Oncometabolites suppress DNA repair by disrupting local chromatin signalling.

Deregulation of metabolism and disruption of genome integrity are hallmarks of cancer1. Increased levels of the metabolites 2-hydroxyglutarate, succinate and fumarate occur in human malignancies owing to somatic mutations in the isocitrate dehydrogenase-1 or -2 (IDH1 or IDH2) genes, or germline mutations in the fumarate hydratase (FH) and succinate dehydrogenase genes (SDHA, SDHB, SDHC and SDHD), respectively2-4. Recent work has made an unexpected connection between these metabolites and DNA repair by showing that they suppress the pathway of homology-dependent repair (HDR)5,6 and confer an exquisite sensitivity to inhibitors of poly (ADP-ribose) polymerase (PARP) that are being tested in clinical trials. However, the mechanism by which these oncometabolites inhibit HDR remains poorly understood. Here we determine the pathway by which these metabolites disrupt DNA repair. We show that oncometabolite-induced inhibition of the lysine demethylase KDM4B results in aberrant hypermethylation of histone 3 lysine 9 (H3K9) at loci surrounding DNA breaks, masking a local H3K9 trimethylation signal that is essential for the proper execution of HDR. Consequently, recruitment of TIP60 and ATM, two key proximal HDR factors, is substantially impaired at DNA breaks, with reduced end resection and diminished recruitment of downstream repair factors. These findings provide a mechanistic basis for oncometabolite-induced HDR suppression and may guide effective strategies to exploit these defects for therapeutic gain.

Multiplexed in-situ mutagenesis driven by a dCas12a-based dual-function base editor.

Mutagenesis driving genetic diversity is vital for understanding and engineering biological systems. However, the lack of effective methods to generate in-situ mutagenesis in multiple genomic loci combinatorially limits the study of complex biological functions. Here, we design and construct MultiduBE, a dCas12a-based multiplexed dual-function base editor, in an all-in-one plasmid for performing combinatorial in-situ mutagenesis. Two synthetic effectors, duBE-1a and duBE-2b, are created by amalgamating the functionalities of cytosine deaminase (from hAPOBEC3A or hAID*Δ ), adenine deaminase (from TadA9), and crRNA array processing (from dCas12a). Furthermore, introducing the synthetic separator Sp4 minimizes interference in the crRNA array, thereby facilitating multiplexed in-situ mutagenesis in both Escherichia coli and Bacillus subtilis. Guided by the corresponding crRNA arrays, MultiduBE is successfully employed for cell physiology reprogramming and metabolic regulation. A novel mutation conferring streptomycin resistance has been identified in B. subtilis and incorporated into the mutant strains with multiple antibiotic resistance. Moreover, surfactin and riboflavin titers of the combinatorially mutant strains improved by 42% and 15-fold, respectively, compared with the control strains with single gene mutation. Overall, MultiduBE provides a convenient and efficient way to perform multiplexed in-situ mutagenesis.

Engineered bacterial orthogonal DNA replication system for continuous evolution.

Continuous evolution can generate biomolecules for synthetic biology and enable evolutionary investigation. The orthogonal DNA replication system (OrthoRep) in yeast can efficiently mutate long DNA fragments in an easy-to-operate manner. However, such a system is lacking in bacteria. Therefore, we developed a bacterial orthogonal DNA replication system (BacORep) for continuous evolution. We achieved this by harnessing the temperate phage GIL16 DNA replication machinery in Bacillus thuringiensis with an engineered error-prone orthogonal DNA polymerase. BacORep introduces all 12 types of nucleotide substitution in 15-kilobase genes on orthogonally replicating linear plasmids with a 6,700-fold higher mutation rate than that of the host genome, the mutation rate of which is unchanged. Here we demonstrate the utility of BacORep-based continuous evolution by generating strong promoters applicable to model bacteria, Bacillus subtilis and Escherichia coli, and achieving a 7.4-fold methanol assimilation increase in B. thuringiensis. BacORep is a powerful tool for continuous evolution in prokaryotic cells.

