Pseudomonas putida as saviour for troubled synechococcus elongatus in a synthetic co-culture – interaction studies based on a multi-omics approach

ABSTRACT In their natural habitats, microbes rarely exist in isolation; instead, they thrive in consortia, where various interactions occur. In this study, a defined synthetic co-culture of

the cyanobacterium _S. elongatus cscB_, which supplies sucrose to the heterotrophic _P. putida cscRABY_, is investigated to identify potential interactions. Initial experiments reveal a

remarkable growth-promoting effect of the heterotrophic partner on the cyanobacterium, resulting in an up to 80% increase in the growth rate and enhanced photosynthetic capacity. Vice versa,

the presence of the cyanobacterium has a neutral effect on _P. putida cscRABY_, highlighting the resilience of pseudomonads against stress and their potential as co-culture partners. Next,

a suitable reference process reinforcing the growth-promoting effect is established in a parallel photobioreactor system, which sets the basis for the analysis of the co-culture at the

transcriptome, proteome, and metabolome levels. In addition to several moderate changes, including alterations in the metabolism and stress response in both microbes, this comprehensive

multi-OMICs approach strongly hints towards the exchange of further molecules beyond the unidirectional feeding with sucrose. Taken together, these findings provide valuable insights into

the complex dynamics between both co-culture partners, indicating multi-level interactions, which can be employed for further streamlining of the co-cultivation system. SIMILAR CONTENT BEING

VIEWED BY OTHERS MIXED AND MEMBRANE-SEPARATED CULTURING OF SYNTHETIC CYANOBACTERIA-YEAST CONSORTIA REVEALS METABOLIC CROSS-TALK MIMICKING NATURAL CYANOLICHENS Article Open access 25 October

2024 SYNTHETIC MICROBIAL COMMUNITIES OF HETEROTROPHS AND PHOTOTROPHS FACILITATE SUSTAINABLE GROWTH Article Open access 30 July 2020 BIOLOGICAL AND METABOLIC EFFECTS OF THE ASSOCIATION

BETWEEN THE MICROALGA _GALDIERIA SULPHURARIA_ AND THE FUNGUS _PENICILLIUM CITRINUM_ Article Open access 31 January 2023 INTRODUCTION Over the last two decades, a paradigm shift has started

in biotechnology, expanding beyond the historically-grown focus on single-species or so-called axenic cultures. This change involves the development of controllable co-cultures comprising

two or more species within the same reaction vessel. Considering that microbes barely live separately in nature expands the bioproduction of value-added compounds through novel pathways and

strategies. Furthermore, co-cultures enable us to understand synergistic effects and uncover otherwise hidden behaviours, allowing for targeted utilisation of microbial capabilities1,2. The

dynamics of a community or a co-culture are determined by interactions of individual cells, which normally act in their local niches. Most microbes have evolved to thrive in the presence of

neighbouring species, which can present either potential threats or offer benefits, and they have adapted accordingly3. Those interactions can be divided into four general classes:

mutualism, neutralism, commensalism, and parasitism4. For instance, a mutualistic interaction, where all community members profit, can appear as cross-feeding or syntrophy. Here, one partner

does not completely metabolise a given substrate, which then, in turn, is accessible to another partner. The latter might remove toxins or harmful gases, creating the environment needed for

the entire community4. The design of a synthetic co-culture is not trivial, as multiple aspects and parameters, such as medium composition, need to be considered5,6, and in the best-case

scenario, resulting co-cultures should align with a sustainable bioprocess. In most cases, it is intended that the microbes live under the premise of the division of labour, which allows

different traits of microbes to complement each other in a profitable way3,7. One example of doing so is pairing heterotrophs with phototrophs8. This composition of microbes is prevalent in

nature, which can be observed in lichens and microbial mats9. Here, the photoautotrophic member, such as cyanobacteria or algae, uses solar power to fix CO2 into organic carbon, a portion of

which then in turn is accessible to the heterotrophic members of the community. In the case of synthetic co-cultures, the heterotrophic partner can be employed to convert the carbon source

provided into value-added products by engineered and optimised metabolic pathways. The model organism _Synechococcus_ PCC 7942 naturally accumulates sucrose as a compatible solute when

exposed to elevated NaCl concentrations. This trait was exploited to construct the sucrose secreting strain _S. elongatus cscB_ by genomic integration of the _cscB_ gene encoding a

H+/sucrose symporter10. The resulting strain secretes sucrose into the surrounding medium with a rate of up to 28 mg L−1 per hour10,11. Up to now, several robust synthetic co-cultures have

been constructed employing _S. elongatus cscB_, and valuable compounds like α-amylase, polyhydroxyalkanoates (PHAs), or isoprene have been successfully produced11,12,13. The metabolic

capacity of the co-culture processes could be expanded depending on the heterotrophic partner used, such as _Escherichia coli_, _Pseudomonas putida_, _Bacillus subtilis_, or _Saccharomyces

cerevisiae_. In contrast to natural communities that are highly complex structures with overlaying interactions, synthetic co-cultures are very well suited to study interactions between and

within the involved species5. The inherent definition of a synthetic defined co-culture is that the partners have not evolved together or, at least, that the connection between them has not

evolved driven by nature. Therefore, we assume that non-engineered feedback and/or communication between the partner organisms have their origin in a general answer adopted from their

individual natural habitats. In this study, we set out to investigate those non-engineered interactions that might guide us towards a better understanding of synthetic co-cultures in general

and how to design them. To this end, we used a synthetic co-culture consisting of the cyanobacterium _S. elongatus cscB_ and the soil-bacterium _P. putida cscRABY_, which was recently

employed for PHA production in our lab12. In this co-culture, _P. putida cscRABY_ has been engineered to transport and metabolise sucrose by the integration of the _cscRABY_ operon into the

chromosome14. In the work described here, first, a suitable reference experiment was set up, which allowed us to compare the co-culture with the respective axenic cultures of _S. elongatus

cscB_ or _P. putida cscRABY_ to identify and investigate the putative interaction of the co-culture partners with each other. Furthermore, we set out to analyse the co-culture not only on a

physiological level but also on the transcriptome, proteome, and metabolome level by employing a multi-OMICs approach, which has been demonstrated to be a powerful tool to decipher hidden

traits of synthetic communities13,15. RESULTS AND DISCUSSION INFLUENCE OF THE CO-CULTURE PARTNERS ON EACH OTHER’S GROWTH It was frequently observed that cyanobacteria grow more efficiently

in co-cultivation with heterotrophic bacteria both in natural9,16 and in synthetic co-cultures11. To this end, we investigated the growth of both strains in the co-culture compared to the

axenic cultures in different scales and conditions (see Supplementary Note S1 and Supplementary Fig. S1). To analyse the influence of _P. putida cscRABY_ on the initial growth of _S.

elongatus cscB_ in 12-well plates at a 1.6 mL scale, we differentiated between a “SuSec-ON” and a “SuSec-OFF” status of the synthetic connection, brought about by the inducible exchange of

sucrose (Fig. 1a, b). In the SuSec-ON situation, the sucrose secretion by _S. elongatus cscB_ is induced, which is not the case in the SuSec-OFF situation. Here, an additional batch of 1 g 

L−1 sucrose was added to all cultures, including the axenically grown _S. elongatus cscB_, to support heterotrophic growth and to identify effects independent of the synthetic connection. To

investigate the influence of different inoculation ratios (phototroph:heterotroph), the co-culture was inoculated with varying amounts of _P. putida cscRABY_ to reach _S. elongatus_ to _P.

