Part:BBa_K2593007

xylR-PxylA-RBS-HasA

This composite part consists of a promoter, an RBS, a coding sequence of HA synthase gene, and a terminator.

xylR-PxylA (BBa_K733002): A xylose inducible promoter with its transcriptional regulator.
This part consists of an xylose inducible promoter, originated from Bacillis megaterium, which is amplified from the integration vector pAX01 and an xylose repressor gene. Promoter PxylA is located within xylose operon, originally to drive the expression of xylA (xylose isomerase coding gene) and xylB (xylulose kinase). xylR with its promoter located at upstream of xylose operon. It encodes xyl repressor which binds to xyl operator in the absence of xylose, repressing transcription activation. In the presence of glucose, glucose-6-phosphate metabolized from glucose can compete with xylose in the binding site of xylose on XylR. In addition, glucose itself is also supposed to be a low efficiency inducer for XylR (DAHL, 1997). Therefore while xylose induces transcription, the existence of glucose, to some extent, represses gene transcription.

RBS(BBa_K2593005): it’s a strong Ribosome Binding Site which is commonly used in bacteria.

HasA gene (BBa_K2593001): HA synthase pathway In Streptococcus species, HA biosynthsis begins with the phosphorylation of glucose by hexokinase to produce the main precursor, glucose-6-phosphate (G6P).From here, HA synthesis pathway can be divided into two distinct pathways that syntheses the two building blocks of HA, glucuronic acid and N-acetylglucosamine (Fig. 2). In the first set of reactions, a-phosphoglucomutase (pgcA) converts glucose-6-phosphate to glucose-1-phosphate before a phosphate group from UTP is transferred to glucose-1-phosphate by UDP-glucose pyrophosphorylase (hasC/gtaB) to produce UDP-glucose. UDP-glucose is oxidised by UDP-glucose dehydrogenase (hasB/tuaB) to yield the first HA precursor, UDP-glucuronic acid (UDP- GlcUA).
In the second set of reactions, G6P is converted into fructose 6-phosphate (F6P) by glucose phosphate isomerase which is encoded by pgi gene. Fructose 6-phosphate (F6P) is then catalyzed stepwide to glucosamine 6-phosphate, glucosamine-1-Pand N-Acetyl Glucosamine-1-P by Glucosamine--fructose-6-phosphateaminotransferase (glmS), Phosphoglucosaminemutase (glmM) and N-acetylglucosamine-1-phosphateuridyltransferase (glmU) respectively, and eventually the second precursor, UDP-GlcNAc, was synthesized. Once the two precursors are synthesised, hyaluronan synthase (hasA) polymerises the two components in an alternate manner to produce the HA polymer.
B. Subtilis, containing all native pathway genes for the biosynthesis for the HA precursors UDP-GlcUA and UDP-GlcNAc, has been regarded as an ideal cell factory for synthetic biology manipulations in HA biosynthesis studies.

Usage:

This device is used for produce high molecular weight HA in B.subtilis. The construct was under the control of promoter PxylA, which is part of xylose inducible expression system. In our experiment, the production of HA was proved by using CTAB method, The results showed that HA production increased at a steady rate as time passed, reaching over 300mg/L (CTAB method) at the 50 h; while bacterial cells maintained normal growth pattern and experienced lag phase dropping in the final hours. These results confirmed the success of HA production by recombinant B.subtilis 168E strain(Fig1-2 ).

In addition, Ubblelohde viscometer method indicated that HA products secreted from these recombinant expression systems were with comparable molecular weights, suggesting that they are all high-molecular-weight HA products (~4.233*106Da)(Fig3)

Figure 1: analysis of HA production level of recombinant hasA and cell growth. A typical time course of recombinant hasA expression in B. subitlis 168E during xylose induction phase. HA production (red, mg/L), and cell growth density (black, OD600 value) were measured at regular intervals.

Figure 2: CTAB analysis of HA concentraton, a. Illustration of the turbidity by mixing different source of HA with CTAB solution. b: effects of overexpressing the precursor genes on HA production in recombinant B. subtilis. 168 strains.

Figure3: molecular weights of HA produced by overexpression of precursor genes in recombinant B. subtilis 168E strains using viscometer analysis.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 847
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1665
  

Reference

Zeigler, D. (2002). Integration Vectors for Gram-Positive Bacteria (7 ed.). Columbus: The Bacillus Genetic Stock Center.
Dahl, M. K., D. Schmiedel, and W. Hillen. 1995. Glucose and glucose-6-phosphate interaction with Xyl repressor proteins from Bacillus spp. May contribute to regulation of xylose utilization. J. Bacteriol. 177:5467–5472.
Peng Jin, Guocheng Du, Zhen Kang. High-yield novel leech hyaluronidase to expedite the preparation of specific hyaluronan oligomers[J].Scientific Reports, 2014 : 1-2
Jinpeng, Kangzhen, Biosynthesis of hyaluronan oligosaccharides and construction of DNA editing and assembly tools[D]Jiangnan University: Jinpeng,2016.9-10.