CRISPR-dCas12a-mediated genetic circuit cascades for multiplexed pathway optimization.

The production efficiency of microbial cell factories is sometimes limited by the lack of effective methods to regulate multiple targets in a coordinated manner. Here taking the biosynthesis of glucosamine-6-phosphate (GlcN6P) in Bacillus subtilis as an example, a 'design-build-test-learn' framework was proposed to achieve efficient multiplexed optimization of metabolic pathways. A platform strain was built to carry biosensor signal-amplifying circuits and two genetic regulation circuits. Then, a synthetic CRISPR RNA array blend for boosting and leading (ScrABBLE) device was integrated into the platform strain, which generated 5,184 combinatorial assemblies targeting three genes. The best GlcN6P producer was screened and engineered for the synthesis of valuable pharmaceuticals N-acetylglucosamine and N-acetylmannosamine. The N-acetylglucosamine titer reached 183.9 g liter-1 in a 15-liter bioreactor. In addition, the potential generic application of the ScrABBLE device was also verified using three fluorescent proteins as a case study.

Histone methyltransferase KMT5C drives liver cancer progression and directs therapeutic response to PARP inhibitors.

BACKGROUND AND AIMS: Epigenetic plasticity is a major challenge in cancer-targeted therapy. However, the molecular basis governing this process has not yet been clearly defined. Despite the considerable success of poly(ADP-ribose) polymerase inhibitors (PARPi) in cancer therapy, the limited response to PARPi, especially in HCC, has been a bottleneck in its clinical implications. Herein, we investigated the molecular basis of the histone methyltransferase KMT5C (lysine methyltransferase 5C) that governs PARPi sensitivity and explored a potential therapeutic strategy for enhancing PARPi efficacy. APPROACH AND RESULTS: We identified KMT5C, a trimethyltransferase of H4K20, as a targetable epigenetic factor that promoted liver tumor growth in mouse de novo MYC/Trp53-/- and xenograft liver tumor models. Notably, induction of KMT5C by environmental stress was crucial for DNA repair and HCC cell survival. Mechanistically, KMT5C interacted with the pivotal component of homologous recombination repair, RAD51, and promoted RAD51/RAD54 complex formation, which was essential for the activation of dsDNA breaks repair. This effect depended on the methyltransferase activity of KMT5C. We further demonstrated that the function of KMT5C in promoting HCC progression was dependent on RAD51. Importantly, either a pharmacological inhibitor (A196) or genetic inhibition of KMT5C sensitized liver cancer cells to PARPi. CONCLUSIONS: KMT5C played a vital role in promoting liver cancer progression by activating the DNA repair response. Our results revealed a novel therapeutic approach using the KMT5C inhibitor A196, concurrent with olaparib, as a potential HCC therapy.

Low-dose decitabine modulates myeloid-derived suppressor cell fitness via LKB1 in immune thrombocytopenia.

Myeloid-derived suppressor cells (MDSCs) are heterogeneous immature cells and natural inhibitors of adaptive immunity. Metabolic fitness of MDSCs is fundamental for its suppressive activity toward effector T cells. Our previous studies showed that the number and inhibitory function of MDSCs were impaired in patients with immune thrombocytopenia (ITP) compared with healthy controls. In this study, we analyzed the effects of decitabine on MDSCs from patients with ITP, both in vitro and in vivo. We found that low-dose decitabine promoted the generation of MDSCs and enhanced their aerobic metabolism and immunosuppressive functions. Lower expression of liver kinase 1 (LKB1) was found in MDSCs from patients with ITP, which was corrected by decitabine therapy. LKB1 short hairpin RNA (shRNA) transfection effectively blocked the function of MDSCs and almost offset the enhanced effect of decitabine on impaired MDSCs. Subsequently, anti-CD61 immune-sensitized splenocytes were transferred into severe combined immunodeficient (SCID) mice to induce ITP in murine models. Passive transfer of decitabine-modulated MDSCs significantly raised platelet counts compared with that of phosphate buffered saline-modulated MDSCs. However, when LKB1 shRNA-transfected MDSCs were transferred into SCID mice, the therapeutic effect of decitabine in alleviating thrombocytopenia was quenched. In conclusion, our study suggests that the impaired aerobic metabolism of MDSCs is involved in the pathogenesis of ITP, and the modulatory effect of decitabine on MDSC metabolism contributes to the improvement of its immunosuppressive function. This provides a possible mechanism for sustained remission elicited by low-dose decitabine in patients with ITP.

