Chromosomal engineering of inducible isopropanol- butanol-ethanol production in Clostridium acetobutylicum.
Chromosomal engineering of inducible isopropanol- butanol-ethanol production in Clostridium acetobutylicum.
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DOI：
10.3389/fbioe.2023.1218099
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发表时间：
2023
影响因子：
5.7
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使用对环境有害的石化原料可以被基于工程微生物底盘的发酵工艺所取代，该工艺将生物质衍生的碳回收到化学品和燃料中。为了扩大产品范围和/或提高生产率，稳定保留引入的基因是必不可少的。因此，我们已经创建了多重标记的丙酮丁醇梭菌营养缺陷型菌株，其提供不同的基因座（pyrE、argH、purD、pheA），在所述基因座处可以使用等位基因偶联交换（ACE）快速整合异源基因。对于每个基因座，ACE介导的插入基于基本培养基上原养型的恢复而方便地选择。编码正交σ因子（TcdR）的艰难梭菌基因（tcdR）在乳糖诱导型bgaR：：P bgaL启动子的控制下整合在pyrE基因座处，以允许同时控制插入在已置于P tcdB启动子控制下的其它不同基因座（purD和pheA）处的基因/操纵子。在对照实验中，随着乳糖浓度的增加，观察到catP报告基因的剂量依赖性表达。在测试的最高剂量（10 mM）下，表达水平比catP直接置于bgaR：：P bgaL的控制下高10倍以上，并且比使用生孢梭菌铁氧还蛋白基因的强P fdx启动子所实现的高2倍以上。该系统在C.在purD基因座处插入合成丙酮操纵子（ctfA/B，Ctf）和在pheA处插入编码二级脱氢酶的基因（sadh）之后，携带tcdR的整合拷贝的丙酮丁醇菌菌株。乳糖诱导（10 mM）导致产生4.4 g/L异丙醇和19.8 g/L异丙醇-丁醇-乙醇混合物。
The use of environmentally damaging petrochemical feedstocks can be displaced by fermentation processes based on engineered microbial chassis that recycle biomass-derived carbon into chemicals and fuels. The stable retention of introduced genes, designed to extend product range and/or increase productivity, is essential. Accordingly, we have created multiply marked auxotrophic strains of Clostridium acetobutylicum that provide distinct loci (pyrE, argH, purD, pheA) at which heterologous genes can be rapidly integrated using allele-coupled exchange (ACE). For each locus, ACE-mediated insertion is conveniently selected on the basis of the restoration of prototrophy on minimal media. The Clostridioides difficile gene (tcdR) encoding an orthogonal sigma factor (TcdR) was integrated at the pyrE locus under the control of the lactose-inducible, bgaR::P bgaL promoter to allow the simultaneous control of genes/operons inserted at other disparate loci (purD and pheA) that had been placed under the control of the P tcdB promoter. In control experiments, dose-dependent expression of a catP reporter gene was observed with increasing lactose concentration. At the highest doses tested (10 mM) the level of expression was over 10-fold higher than if catP was placed directly under the control of bgaR::P bgaL and over 2-fold greater than achieved using the strong P fdx promoter of the Clostridium sporogenes ferredoxin gene. The utility of the system was demonstrated in the production of isopropanol by the C. acetobutylicum strain carrying an integrated copy of tcdR following the insertion of a synthetic acetone operon (ctfA/B, adc) at the purD locus and a gene (sadh) encoding a secondary dehydrogenase at pheA. Lactose induction (10 mM) resulted in the production of 4.4 g/L isopropanol and 19.8 g/L Isopropanol-Butanol-Ethanol mixture.