Abstract
BackgroundMicrobial bioengineering has the potential to become a key contributor to the future development of human society by providing sustainable, novel, and cost-effective production pipelines. However, the sustained productivity of genetically engineered strains is often a challenge, as spontaneous non-producing mutants tend to grow faster and take over the population. Novel strategies to prevent this issue of strain instability are urgently needed.ResultsIn this study, we propose a novel strategy applicable to all microbial production systems for which a genome-scale metabolic model is available that aligns the production of native metabolites to the formation of biomass. Based on well-established constraint-based analysis techniques such as OptKnock and FVA, we developed an in silico pipeline—FRUITS—that specifically ‘Finds Reactions Usable in Tapping Side-products’. It analyses a metabolic network to identify compounds produced in anabolism that are suitable to be coupled to growth by deletion of their re-utilization pathway(s), and computes their respective biomass and product formation rates. When applied to Synechocystis sp. PCC6803, a model cyanobacterium explored for sustainable bioproduction, a total of nine target metabolites were identified. We tested our approach for one of these compounds, acetate, which is used in a wide range of industrial applications. The model-guided engineered strain shows an obligatory coupling between acetate production and photoautotrophic growth as predicted. Furthermore, the stability of acetate productivity in this strain was confirmed by performing prolonged turbidostat cultivations.ConclusionsThis work demonstrates a novel approach to stabilize the production of target compounds in cyanobacteria that culminated in the first report of a photoautotrophic growth-coupled cell factory. The method developed is generic and can easily be extended to any other modeled microbial production system.
Highlights
Microbial bioengineering has the potential to become a key contributor to the future development of human society by providing sustainable, novel, and cost-effective production pipelines
We have developed a new algorithm to analyze genome-scale metabolic model (GSM) that allows identification of target products, along with the necessary modifications to the network to guarantee that their formation is growth coupled
All constraint-based analysis techniques used within this pipeline, namely flux balance analysis (FBA) [31], flux variability analysis (FVA) [32] and OptKnock [33], were performed using PySCeS-CBMPy [34] in combination with ILOG CPLEX Optimization Studio (IBM) under an academic license
Summary
Microbial bioengineering has the potential to become a key contributor to the future development of human society by providing sustainable, novel, and cost-effective production pipelines. Cyanobacteria that can use (sun) light as their sole energy source can be genetically engineered to directly convert CO2 and water into compounds of interest [7]. This is highly relevant for its potential to mitigate climate change and contribute to the increased sustainability of society. We have directly demonstrated that the fitness burden of product synthesis is associated with deviating carbon itself from biomass to product formation [14] This unsettling observation suggests that a certain degree of culture instability is apparently inevitable using the genetic engineering approaches implemented for photoautotrophs far
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