Modular co-culture engineering has been applied to divide labor among different strains in the biomanufacturer of substances through complicated metabolic pathways. However, dynamic instability, uncontrollable population ratios, and lack of mathematical models have hindered its further application. In this study, we built a mathematical model and used it to design a self-stable co-culture system with adjustable population ratios of two engineered Escherichia coli strains. As an indicator product, the yield of chlorogenic acid was successfully increased by 16.5% by adjusting the population ratio in the system. This system provides a robust and scalable production platform for modular co-culture engineering. The co-culture system was confirmed to be a symbiotic system with high carbon atom economy for acetyl-CoA-derived chemicals by optimization of poly-β-hydroxybutyrate production, which was 2.82-fold higher than it was for wild-type E. coli. The conversion rate of glucose to poly-β-hydroxybutyrate also was 3.19-fold higher in the co-culture system. Our results provide new insights into possible future applications of co-culture systems.

Biosynthesis of TCA cycle-derived C4 chemicals through glyoxylate shunt is an attractive metabolic route because it can be drived by TCA-glyoxylate cycle force under aerobic conditions. However, yield of this route is low with at least 1/3 carbon loss from glucose. FAs could sufficiently provide acetyl-CoA by β-oxidation without carbon loss and directly enter the TCA-glyoxylate cycle, which is acknowledged as a promising alternative feedstock. Here β-alanine was selected as the target TCA cycle-derived chemical, of which the theoretical yield is 1.391 g/g FAs, much higher than that of glucose(0.49 g/g). By adopting multi-metabolic engineering strategies and relieving the active oxygen damage caused by FAs utilization, β-alanine production reached 78.05 g/L with a yield of 1.2 g/g, about 86% of theoretical yield. Our study establish a promising bioproduction route of β-alanine from waste FAs (such as gutter oil, palm fatty acid distillate etc.), and more importantly, provide an efficient platform for TCA cycle-derived C4 chemicals biosynthesis.