Maximizing microbial bioproduction from sustainable carbon sources using iterative systems engineering.

Maximizing the production of heterologous biomolecules is a complex problem that can be addressed with a systems-level understanding of cellular metabolism and regulation. Specifically, growth-coupling approaches can increase product titers and yields and also enhance production rates. However, implementing these methods for non-canonical carbon streams is challenging due to gaps in metabolic models. Over four design-build-test-learn cycles, we rewire Pseudomonas putida KT2440 for growth-coupled production of indigoidine from para-coumarate. We explore 4,114 potential growth-coupling solutions and refine one design through laboratory evolution and ensemble data-driven methods. The final growth-coupled strain produces 7.3 g/L indigoidine at 77% maximum theoretical yield in para-coumarate minimal medium. The iterative use of growth-coupling designs and functional genomics with experimental validation was highly effective and agnostic to specific hosts, carbon streams, and final products and thus generalizable across many systems.

Photobiological production of high-value pigments via compartmentalized co-cultures using Ca-alginate hydrogels.

Engineered cyanobacterium Synechococcus elongatus can use light and CO2 to produce sucrose, making it a promising candidate for use in co-cultures with heterotrophic workhorses. However, this process is challenged by the mutual stresses generated from the multispecies microbial culture. Here we demonstrate an ecosystem where S. elongatus is freely grown in a photo-bioreactor (PBR) containing an engineered heterotrophic workhorse (either ß-carotene-producing Yarrowia lipolytica or indigoidine-producing Pseudomonas putida) encapsulated in calcium-alginate hydrogel beads. The encapsulation prevents growth interference, allowing the cyanobacterial culture to produce high sucrose concentrations enabling the production of indigoidine and ß-carotene in the heterotroph. Our experimental PBRs yielded an indigoidine titer of 7.5 g/L hydrogel and a ß-carotene titer of 1.3 g/L hydrogel, amounts 15-22-fold higher than in a comparable co-culture without encapsulation. Moreover, 13C-metabolite analysis and protein overexpression tests indicated that the hydrogel beads provided a favorable microenvironment where the cell metabolism inside the hydrogel was comparable to that in a free culture. Finally, the heterotroph-containing hydrogels were easily harvested and dissolved by EDTA for product recovery, while the cyanobacterial culture itself could be reused for the next batch of immobilized heterotrophs. This co-cultivation and hydrogel encapsulation system is a successful demonstration of bioprocess optimization under photobioreactor conditions.

Tuning a high performing multiplexed-CRISPRi Pseudomonas putida strain to further enhance indigoidine production.

In this study, a 14-gene edited Pseudomonas putida KT2440 strain for heterologous indigoidine production was examined using three distinct omic datasets. Transcriptomic data indicated that CRISPR/dCpf1-interference (CRISPRi) mediated multiplex repression caused global gene expression changes, implying potential undesirable changes in metabolic flux. 13C-metabolic flux analysis (13C-MFA) revealed that the core P. putida flux network after CRISPRi repression was conserved, with moderate reduction of TCA cycle and pyruvate shunt activity along with glyoxylate shunt activation during glucose catabolism. Metabolomic results identified a change in intracellular TCA metabolites and extracellular metabolite secretion profiles (sugars and succinate overflow) in the engineered strains. These omic analyses guided further strain engineering, with a random mutagenesis screen first identifying an optimal ribosome binding site (RBS) for Cpf1 that enabled stronger product-substrate pairing (1.6-fold increase). Then, deletion strains were constructed with excision of the PHA operon (ΔphaAZC-IID) resulting in a 2.2-fold increase in indigoidine titer over the optimized Cpf1-RBS construct at the end of the growth phase (∼6 h). The maximum indigoidine titer (at 72 h) in the ΔphaAZC-IID strain had a 1.5-fold and 1.8-fold increase compared to the optimized Cpf1-RBS construct and the original strain, respectively. Overall, this study demonstrated that integration of omic data types is essential for understanding responses to complex metabolic engineering designs and directly quantified the effect of such modifications on central metabolism.