Publications by authors named "Jingwen Zhou"

204 Publications

The OxrA protein of is required for the oxidative stress response and fungal pathogenesis.

Appl Environ Microbiol 2021 Sep 15:AEM0112021. Epub 2021 Sep 15.

The Key Laboratory of Biotechnology for Medicinal Plants of Jiangsu Province and School of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu 221116, PR China.

An efficient reactive oxygen species (ROS) detoxification system is vital for the survival of the pathogenic fungus within the host high ROS environment of the host. Therefore, identifying and targeting factors essential for oxidative stress response is one approach to develop novel treatments for fungal infections. Oxidation resistance 1 (Oxr1) protein is essential for protection against oxidative stress in mammals, but its functions in pathogenic fungi remain unknown. The present study aimed to characterize the role of an Oxr1 homolog in . The results indicated that the OxrA protein plays an important role in oxidative stress resistance by regulating the catalase function in , and overexpression of catalase can rescue the phenotype associated with OxrA deficiency. Importantly, the deficiency of decreased the virulence of and altered the host immune response. Using the -induced lung infection model, we demonstrated that the mutant strain induced less tissue damage along with decreased levels of LDH and albumin release. Additionally, the mutant caused inflammation at a lower degree, along with a markedly reduced influx of neutrophils to the lungs and a decreased secretion of cytokine usually associated with recruitment of neutrophils in mice. These results characterize for the role of OxrA in , as a core regulator of oxidative stress resistance and fungal pathogenesis. Knowledge of reactive oxygen species (ROS) detoxification in fungal pathogens is useful in the design of new antifungal drugs and could aid in the study of oxidative stress resistance mechanisms. In this study, we demonstrate that OxrA protein localize to the mitochondria and function to protect against oxidative damage. We demonstrate that OxrA contributes to oxidative stress resistance by regulating catalase function, and overexpression of catalase (CatA or CatB) can rescue the phenotype that is associated with OxrA deficiency. Remarkably, a loss of OxrA attenuated the fungal virulence in a mouse model of invasive pulmonary aspergillosis and altered the host immune response. Therefore, our finding indicates that inhibition of OxrA might be an effective approach for alleviating infection. The present study is, to the best of our knowledge, a pioneer in reporting the vital role of Oxr1 protein in pathogenic fungi.
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http://dx.doi.org/10.1128/AEM.01120-21DOI Listing
September 2021

Combinatorial engineering for efficient production of protein-glutaminase in Bacillus subtilis.

Enzyme Microb Technol 2021 Oct 29;150:109863. Epub 2021 Jun 29.

School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China; Science Center for Future Foods, Jiannan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China. Electronic address:

Protein-glutaminase (EC 3.5.1.44, PG) converts protein glutamine residues in proteins and peptides into glutamic acid residue, and markedly improves the solubility, emulsification, and foaming properties of food proteins. However, the source bacteria, Chryseobacterium proteolyticum, have low enzyme production ability, inefficient genetic operation, and high production cost. Therefore, it is critical to establish an efficient expression system for active PG. Here, combinatorial engineering was developed for high-yield production of PG in Bacillus subtilis. First, we evaluated different B. subtilis strains for PG self-activation. Then, combinatorial optimization involving promoters, signal peptides, and culture medium was applied to produce active recombinant PG in a B. subtilis expression system. Through combinatorial engineering, PG enzyme activity reached 3.23 U/mL in shaken-flask cultures. Active PG with the yield of 7.07 U/mL was obtained at 40 h by the P-YdeJ combination in fed-batch fermentation, which is the highest yield of PG in existing reports.
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http://dx.doi.org/10.1016/j.enzmictec.2021.109863DOI Listing
October 2021

Efficient Secretory Expression and Purification of Food-Grade Porcine Myoglobin in .

J Agric Food Chem 2021 Sep 25;69(35):10235-10245. Epub 2021 Aug 25.

Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.

Myoglobin (MG) is one of the eukaryotic heme-binding proteins that is closely associated with the real color and metallic taste of meat and can be used as a color additive in artificial meat alternatives. However, the traditional extraction methods are expensive and time-consuming and the heterologous biosynthesis of MG has never been reported. Herein, we achieved the secretory expression of porcine MG by engineered using the suitable host (X33), signal peptide (α-factor signal peptide), and modified constitutive promoter (G1 promoter). In addition, the fermentation conditions for MG production were optimized at shaking-flask level (BMGY medium with 40 mg/L of hemin, 30 °C) and at fermenter level (30% DO, feeding 150 mg/L of hemin), resulting in the highest titer of 285.42 mg/L MG in fed-batch fermentations. Furthermore, a purification method for food-grade MG was developed, which can obtain 0.22 mol of heme/mol of MG with 88.0% purity and 66.1% recovery rate.
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http://dx.doi.org/10.1021/acs.jafc.1c04124DOI Listing
September 2021

Applied evolution: Dual dynamic regulations-based approaches in engineering intracellular malonyl-CoA availability.

Metab Eng 2021 Sep 17;67:403-416. Epub 2021 Aug 17.

Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China. Electronic address:

Malonyl-CoA is an important building block for microbial synthesis of numerous pharmaceutically interesting or fatty acid-derived compounds including polyketides, flavonoids, phenylpropanoids and fatty acids. However, the tightly regulated intracellular malonyl-CoA availability often impedes overall product formation. Here, in order to unleash this tightly cellular behavior, we present evolution: dual dynamic regulations-based approaches to write artificial robust and dynamic function into intricate cellular background. Firstly, a conserved core domain based evolutionary principles were incorporated into genome mining to explore the biosynthetic diversities of discrete acetyl-CoA carboxylase (ACC) families, as malonyl-CoA is solely derived from carboxylation of acetyl-CoA by ACC in most organisms. A comprehensive phylogenomic and further experimental analysis, which included genomes of 50 strains throughout representative species, was performed to recapitulate the evolutionary history and reveal that previously unnoticed ACC families from Salmonella enterica exhibited the highest activities among all the candidates. A set of orthogonal and bi-functional quorum-sensing (QS)-based regulation tools were further designed and connected with T7 RNA polymerase as genetic amplifier to achieve dual dynamic control in a high dynamic range, which allowed us to efficiently activate and repress different sets of genes dynamically and independently. These genetic circuits were then combined with ACC of S. enterica and CRISPRi system to reprogram central metabolism that rewired the tightly regulated malonyl-CoA pathway to a robust and autonomous behavior, leading to a 29-fold increase of malony-CoA availability. We applied this dual regulation tool to successfully synthesizing malonyl-CoA-derived compound (2S)-naringenin, and achieved the highest production (1073.8 mg/L) reported to date associate with dramatic decreases of by-product formation. Notably, the whole fermentation presents as an autonomous behavior, totally eliminating human supervision and inducer supplementation. Hence, the constructed evolution: dual dynamic regulations-based approaches pave the way to develop an economically viable and scalable procedure for microbial production of malonyl-CoA derived compounds.
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http://dx.doi.org/10.1016/j.ymben.2021.08.004DOI Listing
September 2021

Recent Advances in the Physicochemical Properties and Biotechnological Application of Hemoglobin.

Microorganisms 2021 Jul 7;9(7). Epub 2021 Jul 7.

Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China.

hemoglobin (VHb), the first discovered bacterial hemoglobin, is a soluble heme-binding protein with a faster rate of oxygen dissociation. Since it can enhance cell growth, product synthesis and stress tolerance, VHb has been widely applied in the field of metabolic engineering for microorganisms, plants, and animals. Especially under oxygen-limited conditions, VHb can interact with terminal oxidase to deliver enough oxygen to achieve high-cell-density fermentation. In recent years, with the development of bioinformatics and synthetic biology, several novel physicochemical properties and metabolic regulatory effects of VHb have been discovered and numerous strategies have been utilized to enhance the expression level of VHb in various hosts, which greatly promotes its applications in biotechnology. Thus, in this review, the new information regarding structure, function and expressional tactics for VHb is summarized to understand its latest applications and pave a new way for the future improvement of biosynthesis for other products.
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http://dx.doi.org/10.3390/microorganisms9071455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8306070PMC
July 2021

Non-shivering Thermogenesis Signalling Regulation and Potential Therapeutic Applications of Brown Adipose Tissue.

Int J Biol Sci 2021 13;17(11):2853-2870. Epub 2021 Jul 13.

Guangdong Engineering Research Center of Natural Products and New Drugs, Guangdong Provincial University Engineering Technology Research Center of Natural Products and Drugs, Guangdong Pharmaceutical University, Guangzhou 510006, China.

In mammals, thermogenic organs exist in the body that increase heat production and enhance energy regulation. Because brown adipose tissue (BAT) consumes energy and generates heat, increasing energy expenditure via BAT might be a potential strategy for new treatments for obesity and obesity-related diseases. Thermogenic differentiation affects normal adipose tissue generation, emphasizing the critical role that common transcriptional regulation factors might play in common characteristics and sources. An understanding of thermogenic differentiation and related factors could help in developing ways to improve obesity indirectly or directly through targeting of specific signalling pathways. Many studies have shown that the active components of various natural products promote thermogenesis through various signalling pathways. This article reviews recent major advances in this field, including those the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA), cyclic guanosine monophosphate-GMP-dependent protein kinase G (cGMP-AKT), AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), transforming growth factor-β/bone morphogenic protein (TGF-β/BMP), transient receptor potential (TRP), Wnt, nuclear factor-κ-light-chain-enhancer of activated B cells (NF-κΒ), Notch and Hedgehog (Hh) signalling pathways in brown and brown-like adipose tissue. To provide effective information for future research on weight-loss nutraceuticals or drugs, this review also highlights the natural products and their active ingredients that have been reported in recent years to affect thermogenesis and thus contribute to weight loss via the above signalling pathways.
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http://dx.doi.org/10.7150/ijbs.60354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8326120PMC
July 2021

Growth-coupled evolution and high-throughput screening assisted rapid enhancement for amylase-producing Bacillus licheniformis.

Bioresour Technol 2021 Oct 26;337:125467. Epub 2021 Jun 26.

School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.

Bacillus licheniformis α-amylase is a thermostable enzyme used in industrial starch hydrolysis. However, difficulties in the genetic manipulation of B. licheniformis hamper further enhancement of α-amylase production. In this regard, adaptive evolution is a useful strategy for promoting the productivity of microbial hosts, although the success of this approach requires the application of suitable evolutionary stress. In this study, we designed a growth-coupled adaptive evolution model to enrich B. licheniformis strains with enhanced amylase productivity and utilization capacity of starch substrates. Single cells of high α-amylase-producing B. licheniformis were isolated using a droplet-based microfluidic platform. Clones with 67% higher α-amylase yield were obtained and analyzed by genome resequencing. Our findings confirmed that growth-coupled evolution combined with high-throughput screening is an efficient strategy for enhanced α-amylase production. In addition, we identified several potential target genes to guide further modification of the B. licheniformis host for efficient protein expression.
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http://dx.doi.org/10.1016/j.biortech.2021.125467DOI Listing
October 2021

Identification of microRNA transcriptome throughout the lifespan of yak () corpus luteum.

