La violencia contra las mujeres en El Salvador y la regulación de

Hyaluronic acid (hyaluronan, HA) is a polymer of disaccharide, composed of glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) linked together via alternating β-1,4 and β-1,3 glyco- sidic bonds and it can reach chain lengths of up to 20,000 disaccharide units or higher (8 MDa).

HA is naturally found in various tissues of the body, such as skin, cartilage, and vitreous humor and is one of the chief components of the extracellular matrix. It is known that HA contributes to cell proliferation and migration and may also be involved in the progression of some malignant

tumors. It is therefore well suited to biomedical applications targeting these tissues. The fi rst bio- medical product containing HA was approved for use in eye surgery and it is also used to treat osteoarthritis of the knee (Puhl and Scarf, 1997). Oral use of HA has been suggested, with its use as a dietary supplement (Kajimoto et al., 2001; Satoh et al., 2002). HA is a common ingredient in skin care products.

HA can be obtained by extraction from rooster combs or through fermentation with strains of group C Streptococcus. In rooster combs, HA forms a complex with proteoglycans, which makes the isolation of HA with high purity and high molecular weight costly (O’Regan et al., 1994). In addition, animal-derived products carry the risk of transmitting viruses and other adventitious agents as well as inducing immunogenic and infl ammatory responses. The fermentation method using Streptococcus strain is far from ideal despite its long history because of the diffi culties in genetic engineering.

HA is produced from uridine 5′-diphosphate–N-acetylglucosamine (UDP–GlcNAc) and UDP– GlcUA by hyaluronan synthase. UDP–GlcNAc is a precursor of cell wall biosynthesis and UDP– GlcUA is synthesized from UDP-glucose by NADH-dependent UDP-glucose dehydrogenase.

Recently, metabolically engineered B. subtilis strains have been constructed for HA fermentation (Widner et al., 2005). In these strains, the hasA gene from Streptococcus equisimilis, which encodes hyaluronan synthase, has been expressed under the mutated version of the amyQ promoter from

Bacillus amynoliquefaciens. Artifi cial operons, all of which contain the hasA gene along with one

or more genes encoding enzymes involved in the biosynthesis of UDP–GlcNAc and UDP–GlcUA, were assembled and introduced on the chromosome of B. subtilis (Figure 9.4). It was determined that the production of UDP–GlcUA is limiting in B. subtilis and that overexpressing the hasA gene along with the endogenous tuaD gene encoding UDP-glucose dehydrogenase is suffi cient for high- level production of HA. When the Bacillus strains were cultivated on a minimal medium with sucrose as the carbon source, multigrams of HA were produced in the medium and the molecular weight was in the 1.1–1.2 MDa range.

Although there are no major differences in productivity and molecular weight as compared to the Streptococcus fermentation, Bacillus would be a better production strain because: (1) B. subtilis is generally recognized as safe (GRAS), (2) tools for genetic engineering are well developed in

Bacillus, and (3) HA produced is not cell associated, facilitating the downstream processing.

9.2.5 N-ACETYLGLUCOSAMINE

Glucosamine and GlcNAc are monosaccharide and are known to be precursors in humans of the disaccharide unit in glycosaminoglycans such as HA, chondroitin sulfate, and keratan sulfate, which are necessary to repair and maintain healthy cartilage and joint function. It is estimated that 33 million people suffer from osteoarthritis in the United States, and glucosamine has been

E. coli Glucose Succinate Hydroxylase Hydroxy proline Hydroxyproline l-Pro putA l-Glu l-Pro 2OG TCA cycle

widely used as a dietary supplement for joint health (Hungerford and Jones, 2003, Towheed, 2003). Currently, glucosamine is produced by the acid hydrolysis of chitin, a linear polymer of GlcNAc, which is extracted from crab and shrimp shells. Concentrated hydrochloric acid breaks down the polymer and deacetylates GlcNAc to form glucosamine. Glucosamine production could become limited by variable raw material supply as its demand continues to increase. Moreover, it is pointed out that glucosamine from shellfi sh may not be suitable for people with shellfi sh allergies. Some fi lamentous fungi contain chitosan (a linear copolymer of glucosamine and GlcNAc) in their cell walls, and a method for glucosamine production from the fungal biomass has also been developed.

GlcNAc supplements for joint health are also available, although the market is much smaller than that for glucosamine. GlcNAc is now produced by chemical acetylation of glucosamine using acetic anhydride; however, GlcNAc is synthesized in bacteria, and therefore a novel fermentation method for GlcNAc has been expected.

A metabolically engineered E. coli for the production of GlcNAc was constructed through (1) the overexpression of the biosynthetic genes and (2) the inactivation of the degradation of intermediates for GlcNAc synthesis and GlcNAc transporters (Deng et al., 2005, 2006a,b) (Figure 9.5).

Glucosamine synthase (GlmS), which catalyzes the synthesis of glucosamine-6-phosphate (GlcN-6-P) from fructose-6-P and glutamine, is known to be inhibited by GlcN-6-P. Therefore a mutant enzyme whose sensitivity to GlcN-6-P was signifi cantly reduced was generated. The gene for GlcN-6-P acetyltransferase (GNA1), which converts GlcN-6-P into GlcNAc-6-P, was cloned from Saccharomyces cerevisiae and overexpressed in E. coli. GlcNAc-6-P is then dephosphory- lated and secreted into the medium.

In the producer strain of GlcNAc, transporter proteins of GlcNAc as well as GlcNAc-6-P deacetylase and GlcN-6-P deaminase were deleted. When the metabolically engineered E. coli

Glucose Glc-6-P Fru-6-P GlcN-6-P GlcN-1-P GlcNAc-1-P UDP-GlcNAc Glc-1-P UDP-Glc UDP-GlcUA Hyaluronic acid Hyaluronic acid B. subtilis tuaD hasA

was cultivated on a minimal medium with glucose as the carbon source, GlcNAc accumulated at concentrations greater than 110 g/L for 72 h.