putida_ cell ratios of 1:1, 1:10−3, and 1:10−5 and after 24 h cell counts of both strains were determined. As shown in Fig. 1, in all co-cultures, cyanobacterial cell counts were higher

compared to the axenic culture, suggesting that the presence of _P. putida cscRABY_ promotes the initial growth of _S. elongatus cscB_ (left bar in Fig. 1a, b). This effect was independent

of whether the synthetic connection via the sucrose feed (SuSec) was ON or OFF; however, it was more pronounced in the SuSec-OFF situation. Higher cyanobacterial cell counts are reached in

the SuSec-OFF case due to a general reduction of growth when sucrose secretion is induced. An influence of the inoculation ratio can only be observed in the SuSec-OFF situation, where the

positive impact of the presence of _P. putida cscRABY_ on the initial growth of the cyanobacterium was less pronounced at an inoculation ratio of 1:1 compared to the situation with fewer _P.

putida cscRABY_ cells. A possible explanation might be that the higher _P. putida cscRABY_ cell densities reached within these 24 h caused a more substantial shading on _S. elongatus cscB_,

reducing light availability for photosynthesis in the cyanobacterium. In a co-culture study by Hays et al., _S. elongatus cscB_ was found to have a significant negative impact on various

heterotrophs, particularly on the gram-positive bacterium _Bacillus subtilis_11. In contrast to this, we could not identify an apparent effect of high densities of _S. elongatus cscB_ on the

growth of _P. putida cscRABY_ within 24 h, and, if any, there might be a tendency towards weaker growth of _P. putida cscRABY_ when inoculated with less of cyanobacterial cells (Fig. 1c).

In previous studies, reactive oxidative species (ROS: \({{{{{{{\rm{O}}}}}}}_{2}}^{-}\), \({{{{{{\rm{OH}}}}}}}^{\cdot }\), \({{{{{{\rm{H}}}}}}}_{2}{{{{{{\rm{O}}}}}}}_{2}\)) produced by _S.

elongatus cscB_ were shown to be the most invasive substances for heterotrophic growth11,13,17. Thus, as ROS are side products of photosynthesis, we performed the co-cultivations with or

without light in 12-well plates at 1.6 mL scale, and compared them to axenic cultures of _P. putida cscRABY_ grown under equal conditions. Two different cyanobacterial cell densities were

used for inoculation in the co-culture, as shown in Fig. 1d. Still, no statistically significant difference in the heterotrophic growth could be detected (unpaired T-test, α = 0.05).

Therefore, the formation of ROS through photosynthesis had no detectable influence on the growth of _P. putida cscRABY_ under the conditions tested. INFLUENCE OF ILLUMINATION, INDUCTION, AND

INOCULATION TIME OF _P. PUTIDA CSCRABY_ ON _S. ELONGATUS CSCB_ To analyse the co-culture in more detail, we switched the cultivation to the HD-9.100 CellDeg platform system, which permits

parallel co-cultivations under comparable conditions and ensures high reproducibility. We started by analysing the effect of the illumination profile, the induction of sucrose secretion, and

the time point of induction on the growth of _S. elongatus cscB_ (Fig. 2). With a constant illumination of 150 µmol m−2 s−1 and without IPTG induction, _S. elongatus cscB_ grew with a rate

of 0.064 ± 0.001 h−1. The presence of 0.1 mM IPTG in the culture, however, nearly halved the growth rate to 0.036 ± 0.004 h−1 (Fig. 2a and Supplementary Note S2). This effect is not due to

negative feedback of the sucrose accumulated in the medium, as confirmed by growing the cells in the presence of sucrose (Supplementary Note S3 and Supplementary Fig. S2), but rather

suggests a rechannelling of the fixed carbon into sucrose secretion instead of biomass formation. This is in accordance with previous studies, where it was reported that induction of the

CscB symporter reduces the biomass accumulation of _S. elongatus cscB_ while increasing the total carbon fixation10,18. Next, we analysed the influence of an exponential light profile on the

cyanobacterial growth behaviour (Fig. 2b). As observed with the constant illumination, induction of the sucrose secretion resulted in a decreased growth rate (Supplementary Table S1).

However, here, the effect of induction was way more severe than in conditions of constant light, as cultures did not just have reduced growth rates but also went into a state of

photobleaching manifested as visible pigment loss after 80 h at ~300 µmol m−2 s−1. By shifting the induction to day 2, instead of adding the inducer from the beginning of the cultivation,

this effect could be avoided, and growth rates reverted to what was observed in the absence of IPTG (Supplementary Table S1). Furthermore, a higher amount of sucrose could be measured in the

supernatant of the culture, in which _cscB_ expression was induced on day 2. These cultures accumulated three times more sucrose after 63.5 h than those growing from the beginning in the

presence of the inducer. This trend persisted, and after 87.5 h, the level of sucrose accumulated had risen to 0.75 ± 0.38 g L−1, while the cultures grown in the presence of IPTG from the

beginning exhibited only a slight increase (see Supplementary Note S4 and Supplementary Fig. S3). Taken together, the interplay between the time point of induction and the illumination

profile emerged as a significant factor influencing the growth behaviour of the cyanobacterium. We hypothesise that a prolonged phase after inoculation without induction of _cscB_ expression

facilitated a better adaptation of _S. elongatus cscB_ to the environmental conditions19. This photoacclimatisation, in turn, could lead to notable differences in photosynthetic activity,

resulting in enhanced growth, increased sucrose accumulation, and protection against photobleaching. After analysing the cyanobacterial growth in axenic cultures, we set out to study the

co-cultivation with _P. putida cscRABY_. We observed that under the detrimental conditions of an exponential light profile and initial induction of sucrose secretion, the presence of _P.

putida cscRABY_ in the co-culture rescued _cscB_-expressing _S. elongatus cscB_ from photobleaching and also led to a higher cyanobacterial growth rate (Fig. 2c). Additionally, the time

point of inoculation of _P. putida cscRABY_ had an influence on the growth behaviour and growth rate of _S. elongatus cscB_. The addition of _P. putida cscRABY_ to the culture within the

first 50 h (inoc. day 1, inoc. day 2) had a positive effect on the growth of the cyanobacterium. For instance, when _P. putida cscRABY_ was inoculated on day 1, _S. elongatus cscB_ exhibited

a 32% higher growth rate in comparison to the cultures not expressing _cscB_ and a 61% higher growth rate compared to the cyanobacterial cells expressing _cscB_ (Table 1). Inoculation on

day three, however, could no longer restore the growth behaviour to the one observed with _S. elongatus cscB_ grown without IPTG. These observations prompted the question of why the presence

of _P. putida cscRABY_ has a growth-promoting effect on _S. elongatus cscB_. One possible explanation could be the physical protection from high light intensities. For _S. elongatus_ PCC

7942, the parental strain of the derivative used in this study, light intensities higher than 400 µmol m−2 s−1 are already considered as high light and intensities of 200 µmol m−2 s−1 can

induce oxidative stress, which is noticeably lower than for other cyanobacteria20,21. To test this hypothesis, we chose two non-harmful constant light intensities, 50 µmol m−2 s−1 (low) and

120 µmol m−2 s−1 (medium), and compared the growth of _S. elongatus cscB_ in axenic culture to that in the co-culture (Fig. 2d). We observed a similar growth-promoting effect of the presence

of the co-culture partner, as described above with the exponential light profile, even though the presence of the heterotrophic partner under these low light conditions should rather hamper

the growth of the cyanobacterium due to shading from light. For instance, with a constant illumination of 50 µmol m−2 s−1, the presence of _P. putida cscRABY_ prevented _S. elongatus cscB_

from an early entry into the stationary phase and increased its growth rate by 64% compared to the cells not expressing _cscB_ and even by 82% compared to cells expressing _cscB_ (Table 1).