Anim Biotechnol 2021 Jul 26:1-13. Epub 2021 Jul 26.

Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization, Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization Key Laboratory of Sichuan Province, Ministry of Education, Southwest Minzu University, Chengdu, Sichuan, China.

The corpus luteum (CL) is a temporary organ that plays a critical role for female fertility by maintaining the estrous cycle. MicroRNA (miRNA) is a class of non-coding RNAs involved in various biological processes. However, there exists limited knowledge of the role of miRNA in yak CL. In this study, we used high-throughput sequencing to study the transcriptome dynamics of miRNA in yak early (eCL), middle (mCL) and late-stage CL (lCL). A total of 6,730 miRNAs were identified, including 5,766 known and 964 novels miRNAs. Three miRNAs, including bta-miR-126-3p, bta-miR-143 and bta-miR-148a, exhibited the highest expressions in yak CLs of all the three stages. Most of the miRNAs were 20-24 nt in length and the peak was at 22 nt. Besides, most miRNAs with different lengths displayed significant uracil preference at the 5'-end. Furthermore, 1,067, 280 and 112 differentially expressed (DE) miRNAs were found in eCL vs. mCL, mCL vs. lCL, and eCL vs. lCL, respectively. Most of the DE miRNAs were down-regulated in the eCL vs. mCL and eCL vs. lCL groups, and up-regulated in the mCL vs. lCL group. A total of 18,904 target genes were identified, with 18,843 annotated. Pathway enrichment analysis of the DE miRNAs target genes illustrated that the most enriched cellular process in each group included pathways in cancer, PI3K-Akt pathway, endocytosis, and focal adhesion. A total of 20 putative target genes in 47 DE miRNAs were identified to be closely associated with the formation, function or regression of CL. Three DE miRNAs, including bta-miR-11972, novel-miR-619 and novel-miR-153, were proved to directly bind to the 3'-UTR of their predicated target mRNAs, including , and , respectively. Both of these DE miRNAs and their target mRNAs exhibited dynamic expression profiles across the lifespan of yak CL. This study presents a general basis for understanding of the regulation of miRNA on yak CL and also provides a novel genetic resource for future analysis of the gene network during the estrous cycle in the yak.
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http://dx.doi.org/10.1080/10495398.2021.1946552DOI Listing
July 2021

Simultaneous transformation of five vectors in Gluconobacter oxydans.

Plasmid 2021 Jul 10;117:102588. Epub 2021 Jul 10.

National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China; School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China; Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China; Jiangsu Provisional Research Center for Bioactive Product Processing Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China. Electronic address:

Gluconobacter oxydans is an obligate Gram-negative bacterium that belongs to the family Acetobacteraceae. It is one of the most frequently used microorganisms in industrial biotechnology to produce chemicals related to incomplete oxidation. However, the fine-tuning of G. oxydans is hampered by the lack of efficient genetic tools to enable sophisticated metabolic manipulations. Thus, a series of shuttle vectors for G. oxydans inspired by a series of wild-type plasmids in different G. oxydans strains were constructed. Fifteen shuttle vectors were employed to express mCherry in G. oxydans WSH-003 using the replication origin of these wild-type plasmids. Among them, the intensity of fluorescent proteins expressed by p15-K-mCherry was about 10 times that of fluorescent proteins expressed by p5-K-mCherry. Quantitative real-time polymerase chain reaction showed that the relative copy number of p15-K-mCherry reached 19 and had high stability. In contrast, some of the plasmids had a relative copy number of less than 10. The co-expression of multiple shuttle vectors revealed five shuttle vectors that could be transformed into G. oxydans WSH-003 and could express five different fluorescent proteins. The shuttle vectors will facilitate genetic operations for Gluconobacter strains to produce useful compounds more efficiently.
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http://dx.doi.org/10.1016/j.plasmid.2021.102588DOI Listing
July 2021

A SacB-based system for diverse and multiple genome editing in Gluconobacter oxydans.

J Biotechnol 2021 Sep 8;338:31-39. Epub 2021 Jul 8.

National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China; School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China; Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China; Jiangsu Provisional Research Center for Bioactive Product Processing Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China. Electronic address:

Gluconobacter oxydans is an important industrial bacterial strain widely used to produce a lot of useful products. However, very few gene editing tools are available for G. oxydans. This study aimed to develop an efficient genome editing method for G. oxydans using SacB as a counter-selectable marker. A plasmid that could express the kanamycin resistance gene in both E. coli and G. oxydans was constructed using the screened shuttle promoter P. After optimizing the genome editing conditions, the derivative plasmids could be effectively utilized for diverse genome editing, such as gene deletion, insertion, replacement, and in situ modification in G. oxydans WSH-003. In addition, the SacB-based system also achieved multiple gene editing in G. oxydans. Moreover, the genome of the industrial strain G. oxydans WSH-003 was modified and the growth rate and substrate conversion rate of the strain successfully increased using this system. The system could also have potential to be applied in different G. oxydans strains. The process established in this study also provides a reference for constructing genetic tools for many other genetically recalcitrant bacteria.
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http://dx.doi.org/10.1016/j.jbiotec.2021.07.004DOI Listing
September 2021

[Progress in vitamin C biosynthesis related dehydrogenases].

Sheng Wu Gong Cheng Xue Bao 2021 Jun;37(6):1827-1844

National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, Jiangsu, China.

