Efficient biosynthesis of polysaccharides chondroitin and heparosan by metabolically engineered Bacillus subtilis
Introduction
GAGs (namely Glycosaminoglycans) which locate at the mammalian extracellular matrix and bacteria capsular have attracted intensive research because of the wide biological and physiological functions (DeAngelis, 2002, Linhardt, 2003, Suflita et al., 2015, Yother, 2011). Among them, heparin (HP) and chondroitin sulfate (CS) have been deeply investigated and widely applied in clinic treatments (Ruffell et al., 2011, Wang et al., 2007). For instance, HP was mainly used as anticoagulant (Damus, Hicks, & Rosenberg, 1973) while CS was mainly used as anti-inflammatory drug for the treatment of osteoarthritis and Rheumatism (McAlindon, LaValley, Gulin, & Felson, 2000). In recent years, due to aging of the world population, the market demand of HP and CS has been dramatically increased.
Currently, HP and CS are extracted from animal tissues. However, the disadvantages such as potential risk of interspecies disease and over sulfation raised the concern of the animal sourced HP and CS (Guerrini et al., 2008, Laurencin and Nair, 2008). In view of these problems, development of safe and reliable alternatives to produce HP and CS is always a huge challenge (Laremore, Zhang, Dordick, Liu, & Linhardt, 2009). Accordingly, some de novo chemical synthesis routes for HP and CS with different length and sulfation have been developed (de Paz et al., 2006, Xu et al., 2011). However, it will be unpractical to produce HP and CS in large-scale with these chemical methods because of the complex, time-consuming processes and the rare expensive substrates (Boltje, Buskas, & Boons, 2009). As an alternative approach, chemical synthesis of HP from the precursor heparosan with higher yield has also been reported (Laremore et al., 2009, Zhang et al., 2008). Consequently, semi-chemical synthesis and chemoenzymatic modification of the bioactive HP (which is composed of β-d-glucuronic acid (GlcUA) and N-acetyl-α-d-glucosamine (GlcNAc) repeating disaccharides) or CS (which consists a repeating disaccharide unit of GlcUA and N-acetyl-d-galactosamine, GalNAc) from their precursors heparosan (Fig. 1a) and chondroitin (Fig. 1b) (Bhaskar et al., 2015, Li et al., 2014, Mikami and Kitagawa, 2013, Restaino et al., 2013) will be more attractive. Consequently, achievement of high yield production of the precursors heparosan and chondroitin is the key determinant.
In the past years, it has been found and demonstrated Escherichia coli K5 and K4 produce heparosan and chondroitin respectively (DeAngelis, 2012, DeAngelis et al., 2002, Ninomiya et al., 2002, Zanfardino et al., 2010). Accordingly, many studies on optimization of cultivation process (Cimini et al., 2010, Wang et al., 2010, Wang et al., 2011) and engineering of the pathways (Cimini, De Rosa, Carlino, Ruggiero, & Schiraldi, 2013) have been carried out in these native strains. Nevertheless, the strains E. coli K5 and K4 are pathogenic bacteria and can cause urinary tract infection (Wiles, Kulesus, & Mulvey, 2008). In consideration of this disadvantage, the biosynthetic pathway for synthesis of chondroitin and heparosan have been individually constructed in E. coli BL21 (DE3) (He et al., 2015, Zhang et al., 2012) by introducing the corresponding synthases from E. coli K5 and K4 (Cress et al., 2013a, Cress et al., 2013b). Even though, due to the concern on food safety and the problem of phage contamination (Tanji, Hattori, Suzuki, & Miyanaga, 2008), the engineered E. coli strains will probably be confined in food industry. Consequently, construction of alternative robust engineered strains for producing GAGs at industrial scale should be more promising.