The same trend was observed with a medium light intensity. From these experiments, we concluded that the growth-promoting effect of the presence of _P. putida cscRABY_ on _S. elongatus cscB_

cannot be reduced to the mere protection from high light intensities but rather is the result of a more complex interplay of different factors. As _S. elongatus cscB_ grown under

non-inducing conditions was not drastically affected by the different light profiles, we assumed that sucrose secretion contributes to the stress perceived by _S. elongatus cscB_ that

finally leads to photobleaching and reduced growth. In order to see whether the population ratio provides a hint at the origin of the positive effect on the cyanobacterium, we determined the

cell ratio of phototrophic to heterotrophic cells in the co-cultures (Fig. 2e). Although the initial inoculation ratio was the same with 34% of _P. putida cscRABY_ to 66% of _S. elongatus

cscB_, varying phototroph:heterotroph cell ratios adjusted themselves over time, consequently leading to different shading conditions in each experiment. Nevertheless, the overall behaviour

of the co-cultures was comparable irrespective of the time point of addition of the heterotrophic partner (compare Fig. 2c), hinting again towards more complex processes being involved in

the growth promoting effect observed by the addition _of P. putida cscRABY_. In order to gain a deeper understanding of these processes and the possible interplay between _P. putida cscRABY_

and _S. elongatus cscB_, which extends beyond the mere exchange of sucrose, we chose the exponential light profile for a comprehensive OMICs-driven investigation. CO-CULTURE REFERENCE

EXPERIMENTS FOR MULTI-OMICS ANALYSIS To analyse the interplay between _P. putida cscRABY_ and _S. elongatus cscB_ and to find a hint at what might be the origin of the remarkable increase in

the growth rate of _S. elongatus cscB_ grown in co-culture, we aimed to compare the co-culture with the axenic cultures of _S. elongatus cscB_ and _P. putida cscRABY_, respectively.

Therefore, we set up a reference procedure in the 9-fold parallel photobioreactor system, which allowed us to have comparable conditions in all three different setups, each in biological

triplicates, for the analysis of the transcriptome, proteome, and metabolome (Fig. 3a). To achieve comparable growth rates of _P. putida cscRABY_ in axenic cultures and in the co-culture, we

adjusted an external sucrose feed for axenic _P. putida cscRABY_ cultivations that mimicked the cyanobacterial sucrose secretion. In order to calculate the sucrose feeding rate, the biomass

formation was connected with the sucrose uptake by the heterotrophic partner (calculations see Supplementary Note S5). However, achieving consistency was challenging as the growth rate of

_P. putida cscRABY_ in the co-culture showed a high variance between different experiments, assumingly due to variations in the temperature. Therefore, for the OMICs, two experiments that

showed comparable temperature profiles (see Supplementary Note S6 and Supplementary Fig. S4) have been chosen, from now on referred to as EI and EII (Fig. 3b). Samples for transcriptomics

and proteomics were derived from EII and samples for metabolomics from EI at 27.0 °C or 27.4 °C, respectively. Thus, for each of the OMICs experiments, external conditions, such as light or

temperature, were identical, as all samples were taken from the same experiment conducted in the 9-fold parallel photo-bioreactor. Growth of the phototrophic partner was highly reproducible

in the co-culture compared to the axenic culture (Fig. 3b, Supplementary Note S7 and Supplementary Fig. S5) in both experiments. As observed before, the growth was enhanced in the

co-cultures compared to the axenic cultures. An early entry of the cyanobacterial cells into the stationary phase and visible photobleaching after 85 h was prevented. Furthermore, cells of

_S. elongatus cscB_ grown in co-culture had a smaller size, which fits well with the more than doubled specific growth rate (Table S2 and Supplementary Note S8). The growth of _P. putida

cscRABY_ depends on the cyanobacterial sucrose secretion in the co-culture and on the sucrose feed in the axenic culture. Although the growth rates of _P. putida cscRABY_ were different

between both experiments, they did not differ within the same experiment (Supplementary Table S2). This was important for the -OMICs, as these were the conditions to be compared. No

differences in the cell size of _P. putida cscRABY_ in co-cultures or axenic cultures could be observed, which aligns with the expected outcome due to the same growth rate (Supplementary

Fig. S6). We assumed sucrose to be the growth limiting factor in both cultures, and in fact, in the axenic cultures of _P. putida cscRABY_ and the co-culture, no sucrose could be detected at

the time point when the samples for the OMICs were taken (Supplementary Fig. S7). As _P. putida cscRABY_ is limited by the carbon source in all cultures, we set out to analyse the medium

components citrate and phosphate and other commonly known overflow metabolites of _P. putida_. We detected a transient accumulation of acetate and ethanol, but both substances were

completely taken up again at the end of the process, assumingly by _P. putida cscRABY_ itself, as _S. elongatus cscB_ does not harbour typical genes for acetate uptake. The BG11+ medium

contains citric acid, which was completely consumed within 13.5 hours in all cultures. However, after 40 h, citric acid concentration increased again, particularly in cultures with _P.

putida cscRABY_. At first glance, this is counterintuitive, as under carbon limitation, cells should coordinate their energy and maintenance needs. However, a recent study showed that _E.

coli_ secrets overflow metabolites to address carbon-nutrient imbalances22. Thus, apart from resulting from cell lysis, this might also be the case here (Supplementary Fig. S7). A gross

estimation of the photosynthetic activity of _S. elongatus cscB_ is possible, as the amount of carbon fixed can be approximated by taking into account biomass production and sucrose

accumulation (Supplementary Note S10). It has already been described by others that inducing sucrose secretion resulted in increased overall CO2 fixation in _S. elongatus cscB_10,18,23. This

is explained by the idea that heterologously implemented sucrose production serves as a sink for the carbon captured in the Calvin cycle, thereby alleviating the so-called photosynthetic

sink limitation. Sink limitation describes the situation when photosynthetic activity is reduced due to insufficient withdrawal of products from the Calvin cycle17. In the co-culture, the

constant pull on the sucrose production by the heterotroph seems to lead to a more efficient utilisation of the captured carbon and thereby contributes to overcoming sink limitation. We

observed that in the co-culture, the photosynthetic activity of _S. elongatus cscB_ was even higher than under inducing conditions, as the growth rate surpassed that of the cells not

expressing _cscB_, and as additionally heterotrophic growth was supported by sucrose secretion (Supplementary Table S3 and Supplementary Fig. S8). OVERVIEW OF MULTI-OMICS IN THE CO-CULTURE

PROCESS As described above, the presence of _P. putida cscRABY_ in the co-culture had a positive effect on the growth of _S. elongatus cscB_. In the next step, we aimed to get insights into

the inter-species interaction in the co-culture. This is not only interesting from a fundamental research perspective but will also contribute to enhancing our understanding of co-culture

stability and, eventually, even help to improve the production of value-added products in co-cultures in general. Therefore, transcriptomics, proteomics, and metabolomics were performed (see

Supplementary Note S11). Samples for multi-OMICs were taken at ~60 h and processed as described in the methods section. The time point was chosen to be in the second half of the growth

phase before cells entered the stationary phase to ensure a sufficiently high number of cells for the analysis (compare Fig. 3a). The co-culture was considered as the case of interest and

compared to the axenic cultures, which were considered as the controls. This provided us with a snapshot to describe the cellular status at the time of sampling. In all the datasets, we

could pinpoint distinct clusters that were specific for either the co-culture or the axenic cultures, respectively. Exemplarily, this is demonstrated in Fig. 4a, b for the metabolome data,

which was used for a principal component analysis. Thresholds for differently expressed genes (DEGs) and differently abundant proteins (DAPs) were set at |log2(FC)| >1.0 and a _p_-value

(adjusted, false discovery rate (FDR) corrected) <0.05. For different abundant metabolites, the threshold was the adjusted _p_-value of 0.05 and a mean difference of 0.3. Comparing the

transcriptome of the co-culture to the axenic culture, in _P. putida cscRABY_, a total of 488 differently expressed genes (DEGs) were identified, of which 303 were up-regulated, and 145 were

down-regulated (Fig. 4c). In _S. elongatus cscB_ a total of 790 DEGs were identified. Of these 324 genes were found to be up-regulated, while 466 were down-regulated in comparison to

axenically grown cells (Fig. 4d). Gene set enrichment analysis based on the KEGG Orthology database indicated that in _P. putida cscRABY_, pathways involved in arginine biosynthesis, carbon

metabolism, glyoxylate and dicarboxylate metabolism, as well as two-component systems, were impacted by the presence of the co-culture partner (Supplementary Fig. S9). For _S. elongatus

cscB_, pathways involved in arginine/proline metabolism, photosynthesis, pyruvate, glycolipid, and biosynthesis of secondary metabolites were identified in the analysis to be the most

affected ones. The analysis of the proteome revealed 69 proteins in _P. putida cscRABY_ to be more abundant, and 27 proteins showed less abundance when compared to the axenic culture (Fig. 