Vitamin C is an essential vitamin for human beings. It has a huge market in the fields of food and pharmaceuticals. 2-keto-L-gulonic acid is an important precursor to produce vitamin C by microbial fermentation in industrial. In microbial fermentations, the L-sorbose pathway and the D-gluconate pathway have been the focus of research because of high yield. This article aims at stating recent research progress in dehydrogenases related to biosynthesis of vitamin C in the L-sorbose pathway and the D-gluconate pathway. The properties of dehydrogenase in terms of localization, substrate specificity, cofactors, and electron transport carrier are elaborated. And then, the main problems and strategies are reviewed in the L-sorbose pathway and in the D-gluconate pathway. Finally, future research on the dehydrogenases in the biosynthesis of vitamin C through L-sorbose pathway and D-gluconate pathway is discussed.
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http://dx.doi.org/10.13345/j.cjb.200449DOI Listing
June 2021

Risk Factors, Patterns, and Long-Term Survival of Recurrence After Radiofrequency Ablation With or Without Transarterial Chemoembolization for Hepatocellular Carcinoma.

Front Oncol 2021 27;11:638428. Epub 2021 May 27.

Department of Minimally Invasive Interventional Radiology, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.

Objectives: To classify hepatocellular carcinoma (HCC) recurrence patterns after radiofrequency ablation (RFA) or transarterial chemoembolization (TACE) combined with RFA (TACE-RFA) and analyze their risk factors and impacts on survival.

Methods: We retrospectively evaluated the medical records of HCC patients who underwent RFA or TACE-RFA from January 2006 to December 2016. HCC recurrences were classified into four patterns: local tumor progression (LTP), intra-segmental recurrence, extra-segmental recurrence, and aggressive recurrence. Risk factors, overall survival (OS), and post-recurrence survival of each pattern were evaluated.

Results: A total of 249 patients with a single, hepatitis-B virus (HBV)-related HCC ≤ 5.0 cm who underwent RFA (HCC ≤ 3.0 cm) or TACE-RFA (HCC of 3.1-5.0 cm) were included. During follow-up (median, 53 months), 163 patients experienced HCC recurrence: 40, 43, 62 and 18 patients developed LTP, intra-segmental recurrence, extra-segmental recurrence, and aggressive recurrence, respectively; the median post-recurrence survival was 49, 37, 25 and 15 months, respectively (P < .001); the median OS was 65, 56, 58 and 28 months, respectively (P < .001). Independent risk factors for each pattern were as follows: tumor sized 2.1-3.0 cm undergoing RFA alone and insufficient ablative margin for LTP, periportal tumor and non-smooth tumor margin for intra-segmental recurrence, HBV-DNA ≥ 2000 IU/mL for extra-segmental recurrence, and periportal tumor and α-fetoprotein ≥ 100 ng/mL for aggressive recurrence. Recurrence pattern (P < .001) and Child-Pugh class B (P = .025) were independent predictors for OS.

Conclusions: Based on our classification, each recurrence pattern had different recurrence risk factors, OS, and post-recurrence survival.
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http://dx.doi.org/10.3389/fonc.2021.638428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8191459PMC
May 2021

Efficient Production of Orientin and Vitexin from Luteolin and Apigenin Using Coupled Catalysis of Glycosyltransferase and Sucrose Synthase.

J Agric Food Chem 2021 Jun 1;69(23):6578-6587. Epub 2021 Jun 1.

National Engineering Laboratory for Cereal Fermentation Technology (NELCF), Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.

Orientin and vitexin are flavone 8-glycosides that exhibit many biological characteristics. This study aimed to establish a two-enzyme-coupled catalytic strategy to enhance the biosynthesis of orientin and vitexin from apigenin and luteolin, respectively. The glucosyltransferase (TcCGT1) gene from was cloned and expressed in BL21(DE3). The optimal activity of TcCGT1 was achieved at pH 9.0 and 37 °C. TcCGT1 was relatively stable over the pH range of 7.0-10.0 at a temperature lower than 45 °C. The coupled catalytic strategy of TcCGT1 and different sucrose synthases was adopted to enhance the production of orientin and vitexin. By optimizing the coupling reaction conditions, orientin and vitexin production successfully achieved 2324.4 and 5524.1 mg/L with a yield of 91.4 and 89.3% (mol/mol), respectively. The coupled catalytic strategy proposed in this study might serve as a promising candidate for the large-scale production of orientin and vitexin in the future.
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http://dx.doi.org/10.1021/acs.jafc.1c00602DOI Listing
June 2021

Development of a growth coupled and multi-layered dynamic regulation network balancing malonyl-CoA node to enhance (2S)-naringenin biosynthesis in Escherichia coli.

Metab Eng 2021 Sep 27;67:41-52. Epub 2021 May 27.

National Engineering Laboratory for Cereal Fermentation Technology (NELCF), Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China; Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China; Jiangsu Provincial Research Center for Bioactive Product Processing Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China. Electronic address:

Metabolic heterogeneity and dynamic changes in metabolic fluxes are two inherent characteristics of microbial fermentation that limit the precise control of metabolisms, often leading to impaired cell growth and low productivity. Dynamic metabolic engineering addresses these challenges through the design of multi-layered and multi-genetic dynamic regulation network (DRN) that allow a single cell to autonomously adjust metabolic flux in response to its growth and metabolite accumulation conditions. Here, we developed a growth coupled NCOMB (Naringenin-Coumaric acid-Malonyl-CoA-Balanced) DRN with systematic optimization of (2S)-naringenin and p-coumaric acid-responsive regulation pathways for real-time control of intracellular supply of malonyl-CoA. In this scenario, the acyl carrier protein was used as a novel critical node for fine-tuning malonyl-CoA consumption instead of direct repression of fatty acid synthase commonly employed in previous studies. To do so, we first engineered a multi-layered DRN enabling single cells to concurrently regulate acpH, acpS, acpT, acs, and ACC in malonyl-CoA catabolic and anabolic pathways. Next, the NCOMB DRN was optimized to enhance the synergies between different dynamic regulation layers via a biosensor-based directed evolution strategy. Finally, a high producer obtained from NCOMB DRN approach yielded a 8.7-fold improvement in (2S)-naringenin production (523.7 ± 51.8 mg/L) with a concomitant 20% increase in cell growth compared to the base strain using static strain engineering approach, thus demonstrating the high efficiency of this system for improving pathway production.
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http://dx.doi.org/10.1016/j.ymben.2021.05.007DOI Listing
September 2021

Optimization of CRISPR-Cas9 through promoter replacement and efficient production of L-homoserine in Corynebacterium glutamicum.

