Engineering and systems-level analysis of Saccharomyces cerevisiae for production of 3-hydroxypropionic acid via malonyl-CoA reductase-dependent pathway.
Engineering and systems-level analysis of Saccharomyces cerevisiae for production of 3-hydroxypropionic acid via malonyl-CoA reductase-dependent pathway.
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DOI：
10.1186/s12934-016-0451-5
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发表时间：
2016-03-15
影响因子：
6.4
通讯作者：
Borodina I
中科院分区：
文献类型：
--
作者：
Kildegaard KR;Jensen NB;Schneider K;Czarnotta E;Özdemir E;Klein T;Maury J;Ebert BE;Christensen HB;Chen Y;Kim IK;Herrgård MJ;Blank LM;Forster J;Nielsen J;Borodina I
在未来，石油和天然气衍生的聚合物可能会被生物基聚合物取代，生物基聚合物使用工程细胞工厂从可再生原料中生产。到2017年，估计世界年产量约为600万吨的丙烯酸和丙烯酸酯可以衍生自3-羟基丙酸（3 HP），其可以通过微生物发酵生产。对于经济上可行的方法，3 HP必须以高滴度、速率和产率生产，并且优选在低pH下生产，以使下游加工成本最小化。在这里，我们描述了面包酵母酿酒酵母通过丙二酰辅酶A还原酶（MCR）依赖性途径生物合成3 HP的代谢工程。整合多个拷贝的MCR从橙黄绿弯藻和磷酸化缺陷的乙酰辅酶A羧化酶ACC 1基因到酵母的基因组中增加3 HP滴度5倍，与单一整合相比。此外，我们通过过表达来自沙门氏菌SEacsL 641 P的天然丙酮酸脱羧酶PDC 1、醛脱氢酶ALD 6和乙酰辅酶A合酶来优化乙酰辅酶A的供应。最后，我们设计了甘油醛-3-磷酸脱氢酶的辅因子特异性，以牺牲NADH增加NADPH的细胞内产生，从而提高3 HP的产生并减少作为副产物的甘油的形成。最终菌株在pH 5的碳限制补料分批培养100小时后产生9.8 ± 0.4 g L−1 3 HP，产量为13 % C-mol C-mol−1葡萄糖。通过13 C代谢通量分析和转录组分析对3 HP产生菌进行了表征，揭示了所采取的代谢工程策略的一些意想不到的后果，并基于这些数据，提出了未来的代谢工程方向。在本研究中，S.通过增加生物合成基因的拷贝数和改善朝向前体和氧化还原辅因子的通量，将酿酒酵母工程化用于高水平生产3 HP。该菌株代表了进一步优化3 HP生产的良好平台，因此是迈向3 HP的潜在商业生物基生产的重要一步。本文的在线版本（doi：10.1186/s12934-016-0451-5）包含补充材料，可供授权用户使用。
In the future, oil- and gas-derived polymers may be replaced with bio-based polymers, produced from renewable feedstocks using engineered cell factories. Acrylic acid and acrylic esters with an estimated world annual production of approximately 6 million tons by 2017 can be derived from 3-hydroxypropionic acid (3HP), which can be produced by microbial fermentation. For an economically viable process 3HP must be produced at high titer, rate and yield and preferably at low pH to minimize downstream processing costs. Here we describe the metabolic engineering of baker’s yeast Saccharomyces cerevisiae for biosynthesis of 3HP via a malonyl-CoA reductase (MCR)-dependent pathway. Integration of multiple copies of MCR from Chloroflexus aurantiacus and of phosphorylation-deficient acetyl-CoA carboxylase ACC1 genes into the genome of yeast increased 3HP titer fivefold in comparison with single integration. Furthermore we optimized the supply of acetyl-CoA by overexpressing native pyruvate decarboxylase PDC1, aldehyde dehydrogenase ALD6, and acetyl-CoA synthase from Salmonella entericaSEacsL641P. Finally we engineered the cofactor specificity of the glyceraldehyde-3-phosphate dehydrogenase to increase the intracellular production of NADPH at the expense of NADH and thus improve 3HP production and reduce formation of glycerol as by-product. The final strain produced 9.8 ± 0.4 g L−1 3HP with a yield of 13 % C-mol C-mol−1 glucose after 100 h in carbon-limited fed-batch cultivation at pH 5. The 3HP-producing strain was characterized by 13C metabolic flux analysis and by transcriptome analysis, which revealed some unexpected consequences of the undertaken metabolic engineering strategy, and based on this data, future metabolic engineering directions are proposed. In this study, S. cerevisiae was engineered for high-level production of 3HP by increasing the copy numbers of biosynthetic genes and improving flux towards precursors and redox cofactors. This strain represents a good platform for further optimization of 3HP production and hence an important step towards potential commercial bio-based production of 3HP. The online version of this article (doi:10.1186/s12934-016-0451-5) contains supplementary material, which is available to authorized users.