GLI1 polymorphisms influence remission rate and prognosis of young de novo acute myeloid leukemia patients treated with cytarabine-based chemotherapy.

Acute myeloid leukemia (AML) is a highly heterogeneous hematological malignancy. Cytarabine (Ara-C)-based chemotherapy is the primary treatment for AML, but currently known prognostic risk stratification factors cannot fully explain the individual differences in outcome of patients. In this article, we reported that patients with homozygous GLI1 rs2228224 mutation (AA genotype) had a significantly lower complete remission rate than those with GG wild type (54.17% vs.76.02%, OR = 1.993, 95% CI: 1.062-3.504, P = 0.031). GLI1 rs2229300 T allele carriers had remarkably shorter overall survival (513 vs. 645 days, P = 0.004) and disease-free survival (342 vs. 456 days, P = 0.033) than rs2229300 GG carriers. Rs2229300 G > T variation increased the transcriptional activity of GLI1. CCND1, CD44 and PROM1 were potential target genes differentially regulated by GLI1 rs2229300. Our results demonstrated for the first time that GLI1 polymorphisms influence chemosensitivity and prognosis of young de novo AML patients treated with Ara-C.

Combinatorial metabolic engineering of Bacillus subtilis for menaquinone-7 biosynthesis.

Menaquinone-7 (MK-7), a form of vitamin K2, supports bone health and prevents arterial calcification. Microbial fermentation for MK-7 production has attracted widespread attention because of its low cost and short production cycles. However, insufficient substrate supply, unbalanced precursor synthesis, and low catalytic efficiency of key enzymes severely limited the efficiency of MK-7 synthesis. In this study, utilizing Bacillus subtilis BSAT01 (with an initial MK-7 titer of 231.0 mg/L) obtained in our previous study, the glycerol metabolism pathway was first enhanced to increase the 3-deoxy-arabino-heptulonate 7-phosphate (DHAP) supply, which led to an increase in MK-7 titer to 259.7 mg/L. Subsequently, a combination of knockout strategies predicted by the genome-scale metabolic model etiBsu1209 was employed to optimize the central carbon metabolism pathway, and the resulting strain showed an increase in MK-7 production from 259.7 to 318.3 mg/L. Finally, model predictions revealed the methylerythritol phosphate pathway as the major restriction pathway, and the pathway flux was increased by heterologous introduction (Introduction of Dxs derived from Escherichia coli) and fusion expression (End-to-end fusion of two enzymes by a linker peptide), resulting in a strain with a titer of 451.0 mg/L in a shake flask and 474.0 mg/L in a 50-L bioreactor. This study achieved efficient MK-7 synthesis in B. subtilis, laying the foundation for large-scale MK-7 bioproduction.

METTL14-mediated lncRNA XIST silencing alleviates GDM progression by facilitating trophoblast cell proliferation and migration via the miR-497-5p/FOXO1 axis.

