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      Construction of efficient Streptococcus zooepidemicus strains for hyaluoronic acid production based on identification of key genes involved in sucrose metabolism

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          Abstract

          Biosynthesis of polysaccharide hyaluoronic acid (HA) by Streptococcus zooepidemicus is a carbon-intensive process. The carbon flux and factor(s) restricting HA yield were not well understood. Here, we investigated the function of genes involved in sucrose metabolism and identified targets limiting HA yield, which were exploited to construct efficient S. zooepidemicus strains for HA production. The sucrose uptake was addressed by deletion of scrA and scrB, which encodes sucrose-PTS permease and sucrose-6-phosphate hydrolase, respectively. We found that scrB was essential for the growth of S. zooepidemicus and HA biosynthesis, and accumulation of sucrose-6-phosphate was toxic. Δ scrB could not grow in THY-sucrose medium, while Δ scrA and Δ scrAΔ scrB showed negligible growth defects. Overexpression of scrA significantly reduced biomass and HA production, while overexpression of scrB resulted in 26% increase of biomass and 30% increase of HA yield. We revealed that fructose-6-phosphate for HA biosynthesis mainly originates from glucose-6-phosphate. Deletion of scrK, a gene encoding hexokinase, led to 11% reduction of biomass and 12% decrease of HA yield, while deletion of hasE, a gene encoding phosphoglucoisomerase, resulted in the abolishment of HA biosynthesis and a significantly slow growth. We found that HA biosynthesis could be improved by directing carbon flux to fructose-6-phosphate. Deletion of fruA encoding the EII of fructose-PTS and fruK encoding phosphofructokinase showed no apparent effect on cell growth, but resulted in 22 and 27% increase of HA yield, respectively. Finally, a strain with 55% increase of HA was constructed by overexpression of scrB in Δ fruK. These results provide a solid foundation for further metabolic engineering of S. zooepidemicus for highly efficient HA production.

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          The online version of this article (doi:10.1186/s13568-016-0296-7) contains supplementary material, which is available to authorized users.

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          Hyaluronic acid: a natural biopolymer with a broad range of biomedical and industrial applications.

          Hyaluronic acid (hyaluronan, HA) is a linear polysaccharide formed from disaccharide units containing N-acetyl-D-glucosamine and glucuronic acid. It has a high molecular mass, usually in the order of millions of Daltons, and interesting viscoelastic properties influenced by its polymeric and polyelectrolyte characteristics. HA is present in almost all biological fluids and tissues. In clinical medicine, it is used as a diagnostic marker for many diseases including cancer, rheumatoid arthritis and liver pathologies, as well as for supplementation of impaired synovial fluid in arthritic patients by means of intra-articular injections. It is also used in certain ophthalmological and otological surgeries and cosmetic regeneration and reconstruction of soft tissue. Herein we present an overview of the occurrence and physiological properties of HA, as well as of the recent advances in production biotechnology and preparation of the HA-based materials for medical application.
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            Microbial production of hyaluronic acid: current state, challenges, and perspectives

            Hyaluronic acid (HA) is a natural and linear polymer composed of repeating disaccharide units of β-1, 3-N-acetyl glucosamine and β-1, 4-glucuronic acid with a molecular weight up to 6 million Daltons. With excellent viscoelasticity, high moisture retention capacity, and high biocompatibility, HA finds a wide-range of applications in medicine, cosmetics, and nutraceuticals. Traditionally HA was extracted from rooster combs, and now it is mainly produced via streptococcal fermentation. Recently the production of HA via recombinant systems has received increasing interest due to the avoidance of potential toxins. This work summarizes the research history and current commercial market of HA, and then deeply analyzes the current state of microbial production of HA by Streptococcus zooepidemicus and recombinant systems, and finally discusses the challenges facing microbial HA production and proposes several research outlines to meet the challenges.
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              Hyaluronic acid capsule is a virulence factor for mucoid group A streptococci.

              Mucoid strains of group A Streptococcus have been associated with recent outbreaks of acute rheumatic fever. The mucoid colony morphology of these strains is a result of abundant production of capsular polysaccharide, which is composed of hyaluronic acid. To study the role of the hyaluronic acid capsule in virulence, we derived an acapsular mutant from a mucoid strain of group A Streptococcus by transposon mutagenesis. M protein expression was not altered in the mutant strain. The mucoid wild-type strain grew in fresh human blood and was resistant to phagocytic killing in vitro. In contrast, the acapsular mutant failed to grow in fresh human blood and was sensitive to phagocytic killing in vitro. Loss of capsule was associated with a 100-fold reduction in virulence of the organisms in mice. We conclude that the hyaluronic acid capsule protects mucoid group A streptococci from phagocytosis and has an important role in virulence.
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                Author and article information

                Contributors
                15022010757@139.com
                13502069697@163.com
                1163976479@qq.com
                fulixia321@163.com
                caowei040108@tust.edu.cn
                liuhao@tust.edu.cn
                Journal
                AMB Express
                AMB Express
                AMB Express
                Springer Berlin Heidelberg (Berlin/Heidelberg )
                2191-0855
                28 November 2016
                28 November 2016
                2016
                : 6
                : 121
                Affiliations
                MOE Key Lab of Industrial Fermentation Microbiology, College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457 China
                Article
                296
                10.1186/s13568-016-0296-7
                5125315
                27896786
                bbcbdfdf-b25c-4069-bfb1-7a5ff8c96afa
                © The Author(s) 2016

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

                History
                : 20 November 2016
                : 22 November 2016
                Funding
                Funded by: Tianjin Science and Technology Commission
                Award ID: 13RCGFSY19400
                Award Recipient :
                Funded by: Tianjin Municipal High School Science and Technology Development Fund
                Award ID: 20130602
                Award Recipient :
                Categories
                Original Article
                Custom metadata
                © The Author(s) 2016

                Biotechnology
                hyaluoronic acid,metabolic engineering,streptococcus zooepidemicus
                Biotechnology
                hyaluoronic acid, metabolic engineering, streptococcus zooepidemicus

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