Biotechnol J 2021 Aug 3;16(8):e2100093. Epub 2021 Jun 3.

National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, Jiangsu, China.

Background: Corynebacterium glutamicum is an important chassis for industrial applications. The low efficiency of commonly used genome editing methods for C. glutamicum limits the rapid multiple engineering of the bacterium.

Main Methods And Major Results: In this study, chromosome-borne expression of cas9 and recET from Escherichia coli K12-MG1655 was achieved to avoid toxicity to the strain, increase the probability of homologous recombination, and reduce loss of viability caused by double-strand breaks. Constitutive strong promoters, such as P , P , and P , were used to replace P and to expand the application of the CRISPR-Cas9 system. By using this system, a C. glutamicum strain producing L-homoserine to 22.1 g per L in a 5-L bioreactor after 96 h was obtained.

Conclusions And Implications: Through the application of visualized fluorescent protein, the process of plasmid curing was optimized, obtain a continuous and rapid CRISPR-Cas9 genome editing system. The method described here could be useful to construct C. glutamicum mutant rapidly.
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http://dx.doi.org/10.1002/biot.202100093DOI Listing
August 2021

Structure-based engineering of substrate specificity for pinoresinol-lariciresinol reductases.

Nat Commun 2021 05 14;12(1):2828. Epub 2021 May 14.

Research and Development Center of Chinese Medicine Resources and Biotechnology, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai, China.

Pinoresinol-lariciresinol reductases (PLRs) are enzymes involved in the lignan biosynthesis after the initial dimerization of two monolignols, and this represents the entry point for the synthesis of 8-8' lignans and contributes greatly to their structural diversity. Of particular interest has been the determination of how differing substrate specificities are achieved with these enzymes. Here, we present crystal structures of IiPLR1 from Isatis indigotica and pinoresinol reductases (PrRs) AtPrR1 and AtPrR2 from Arabidopsis thaliana, in the apo, substrate-bound and product-bound states. Each structure contains a head-to-tail homodimer, and the catalytic pocket comprises structural elements from both monomers. β4 loop covers the top of the pocket, and residue 98 from the loop governs catalytic specificity. The substrate specificities of IiPLR1 and AtPrR2 can be switched via structure-guided mutagenesis. Our study provides insight into the molecular mechanism underlying the substrate specificity of PLRs/PrRs and suggests an efficient strategy for the large-scale commercial production of the pharmaceutically valuable compound lariciresinol.
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http://dx.doi.org/10.1038/s41467-021-23095-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8121951PMC
May 2021

Optimum chalcone synthase for flavonoid biosynthesis in microorganisms.

Crit Rev Biotechnol 2021 May 12:1-15. Epub 2021 May 12.

National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, China.

Chalcones and the subsequently generated flavonoids, as well as flavonoid derivatives, have been proven to have a variety of physiological activities and are widely used in: the pharmaceutical, food, feed, and cosmetic industries. As the content of chalcones and downstream products in native plants is low, the production of these compounds by microorganisms has gained the attention of many researchers and has a history of more than 20 years. The mining and engineering of chalcone synthase (CHS) could be one of the most important ways to achieve more efficient production of chalcones and downstream products in microorganisms. CHS has a broad spectrum of substrates, and its enzyme activity and expression level can significantly affect the efficiency of the biosynthesis of flavonoids. This review summarizes the recent advances in the: structure, mechanism, evolution, substrate spectrum, transformation, and expression regulation in the flavonoid biosynthesis of this vital enzyme. Future development directions were also suggested. The findings may further promote the research and development of flavonoids and health products, making them vital in the fields of human diet and health.
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http://dx.doi.org/10.1080/07388551.2021.1922350DOI Listing
May 2021

Identification of Gradient Promoters of and Their Applications in the Biosynthesis of 2-Keto-L-Gulonic Acid.

Front Bioeng Biotechnol 2021 9;9:673844. Epub 2021 Apr 9.

Key Laboratory of Industrial Biotechnology, Ministry of Education and School of Biotechnology, Jiangnan University, Wuxi, China.

The acetic acid bacterium is known for its unique incomplete oxidation and therefore widely applied in the industrial production of many compounds, e.g., 2-keto-L-gulonic acid (2-KLG), the direct precursor of vitamin C. However, few molecular tools are available for metabolically engineering , which greatly limit the strain development. Promoters are one of vital components to control and regulate gene expression at the transcriptional level for boosting production. In this study, the low activity of SDH was found to hamper the high yield of 2-KLG, and enhancing the expression of SDH was achieved by screening the suitable promoters based on RNA sequencing data. We obtained 97 promoters from 's genome, including two strong shuttle promoters and six strongest promoters. Among these promoters, P and P revealed strong activities in both and , and the activity of the strongest promoter (P) was about threefold that of the other reported strong promoters of . These promoters were used to overexpress SDH in WSH-003. The titer of 2-KLG reached 3.7 g/L when SDH was under the control of strong promoters P and P. This study obtained a series of gradient promoters, including two strong shuttle promoters, and expanded the toolbox of available promoters for the application in metabolic engineering of for high-value products.
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http://dx.doi.org/10.3389/fbioe.2021.673844DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8064726PMC
April 2021

Meikin synergizes with shugoshin to protect cohesin Rec8 during meiosis I.

