A recombinant E. coli bioprocess for hyaluronan synthesis
A recombinant E. coli bioprocess for hyaluronan synthesis
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DOI：
10.1007/s00253-009-1963-2
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发表时间：
2009-08-01
影响因子：
5
通讯作者：
Chen, Rachel
中科院分区：
文献类型：
--
作者：
Mao, Zichao;Shin, Hyun-Dong;Chen, Rachel
以大肠杆菌JM 109为出发菌株，通过共表达来自革兰氏阴性杆菌（Pasteurella multocida）的II类透明质酸合成酶和来自大肠杆菌（E. coli K5菌株。该工程菌在摇瓶培养中产生约0.5g/L HA，在1 L生物反应器中的补料分批发酵过程中产生约2.0- 3.8g/L HA。与HA积累相关的粘度的急剧增加需要纯氧补充以维持好氧状态下的发酵。通过葡萄糖胺补充剂探测HA合成过程中的前体供应，其缩短了生物合成途径并消除了需要ATP的一个步骤。HA合成随着葡萄糖胺补充从2.7 g/L增加到3.7 g/L（37%），这与伴随的纯氧输入减少42%相对应，表明能量代谢和前体供应之间存在密切联系。通过使用磷霉素（细胞壁合成抑制剂）将HA合成与细胞生长脱钩，导致HA合成增加70%，这表明通过前体竞争对细胞生长的HA合成产生不利影响。本研究证明了基于重组E. coli菌株。此外，在这项研究中确定的前体供应限制表明，在随后的代谢工程的努力新的工程目标。
An Escherichia coli strain, JM109, was successfully engineered into an efficient hyaluronic acid (HA) producer by co-expressing the only known class-II HA synthase from a Gram-negative bacterium (Pasteurella multocida) and uridine diphosphate-glucose dehydrogenase from E. coli K5 strain. The engineered strain produced about 0.5 g/L HA in shake flask culture and about 2.0-3.8 g/L in a fed-batch fermentation process in a 1-L bioreactor. The sharp increase in viscosity associated with HA accumulation necessitated pure oxygen supplement to maintain fermentation in aerobic regime. Precursor supply during HA synthesis was probed by glucosamine supplement, which shortens biosynthesis pathway and eliminates one step requiring ATP. HA synthesis was increased with glucosamine supplement from 2.7 to 3.7 g/L (37%), which was mirrored with a concomitant 42% decrease in pure oxygen input, suggesting a close connection between energy metabolism and precursor supply. Decoupling HA synthesis from cell growth by using fosfomycin (an inhibitor for cell wall synthesis) led to a 70% increase in HA synthesis, suggesting detrimental effects on HA synthesis from cell growth via precursor competition. This study demonstrates a potentially viable process for HA based on a recombinant E. coli strain. In addition, the precursor supply limitation identified in this study suggests new engineering targets in subsequent metabolic engineering efforts.