Enhanced Astaxanthin Production in Escherichia coli via Morphology and Oxidative Stress Engineering

Abstract

Astaxanthin is a carotenoid of high commercial value because of its excellent antioxidative, anti-inflammatory, and anticancer properties. Here, we developed a novel strategy for improving the production of astaxanthin via morphology and oxidative stress engineering. First, we identified the morphology-/membrane- and oxidative stress-related genes, which should be knocked down, using the CRISPRi system. Deleting the morphology-/membrane-related genes (lpp and bamB) and the oxidative stress-related genes (uspE and yggE) generated longer and larger cells with higher reactive oxygen species (ROS) levels, thus enhancing the production of astaxanthin and decreasing cell growth. To not only improve cell growth but also obtain longer and larger cells with higher ROS levels, a complementary expression system using a temperature-sensitive plasmid was established. Complementarily expressing the morphology-/membrane-related genes (lpp and bamB) and the oxidative stress-related genes (uspE and yggE) further improved the production of astaxanthin to 11.92 mg/g dry cell weight in shake flask cultures.

Adaptive laboratory evolution and shuffling of Escherichia coli to enhance its tolerance and production of astaxanthin

“…Gene knockout or replacement was performed according to the CRISPR-Cas method as previously described [ 17 , 34 , 43 , 44 ]. The sgRNA plasmid pTargetB-yadC for replacement was obtained as described above for the CRISPRi system.…”

“…Cells were extracted with acetone to isolate carotenoids as previously described [ 9 , 17 ]. E. coli cultures (250 μL) were harvested by centrifugation at 13,523× g for 5 min.…”

“…To lower the cost, many studies focused on the production of astaxanthin from an inexpensive carbon source using microbes. Escherichia coli [8][9][10][11][12][13][14][15][16][17], Saccharomyces cerevisiae [18][19][20][21][22][23][24], Corynebacterium glutamicum [25] and Yarrowia lipolytica [26] have been used as host…”

“…To lower the cost, many studies focused on the production of astaxanthin from an inexpensive carbon source using microbes. Escherichia coli [ 8 – 17 ], Saccharomyces cerevisiae [ 18 – 24 ], Corynebacterium glutamicum [ 25 ] and Yarrowia lipolytica [ 26 ] have been used as host strains for astaxanthin production by the introduction of the astaxanthin biosynthesis pathway. In our study [ 9 ], we engineered an E. coli that produced 7.4 mg/g dry cell weight (DCW) of astaxanthin as the predominant carotenoid (96.6%) by coordinately expression of β-carotene hydroxylase (CrtZ) and ketolase (CrtW).…”

“…In our study [ 9 ], we engineered an E. coli that produced 7.4 mg/g dry cell weight (DCW) of astaxanthin as the predominant carotenoid (96.6%) by coordinately expression of β-carotene hydroxylase (CrtZ) and ketolase (CrtW). Further morphology and oxidative stress engineering increased the yields to 11.92 mg/g DCW via [ 17 ]. The highest astaxanthin yield in E. coli and S. cerevisiae was 15.1 mg/g DCW [ 15 ] and 13.8 mg/g DCW [ 18 ] so far, respectively.…”