Genes Dev 2021 05 22;35(9-10):692-697. Epub 2021 Apr 22.

Science Center for Future Foods, Jiangnan University, Wuxi, Jiangsu 214122, China.

The conserved meiosis-specific kinetochore regulator, meikin (Moa1 in fission yeast) plays a central role in establishing meiosis-specific kinetochore function. However, the underlying molecular mechanisms remain elusive. Here, we show how Moa1 regulates centromeric cohesion protection, a function that has been previously attributed to shugoshin (Sgo1). Moa1 is known to associate with Plo1 kinase. We explore Plo1-dependent Rec8 phosphorylation and identify a key phosphorylation site required for cohesion protection. The phosphorylation of Rec8 by Moa1-Plo1 potentiates the activity of PP2A associated with Sgo1. This leads to dephosphorylation of Rec8 at another site, which thereby prevents cleavage of Rec8 by separase.
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http://dx.doi.org/10.1101/gad.348052.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8091969PMC
May 2021

Systematically Engineered Fatty Acid Catabolite Pathway for the Production of (2)-Naringenin in .

ACS Synth Biol 2021 05 20;10(5):1166-1175. Epub 2021 Apr 20.

Key Laboratory of Industrial Biotechnology, Ministry of Education and School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.

The (2)-naringenin is an important natural flavonoid with several bioactive effects on human health. It is also a key precursor in the biosynthesis of other high value compounds. The production of (2S)-naringenin is significantly influenced by the acetyl-CoA available in the cytosol. In this study, we increased the acetyl-CoA supply via the β-oxidation of fatty acids in the peroxisomes of . Several lipases from different sources and , , , and , the key genes of the fatty acid β-oxidation pathway, were overexpressed during the production of (2)-naringenin in yeast. The level of acetyl-CoA was 0.205 nmol higher than that in the original strain and the production of (2)-naringenin increased to 286.62 mg/g dry cell weight when was overexpressed in strain L07. Remarkable (2)-naringenin production (1129.44 mg/L) was achieved with fed-batch fermentation, with the highest titer reported in any microorganism. Our results demonstrated the use of fatty acid β-oxidation to increase the level of cytoplasmic acetyl-CoA and the production of its derivatives.
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http://dx.doi.org/10.1021/acssynbio.1c00002DOI Listing
May 2021

Zi Qi Decoction Alleviates Liver Fibrosis by Inhibiting the Toll-Like Receptor 4 (TLR4)-Related Nuclear Factor kappa b (NF-κB) and Mitogen-Activated Protein Kinase (MAPK) Signaling Pathways.

Med Sci Monit 2021 Apr 14;27:e929438. Epub 2021 Apr 14.

The First Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China (mainland).

BACKGROUND Hepatic stellate cells (HSCs) play a vital role in hepatic fibrogenesis. Our recent clinical study indicated that the Zi Qi decoction, a Traditional Chinese Medicine formula, exhibited good efficacy in alleviating liver fibrosis, but the underlying mechanism remains elusive. MATERIAL AND METHODS Rats repeatedly injected with CCl₄ and cells stimulated with lipopolysaccharide were used as in vivo and in vitro models for liver fibrosis, respectively. The viability of LX-2 cells was evaluated with MTT assay. Relative messenger RNA (mRNA) expression of representative extracellular matrix (ECM) components was detected with real-time quantitative polymerase chain reaction (RT-qPCR). Moreover, total and phosphorylation levels of ECM proteins and pathway-related proteins were detected with western blotting. Immunofluorescent staining was used to show the nuclear translocation of nuclear factor kappa b (NF-kappaB) p65. Hematoxylin & eosin (H&E) and Masson trichrome staining and immunohistochemistry were performed to evaluate the extent of liver fibrosis. The levels of alanine transaminase (ALT), aspartate transaminase (AST), gamma-glutamyl transpeptidase (GGT), Hyp, tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) were tested with an enzyme-linked immunosorbent assay. In addition, 7.0T micro-magnetic resonance imaging (micro-MRI) was used to evaluate the severity of hepatic damage. RESULTS The Zi Qi decoction inhibited lipopolysaccharide-mediated upregulation of mRNA and protein levels of representative ECM proteins both in vivo and in vitro. The Zi Qi decoction also suppressed activation of the Toll-like receptor 4 (TLR4)-related NF-kappaB signaling pathway and subsequently inhibited the nuclear translocation of activated NF-kappaB. Moreover, another TLR4 downstream pathway, mitogen-activated protein kinase (MAPK), was simultaneously restrained. The results of liver pathology and MRI in rat models also suggested the efficacy of the Zi Qi decoction in attenuating liver damage. CONCLUSIONS The Zi Qi decoction inhibited liver fibrosis by inhibiting the TLR4-related NF-kB and MAPK signaling pathways and preventing activation of HSCs.
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http://dx.doi.org/10.12659/MSM.929438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8054620PMC
April 2021

[Progress in gene editing technologies for Saccharomyces cerevisiae].

Sheng Wu Gong Cheng Xue Bao 2021 Mar;37(3):950-965

National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, Jiangsu, China.