4e). In _S. elongatus cscB_, a total of 92 proteins were identified to be more abundant in co-culture, and 91 proteins were less abundant (Fig. 4f). The majority of proteins identified in

_P. putida cscRABY_ belong to the category amino acid metabolism and transport, and the majority identified in _S. elongatus cscB_ belong to the group of photosynthesis or the category

stress. This trend was also observed in the transcriptome (see above). In general, the match between transcriptomics and proteomics regarding the identification of processes and direction of

regulation is the range of what is described as the regular magnitude24 (Supplementary Fig. S10). Analysing the metabolome, in total, 876 features could be identified in the cells with the

HILIC-MS measurements (− and + MS-mode), and 1013 features were identified with RP-MS (− & + MS-mode). When comparing the features obtained in the co-culture grown cells to those

identified _P. putida cscRABY_ grown in axenic culture, 336 features for HILIC (− & + MS-mode) and 427 for RP (− & + MS-mode) fulfilled the conditions set (see Supplementary Fig. S11

for Volcano plots). A comparison of the features identified in the co-culture grown cells to those obtained in _S. elongatus cscB_ grown in axenic culture revealed 143 features that

fulfilled the threshold set for HILIC-MS measurement (− & + MS-mode) and 254, which fulfilled it for the RP-MS (− & + MS-mode). Most features were more abundant in the co-culture

than in the respective axenic culture. By reference measurements, some metabolites could be identified (Supplementary Fig. S12). Most of them participate in sugar metabolism or belong to the

group of phospholipids, amino acids, or fatty acids. CELLULAR PROCESSES AFFECTED BY THE CO-CULTURE PARTNER: CORE METABOLISM AND PHOTOSYNTHESIS The analysis of the multi-OMICs data yielded a

large number of genes and proteins that were differentially expressed or abundant when comparing co-cultivated cells to those cultivated in axenic cultures. Additionally, on the metabolome

level, some differences were identified. To get a first overview of the cellular processes that were mainly affected by the presence of the respective co-culture partner, we sorted the DEGs,

DAPs, and metabolites into different groups according to their putative function (Fig. 5). In _P. putida cscRABY_ the presence of the phototrophic partner led to changes in various cellular

processes, namely in the core metabolism, transport of amino acids, nitrogen, small acids, and sugars, but also in the general stress response, detoxification, and degradation. In _S.

elongatus cscB_, we found that the presence of the heterotrophic partner likewise had an effect on the core metabolism but also on photosynthesis, which was somehow expected as _S. elongatus

cscB_ exhibited a higher growth rate in the co-culture. Furthermore, other processes connected to stress, detoxification, or transport of sulphur and iron were affected. In the following,

these processes will be discussed in more detail. In _P. putida cscRABY_ several genes, proteins, and metabolites with a potential function in the core metabolism were identified to be

affected by the presence of _S. elongatus cscB_ in the co-cultivation. More specifically, on the proteome as well as on the transcriptome level, processes that are connected to the amino

acid (AA) synthesis or degradation, the TCA cycle, or to the fatty acid (FA) metabolism were affected (Fig. 6a, b). Additionally, some genes encoding proteins involved in the

Entner-Doudoroff-Embden-Meyerhof-Parnas (EDEMP) cycle were differentially regulated (Fig. 6b). On the metabolome level, the metabolites identified mainly belonged to the group of amino acids

(Fig. 6c). As a rhizobacterium, _P. putida_ is specialised for the uptake and metabolisation of amino acids25. In line with this, it was not surprising that the expression of genes, as well

as the abundance of proteins connected to amino acid metabolism, was affected by the presence of the co-culture partner. On the proteome level, the asparagine synthetase AsnB, responsible

for the conversion of aspartic acid into asparagine, showed the biggest differences with a log2-FC 3.9 (Fig. 6a). The corresponding transcript _asnB_, however, was down-regulated (Fig. 6b,

PP_2453 log2-FC -3.1). This demonstrates a prevailing issue of multi-OMICs analysis, which is that the correlations between proteomics and transcriptomics are only moderate24. However, these

differences may also indicate a post-transcriptional control. On the metabolome level, the amino acids L-phenylalanine, L-glutamic acid, L-aspartic acid and L-glutamine were identified to

be more abundant in the co-culture compared to _P. putida cscRABY_ grown axenically (Fig. 6c and Supplementary Fig. S12). However, the metabolites were identified in all cells grown in

co-culture and, therefore, cannot be assigned specifically to one of the co-culture partners. Another sector of the core metabolism that was affected is the TCA cycle. On the proteome level,

lower protein abundances of Idh and SucA/B/D went together with a higher abundance of the proteins AceA and GlcB, hinting towards a shut-down of the TCA cycle and a redirection of the

metabolic flux through the glyoxylate cycle (Fig. 6a). This is known to happen in _P. putida_ when degradation of aromatics or xenobiotics is necessary 26. In the transcriptome, the contrary

is observed with a slight up-regulation of _Idh_ (PP_4012 log2-FC 1.9) combined with the down-regulation of the transcript encoding AceA (PP_4116 log2-FC -2.64). Taken together, the core

metabolism of _P. putida cscRABY_ is influenced by the presence of the co-culture partner, particularly affecting processes belonging to the amino acid metabolism and the TCA cycle. In

general, the central metabolism can reflect different metabolic states of the cell, as it was described for cells growing on mixtures of carbon sources26. Differences in the environmental

conditions, brought about by cultivation in the co-culture, might, therefore, lead to changes in the core carbon metabolism or in its periphery, such as the amino acid or fatty acid

metabolism. Alterations in the metabolism were further corroborated by the identification of secretion and re-uptake of some metabolites, such as transient accumulation of ethanol, acetate,

and citrate in the supernatant during different cultivation phases (Supplementary Note S9). When looking at the processes affected in _S. elongatus cscB_ by the presence of _P. putida

cscRABY_, it has to be kept in mind that the growth rates of axenically grown cells and cells grown in co-culture differ by a factor of about two. Variations in transcripts or proteins can

be the consequence of the higher growth rate or arise from the interaction with the co-culture partner, which encompasses specific interactions as well as non-specific effects, such as

shading or the response to metabolic signals, which might arise from secreted metabolites and/or consumed resources. These effects might also be entangled, as a positive interaction could

lead to a higher growth rate. The higher growth rate of _S. elongatus cscB_ in the co-culture is reflected by an up-regulation of many growth-associated genes, such as ribosomes, tRNAs, and

polymerases, as it was observed at the transcriptional level (Supplementary Note S12 and Supplementary Fig. S13). This was not the case for _P. putida cscRABY_, showing that a similar growth

rate in axenic culture and co-culture leads to a similar expression pattern of these genes. The higher abundance of amino acids in the metabolome of the co-culture, which was already

