Key Laboratory for Industrial Biocatalysis of the Ministry of Education, Department of Chemical Engineering, Tsinghua University, Beijing 100084, P. R. China.
Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, P. R. China.
Biotechnol J. 2017 Oct;12(10). doi: 10.1002/biot.201700191. Epub 2017 Sep 28.
Hyaluronic acid (HA) is a polysaccharide used in many industries such as medicine, surgery, cosmetics, and food. To avoid potential pathogenicity caused by its native producer, Streptococcus, efforts have been made to create a recombinant host for HA production. In this work, a GRAS (generally recognized as safe) strain, Corynebacterium glutamicum, is engineered for enhanced biosynthesis of HA via metabolic pathway regulation. Five enzymes (HasA-HasE) involved in the HA biosynthetic pathway are highlighted, and eight diverse operon combinations, including HasA, HasAB, HasAC, HasAD, HasAE, HasABC, HasABD, and HasABE, are compared. HasAB and HasABC are found to be optimal for HA biosynthesis in C. glutamicum. To meet the energy demand for HA synthesis, the metabolic pathway that produces lactate is blocked by knocking out the lactate dehydrogenase (LDH) gene using single crossover homologous recombination. Engineered C. glutamicum/Δldh-AB is superior and had a significantly higher HA titer than C. glutamicum/Δldh-ABC. Batch and fed-batch cultures of C. glutamicum/Δldh-AB are performed in a 5-L fermenter. Using glucose feeding, the maximum HA titer reached 21.6 g L , more than threefolds of that of the wild-type Streptococcus. This work provides an efficient, safe, and novel recombinant HA producer, C. glutamicum/Δldh-AB, via metabolic pathway regulation.
透明质酸（HA）是一种多糖，广泛应用于医学、手术、化妆品和食品等多个领域。为避免其天然产生菌链球菌引起的潜在致病性，人们努力开发用于 HA 生产的重组宿主。本工作通过代谢途径调控，对 GRAS（一般认为安全）菌株谷氨酸棒杆菌进行工程化改造，以增强其 HA 的生物合成。强调了参与 HA 生物合成途径的 5 种酶（HasA-HasE），并比较了 8 种不同的操纵子组合，包括 HasA、HasAB、HasAC、HasAD、HasAE、HasABC、HasABD 和 HasABE。发现 HasAB 和 HasABC 最有利于谷氨酸棒杆菌中 HA 的生物合成。为满足 HA 合成的能量需求，通过单交换同源重组敲除乳酸脱氢酶（LDH）基因阻断产生乳酸的代谢途径。工程化的谷氨酸棒杆菌/Δldh-AB 优于谷氨酸棒杆菌/Δldh-ABC，其 HA 产量显著更高。在 5-L 发酵罐中进行了谷氨酸棒杆菌/Δldh-AB 的分批和补料分批培养。通过葡萄糖补料，HA 的最大产量达到 21.6 g/L，是野生型链球菌的三倍多。本工作通过代谢途径调控，提供了一种高效、安全且新颖的重组 HA 生产菌株谷氨酸棒杆菌/Δldh-AB。
