Yidi Liu1, Xinlei Mao1, Baoqi Zhang1, Jinping Lin2, Dongzhi Wei1. 1. State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China. 2. State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China. jplin@ecust.edu.cn.
Abstract
OBJECTIVES: 3,4-Dihydroxybutyric acid (3,4-DHBA) is a multifunctional C4 platform compound widely used for the synthesis of various materials, including pharmaceuticals. Although, a biosynthetic pathway for 3,4-DHBA production has been developed, its low yield still precludes large-scale use. Here, a heterologous four-step biosynthetic pathway was established in recombinant Escherichia coli (E. coli) using a combinatorial strategy. RESULTS: Several aldehyde dehydrogenases (ALDHs) were screened, using in vitro enzyme assays, to identify suitable catalysts for the dehydrogenation of 3,4-dihydroxybutanal (3,4-DHB) to 3,4-DHBA. A pathway containing glucose dehydrogenase (BsGDH) from Bacillus subtilis, D-xylonate dehydratase (YagF) from E. coli, benzoylformate decarboxylase (PpMdlC) from Pseudomonas putida and ALDH was introduced into E. coli, generating 3.04 g/L 3,4-DHBA from D-xylose (0.190 g 3,4-DHBA/g D-xylose). Disruption of competing pathways by deleting xylA, ghrA, ghrB and adhP contributed to an 87% increase in 3,4-DHBA accumulation. Expression of a fusion construct containing PpMdlC and YagF enhanced the 3,4-DHBA titer, producing the highest titer and yield reported thus far (7.71 g/L; 0.482 g 3,4-DHBA/g D-xylose). CONCLUSIONS: These results showed that deleting genes from competing pathways and constructing fusion proteins significantly improved the titer and yield of 3,4-DHBA in engineered E. coli.
OBJECTIVES: 3,4-Dihydroxybutyric acid (3,4-DHBA) is a multifunctional C4 platform compound widely used for the synthesis of various materials, including pharmaceuticals. Although, a biosynthetic pathway for 3,4-DHBA production has been developed, its low yield still precludes large-scale use. Here, a heterologous four-step biosynthetic pathway was established in recombinant Escherichia coli (E. coli) using a combinatorial strategy. RESULTS: Several aldehyde dehydrogenases (ALDHs) were screened, using in vitro enzyme assays, to identify suitable catalysts for the dehydrogenation of 3,4-dihydroxybutanal (3,4-DHB) to 3,4-DHBA. A pathway containing glucose dehydrogenase (BsGDH) from Bacillus subtilis, D-xylonate dehydratase (YagF) from E. coli, benzoylformate decarboxylase (PpMdlC) from Pseudomonas putida and ALDH was introduced into E. coli, generating 3.04 g/L 3,4-DHBA from D-xylose (0.190 g 3,4-DHBA/g D-xylose). Disruption of competing pathways by deleting xylA, ghrA, ghrB and adhP contributed to an 87% increase in 3,4-DHBA accumulation. Expression of a fusion construct containing PpMdlC and YagF enhanced the 3,4-DHBA titer, producing the highest titer and yield reported thus far (7.71 g/L; 0.482 g 3,4-DHBA/g D-xylose). CONCLUSIONS: These results showed that deleting genes from competing pathways and constructing fusion proteins significantly improved the titer and yield of 3,4-DHBA in engineered E. coli.