mentioned above, could also be the consequence of the increased metabolic activity of the cyanobacterium. However, attributing core metabolites to a specific co-culture partner is not

possible. Analysing the core metabolism of _S. elongatus cscB_ in more detail, only a few proteins showed different abundance. They can be grouped into proteins being involved in the

porphyrin metabolism, the Calvin cycle, or photosynthesis (Fig. 7a). Interestingly, the heterologously expressed sucrose transport protein CscB was less abundant in _S. elongatus cscB_ when

grown in co-culture. This is on the first glace counterintuitive, as its transcription is regulated by the IPTG inducible _P__lacUV5_ promoter and should, therefore, be constant. No

information on its transcript level is available, as the _cscB_ gene was not included in the transcriptomic analysis. We explain the lower abundance of the CscB protein by the higher growth

rate of _S. elongatus cscB_ in the co-culture. Assuming the total amount of the CscB protein produced remains constant, but cells divide more rapidly, the CscB protein is distributed across

a larger number of cells, which results in a lower abundance. In the metabolome of the co-culture grown cells, we detected a higher amount of disaccharide, which could be sucrose, which

might hint towards a higher accumulation of sucrose in the cytoplasm of the cyanobacterial cells due to decreased export activity. Other metabolites identified in the metabolome of

co-culture grown cells include compounds assigned to the biosynthesis of amino acids or purine and pyrimidine metabolism (Fig. 7c). Looking at genes and proteins involved in photosynthesis,

the effect of the different growth rates, when grown axenically or in co-culture, becomes even more obvious. In the proteome, the most pronounced change was the increased abundance of

phycobiliproteins. Additionally, the pigment-proteins phycocyanin and allophycocyanin, which are present in the light-harvesting complex, were also more abundant (Fig. 7b). In general, the

light harvesting complexes are connected to photosynthetic activity and growth. However, here, only a few proteins of the Photosystem I (PSI), Photosystem II (PSII), and the connecting

electron chain consisting of the NAD(P)H-dehydrogenase-like complex (NDH) and Plastocyanin (PQ) were detected. In the transcriptome, the opposite effect was observed: Genes encoding proteins

forming the PSI and PSII and the phycobiliproteins were down-regulated, whereby genes coding for the connecting NDH/PQ complex were up-regulated (Fig. 7d). A likely explanation for the

difference observed is provided again by the different growth rates observed in the two culture conditions. The axenically grown cells of _S. elongatus cscB_ displayed reduced growth and

manifested phenotypically evident stress effects. At the time point of sampling, cells grew linearly, suggesting a non-constant growth rate and, consequently, a dynamic state of cellular

processes, whereas the cells grown in co-culture exhibited exponential growth with a constant growth rate, indicating an intracellular steady state of transcripts, proteins, and metabolites.

As a result, transcripts and proteins may be differently affected when comparing the co-culture to the axenic cultures of _S. elongatus cscB_. Photosynthesis is highly regulated, for

instance, by the PSI:PII ratio27. In order to cope with excess energy, photosynthetic organisms regulate their electron transport chain (ETC) to prevent the production of ROS. Another

mechanism for encountering photooxidative stress in high-light conditions involves the protein pair IsiA and IsiB. Both proteins were more abundant in the co-culture, IsiA with a log2-FC of

5.7 and IsiB with a log2-FC of 4.5, which was amongst the highest increases detected at the protein level (Fig. 7b). IsiA is annotated as an iron stress induced chlorophyll-binding protein

and IsiB as a flavodoxin. Consistent with iron induced stress, two other proteins linked to iron limitation, IdiA and IrpA were notably more abundant in the co-culture grown cyanobacteria

(see Table 2). However, we could not detect a stronger iron limitation for _S. elongatus cscB_ in the co-culture, as iron supplementation or limitation had no discernible effect on the

cultures (Supplementary Note S13 and Supplementary Fig. S14), nor was the higher protein abundance of IsiA and IsiB reflected in the transcriptome. Additionally, at the proteome level, some

proteins associated with porphyrin biosynthesis were identified, with the majority showing higher abundance (Fig. 7a), potentially linked to observed variations in photosynthesis. CELLULAR

PROCESSES AFFECTED BY THE CO-CULTURE PARTNER: TRANSPORT Microbes often rely on their capacity to efficiently utilise a wide range of resources, which can be a critical factor in their

competitiveness and overall performance in relation to other microorganisms. Consequently, they have developed numerous strategies for acquiring compounds from their surrounding environment.

Most microbial interactions require a form of uptake of substrates, signals in diverse forms, or toxins. In line with this, we have identified various transporters to be differentially

regulated in the co-culture versus axenic cultures (Fig. 8). In general, we observed that in _P. putida cscRABY_, transporters are more likely up-regulated, whereas the opposite is the case

in _S. elongatus cscB_. This might originate in the individual lifestyles of each of the co-culture partners, as a strict autotrophic and anabolic mode needs less transport of organic carbon

compounds compared to a heterotrophic lifestyle. In _P. putida cscRABY_ at the transcriptome level, the DEGs related to transport are diverse and include genes encoding putative

transporters for amino acids, nitrogen, small acids, and sugars (Fig. 8a). While in the group of amino acid transporters, the transcription of the corresponding genes showed regulation in

both directions, in the group of nitrogen transport transcription was down-regulated and in the groups of small acids and sugar transport, transcription was mainly induced. The most highly

up-regulated transcript was PP_4604, found in the group of amino acid transport. It encodes a putative transporter belonging to the EamA family, which in _E. coli_ is related to transport of

cysteine-derivatives28. Directly downstream of this gene, a gene encoding an AraC-type regulator (PP_4605, log2-FC 5.3), was found to be highly up-regulated as well. A putative connection

between these two genes is predicted by the string-database. Down-regulation was observed for some genes encoding ABC-transporters for glutamate/aspartate uptake (_gltJ_ log2-FC -2.6, _gltP_

log2-FC −2.4, and _gltK_ log2-FC −2.7), whereas genes encoding proteins connected to the transport of other amino acids derivatives, such as putrescine or spermidine (_potA_ ATB-binding and

PP_0412 substrate binding with a log2-FC of 1.5 and 1.0) were up-regulated. At the proteome level, the latter one, PP_0412, was also identified to be more abundant. Additionally, the

proteins SpuD and YhdW, annotated as polyamide transporter, were found to be more abundant in _P. putida cscRABY_ (Fig. 8c). The down-regulation of the transcription of genes encoding

nitrogen and urea transporter, for instance _amtB_ encoding an ammonium transporter, or _urtABC_, coding for a urea transporter, fit well to the downregulation of the _ureABCD_ cluster

encoding a urease for urea degradation. In line with this, at the protein level, the global regulators NtrB and NtrC, which are responsible for nitrogen regulation, are less abundant. At

first glance, it is not intuitive, that many genes involved in transport are differentially regulated in the co-culture, as, neglecting the small amount of citrate in BG11+ medium, the sole

carbon source is sucrose secreted by the phototrophic partner. However, at a global ecological scale, cyanobacteria including _Synechococcus spp_., are well-known to drive marine bacterial

communities because they are the main suppliers of organic matter due to cell death, cell lysis and leakiness to photosynthate or exudates16,29. In artificial seawater medium (nutrient rich)

_Synechococcus_ cultures accumulated up to 200 µg mL−1 carbohydrates and 400 µg mL−1 proteins16. Furthermore, it is described that cyanobacteria can secret amino acids and other components.

