Yoshimura Tomohiro, Shibata Nobuyuki, Hamano Yoshimitsu, Yamanaka Kazuya
Yokohama Research Center, JNC Corporation, Yokohama, Kanagawa, Japan.
Department of Bioscience, Fukui Prefectural University, Eiheiji-cho, Yoshida-gun, Fukui, Japan.
Appl Environ Microbiol. 2015 Jun;81(11):3631-40. doi: 10.1128/AEM.00269-15. Epub 2015 Mar 20.
Hyaluronic acid (HA) is used in a wide range of medical applications, where its performance and therapeutic efficacy are highly dependent on its molecular weight. In the microbial production of HA, it has been suggested that a high level of intracellular ATP enhances the productivity and molecular weight of HA. Here, we report on heterologous HA production in an ε-poly-l-lysine producer, Streptomyces albulus, which has the potential to generate ATP at high level. The hasA gene from Streptococcus zooepidemicus, which encodes HA synthase, was refactored and expressed under the control of a late-log growth phase-operating promoter. The expression of the refactored hasA gene, along with genes coding for UDP-glucose dehydrogenase, UDP-N-acetylglucosamine pyrophosphorylase, and UDP-glucose pyrophosphorylase, which are involved in HA precursor sugar biosynthesis, resulted in efficient production of HA in the 2.0 MDa range, which is greater than typical bacterial HA, demonstrating that a sufficient amount of ATP was provided to support the biosynthesis of the precursor sugars, which in turn promoted HA production. In addition, unlike in the case of streptococcal HA, S. albulus-derived HA was not cell associated. Based on these findings, our heterologous production system appears to have several advantages for practical HA production. We propose that the present system could be applicable to the heterologous production of a wide variety of molecules other than HA in the case their biosynthesis pathways require ATP in vivo.
透明质酸(HA)被广泛应用于众多医学领域,其性能和治疗效果高度依赖于分子量。在微生物生产HA的过程中,有人提出细胞内高水平的ATP可提高HA的产量和分子量。在此,我们报道了在一种能高水平产生ATP的ε-聚-L-赖氨酸产生菌——白色链霉菌中进行异源HA生产的情况。对来自兽疫链球菌的编码HA合酶的hasA基因进行了改造,并在对数生长后期起作用的启动子控制下进行表达。改造后的hasA基因与参与HA前体糖生物合成的UDP-葡萄糖脱氢酶、UDP-N-乙酰葡糖胺焦磷酸化酶和UDP-葡萄糖焦磷酸化酶的编码基因一起表达,使得能够高效生产分子量在2.0 MDa范围内的HA, 这一分子量大于典型的细菌HA,表明提供了足够量的ATP来支持前体糖的生物合成,进而促进了HA的生产。此外,与链球菌HA不同,白色链霉菌产生的HA不与细胞结合。基于这些发现,我们的异源生产系统在实际生产HA方面似乎具有几个优势。我们提出,在各种分子的生物合成途径在体内需要ATP的情况下,本系统可能适用于除HA之外的多种分子的异源生产。