Gestational diabetes mellitus (GDM), a prevalent complication during the gestation period, has been linked to impaired proliferation and migration of trophoblasts causing placental maldevelopment. We previously found that lncRNA X-inactive specific transcript (XIST) played an essential role in GDM progression. Here, we investigated the precise biological functions as well as the upstream and downstream regulatory mechanisms of XIST in GDM. We found that XIST and forkhead box O1 (FOXO1) were conspicuously upregulated and miR-497-5p and methyltransferase-like 14 (METTL14) were downregulated in the placentas of GDM patients. XIST silencing facilitated proliferation and migration and inhibited cell apoptosis and cell cycle arrest in HG-cultured HTR8/SVneo cells. METTL14 inhibited XIST expression through m6A methylation modification. XIST overexpression abrogated the positive effect of METTL14 overexpression on HG-cultured HTR8/SVneo cell progression. MiR-497-5p and FOXO1 are downstream regulatory genes of XIST in HTR8/SVneo cells. Reverse experiments illustrated that XIST mediated HTR8/SVneo cell functions by regulating the miR-497-5p/FOXO1 axis. Additionally, XIST silencing augmented glucose tolerance and alleviated fetal detrimental changes in GDM rats. To conclude, METTL14-mediated XIST silencing facilitated proliferation and migration and inhibited cell apoptosis and cell cycle arrest in HG-cultured HTR8/SVneo cells via the miR-497-5p/FOXO1 axis, thereby alleviating GDM progression in rats.

JinJiang ablation catheter is no inferior than Johnson ablation catheter in radiofrequency ablation of paroxysmal supraventricular tachycardia.

BACKGROUND: Paroxysmal supraventricular tachycardia (PSVT) is characterized by episodes of rapid tachycardia with sudden onset and sudden termination. PSVT treatment has evolved considerably over the past 30 years. Currently, radiofrequency catheter ablation is the first-line treatment. HYPOTHESIS: We conducted a randomized controlled trial to compare safety and effectiveness of PSVT ablation between the Jinjiang and Johnson (J&J) catheters in 57 patients in our hospital. METHODS AND RESULTS: Patients were randomly assigned to ablation procedures using either the Jinjiang system or the J&J Carto system. Follow-up was performed 3 days, 1, and 6 months after the procedure. Success rate, ablation time, frequency of ablation, and rates of complications and recurrence did not significantly differ between the groups. One Jinjiang group patient (3.6%) experienced arrhythmia recurrence during the 6-month follow-up. CONCLUSIONS: The Jinjiang catheter for radiofrequency ablation of PSVT is as safe and effective as the J&J catheter.

Genome-wide characterization of Remorin gene family and their responsive expression to abiotic stresses and plant hormone in Brassica napus.

KEY MESSAGE: Remorin proteins could be positively related to salt and osmotic stress resistance in rapeseed. Remorins (REMs) play a crucial role in adaptations to adverse environments. However, their roles in abiotic stress and phytohormone responses in oil crops are still largely unknown. In this study, we identified 47 BnaREM genes in the B.napus genome. Phylogenetic relationship and synteny analysis revealed that they were categorized into 5 distinct groups and have gone through 55 segmental duplication events under purifying selection. Gene structure and conserved domains analysis demonstrated that they were highly conserved and all BnaREMs contained a conserved Remorin_C domain, with a variable N-terminal region. Promoter sequence analysis showed that BnaREM gene promoters contained various hormones and stress-related cis-acting elements. Transcriptome data from BrassicaEDB database exhibited that all BnaREMs were ubiquitously expressed in buds, stamens, inflorescences, young leaves, mature leaves, roots, stems, seeds, silique pericarps, embryos and seed coats. The qRT-PCR analysis indicated that most of them were responsive to ABA, salt and osmotic treatments. Further mutant complementary experiments revealed that the expression of BnaREM1.3-4C-1 in the Arabidopsis rem1.3 mutant restored the retarded growth phenotype and the ability to resistance to salt and osmotic stresses. Our findings provide fundamental information on the structure and evolutionary relationship of the BnaREM family genes in rapeseed, and reveal the potential function of BnaREM1.3-4C-1 in stress and hormone response.

A pathway independent multi-modular ordered control system based on thermosensors and CRISPRi improves bioproduction in Bacillus subtilis.

