Jeong Euijoon, Shim Woo Yong, Kim Jung Hoe
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 291 Daehak-ro (373-1 Guseong-dong), Yuseong-gu, Daejeon 305-701, Republic of Korea.
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 291 Daehak-ro (373-1 Guseong-dong), Yuseong-gu, Daejeon 305-701, Republic of Korea.
J Biotechnol. 2014 Sep 20;185:28-36. doi: 10.1016/j.jbiotec.2014.05.018. Epub 2014 Jun 2.
The high molecular weight (>1 MDa) of hyaluronic acid (HA) is important for its biological functions. The reported limiting factors for the production of HA with high molecular weight (MW) by microbial fermentation are the insufficient HA precursor pool and cell growth inhibition. To overcome these issues, the Xenopus laevis xhasA2 and xhasB genes encoding hyaluronan synthase 2 (xhasA2) and UDP-glucose dehydrogenase (xhasB), were expressed in Pichia pastoris widely used for production of heterologous proteins. In this study, expression vectors containing various combination cassettes of HA pathway genes including xhasA2 and xhasB from X. laevis as well as UDP-glucose pyrophosphorylase (hasC), UDP-N-acetylglucosamine pyrophosphorylase (hasD) and phosphoglucose isomerase (hasE) from P. pastoris were constructed and tested. First, HA pathway genes were overexpressed using pAO815 and pGAPZB vectors, resulting in the production of 1.2 MDa HA polymers. Second, in order to decrease hyaluronan synthase expression a strong AOX1 promoter in the xhasA2 gene was replaced by a weak AOX2 promoter which increased the mean MW of HA to 2.1 MDa. Finally, the MW of HA polymer was further increased to 2.5 MDa by low-temperature cultivation (26 °C) which reduced cell growth inhibition. The yield of HA production by the P. pastoris recombinant strains in 1L of fermentation culture was 0.8-1.7 g/L.
透明质酸（HA）的高分子量（>1 MDa）对其生物学功能至关重要。据报道，微生物发酵生产高分子量（MW）HA的限制因素是HA前体库不足和细胞生长抑制。为克服这些问题，编码透明质酸合酶2（xhasA2）和UDP-葡萄糖脱氢酶（xhasB）的非洲爪蟾xhasA2和xhasB基因在广泛用于生产异源蛋白的毕赤酵母中表达。在本研究中，构建并测试了包含HA途径基因各种组合盒的表达载体，这些基因包括来自非洲爪蟾的xhasA2和xhasB，以及来自毕赤酵母的UDP-葡萄糖焦磷酸化酶（hasC）、UDP-N-乙酰葡糖胺焦磷酸化酶（hasD）和磷酸葡萄糖异构酶（hasE）。首先，使用pAO815和pGAPZB载体过表达HA途径基因，产生了1.2 MDa的HA聚合物。其次，为了降低透明质酸合酶的表达，xhasA2基因中的强AOX1启动子被弱AOX2启动子取代，这将HA的平均分子量提高到了2.1 MDa。最后，通过低温培养（26°C）进一步提高了HA聚合物的分子量至2.5 MDa，这减少了细胞生长抑制。毕赤酵母重组菌株在1L发酵培养物中的HA产量为0.8 - 1.7 g/L。