Saccharomyces cerevisiae is one of the most important hosts in metabolic engineering. Advanced gene editing technology has been widely used in the design and construction of S. cerevisiae cell factories. With the rapid development of gene editing technology, early gene editing technologies based on recombinase and homologous recombination have been gradually replaced by new editing systems. In this review, the principle and application of gene editing technology in S. cerevisiae are summarized. Here, we first briefly describe the classical gene editing techniques of S. cerevisiae. Then elaborate the genome editing system of MegNs, ZFNs and TALENs based on endonuclease. The latest research progress is especially introduced and discussed, including the CRISPR/Cas system, multi-copy integration of heterologous metabolic pathways, and genome-scale gene editing. Finally, we envisage the application prospects and development directions of Saccharomyces cerevisiae gene editing technology.
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http://dx.doi.org/10.13345/j.cjb.200542DOI Listing
March 2021

The efficacy of novel metabolic targeted agents and natural plant drugs for nonalcoholic fatty liver disease treatment: A PRISMA-compliant network meta-analysis of randomized controlled trials.

Medicine (Baltimore) 2021 Mar;100(12):e24884

The First Clinical Medical College of Nanjing University of Chinese Medicine, Nanjing.

Background: Nonalcoholic fatty liver disease (NAFLD) is a highly prevalent chronic liver disease characterized by excess accumulation of fat in hepatocytes. Because no drug has been approved for NAFLD treatment, this work analyzed the effects of agents resulting from 2 research hotspots, metabolic target agents, and natural plant drugs, on NAFLD with network meta-analysis.

Methods: Public databases were searched through August 14, 2020. Randomized controlled trials that compared obeticholic acid, elafibranor, cenicriviroc, selonsertib, curcumin, silymarin, and resveratrol to placebo were included. Liver pathology improvement, hepatic biochemical indicators, and lipid metabolism indicators were analyzed.

Results: Thirty-five studies were included in the meta-analysis. Obeticholic acid was found to significantly increase the frequency of liver biopsy improvement compared to placebo (OR: 2.10; 95% CI: 1.60, 2.77). The ranking results among the hepatic biochemical indicators showed that obeticholic acid (94.9%) and elafibranor (86.3%) have a relative advantage in reducing alanine aminotransferase (ALT) levels, and obeticholic acid also had an advantage (95.4%) in reducing aspartate aminotransferase (AST) levels. Considering lipid metabolic indicators, elafibranor (expSMD: 0.01; 95% CI: 0.00, 0.05; SUCRA: 100%), and obeticholic acid (expSMD: 0.48; 95% CI: 0.28,0.84; SUCRA: 75.6%) significantly reduced triglyceride (TG) levels compared with placebo; moreover, obeticholic acid, but not elafibranor, caused a serious increase in total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels and a decrease in high-density lipoprotein cholesterol (HDL-C) levels.

Conclusions: Novel metabolic targeted agents generally have better effects than natural plant drugs, especially obeticholic acid, and elafibranor. However, obeticholic acid showed serious adverse effects such as increasing LDL-C levels and decreasing HDL-C levels. Curcumin showed potential advantages for NAFLD but lacked statistical significance.
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http://dx.doi.org/10.1097/MD.0000000000024884DOI Listing
March 2021

Enhanced Production of Transglutaminase in through Random Mutagenesis and Site-Directed Genetic Modification.

J Agric Food Chem 2021 Mar 2;69(10):3144-3153. Epub 2021 Mar 2.

National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.

transglutaminase (TGase) is widely used to improve food texture properties. In this study, random mutagenesis and site-directed genetic modification were used to improve the production of TGase in . First, DSM40587 (smWT) was subjected to atmospheric and room-temperature plasma mutagenesis, and then a mutant (smY2019) with a 5.5-fold increase in TGase yield was screened from approximately 3000 × 25 (round) mutants. Compared to smWT, smY2019 exhibits a 3.2-fold higher TGase mRNA level and two site mutations within the -10 region of the TGase promoter. The recombinant expression analysis in the TGase-deficient suggests that the mutated TGase promoter is more robust than the wild-type one. Finally, we integrated two additional TGase expression cassettes into the smY2019 genome, yielding the recombinant strain smY2019-3C with a 103% increase in TGase production compared to smY2019. The smY2019-3C strain with 40 U/mL of TGase yield could be a suitable candidate for the industrial production of TGase.
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http://dx.doi.org/10.1021/acs.jafc.1c00645DOI Listing
March 2021

[Interdisciplinary education of fermentation engineering graduates].

Sheng Wu Gong Cheng Xue Bao 2021 Feb;37(2):689-695

Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.

Fermentation engineering is an industrial process that uses the transformation of microorganisms or other cells to produce a specific product in a specific bioreactor. Fermentation engineering has developed from an ancient food fermentation relying solely on experience accumulation to an important production mode of food, agriculture, medicine, chemical industry and other means of production and life. It has become a key technology to support the sustainable development of human beings, and is inseparable from the continuous progress of interdisciplinary technology. The interdisciplinary integration and the continuous upward movement of China's global industrial chain will inevitably put forward higher requirements for the cultivation of fermentation engineering composite talents in the new situation. In order to constantly improve the interdisciplinary fermentation engineering compound talent training system, in recent years, the research lab has been refining and improving the concept of talent training, and actively deepening the reform of talent training system. Systematic research and practice have been carried out around the aspects of training program, enrollment system, teacher background, subject setting, scientific research practice, evaluation system, etc., which has promoted the technological progress of fermentation engineering and related supporting industries, and contributed an important force to the transformation of China from a big fermentation country to a powerful fermentation country.
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http://dx.doi.org/10.13345/j.cjb.200288DOI Listing
February 2021

[Bioethical considerations of cell-cultured meat].

Sheng Wu Gong Cheng Xue Bao 2021 Feb;37(2):378-383

Science Center for Future Foods, Jiangnan University, Wuxi 214122, Jiangsu, China.