Bacillus subtilis, the best-characterized gram-positive bacterium, is regarded as GRAS (generally recognized as safe) strain (Kang et al., 2014, van Dijl and Hecker, 2013, Westers et al., 2004) and has been widely used for the production enzymes and chemicals that used in food industries (Shi et al., 2009, Song et al., 2015, Wang et al., 2012, Yang et al., 2015). Compared with E. coli, B. subtilis has no significant codon bias and shows stronger tolerance to different environments. In addition, according to the genetic information (Kunst et al., 1997) and previous study on hyaluronan (Widner et al., 2005), B. subtilis hosts seem unlikely to encode enzymes degrading heparosan and chondroitin which will benefit the accumulation of heparosan and chondroitin.
In the present study, we firstly constructed and investigated the heparosan and chondroitin biosynthetic pathways in B. subtilis. By further optimization of the synthetic pathway, the production of heparosan and chondroitin were enhanced to 5.82 g L−1 and 5.22 g L−1, respectively. The present work paved the way for large-scale production of heparosan and chondroitin and its derivatives in the GRAS B. subtilis strain.
Section snippets
Strains and plasmids construction
The bacterial strains, plasmids, and primers used in this study were listed in Table 1, Table 2, respectively. Molecular cloning and manipulation of plasmids were done with B. subtilis 168. The polymerase chain reaction (PCR) was performed in 50-μL volumes using 1 μL DNA template, 10 pmol of each primer, 25 μL 2× Super Pfu PCR Master Mix (Hangzhou Biosci Co., Ltd, China) under the following conditions: 94 °C for 3 min; 32 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1.5 min; 72 °C for 5 min. The
Construction and integration of expression cassettes in B. subtilis 168
To construct the polysaccharides heparosan and chondroitin biosynthetic pathway (Fig. 2a) in B. subtilis, firstly, the heparosan synthase encoding genes kfiA and kfiC from E. coli K5 were cloned and inserted into the integration vector pAX01 to yield plasmid pAX01-kfiC–kfiA; the chondroitin pathway genes kfoA and kfoC were amplified from E. coli K4 and subcloned into the integration vector pAX01 to yield plasmid pAX01-kfoC–kfoA (Fig. 2b). Then the plasmids were transformed into B. subtilis and
Conclusion
In the present study, a recombinant B. subtilis platform was developed to produce chondroitin and heparosan from inexpensive sucrose. Over the course this work, the production of chondroitin and heparosan were increased to 5.22 g L−1 and 5.82 g L−1 respectively by a metabolic engineering and optimization strategy. Compared with the recombinant E. coli strains and the native producing strains, B. subtilis represents an ideal alternative for efficiently producing chondroitin and heparosan because of
Conflict of interest
The authors declare that there is no conflict of interest.
Acknowledgements
We appreciate Professor Shunpeng Li (Nanjing Agricultural University, China) for supplying the plasmid pP43NMK. This work was financially supported by a grant from the Key Technologies R&D Program of Jiangsu Province, China (BE2014607); Program for Changjiang Scholars and Innovative Research Team in University (no. IRT_15R26); the Natural Science Foundation of Jiangsu Province (BK20141107); China Postdoctoral Science Foundation funded project (125960) and 111 Project.
References (60)
- et al.
Ratio of intracellular precursors concentration and their flux influences hyaluronic acid molecular weight in Streptococcus zooepidemicus and recombinant Lactococcus lactis
Bioresource Technology
(2014) - et al.
Combinatorial one-pot chemoenzymatic synthesis of heparin
Carbohydrate Polymers
(2015) - et al.
A modified uronic acid carbazole reaction
Analytical Biochemistry
(1962) - et al.
Hyaluronan molecular weight is controlled by UDP-N-acetylglucosamine concentration in Streptococcus zooepidemicus
Journal of Biological Chemistry
(2009) - et al.
Identification of the capsular polysaccharides of Type D and F Pasteurella multocida as unmodified heparin and chondroitin, respectively
Carbohydrate Research
(2002) - et al.