For example, in the supernatant of _S. elongatus_ CCMP 1631 tryptophan and phenylalanine were found5,30. Moreover, _P. putida_ is able to colonise plant roots and was shown to exhibit

advanced chemotaxis towards polyamides, which are a component of complex root extrudates31,32. Thus, in a more general view, it is plausible that transporters for carbon, carbon-nitrogen

compounds, or nitrogen in _P. putida cscRABY_ are affected by the presence of the phototrophic co-culture partner. In _S. elongatus cscB_ the DEGs encoding proteins related to transport

mainly belong to the group of transporters for iron or sulphur (Fig. 8b). Almost all of them were down-regulated, with the exception of Synpcc7942_0197, which was the most highly

up-regulated gene, encoding a putative folate/biopterin family MFS transporter (log2-FC 2.8). Pterins are ubiquitously occurring molecules, which are needed by cyanobacteria for pigment

generation, phototaxis, and UV protection33. In the group of genes related to iron transport, the _futABC_ operon encoding siderophores responsible for iron uptake (_futA2_ log2-FC −1.6,

_futB_, log2-FC −3.1, and _futC_ log2-FC −1.74) was down-regulated (Fig. 8b). In the group of genes encoding sulphur transporters, the strongest down-regulation was observed for

Synpcc7942_1681, annotated to encode a sulphate/sulfonate transporter. Synpcc7942_1682, and Synpcc7942_1722, also encoding putative sulphate/sulfonate transporters, were likewise

down-regulated. Other genes encoding putative sulphite exporters were slightly up-regulated (Synpcc7942_0935, Synpcc7942_0238). Sulphur is an essential element for microbes and participates

in iron-sulphur clusters, a common co-factor of proteins, in many important physiological processes including photosynthesis, DNA/RNA modification, and purine metabolism34. Sulphite is cell

toxic and arises from the intracellular breakdown of metabolic products, including sulphur-containing amino acids, which boosts ROS generation35. Regulation of the transcription of genes

potentially involved in sulphur or sulphite transport hints towards differences in the complex processes of sulphur homeostasis in _S. elongatus cscB_, when grown in co-culture with _P.

putida cscRABY_. In conclusion, iron and sulphur transport seems to be down-regulated in _S. elongatus cscB_ when growing together with the heterotrophic partner. On the protein level, only

three proteins associated with transport were identified to be differentially abundant. Two of them, annotated as substrate-binding protein of an iron transport system (Synpcc7942_1409) and

as a hypothetical porin (major outer membrane protein, Synpcc7942_1607) showed a higher abundance whereas another porin (Synpccc7942_1635) was less abundant in the co-culture grown _S.

elongatus cscB_ cells (Fig. 8c). CELLULAR PROCESSES AFFECTED BY THE CO-CULTURE PARTNER: DETOXIFICATION, DEGRADATION AND STRESS Next, we analysed the group of regulated genes and proteins,

that can be functionally related to detoxification, degradation, and stress. Hays et al. had observed a negative effect of _S. elongatus cscB_ on the growth of respective heterotrophic

partner11, however as described above, we have not seen this effect on _P. putida cscRABY_ in small-scale experiments. In the co-culture setup, both organisms experience multiple situations

that could cause different types of stress. One situation they have to cope with is the increased salinity conferring high ionic strength and external osmotic pressure. Additionally, light

can induce oxidative stresses and the adaptation to changes in the illumination can be a further stress factor. However, these external factors are comparable in both conditions, the axenic

cultures and the co-cultivation, thus differences that are identified in transcript or protein abundance related to stress signals are regarded to be specific for the presence of the

respective co-culture partner. Our data indicate that several processes assumingly connected to stress in _P. putida cscRABY_ are affected by the presence of _S. elongatus cscB_ (Fig. 9).

More specifically, processes involved in the degradation of compounds, in the stress response induced by light, in the efflux of (toxic) substances, or in the general stress response were

impacted. A general trend towards up-regulation of transcription was observed. For instance, the transcription of the genes belonging to the _ben_- and _cat_-operons encoding enzymes

responsible for the degradation of benzoate were up-regulated (Fig. 9a, b). The gene encoding the AraC-type regulator BenR, however, was slightly down-regulated (log2-FC −1.2). The

transcription of a gene cluster (PP_0738 to PP_0742) that might be connected to light-induced stress was found to be up-regulated (Fig. 9b): One of its genes (PP_0739) encodes a putative

deoxyribodipyrimidine photolyase and another PP_0740 encodes a putative MerR family transcriptional regulator of light-inducible genes, known as PplR136. As the illumination was identical

for the axenic culture of _P. putida cscRABY_ and the co-culture, the light intensity per cell assumingly was lower in the co-culture due to the higher cell densities. Therefore, changes in

the expression of these genes might be traced back to a different stress situation caused by the presence of the co-culture partner. Most of the genes that were differentially expressed and

are associated with the efflux of substances are up-regulated (Fig. 9c). Many are annotated to encode putative resistance-nodulation-division (RND) efflux pumps, such as Mex-RND and

TolC-RND, which are responsible for the removal of toxic compounds. The highest up-regulation of transcription was detected for the genes PP_2817 and PP_2731 encoding putative multidrug

efflux pumps with a log2-FC of 2.1 or 3.2, respectively. Furthermore, genes encoding proteins that can be connected to perceiving and combatting stress were differentially regulated, most of

them showed upregulation in the co-culture. As mentioned above, ROS derived from the cyanobacterium’s photosynthesis is likely to be one of the major stress factors for heterotrophic

partners. In line, one gene encoding a catalase (PP_2887 log2-FC 1.2) was found to be slightly up-regulated and, on the protein level, the catalase KatG was more abundant. However, only one

of the two major cellular ROS degrading regulators, SoxR (PP_2060 log2-FC 1.1), was found to be marginally up-regulated. Another mechanism in the antioxidant defence is the glutathione

metabolism11,37. However, no genes encoding proteins associated with glutathione metabolism could be identified to be differentially regulated, though, on the metabolome level, metabolites

belonging to the glutathione metabolism were detected in the co-culture cells (Fig. 9e). The transcription of genes belonging to the _cop_ and _czc_-operons was mainly up-regulated

(Supplementary Note S14 and Supplementary Table S4). Their gene products are involved in copper homeostasis and the cytoplasmic detoxification of copper and silver ions, a vital process

controlled by the CopR/CopS two-component system (Supplementary Fig. S15). Taken together, these findings indicate that _P. putida cscRABY_ experienced a general stress situation, which is

also corroborated by the up-regulation of genes encoding putative transcriptional regulators connected to stress38 (e.g. PP_0740 log2-FC of 3.3). We assume that the heterotrophic partner

still has some capacity left to react to stresses, as certain stress answers, for example the glutathione metabolism, do not yet seem to be affected by the co-cultivation. This underlines

that _P. putida cscRABY_ is a well-fitting co-culture partner for _S. elongatus cscB_ due to its natural tolerance towards all different kind of stresses. By analysing the genes, proteins,

and metabolites related to the stress response in _S. elongatus cscB_ we have identified processes involved in redox reactions, efflux, general stress and ion homeostasis (Supplementary Note

S15 and Supplementary Table S5). As already mentioned above, one of the key compounds to combat redox stress is glutathione, and oxidised glutathione (GSSG), L-glutamate and

γ-glutamylglutamic acid and these were more abundant in the co-culture cells compared to either axenic culture (Fig. 9d). the transcription of genes encoding putative glutathione peroxidases

(Synpcc7942_0437 and H6G84_RS08920) or a thioredoxin peroxidase _tpxA_ (log2-FC 1.3) were up-regulated in cyanobacterial cells grown in the co-culture (Fig. 9e). Another way to handle

oxidative stress is by the Glutathione-independent degradation of H2O2, performed enzymatically by catalases, peroxidases, and superoxide dismutase. Interestingly, the transcript of the

catalase KatG was considerably down-regulated with a log2-FC -5.36 in the co-culture growing cyanobacterium. However, the superoxide dismutase SodB was up-regulated with a log2-FC 1.5 in _S.

elongatus cscB_ grown in co-culture (Fig. 9f). As already observed for the heterotrophic partner, alterations were also found in the sector of efflux processes and general stress (Fig. 9f).