Dynamic regulation is an effective strategy for control of gene expression in microbial cell factories. In some pathway contexts, several metabolic modules must be controlled in a time dependent or ordered manner to maximize production, while the creation of genetic circuits with ordered regulation capacity still remains a great challenge. In this work, we develop a pathway independent and programmable system that enables multi-modular ordered control of metabolism in Bacillus subtilis. First, a series of thermosensors were created and engineered to expand their thresholds. Then we designed single-input-multi-output circuits for ordered control based on the use of thermosensors with different transition points. Meanwhile, a repression circuit was constructed by combining CRISPRi-based NOT gates. As a proof-of-concept, these genetic circuits were applied for multi-modular ordered control of 2'-fucosyllactose (2'-FL) biosynthesis, resulting in a production of 1839.7 mg/l in shake flask, which is 5.16-times that of the parental strain. In a 5-l bioreactor, the 2'-FL titer reached 28.2 g/l with down-regulation of autolysis. Taken together, this work provides programmable and versatile thermosensitive genetic toolkits for dynamic regulation in B. subtilis and a multi-modular ordered control framework that can be used to improve metabolic modules in other chassis cells and for other compounds.

Study on the dynamic effects of plateau hypoxic and cold environment on the thermal adaptation of short-term sojourners in Xizang.

The plateau hypoxic environment can affect the thermoregulation process of the human body, and due to the different acclimatization ability to the hypoxic environment, the thermal requirements among the people who enter Xizang at different times may be different. Accordingly, this study aims to clarify how plateau hypoxic environments influence the physiological and subjective responses of people entering Xizang at different times. And field experiments were conducted in Xi'an and Lhasa, respectively, to compare the thermal responses and oxygen responses of the subjects under different temperature conditions on the plain, the first day of entering Xizang and the 15th day of entering Xizang. The results showed that under the hypoxic environment, the thermal sensation of the subjects decreased. With the extension of the time entering Xizang, the influence of the hypoxic environment on thermal comfort was gradually weakened, but under the low temperature environment, the effect of hypoxia on thermal response was not significantly reduced. The results of this study can help to reveal how plateau hypoxic environments affect human thermal comfort and provide a theoretical basis for the design of indoor thermal environment parameters suitable for sojourners entering Xizang at different times.

Managing Interface Defects via Mixed-Salt Passivation toward Efficient and Stable Perovskite Solar Cells.

Iodine vacancies and uncoordinated Pb0 defects existing at the perovskite surface have been widely demonstrated to induce deep-level defects, which can greatly limit improvement of the efficiency and stability of perovskite solar cells (PSCs). In this work, a novel strategy is proposed for functionalizing perovskite surface by using trimethylsulfoxonium iodide (TMSI), which can enhance the defect formation energy and inhibit Pb0 defects. Meanwhile, TMSI modification also can fill the iodine vacancies of perovskite surface-terminating ends. Consequently, the optimized device shows the improved charge dynamics and the reduced energy losses, achieving a champion efficiency of up to 24.03% along with excellent air-storage and thermal stabilities. This work offers guidelines for more efficient and stable PSCs based on the management of interface defects.

Enhancing regulatory T-cell function via inhibition of high mobility group box 1 protein signaling in immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is the most common acquired autoimmune bleeding disorder. Abnormally increased levels of High Mobility Group Box 1 (HMGB1) protein associate with thrombocytopenia and therapeutic outcome in ITP. Previous studies proposed that a natural inhibitor of HMGB1, 18ß-glycyrrhetinic acid (18ß-GA), could be used for its anti-inflammatory and immune-modulatory effects, although its ability to correct immune balance in ITP is unclear. In this study, we showed that plasma HMGB1 correlated negatively with platelet counts in ITP patients, and confirmed that 18ß-GA stimulated the production of regulatory T cells (Treg), restored the balance of CD4+ T-cell subsets and enhanced the suppressive function of Treg through blocking the effect on HMGB1 in patients with ITP. HMGB1 short hairpin RNA interference masked the effect of 18ß-GA in Treg of ITP patients. Furthermore, we found that 18ß-GA alleviated thrombocytopenia in mice with ITP. Briefly, anti-CD61 immune-sensitized splenocytes were transferred into severe combined immunodeficient mice to induce a murine model of severe ITP. The proportion of circulating Treg increased significantly, while the level of plasma HMGB1 and serum antiplatelet antibodies decreased significantly in ITP mice along 18ß-GA treatment. In addition, 18ß-GA reduced phagocytic activity of macrophages towards platelets both in ITP patients and ITP mice. These results indicate that 18ß-GA has the potential to restore immune balance in ITP via inhibition of HMGB1 signaling. In short, this study reveals the role of HMGB1 in ITP, which may serve as a potential target for thrombocytopenia therapy.

