Part:BBa_K4907145

J23100-B0034-hasA-B0015

Biology

hasA encodes the hyaluronan synthase (HAS) from S. zooepidemicus ATCC 39920. Specifically, HAS is a glycosyltransferase, that utilizes the activated glycosyl donors to produce Hyaluronic acid (HA). HA is polymerized on the cytoplasmic side of the plasma membrane by a membrane-associated processive synthase with four transmembranes and two membrane-associated domains (1). It is reported that hasA is the only gene required for hyaluronic acid synthesis in E. coli. HA is a high molecular weight polymer and the major component of mucoid capsules in bacteria, as well as the extracellular matrix (ECM) in vertebrate tissue (2).

Usage and design

Since the metabolic pathway for cell wall biosynthesis in E. coli is very similar to the pathway of HA synthesis in Streptococcus spp (1), studies have shown the heterologous expression of the hasA gene alone is sufficient to promote the production of HA in E. coli BL21(DE3) (2). In our project, the hasA gene controlled by the constant promoter BBa_J23100 and the RBS BBa_B0034 as well as the terminator BBa_B0015 was assembled into the bacterial expression vector pSB1A2 and transformed into E. coli BL21(DE3). As we want to produce a kind of water-retention material, the in-situ modification of bacterial cellulose (BC) with HA can significantly improve the water-holding capacity (WHC) (3), and the co-culture system of E. coli BL21(DE3) and Nissle 1917 was proposed. For more details please see Design.

Fig. 1 Gene circuit of hasA

Characterization

Agarose gel electrophoresis (AGE)

Colony PCR and gene sequencing were used to verify that the transformants were correct when constructing this circuit. Target bands (1690 bp) can be observed between 2000 bp and 1500 bp (Fig. 2).

Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4907035_pSB1A2

Standard curve of HA

CTAB can combine with HA to form a precipitate, which has an absorption of 400 nm. Prepare a standard solution of HA with an initial concentration of 0~700 mg/L, mix 150 µL of standard solution with 350 µL acetic acid buffer and 1 ml of 2.5 g/L CTAB solution at room temperature for 5 min, and use the modified CTAB method to determine the standard curve of OD₄₀₀ and HA concentration. The result is shown in Fig. 3.

Fig. 3 Standard curve of HA (R²=0.9490)

Production of HA

We wanted to test if our E. coli BL21(DE3) strain carrying the hasA gene would produce more HA. For this, we transformed the plasmid pTet-B0034-ecfp-B0015_pSB1A2 into E. coli BL21(DE3) as a negative control. Both bacteria were grown in 150 mL of LB broth with 5% glucose for 24 hours and the production of HA is measured via the CTAB method. As shown in Fig. 4, there was a significant difference in HA production between the positive and negative samples.

Fig. 4 Comparison of the HA concentration (p=0.0001)

Measurement of the growth curve

In consideration of the consequent regulating and module constructing of our co-culture system, we also measured the OD₆₀₀ for the growth state (Fig. 5a) and OD₄₀₀ for HA concentration (Fig. 5b) of experimental and negative E. coli BL21(DE3) groups every 4 hours in 37 ℃.

Fig. 5 The growth curve

According to the growth curve analysis, the positive bacteria's growth rate is slightly slower than that of the negative control due to metabolic stress caused by HA production. The production curve indicates that the concentration of HA increases as the bacteria continue to grow. After 20 hours of growth, the bacteria's HA production reaches a peak of about 180 mg/L. For more specific details, please refer to Results.

Reference

1. B. F. Chong, L. M. Blank, R. Mclaughlin, L. K. Nielsen, Microbial hyaluronic acid production. Appl. Microbiol. Biotechnol. 66, 341-351 (2005).
2. Z. Lai, C. Teo, Cloning and expression of hyaluronan synthase (hasA) in recombinant Escherichia coli BL21 and its hyaluronic acid production in shake flask culture. J. Microbiol. 15, 575-582 (2019).
3. K. Liu, J. M. Catchmark, Bacterial cellulose/hyaluronic acid nanocomposites production through co-culturing Gluconacetobacter hansenii and Lactococcus lactis under different initial pH values of fermentation media. Cellulose. 27, 2529-2540 (2020).

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 386
  Illegal AgeI site found at 614
  
• 1000
  COMPATIBLE WITH RFC[1000]