DEGs encoding different types of efflux transporters, such as HlyD-family efflux transporters (Synpcc7942_1224) or RND efflux transporters (Synpcc7942_1869, Synpcc7942_1870) were mostly

up-regulated in the co-culture, as was the transcription of genes that encode proteins putatively involved in stress response. In general, in cyanobacteria, high-light-inducible proteins

(Hlip) are expressed in response to various exogenous stresses, including already moderate light intensity39,40. In this study, two transcripts encoding these proteins were also found to be

up-regulated (Synpcc7942_1997 and Synpcc7942_1120) (Fig. 9f). Three FtsH proteases, responsible for protein homeostasis of the thylakoid membrane in photooxidative stress situations, were

up-regulated on the transcriptional level (Synpcc7942_1820, Synpcc7942_0998, and Synpcc7942_0942), but on the protein level the proteins FtsH, FtsH.1, and FtsH.3 (Synpcc7942_0297,

Synpcc7942_0942, and Synpcc7942_0998) were less abundant in the co-culture (Fig. 9f for transcripts and Supporting Information S15 for proteins). This is another example of the discrepancies

between transcriptomics and proteomics results24. Taken together, both co-culture partners showed differential regulation in processes connected to various stresses, detoxification and

degradation when grown together. Even though _S. elongatus cscB_ suffered from severe photobleaching and reduced growth in the axenic culture, the transcription of many genes encoding

proteins involved in stress response was mostly up-regulated in the co-culture and not vice versa. In summary, the multi-OMICs analysis of the co-culture provided us with a snapshot of the

cellular status at the time of sampling and revealed multi-layered signals of small changes. Thus, in addition to the synthetic connection by sucrose, more links have to be integrated into

the mechanistic model of the co-culture. We propose to incorporate the competition for common resources, such as medium components, including citrate and various salts, as we have observed

transient uptake or accumulation of citrate, acetate, and ethanol. Furthermore, in both organisms, the ion homeostasis was unbalanced, which might indicate limitations or reduced

accessibility of ions through advanced scavenging strategies of the respective co-culture partner. This highlights the requirement for careful medium optimisation in co-cultures in general.

The up-regulation of transport processes, particularly for amino acids and degradation of aromatic compounds in _P. putida cscRABY_, suggests the exchange of molecules belonging to these

groups. Further studies will be directed to investigate the metabolites in the supernatant to determine whether amino acids or other compounds accumulate. As this phenomenon has been

previously reported in growing cultures of _S. elongatus_ CCMP 163130 and is commonly observed in marine cyanobacteria consortia16. Setting up a precise mechanistic model of co-cultures will

contribute to better controllability and stability in multi-species processes and enable upscaling and exploitation for biotechnological applications. However, it is challenging to

translate the results of a general grow-associated classification of microbial interactions, e.g., positive/neutral/negative, and the comprehensive results obtained by a multi-OMICs analysis

into quantitative, predictive models. Nevertheless, combining phototrophic and heterotrophic organisms holds great potential for co-culture applications, as it combines different metabolic

regimes and thus can link CO2 fixation to diverse metabolic traits. The ability to efficiently utilise and recycle carbon offers innovative solutions to address environmental and industrial

challenges, making these partnerships a promising avenue for future biotechnological advancements. Our findings contribute to a deeper understanding of co-culture dynamics and may, at the

end of the day, contribute to harnessing the benefits of synergistic interactions between different microorganisms in biotechnological endeavours. MATERIALS AND METHODS STRAINS AND CULTURE

PREPARATION The sucrose metabolising strain, _Pseudomonas putida_ EM178 _att::Tn7 cscRABY_14 harbouring the _cscRABY_-operon was used as the co-culture partner for _Synechococcus elongatus_

PCC 7942 _cscB_10_. S. elongatus cscB_ pre-cultures were first grown in BG11+ medium41 under continuous illumination of 22 µmol photons s−1 m−2, 30 °C, and 120 rpm in an orbital shaker

(Multitron Pro from Infors HT, Switzerland) without additional aeration. After reaching the stationary phase, the cultures were transferred to BG11+ medium supplemented with 150 mM NaCl,

inoculated with a 1:20 ratio, and grown under the same conditions. These salt-adapted phototrophic cultures were used for all experiments. Pre-cultures of _P. putida cscRABY_ were grown in 3

 mL LB-medium at 30 °C and 220 rpm14, and subsequently transferred to a second pre-culture consisting of 3 mL BG11+ medium with 3 g L−1 sucrose. In the reference experiments, the stationary

_P. putida cscRABY_ cultures were transferred into BG11+ medium supplemented with 150 mM NaCl and 1–3 g L−1 sucrose in 100 mL shake flasks and grown under the same conditions as the

pre-cultures. The cultures were centrifuged at 4000 × _g_ for 5 min. and then resuspended in fresh BG11+ supplemented with 150 mM NaCl before being added to the process vessels.

PHYSIOLOGICAL INVESTIGATION OF CO-CULTURE IN 12-WELL PLATES (1.6 ML SCALE) To investigate potential interactions, experiments were performed in 12-well plates (Brand GmbH, Germany) at a 1.6 

mL scale. _S. elongatus cscB_ was inoculated to an OD750 of 0.05 in BG11+ medium supplemented with 150 mM NaCl, and cells were acclimated for two days to salt and other conditions (25–30 °C,

120 rpm, 20 photons µmol m−2 s−2, incubator Multitron Pro from Infors HT from Switzerland). No additional aeration was provided, and the plates were sealed with laboratory film to prevent

water evaporation. Water loss was considered by verifying the volume left in the wells at the end of the experiment. Gene expression of the transporter CscB in cyanobacterial cultures was

induced with 0.1 mM Isopropyl ß-D-1-thiogalactopyranoside (IPTG), and an extra amount of sucrose of 1 g L−1 was added to support heterotrophic growth at the beginning of the experiment.

Co-cultures were started by inoculating different cell counts of _P. putida cscRABY_ to achieve different phototroph:heterotroph ratios. For experiments in darkness, the plates were covered

in tinfoil. PHYSIOLOGICAL INVESTIGATION OF THE CO-CULTURE IN THE MEMBRANE REACTOR The CellDEG HDC 9.100 Universal Platform (CellDEG GmbH, Germany) consisting of 9 cultivators (HD100

Cultivator) mounted to the platform, an orbital-shaker, and a control unit was used. A partial CO2 pressure of 2% and different light profiles for the high-power LED light sources (RX-400

LED light Source from Valoya) were implemented (e.g. constant light of 50 or 120 µmol photons s−1 m−2 and exponential light td = 52 h). Cells were grown in BG11+ supplemented with 150 mM

NaCl and 0.1 mM IPTG for induction of sucrose permease CscB of the phototrophic partner. At the beginning of the process, the pH was set to 7.5, and no further control occurred. The overall

volume of each reactor was 95 mL, and water loss through condensation was considered by monitoring the weight of the membrane reactors during the processes. The process was started by

inoculating the cyanobacterium from a salt-adapted culture to an OD750 of 0.1–0.2 in the cultivation vessel. _P. putida cscRABY_ was added to the co-cultures to an OD600 of 0.05–0.01. Cell

count, optical density, and sucrose concentration were analysed by daily sampling of 1–2 mL of the culture broth. REFERENCE EXPERIMENT WITH DIFFERENT SETTINGS FOR COMPARATIVE OMICS ANALYSIS