E2F1 rs3213150 polymorphism influences cytarabine sensitivity and prognosis in patients with acute myeloid leukemia.

Cytarabine (Ara-C) plays an irreplaceable role in the treatment of acute myeloid leukemia (AML). However, there are significant differences in efficacy among patients. Our previous studies found that E2F1 rs3213150 polymorphism was associated with remission rate of Ara-C chemotherapy, but the specific mechanism is not clear. This study aimed to further confirm the correlation between E2F1 rs3213150 polymorphism and Ara-C resistance and prognosis in AML patients, and to provide valuable information for elucidating the molecular mechanisms involved. METHODS: Rs3213150 genotyping was performed in 922 AML patients by Sanger sequencing, and the effects of different genotypes on chemosensitivity and prognosis were analyzed by Logistic regression and Cox regression. Meanwhile, a prediction model of Ara-C chemotherapy resistance was established. The impact of rs3213150 polymorphism on E2F1 expression level was determined by luciferase reporter gene assay, and differentially expressed genes between patients with different genotypes were identified by RNA sequencing. RESULTS: Compared with rs3213150 G allele carriers, patients with AA genotype had more obvious Ara-C resistance (41.94% vs. 27.94%, P = 0.002), shorter overall survival (529 d vs. 644 d, P = 0.008) and disease-free survival (519 d vs. 556 d, P = 0.023). Rs3213150G > A mutation resulted in decreased E2F1 expression. CONCLUSION: E2F1 rs3213150 polymorphism influences the chemosensitivity and prognosis of Ara-C in Chinese AML patients.

etiBsu1209: A comprehensive multiscale metabolic model for Bacillus subtilis.

Genome-scale metabolic models (GEMs) have been widely used to guide the computational design of microbial cell factories, and to date, seven GEMs have been reported for Bacillus subtilis, a model gram-positive microorganism widely used in bioproduction of functional nutraceuticals and food ingredients. However, none of them are widely used because they often lead to erroneous predictions due to their low predictive power and lack of information on regulatory mechanisms. In this work, we constructed a new version of GEM for B. subtilis (iBsu1209), which contains 1209 genes, 1595 metabolites, and 1948 reactions. We applied machine learning to fill gaps, which formed a relatively complete metabolic network able to predict with high accuracy (89.3%) the growth of 1209 mutants under 12 different culture conditions. In addition, we developed a visualization and code-free software, Model Tool, for multiconstraints model reconstruction and analysis. We used this software to construct etiBsu1209, a multiscale model that integrates enzymatic constraints, thermodynamic constraints, and transcriptional regulatory networks. Furthermore, we used etiBsu1209 to guide a metabolic engineering strategy (knocking out fabI and yfkN genes) for the overproduction of nutraceutical menaquinone-7, and the titer increased to 153.94 mg/L, 2.2-times that of the parental strain. To the best of our knowledge, etiBsu1209 is the first comprehensive multiscale model for B. subtilis and can serve as a solid basis for rational computational design of B. subtilis cell factories for bioproduction.

Analysis of acid-tolerance mechanism based on membrane microdomains in Saccharomyces cerevisiae.

