Kildegaard Kanchana R, Jensen Niels B, Schneider Konstantin, Czarnotta Eik, Özdemir Emre, Klein Tobias, Maury Jérôme, Ebert Birgitta E, Christensen Hanne B, Chen Yun, Kim Il-Kwon, Herrgård Markus J, Blank Lars M, Forster Jochen, Nielsen Jens, Borodina Irina
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark.
Evolva Biotech A/S, Lersø Park Allé 42-44, 2100, Copenhagen, Denmark.
Microb Cell Fact. 2016 Mar 15;15:53. doi: 10.1186/s12934-016-0451-5.
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 enterica SEacs (L641P). 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 (13)C 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.
未来，石油和天然气衍生的聚合物可能会被生物基聚合物所取代，这些生物基聚合物由可再生原料通过工程细胞工厂生产。到2017年，估计全球年产量约为600万吨的丙烯酸和丙烯酸酯可由3-羟基丙酸（3HP）衍生而来，3HP可通过微生物发酵生产。对于一个经济可行的工艺来说，3HP必须以高滴度、高速度和高产量生产，并且最好在低pH值下生产，以尽量降低下游加工成本。
在此，我们描述了酿酒酵母（Saccharomyces cerevisiae）的代谢工程，用于通过依赖丙二酰辅酶A还原酶（MCR）的途径生物合成3HP。将来自橙黄嗜热栖热菌（Chloroflexus aurantiacus）的多个MCR拷贝以及磷酸化缺陷型乙酰辅酶A羧化酶ACC1基因整合到酵母基因组中，与单拷贝整合相比，3HP滴度提高了五倍。此外，我们通过过表达天然丙酮酸脱羧酶PDC1、醛脱氢酶ALD6和来自肠炎沙门氏菌（Salmonella enterica）的乙酰辅酶A合酶SEacs（L641P）来优化乙酰辅酶A的供应。最后，我们改造了甘油醛-3-磷酸脱氢酶的辅因子特异性，以增加细胞内以NADH为代价的NADPH产量，从而提高3HP产量并减少副产物甘油的形成。在pH 5的碳限制补料分批培养100小时后，最终菌株产生了9.8±0.4 g L(-1)的3HP，产率为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3HP生产菌株进行了表征，这揭示了所采用的代谢工程策略的一些意外结果，并基于这些数据提出了未来的代谢工程方向。
在本研究中，通过增加生物合成基因的拷贝数并改善通向前体和氧化还原辅因子的通量，对酿酒酵母进行了工程改造以实现3HP的高水平生产。该菌株是进一步优化3HP生产的良好平台，因此是迈向3HP潜在商业生物基生产的重要一步。