The reference experiment consisted of three different settings in biological triplicates. The setting were the axenic cultures of _S. elongatus cscB_, the axenic culture of _P. putida

cscRABY_ and the co-cultures, thus 9 samples in total. The experiment started with an acclimatisation phase for the phototrophic partner (Start OD750 of 0.1–0.2) under constant light (120 

µmol photons s−1 m−2). After 14–18 h, the co-culture was started by inoculating _P. putida cscRABY_ to an OD600 of 0.05. A sucrose feed supplied _P. putida cscRABY_ axenic cultures with

external carbon. Therefore, a cap was designed to enable feeding and in situ sampling from the membrane reactor. The sucrose secreted by _S. elongatus cscB_ grown in co-culture was estimated

and used to define the sucrose feeding rate for the axenic cultures of _P. putida cscRABY_ (see Supplementary Note S5 for the calculation). A batch sucrose of 0.1 g L−1 was provided at the

beginning, mimicking the initial sucrose production of the phototrophic partner. The axenic cultures of _S. elongatus cscB_ were handled as described above. After ~60 h samples for

multi-OMICs analysis were taken, centrifuged at 4000 rpm for 5 min (10 mL proteomics) or 13,000 rpm 1 min (1 mL metabolomics and transcriptomics) at 4 °C in a centrifuge 5418R from Eppendorf

and subsequently stored at – 80 °C. SAMPLE PREPARATION AND ANALYTICAL METHODS Samples of the processes were directly used to determine the optical density at 750 nm (600 nm) and cell

counts. For further analysis, cells were separated from the medium by centrifugation at 13,000 rpm for 30 s. in a centrifuge 5418R from Eppendorf. Cell counting was carried out as previously

described12. High-performance liquid chromatography (HPLC) was used to quantify sugars, medium components and common overflow metabolites. The Agilent 1100 series, Waldbronn, Germany with a

Shodex SH 1011 column was used for sugar analysis and a Shimadzu LC2030C Plus with a Bio Rad aminex HPX-87H column for other metabolites. The flow rate for the sugar analysis was 0.45 mL 

min−1 with 0.5 mM sulfuric acid, the column was heated to 30 °C, and the refractive index (RI) detector to 50 °C. For analysing medium components and overflow metabolites, a flow rate of 0.6

 mL min−1 was used with the same aqueous solvent and an RI temperature of 40 °C. Concentrations were calculated by integration of the peak area of each peak and correlation to the

corresponding standards. MULTI OMICS METHODS TRANSCRIPTOMICS Samples were sent on dry ice to Eurofins genomic in Konstanz, Germany, for RNA isolation, sequencing, and initial bioinformatic

analysis. Results were verified and visualised using the Galaxy platform and R-studio. METABOLOMICS Samples were extracted from the cell pellets and separated using two types of columns. A

UPLC BEH Amide 2.1 × 100 mm, 1.7 µm analytic column (Waters, Eschborn Germany) with a 400 µL min−1 flow rate for hydrophilic interaction liquid chromatography (HILIC) and a Kinetex XB18 2.1

×100 mm, 1.7 µm (Phenomenex, Aschaffenburg Germany) for reverse phase chromatography (RP) with a 300 µL min−1 flow rate. A volume of 5 µL per sample was injected. The autosampler was cooled

to 10 °C, and the column oven heated to 40 °C. MS settings in the positive mode were as follows: Gas 1 55 psi, Gas 2 65 psi, Curtain gas 35 psi, temperature 500 °C, Ion Spray Voltage 5500 V,

declustering potential 80 V. The mass range of the TOF MS and MS/MS scans were 50–2000 _m/z_ and the collision energy was ramped from 15–55 V. MS settings in the negative mode were as

follows: Gas 1 55 psi, Gas 2 65 psi, Cur 35 psi, temperature 500 °C, Ion Spray Voltage –4500 V, declustering potential –80 V. The mass range of the TOF MS and MS/MS scans were 50–2000 _m/z_

and the collision energy was ramped from –15–55 V. The data was collected in the data-dependent-acquisition mode. A more detailed description of the procedure and data analysis can be found

in Supplementary Note S16. PROTEOMICS Proteins were extracted from cell pellets and trypsin-digested peptide desalting was processed using Bond Elut OMIX C18 tips (Agilent Technologies)

following the manufacturer’s instructions. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteome analysis was performed using reverse-phase LC on a Dionex Ultimate 3000 RSLC

nano 2 system coupled online to a Q Exactive HF mass spectrometer (Thermo Scientific). A more detailed description of the procedure and data analysis can be found in Supplementary Note S16.

STATISTICS AND REPRODUCIBILITY All data shown in this work is derived from three biological replicates, i.e. three different cultures that were inoculated from three different precultures,

each derived from an individual clone. The mean and the standard deviation were calculated from these three replicates. The 9-fold parallel membrane reactor system allowed to grow all

cultures at the same time, reducing variability due to other environmental factors as temperature. The reference experiment was run twice (EI and EII), and data are shown for both

experiments (Fig. 3 and Supplementary Note S7). OMICs data is also derived from three biological replicates each, and details on the data analysis are specified in Supplementary Note S16.

REPORTING SUMMARY Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. DATA AVAILABILITY The data that supports the findings

of this study are available in the supplementary material of this article. All data for transcriptomics and proteomics are presented in the supplementary file Supplementary Data 1 and

metabolomics data can be found at MassIVE (https://massive.ucsd.edu) using the data identifier MSV000092369. Source data behind the diagrams and graphs of Figs. 1, 2, and 3 is presented in

the supplementary file Supplementary Data 2. REFERENCES * Nai, C. & Meyer, V. From axenic to mixed multures: Technological advances accelerating a paradigm shift in microbiology. _Trends

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ACKNOWLEDGEMENTS This work was funded by the DFG priority program SPP2170 (InterZell) (Project Number 427887573). We thank Prof. Karl Forchhammer and Dr. Joachim Kopka for discussions.

FUNDING Open Access funding enabled and organized by Projekt DEAL. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Professorship for Systems Biotechnology, TUM School of Engineering and

Design, Technical University of Munich, Garching, Germany Franziska Kratzl, Marlene Urban, Andreas Kremling & Katharina Pflüger-Grau * Department of Chemical and Biological Engineering,

University of Sheffield, Sheffield, United Kingdom Jagroop Pandhal & Mengxun Shi * Bavarian Center for Biomolecular Mass Spectrometry (BayBioMS), TUM School of Life Sciences, Technical

University of Munich, Freising, Germany Chen Meng & Karin Kleigrewe Authors * Franziska Kratzl View author publications You can also search for this author inPubMed Google Scholar *

Marlene Urban View author publications You can also search for this author inPubMed Google Scholar * Jagroop Pandhal View author publications You can also search for this author inPubMed 

Google Scholar * Mengxun Shi View author publications You can also search for this author inPubMed Google Scholar * Chen Meng View author publications You can also search for this author

inPubMed Google Scholar * Karin Kleigrewe View author publications You can also search for this author inPubMed Google Scholar * Andreas Kremling View author publications You can also search

for this author inPubMed Google Scholar * Katharina Pflüger-Grau View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS F.K., A.K. and K.P.G.

designed the experiments. F.K. and M.U. performed the experiments. J.P. and M.S. designed and performed the proteomics and K.K. and C.M. designed and performed the metabolomics. F.K. and

K.P.G. wrote the manuscript. A.K., J.P. and K.K. carefully proofread the manuscript. All authors read and approved the final manuscript. CORRESPONDING AUTHOR Correspondence to Katharina

Pflüger-Grau. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION Communications Biology thanks Stephan Klaehn, Katja

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troubled _Synechococcus elongatus_ in a synthetic co-culture – interaction studies based on a multi-OMICs approach. _Commun Biol_ 7, 452 (2024). https://doi.org/10.1038/s42003-024-06098-5

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