BACKGROUND: Saccharomyces cerevisiae has been used in the biosynthesis of acid products such as organic acids owing to its acid tolerance. Improving the acid tolerance of S. cerevisiae is beneficial for expanding its application range. Our previous study isolated the TAMC strain that was tolerant to a pH 2.3 through adaptive laboratory evolution; however, its mechanism underlying tolerance to low pH environment remains unclear. RESULTS: In this study, through visual observation and order analysis of plasma membrane and membrane microdomains, we revealed that the membrane microdomains of TAMC strain play an indispensable role in acid tolerance. Transcriptomic analysis showed an increase in the expression of genes related to key components of membrane microdomains in TAMC strain. Furthermore, an obvious reduction was observed in the acid tolerance of the strain with sterol C-24 methyltransferase encoding gene ERG6 knockout for inhibiting membrane microdomain formation. Finally, colocalization analysis of H+-ATPase PMA1 and plasma membrane protein PMP1 showed that disruption of membrane microdomains could inhibit the formation of the H+-ATPase complex. CONCLUSIONS: Membrane microdomains could provide a platform for forming H+-ATPase complexes to facilitate intracellular H+ homeostasis, and thereby improve cell acid resistance. This study proposed a novel acid tolerance mechanism, providing a new direction for the rational engineering of acid-tolerant strains.

Recent progress in CRISPR-based bioengineering of microbial cell factories for important nutraceuticals synthesis.

Nutraceuticals are defined as food or food components with therapeutic capabilities that have few side effects and are regarded as a natural therapy for preventing the onset of several life-threatening illnesses. The use of microbial cell factories to produce nutraceuticals is considered to be sustainable and promising for meeting market demand. Among the diverse strategies for optimizing microbial cell factories, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system has emerged as a valuable tool for gene integration, deletion, activation, and downregulation. With the advent of multiplexed and precise CRISPR strategies, optimized microbial cell factories are revolutionizing the yield of nutraceuticals. This review focuses on the development of highly adaptable CRISPR strategies to optimize the production in microbial cell factories of some important nutraceuticals (belonging to the class of carotenoids, flavonoids, stilbenoids, polysaccharides, and nonprotein amino acids). Further, we highlighted current challenges related to the efficiency of CRISPR strategies and addressed potential future directions to fully harness CRISPR strategies to make nutraceutical synthesis in microbial cell factories an industrially favorable method.

Combinatorial metabolic engineering enables high yield production of α-arbutin from sucrose by biocatalysis.

α-Arbutin has been widely used as a skin-whitening ingredient. Previously, we successfully produced α-arbutin via whole-cell biocatalysis and found that the conversion rate of sucrose to α-arbutin was low (~45%). To overcome this issue, herein, we knocked out the genes of enzymes related to the sucrose hydrolysis, including sacB, sacC, levB, and sacA. The sucrose consumption was reduced by 17.4% in 24 h, and the sucrose conversion rate was increased to 51.5%. Furthermore, we developed an inducible protein degradation system with Lon protease isolated from Mesoplasma florum (MfLon) and proteolytic tag to control the PfkA activity, so that more fructose-6-phosphate (F6P) can be converted into glucose-1-phosphate (Glc1P) for α-arbutin synthesis, which can reduce the addition of sucrose and increase the sucrose conversion efficiency. Finally, the pathway of F6P to Glc1P was enhanced by integrating another copy of glucose 6-phosphate isomerase (Pgi) and phosphoglucomutase (PgcA); a high α-arbutin titer (~120 g/L) was obtained. The sucrose conversion rate was increased to 60.4% (mol/mol). In this study, the substrate utilization rate was boosted due to the attenuation of its hydrolysis and the assistance of the intracellular enzymes that converted the side product back into the substrate for α-arbutin synthesis. This strategy provides a new idea for the whole-cell biocatalytic synthesis of other products using sucrose as substrate, especially valuable glycosides.Key points The genes of sucrose metabolic pathway were knocked out to reduce the sucrose consumption. The by-product fructose was reused to synthesize α-arbutin. The optimized whole-cell system improved sucrose conversion by 15.3%.