Elsevier

Carbohydrate Polymers

Volume 140, 20 April 2016, Pages 424-432
Carbohydrate Polymers

Efficient biosynthesis of polysaccharides chondroitin and heparosan by metabolically engineered Bacillus subtilis

https://doi.org/10.1016/j.carbpol.2015.12.065Get rights and content

Highlights

  • First report of chondroitin and heparosan production in metabolically engineered Bacillus subtilis.

  • The expression cassettes kfoCkfoA and kfiCkfiA regulated by xylose were constructed and integrated onto genome.

  • Up-regulation of tuaD significantly increased the production and molecular weights of chondroitin and heparosan.

  • Highest titers of chondroitin and heparosan (5.22 g L−1 and 5.82 g L−1, respectively) was obtained in fed-batch cultures.

Abstract

Chondroitin and heparosan, important polysaccharides and key precursors of chondroitin sulfate and heparin/heparan sulfate, have drawn much attention due to their wide applications in many aspects. In this study, we designed two independent synthetic pathways of chondroitin and heparosan in food-grade Bacillus subtilis, integrating critical synthases genes derived from Escherichia coli into B. subtilis genome. By RT-PCR analysis, we confirmed that synthases genes transcripted an integral mRNA chain, suggesting co-expression. In shaken flask, chondroitin and heparosan were produced at a level of 1.83 g L−1 and 1.71 g L−1, respectively. Since B. subtilis endogenous tuaD gene encodes the limiting factor of biosynthesis, overexpressing tuaD resulted in enhanced chondroitin and heparosan titers, namely 2.54 g L−1 and 2.65 g L−1. Moreover, production reached the highest peaks of 5.22 g L−1 and 5.82 g L−1 in 3-L fed-batch fermentation, respectively, allowed to double the production that in shaken flask. The weight-average molecular weight of chondroitin and heparosan from B. subtilis E168C/pP43-D and E168H/pP43-D were 114.07 and 67.70 kDa, respectively. This work provided alternative safer synthetic pathways for metabolic engineering of chondroitin and heparosan in B. subtilis and a useful approach for enhancing production, which can be optimized for further improvement.

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.

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    Both authors contributed equally to this work.

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