Production of chondroitin in metabolically engineered E. coli
Metabolic Engineering
(2015) - et al.
Identification that KfiA, a protein essential for the biosynthesis of the Escherichia coli K5 capsular polysaccharide, is an alpha-UDP-GlcNAc glycosyltransferase. The formation of a membrane-associated K5 biosynthetic complex requires KfiA, KfiB, and KfiC
Journal of Biological Chemistry
(2000) - et al.
Influence of competing metabolic processes on the molecular weight of hyaluronic acid synthesized by Streptococcus zooepidemicus
Biochemical Engineering Journal
(2010) - et al.
Recent progress and applications in glycosaminoglycan and heparin research
Current Opinion in Chemical Biology
(2009) - et al.
Biosynthesis and function of chondroitin sulfate
Biochimica et Biophysica Acta-general Subjects
(2013)
Molecular cloning and characterization of chondroitin polymerase from Escherichia coli strain K4
Journal of Biological Chemistry
Differential use of chondroitin sulfate to regulate hyaluronan binding by receptor CD44 in inflammatory and interleukin 4-activated macrophages
Journal of Biological Chemistry
A 3D-structural model of unsulfated chondroitin from high-field NMR: 4-sulfation has little effect on backbone conformation
Carbohydrate Research
Transcriptome analysis guided metabolic engineering of Bacillus subtilis for riboflavin production
Metabolic Engineering
Deleting multiple lytic genes enhances biomass yield and production of recombinant proteins by Bacillus subtilis
Microbial Cell Factories
Bacillus subtilis as cell factory for pharmaceutical proteins: A biotechnological approach to optimize the host organism
Biochimica et Biophysica Acta Molecular Cell Research
Origins and virulence mechanisms of uropathogenic Escherichia coli
Experimental and Molecular Pathology
Metabolic engineering of Escherichia coli for biosynthesis of hyaluronic acid
Metabolic Engineering
Enzymatic production of specifically distributed hyaluronan oligosaccharides
Carbohydrate Polymers
Metabolic engineering of Escherichia coli BL21 for biosynthesis of heparosan, a bioengineered heparin precursor
Metabolic Engineering
Opportunities and challenges in synthetic oligosaccharide and glycoconjugate research
Nature Chemistry
Homologous overexpression of rfaH in E. coli K4 improves the production of chondroitin-like capsular polysaccharide
Microbial Cell Factories
Production of capsular polysaccharide from Escherichia coli K4 for biotechnological applications
Applied Microbiology and Biotechnology
Draft genome sequence of Escherichia coli strain ATCC 23502 (Serovar O5: K4: H4)
Genome Announcements
Draft genome sequence of Escherichia coli strain ATCC 23506 (Serovar O10: K5: H4)
Genome Announcements
Anticoagulant action of heparin
Nature
Microarrays of synthetic heparin oligosaccharides
Journal of the American Chemical Society
Evolution of glycosaminoglycans and their glycosyltransferases: Implications for the extracellular matrices of animals and the capsules of pathogenic bacteria
Anatomical Record
Glycosaminoglycan polysaccharide biosynthesis and production: Today and tomorrow
Applied Microbiology and Biotechnology
Enzymatic assembly of DNA molecules up to several hundred kilobases
Nature Methods
Cited by (83)
Advancements in heparosan production through metabolic engineering and improved fermentation
2024, Carbohydrate PolymersAdvances and challenges in biotechnological production of chondroitin sulfate and its oligosaccharides
2023, International Journal of Biological MacromoleculesProduction of different molecular weight glycosaminoglycans with microbial cell factories
2023, Enzyme and Microbial TechnologyBiotechnological advances in the synthesis of modified chondroitin towards novel biomedical applications
2023, Biotechnology AdvancesMicrobial synthesis of glycosaminoglycans and their oligosaccharides
2023, Trends in Microbiology
- 1
Both authors contributed equally to this work.