Itaconic acid production from glycerol using Escherichia coli harboring a random synonymous codon-substituted 5′-coding region variant of the cadA gene
Itaconic acid production from glycerol using Escherichia coli harboring a random synonymous codon-substituted 5′-coding region variant of the cadA gene
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DOI：
10.1002/bit.25914
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发表时间：
2016-07-01
影响因子：
3.8
通讯作者：
Choi, Jong Hyun
中科院分区：
文献类型：
--
作者：
Jeon, Ho-Geun;Cheong, Dae-Eun;Choi, Jong Hyun
土曲霉cadA编码顺乌头酸脱羧酶，是衣康酸（IA）生物合成的必需基因，但在大多数工业宿主中主要以不溶性聚集体形式表达。这一直是开发IA生产的重组策略的障碍。在这里，我们创建了cadA基因的同义密码子变体（scv）库，其中前10个密码子（除ATG）中含有同义密码子，并在大肠杆菌中进行筛选。在阳性克隆中，E. coli scvCadA_No8的可溶性组分中表达的CadA超过95%，并且在生产运行中，产生的IA比野生型E.大肠杆菌中的Luria-Bertani肉汤补充0.5%葡萄糖。在含有0.85g/L柠檬酸和1%甘油的M9基本培养基中，E. coliscvCadA_No8经异丙基-D-1-硫代半乳糖苷诱导后，在72小时内产生985.6 ± 33.4mg/L的IA。在2L的分批补料发酵中，采用E.通过仅导入scv_cadA基因和优化IA产生的培养条件，将其转化到大肠杆菌中。这些结果可以与代谢工程相结合，产生一个E。大肠杆菌菌株作为工业IA生产者。Biotechnol. Bioeng. 2016;113：1504-1510. (c)2015 Wiley Periodicals，Inc.
Aspergillus terreus cadA, encoding cis-aconitate decarboxylase, is an essential gene for itaconic acid (IA) biosynthesis, but it is primarily expressed as insoluble aggregates in most industrial hosts. This has been a hurdle for the development of recombinant strategies for IA production. Here, we created a library of synonymous codon variants (scv) of the cadA gene containing synonymous codons in the first 10 codons (except ATG) and screened it in Escherichia coli. Among positive clones, E. coli scvCadA_No8 showed more than 95% of expressed CadA in the soluble fraction, and in production runs, produced threefold more IA than wild-type E. coli in Luria-Bertani broth supplemented with 0.5% glucose. In M9 minimal media containing 0.85g/L citrate and 1% glycerol, E. coli scvCadA_No8 produced 985.6 +/- 33.4mg/L IA during a 72-h culture after induction with isopropyl -D-1-thiogalactopyranoside. In a 2-L fed-batch fermentation consisting of two stages (growth and nitrogen limitation conditions), we obtained 7.2g/L IA by using E. coli by introducing only the scv_cadA gene and optimizing culture conditions for IA production. These results could be combined with metabolic engineering and generate an E. coli strain as an industrial IA producer. Biotechnol. Bioeng. 2016;113: 1504-1510. (c) 2015 Wiley Periodicals, Inc.