代谢工程改造甘油代谢途径提高β-胡萝卜素产量

Improving β-carotene production in Escherichia coli by metabolic engineering of glycerol utilization pathway

摘要:

甘油是生物柴油的副产物，因其价格低廉和高还原性，成为生物发酵的重要碳源。为了进一步提高工程菌对甘油的利用能力，从而提高萜类化合物的合成能力，本研究从β-胡萝卜素高产菌CAR015出发，对其甘油代谢途径的多个基因进行了调控。首先敲除了编码3-磷酸甘油抑制子的glpR基因，然后分别用M1-37、M1-46和M1-93三个不同强度的人工调控元件对glpFK、glpD和tpiA三组基因进行单基因调控和多基因组合调控。研究发现用M1-46调控glpD基因后β-胡萝卜素产量达到了64.82 mg/L，是CAR015的4.86倍，甘油消耗速率也提高了100%；调控tpiA基因后β-胡萝卜素产量略有提高；调控glpFK基因后β-胡萝卜素产量略有降低。说明GlpD是甘油代谢途径中的关键限速步骤。Q-PCR结果表明，降低甘油代谢途径的glpD和glpFK基因转录水平，增加tpiA基因转录水平，可以增加细胞生长速度、提高β-胡萝卜素产量，可能是因为减少了丙酮醛毒性所致。组合调控glpD和tpiA基因，获得β-胡萝卜素产量最高菌株Gly003，其β-胡萝卜素产量达72.45 mg/L、产率达18.65 mg/g每克干细胞，分别是出发菌株CAR015的5.23倍和1.99倍。总之，GlpD是甘油代谢途径中的关键限速步骤，适当强度调控glpD，可以有效提高重组大肠杆菌的β-胡萝卜素产量。

Abstract:

Glycerol is a byproduct during biodiesel production. It is an important feedstock for fermentation due to its low price and high reduced status. Multiple genes of the glycerol utilization pathway were modulated in a previously engineered high β-carotene producing Escherichia coli strain CAR015 to enhance glycerol utilization capability for improving isoprenoids production. The glpR gene, encoding glycerol 3-phosphate repressor, was firstly deleted. The glpFK, glpD and tpiA genes were then modulated by three artificial regulatory parts, M1-37, M1-46 and M1-93, respectively. β-carotene titer reached 64.82 mg/L after modulating glpD with M1-46, which was 4.86 times higher than that of CAR015, and glycerol consumption rate also increased 100%. Modulating tpiA led to a little increase of β-carotene titer, whereas modulating glpFK led to a little decrease of β-carotene titer. This demonstrated that GlpD was a rate-limiting step in glycerol utilization pathway. Q-PCR of glpF, glpK, glpD and tpiA results showed that decrease the transcription level of glpF, glpK, glpD, or decrease the transcription level of tpiA could increase the cell growth and β-carotene production, probably for the decrease of methylglyoxal toxicity. Modulating glpD and tpiA genes in combination resulted in the best strain Gly003, which produced 72.45 mg/L β-carotene with a yield of 18.65 mg/g dry cell weight. The titer was 5.23 and yield 1.99 times of that of the parent strain CAR015. Our work suggested that appropriate activation of glpD and tpiA genes in glycerol utilization pathway could effectively improve β-carotene production. This strategy can be used for production of other terpenoids in E. coli.