In recent years, to solve the increasingly prominent problem of the contradiction between human social development and environmental resources, artificial meat has appeared in public view more and more. Generally speaking, the artificial meat can be divided into vegetable protein meat and cell cultured meat. Among them, vegetable protein meat has gradually begun to be commercialized, and cell cultured meat is cultured with animal cells, which is more similar to the real meat. Based on the analysis of the essence of cell cultured meat, we explore the positive significance of cell cultured meat technology for the meat production industry, consumer groups, and the sustainable development of mankind in the future. From the perspective of bioethics, the research, development and production of cell cultured meat can help ensure the sustainable development of human society, improve animal welfare, reduce resource demand, improve the nutritional function of meat products, and provide new growth points for the development of other industries. In addition, the ethical risks of food safety, technology abuse and technical supervision involved in cell cultured meat production are put forward for deep consideration, hoping to provide reference for the sustainable development of artificial meat industry from the perspective of bioethics.
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http://dx.doi.org/10.13345/j.cjb.200278DOI Listing
February 2021

A High-Efficiency Artificial Synthetic Pathway for 5-Aminovalerate Production From Biobased L-Lysine in .

Front Bioeng Biotechnol 2021 9;9:633028. Epub 2021 Feb 9.

National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, China.

Bioproduction of 5-aminovalerate (5AVA) from renewable feedstock can support a sustainable biorefinery process to produce bioplastics, such as nylon 5 and nylon 56. In order to achieve the biobased production of 5AVA, a 2-keto-6-aminocaproate-mediated synthetic pathway was established. Combination of L-Lysine α-oxidase from , α-ketoacid decarboxylase from and aldehyde dehydrogenase from could achieve the biosynthesis of 5AVA from biobased L-Lysine in . The HO produced by L-Lysine α-oxidase was decomposed by the expression of catalase KatE. Finally, 52.24 g/L of 5AVA were obtained through fed-batch biotransformation. Moreover, homology modeling, molecular docking and molecular dynamic simulation analyses were used to identify mutation sites and propose a possible trait-improvement strategy: the expanded catalytic channel of mutant and more hydrogen bonds formed might be beneficial for the substrates stretch. In summary, we have developed a promising artificial pathway for efficient 5AVA synthesis.
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http://dx.doi.org/10.3389/fbioe.2021.633028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900509PMC
February 2021

Comparative analysis of the chemical and biochemical synthesis of keto acids.

Biotechnol Adv 2021 Mar-Apr;47:107706. Epub 2021 Feb 4.

National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China; Jiangsu Provisional Research Center for Bioactive Product Processing Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China. Electronic address:

Keto acids are essential organic acids that are widely applied in pharmaceuticals, cosmetics, food, beverages, and feed additives as well as chemical synthesis. Currently, most keto acids on the market are prepared via chemical synthesis. The biochemical synthesis of keto acids has been discovered with the development of metabolic engineering and applied toward the production of specific keto acids from renewable carbohydrates using different metabolic engineering strategies in microbes. In this review, we provide a systematic summary of the types and applications of keto acids, and then summarize and compare the chemical and biochemical synthesis routes used for the production of typical keto acids, including pyruvic acid, oxaloacetic acid, α-oxobutanoic acid, acetoacetic acid, ketoglutaric acid, levulinic acid, 5-aminolevulinic acid, α-ketoisovaleric acid, α-keto-γ-methylthiobutyric acid, α-ketoisocaproic acid, 2-keto-L-gulonic acid, 2-keto-D-gluconic acid, 5-keto-D-gluconic acid, and phenylpyruvic acid. We also describe the current challenges for the industrial-scale production of keto acids and further strategies used to accelerate the green production of keto acids via biochemical routes.
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http://dx.doi.org/10.1016/j.biotechadv.2021.107706DOI Listing
April 2021

[Discovery and functional verification of endogenous glucanases for scleroglucan hydrolysis in Sclerotium rolfsii].

Sheng Wu Gong Cheng Xue Bao 2021 Jan;37(1):207-217

Science Center for Future Foods, Jiangnan University, Wuxi 214122, Jiangsu, China.

Scleroglucan is a high-molecular water-soluble microbial exopolysaccharide and mainly applied in the fields of petroleum, food, medicine and cosmetics. The high molecular weight of scleroglucan produced by microbial fermentation leads to low solubility, high viscosity and poor dispersibility, thus bringing a series of difficulties to extraction, preservation and application. It is important to explore suitable degradation method to adjust the molecular weight of scleroglucan for expanding its industrial application. Taking Sclerotium rolfsii WSH-G01 as a model strain, in which functional annotations of the glucanase genes were conducted by whole genome sequencing. Based on design of culture system for culture system for differential expression of β-glucanase, endogenous β-glucanase genes in S. rolfsii WSH-G01 were excavated by transcriptomics analysis. Functions of these potential hydrolases were further verified. Finally, 14 potential endogenous hydrolase genes were obtained from S. rolfsii. After heterologous overexpression in Pichia pastoris, 10 soluble enzymes were obtained and 5 of them had the activity of laminarin hydrolysis by SDS-PAGE and enzyme activity analysis. Further investigation of the 5 endogenous hydrolases on scleroglucan degradation showed that enzyme GME9860 has positive hydrolysis effect. The obtained results provide references not only for obtaining low and medium molecular weight of scleroglucan with enzymatic hydrolysis, but also for producing different molecular weight of scleroglucan during S. rolfsii fermentation process with metabolic engineering.
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http://dx.doi.org/10.13345/j.cjb.200236DOI Listing
January 2021
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