine export. Deletion of one L-serine exporter upregulated the transcription levels of the other three, which might be coursed by increased intracellular concentrations of L-serine. Overexpression of NCgl0254 and NCgl0255 increased L-serine titer by 20.8% in C. glutamicum A36, while overexpression of the four L-serine exporters increased L-serine production by 31.9% (41.1 g·L-1 ) in C. glutamicum A36. The identification of novel L-serine exporters in C. glutamicum will help to improve industrial production of L-serine.

Production of L-serine and its derivative L-cysteine from renewable feedstocks using Corynebacterium glutamicum: advances and perspectives.

L-serine and its derivative L-cysteine have broad industrial applications, and their direct fermentative production from renewable biomass is gaining increasing attention. Corynebacterium glutamicum is an extensively studied and well-established industrial microorganism, which is a predominant microbial host for producing amino acids. In this review, updated information on the genetics and molecular mechanisms underlying L-serine and L-cysteine production using C. glutamicum is presented, including their synthesis and degradation pathways, and other intracellular processes related to their production, as well as the mechanisms underlying substrate import and product export are also analyzed. Furthermore, metabolic strategies for strain improvement are systematically discussed, and conclusions and future perspectives for bio-based L-serine and L-cysteine production using C. glutamicum are presented. This review can provide a thorough understanding of L-serine and L-cysteine metabolic pathways to facilitate metabolic engineering modifications of C. glutamicum and development of more efficient industrial fermentation processes for L-serine and L-cysteine production.

Long non-coding RNAs and tyrosine kinase-mediated drug resistance in pancreatic cancer.

Pancreatic cancer (PC) is one of the most malignant tumors with a dismal survival rate, this is primarily due to inevitable chemoresistance. Dysfunctional tyrosine kinases (TKs) and long non-coding RNAs (lncRNAs) affect the drug resistance and prognosis of PC. Here, we summarize the mechanisms by which TKs or lncRNAs mediate drug resistance and other malignant phenotypes. We also discuss that lncRNAs play oncogenic or tumor suppressor roles and different mechanisms including lncRNA-proteins/microRNAs to mediate drug resistance. Furthermore, we highlight that lncRNAs serve as upstream regulators of TKs mediating drug resistance. Finally, we display the clinical significance of TKs (AXL, EGFR, IGF1R, and MET), clinical trials, and lncRNAs (LINC00460, PVT1, HIF1A-AS1). In the future, TKs and lncRNAs may become diagnostic and prognostic biomarkers or drug targets to overcome the drug resistance of PC.

Engineering of redox partners and cofactor NADPH supply of CYP68JX for efficient steroid two-step ordered selective hydroxylation activity.

CYP68JX, a P450 hydroxylase, derived from Colletotrichum lini ST-1 is capable of biotransforming dehydroepiandrosterone (DHEA) to 3ß,7α,15α-trihydroxy-5-androstene-17-one (7α,15α-diOH-DHEA). Redox partners and cofactor supply are important factors affecting the catalytic activity of CYP68JX. In this study, the heterologous expression of CYP68JX in Saccharomyces cerevisiae BY4741 was realized resulting in a 17.1% target product yield. In order to increase the catalytic efficiency of CYP68JX in S. cerevisiae BY4741, a complete cytochrome P450 redox system was constructed. Through the combination of CYP68JX and heterologous CPRs, the yield of the target product 7α,15α-diOH-DHEA in CYP68JX recombinant system was increased to 37.8%. Furthermore, by adding NADPH coenzyme precursor tryptophan of 40 mmol/L and co-substrate fructose of 20 g/L during the conversion process, the catalytic efficiency of CYP68JX was further improved, the target product yield reached 57.9% which was 3.39-fold higher than initial yield. Overall, this study provides a reference for improving the catalytic activity of P450s.