Intensification of 2'-Fucosyllactose Biosynthesis pathway by using a novel fucosyltransferase from Bacillus cereus

Kainuo Zhang¹Miaomiao Gao¹Chenqi Cao¹Mengxin Zhang¹Waqar Ahmad¹Ahmed Rady²Badr Aldahmash²Tianze Zhu³Shahin Shah Khan¹Luo Liu^1*

• ¹Beijing University of Chemical Technology, Beijing, China
• ²King Saud University, Riyadh, Riyadh, Saudi Arabia
• ³Beijing Zeno Biotechnology Development Co. Ltd, Beijing, China

Introduction: 2′-Fucosyllactose (2′-FL) is an oligosaccharide that can be synthesized in the human body and is known for its health-promoting and prebiotic effects. The biosynthesis of 2′-FL using microorganisms has received attention recently due to its increased application in nutritional and medical infant formulations.Methods: This work attempts the new application of Bacillus cereus α-1,2-fucosyltransferase (FutCB) in the de novo synthesis of 2′-FL in Escherichia coli (E. coli). Additionally, knocking out the lacZ and waaF genes alongside overexpression of the key gmd, manB, wcaG, and manC genes enhances the availability of the necessary precursors GDP-L-fucose and lactose for the synthesis of 2′-FL.The use of constitutive promoters achieved better control over the production of 2′-FL during fed-batch fermentation. After 64 hours of fermentation, the modified E. coli strains produced 121.4 g/L 2′-FL with a yield of 1.90 g/L/h, resulting in an impressive 2′-FL output. These results together indicate the potential of large-scale, high-yield production of 2′-FL and form a basis of much more refinement to be done. The next step will focus on maximum substrate utilization, alteration of gene regulation, and improvement of commercial-scale synthesis. 1) E. coli based microbial cell factory was used for de novo biosynthesis of 2′-FL from Bacillus cereus by α-1,2-fucosyltransferase (FutCB).2) By knocking out lacZ and waaF along with overexpressing manB, manC, gmd, and wcaG, lactose and GDP-L-fucose were accumulated to enhance 2'-fucosyllactose (2'-FL) biosynthesis3) The incorporation of a constitutive promoter significantly enhanced the stability of 2′-FL biosynthesis during fed-batch cultivation.4) The fed-batch cultivation achieved a peak 2′-FL production of 121.4 g/L after 64 hours, with a production rate of 1.90 g/L/h.