One-Stage vs. Two-Stage Bioprocesses: Comparative Computational and Experimental Studies and Consequences for Strain Design

Abstract

Biotechnological processes for the production of (high-volume) chemicals are often based on one-stage or two-stage processes (OSPs and TSPs, respectively). Many classical bioprocesses are OSPs in which growth and product synthesis are coupled, however, OSPs often exhibit low volumetric productivities due to inherent trade-offs between biomass and product yields. Here, TSPs, where growth and production are separated, may help to improve the productivity of bioprocesses and with rapidly growing genetic toolboxes for dynamic metabolic control TSPs are becoming more attractive. However, there are only few theoretical and experimental studies systematically comparing OSPs and TSPs in terms of their process metrics (productivity, yield etc.).We have constructed E. coli strains for heterologous production of itaconate for both OSP and TSP. We used computer-aided design to build a mutant strain (ita23) for growth-coupled synthesis of itaconate. In an OSP, this strain reached the highest product yield and titer that have ever been reported for heterologous itaconate production. For increasing the volumetric productivity via a TSP, we constructed a new strain (ita36A) allowing a temperature-dependent switch from fast growth to production. Indeed, a TSP with ita36A increased the peak productivity of ita23 by 91% and the titer by 46%. Despite of this successful implementation of a TSP, we observed that the productivity, after reaching a peak value, decreased quickly in the production phase due to low substrate uptake and product synthesis rates. We therefore used mathematical models to systematically compare productivities of TSPs and OSPs. Our simulations show that the overall volumetric productivity of a TSP can in fact be lower than of a corresponding OSP due to the sharp decrease of the specific substrate uptake rate in (non-growth) TSP production phases. This raises a (often neglected) big challenge for designing competitive TSPs. We propose approaches to improve substrate utilization rates in the production phase by which TSPs really outperform OSPs. In conclusion, a careful assessment of the trade-offs between substrate uptake rates, yields, and productivity is necessary when deciding for OSP vs. TSP.