Hyaluronan synthase genes and expression thereof in Bacillus hosts

Abstract

The present invention relates to a recombinant Bacillus host cell containing a recombinant vector including a nucleic acid segment having a coding region segment encoding enzymatically active hyaluronan synthase (HAS). The recombinant Bacillus host cell is utilized in a method for producing hyaluronic acid (HA).

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. Ser. No. 11 / 474,663, filed Jun. 26, 2006; which is a continuation of U.S. Ser. No. 10 / 981,632, filed Nov. 5, 2004, now U.S. Pat. No. 7,091,008, issued Aug. 15, 2006; which is a divisional of U.S. Ser. No. 10 / 172,527, filed Jun. 13, 2002, now U.S. Pat. No. 6,951,743, issued Oct. 4, 2005; which is a continuation-in-part of U.S. Ser. No. 09 / 469,200, filed Dec. 21, 1999, now U.S. Pat. No. 6,833,264, issued Dec. 21, 2004; which is a continuation of U.S. Ser. No. 09 / 178,851, filed Oct. 26, 1998, now abandoned; and which also claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Application U.S. Ser. No. 60 / 064,435, filed Oct. 31, 1997. Said U.S. Ser. No. 10 / 172,527 is also a continuation-in-part of U.S. Ser. No. 09 / 146,893, filed Sep. 3, 1998, now U.S. Pat. No. 6,455,304, issued Sep. 24, 2002; which is a continuation of U.S. Ser. No. 08 / 270,581, filed Jul. 1, 1994, now abandoned. The contents...

AI Technical Summary

Benefits of technology

This approach allows for the production of high molecular weight HA with improved purity and ease of preparation, addressing the limitations of existing methods by enabling large-scale production of HA with controlled size distribution and structure, suitable for various applications including cosmetics and medical uses.

Problems solved by technology

The incidence of streptococcal infections is a major health and economic problem worldwide, particularly in developing countries.

Although the HA biosynthetic system was one of the first membrane heteropolysaccharide synthetic pathways studied, the mechanism of HA synthesis is still not well understood.

This may be because in vitro systems developed to date are inadequate in that de novo biosynthesis of HA has not been accomplished.

The extrusion of the growing chain into the extracellular space also allows for unconstrained polymer growth, thereby achieving the exceptionally large size of HA, whereas confinement of synthesis within a Golgi or post-Golgi compartment limits the overall amount or length of the polymers formed.

High concentrations of HA within a confined lumen may also create a high viscosity environment that might be deleterious for other organelle functions.

Although the streptococcal and murine oligodendroglioma enzymes were successfully detergent-solubilized and studied, efforts to purify an active HAS for further study or molecular cloning remained unsuccessful for decades.

This led to a report claiming that the Group C streptococcal HAS had been cloned, which was unfortunately erroneous.

Despite these efforts, progress in understanding the regulation and mechanisms of HA synthesis was essentially stalled, since there were no molecular probes for HAS mRNA or HAS protein.

Unfortunately, several studies have employed antibodies to this uncharacterized 52-kDa streptococcal protein to investigate what was believed to be eukaryotic HAS.

It is generally felt that isolation of HA from rooster combs is laborious and difficult, since one starts with HA in a less pure state.

Unfortunately, very high molecular weight HA, such as that ranging up to 107, has been difficult to obtain by currently available isolation procedures.

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