Streamlining of a synthetic co‐culture towards an individually controllable one‐pot process for polyhydroxyalkanoate production from light and CO₂

Abstract

Rationally designed synthetic microbial consortia carry a vast potential for biotechnological applications. The application of such a consortium in a bioprocess, however, requires tight and individual controllability of the involved microbes. Here, we present the streamlining of a co-cultivation process consisting of Synechococcus elongatus cscB and Pseudomonas putida for the production of polyhydroxyalkanoates (PHA) from light and CO 2 . First, the process was improved by employing P. putida cscRABY, a strain with a higher metabolic activity towards sucrose. Next, the individual controllability of the co-culture partners was addressed by providing different nitrogen sources, each exclusively available for one strain. By this, the growth rate of the co-culture partners could be regulated individually, and defined conditions could be set. The molC/molN ratio, a key value for PHA accumulation, was estimated from the experimental data, and the necessary feeding rates to obtain a specific ratio could be predicted. This information was then implemented in the co-cultivation process, following the concept of a DBTL-cycle. In total, the streamlining of the process resulted in an increased maximal PHA titer of 393 mg/L and a PHA production rate of 42.1 mg/(L•day).

Cyanobacteria as cell factories for the photosynthetic production of sucrose

“…Francis (2016 10.3389/fmicb.2023.1126032 Frontiers in Microbiology 15 frontiersin.org modest (Löwe et al, 2017;Fedeson et al, 2020). Additional expression of a sucrose porin (cscY) and a sucrose operon repressor (cscR) further improved sucrose utilization (Löwe et al, 2020), while further optimization of the nitrogen-deficiency response pathway (Hobmeier et al, 2020) and culture conditions could boost PHA titer further (Kratzl et al, 2023; Table 2). Other co-culture products include the metabolites ethylene, isoprene, 3-hydroxypropionic acid (3-HP), and 2,3-butanediol (Table 2), which are compounds in a broader class of industrially relevant precursors widely used for chemical synthesis (e.g., diols, organic acids, gaseous alkenes; Cui et al, 2022;Li C. et al, 2022;Ma et al, 2022).…”

“…Indeed, initial reports demonstrated that P. putida expressing cscAB was capable of growing solely on sucrose provided by S. elongatus PCC 7942 and accumulated PHA in co-culture, though sucrose utilization was incomplete and productivities were modest ( Löwe et al, 2017 ; Fedeson et al, 2020 ). Additional expression of a sucrose porin ( cscY ) and a sucrose operon repressor ( cscR ) further improved sucrose utilization ( Löwe et al, 2020 ), while further optimization of the nitrogen-deficiency response pathway ( Hobmeier et al, 2020 ) and culture conditions could boost PHA titer further ( Kratzl et al, 2023 ; Table 2 ).…”

“…A number of cyanobacterial species have been effectively engineered to produce and secrete large amounts of sucrose by taking advantage of cyanobacterial sucrose biosynthesis pathways and heterologous co-expression of sucrose permease (CscB, Ducat et al, 2012 ; Du et al, 2013 ; Abramson et al, 2016 ; Kirsch et al, 2018 ; Lin P. C. et al, 2020 ) to export sucrose from the cell. In addition to batch cultures, there are increasing examples of real-time conversion of the carbohydrate feedstock through the direct co-culture of microbial partner strains that metabolize the secreted bacterial sucrose to higher-value products ( Smith and Francis, 2016 ; Hays et al, 2017 ; Löwe et al, 2017 ; Weiss et al, 2017 ; Fedeson et al, 2020 ; Hobmeier et al, 2020 ; Zhang et al, 2020 ; Ma et al, 2022 ; Kratzl et al, 2023 ), potentially bypassing the costly processes of purifying and concentrating sucrose ( Radakovits et al, 2010 ). However, to further use these synthetic light-driven microbial consortia in industrial applications, a number of challenges need to be overcome, such as long-term production stability, vulnerability to invasion by opportunistic microbial or viral contaminants, and imbalances in attributes of consortia that can contribute to inefficiencies ( Hays and Ducat, 2015 ; Gao et al, 2022 ).…”

Light‐driven synthetic microbial consortia: playing with an oxygen dilemma

“…Once co-cultured with cyanobacteria, the microbial PHA producer will be benefited from the oxygen generated from photosynthesis. The microbial consortia containing sucrosesecreting S. elongatus and PHA producer P. putida were constructed for PHA production [26][27][28].…”

“…, where an engineered P. putida capable of efficiently utilizing sucrose was used [28]. In addition, another engineered P. putida capable of degrading 2,4-dinitrotoluene was used in microbial consortia, allowing simultaneous removal of 2,4-dinitrotoluene and accumulation of PHA from CO2 and light [27].…”