Prospective CO2 and CO bioconversion into ectoines using novel microbial platforms

Microbial conversion of CO2 and CO into chemicals is a promising route that can contribute to the cost-effective reduction of anthropogenic green house and waste gas emissions and create a more circular economy. However, the biotechnological valorization of CO2 and CO into chemicals is still restricted by the limited number of model microorganisms implemented, and the small profit margin of the products synthesized. This perspective paper intends to explore the genetic potential for the microbial conversion of CO2 and CO into ectoines, in a tentative to broaden bioconversion platforms and the portfolio of products from C1 gas fermentations. Ectoine and hydroxyectoine can be produced by microorganisms growing at high salinity. They are high-value commodities for the pharmaceutical and medical sectors (1000–1200 €/kg). Currently microbial ectoine production is based on sugar fermentations, but expansion to other more sustainable and cheaper substrates is desirable. In this work, a literature review to identify halophilic microbes able to use CO2 and CO as a carbon source was performed. Subsequently, genomes of this poll of microbes were mined for genes that encode for ectoine and hydroxyectoine synthesis (ectABCD, ask, asd and ask_ect). As a result, we identified a total of 31 species with the genetic potential to synthesize ectoine and 14 to synthesize hydroxyectoine. These microbes represent the basis for the creation of novel microbial-platforms that can promote the development of cost-effective and sustainable valorization chains of CO2 and CO in different industrial scenarios.

worth more than 3.6 trillion € per year globally, but less than 5% of the chemicals are produced using biotechnological processes. Most of the existing biotechnologies use sugars as feedstock, whose price accounts for up to 50% of the production costs (Zeng 2019). Besides, utilization of sugars for the production of chemicals directly competes with food markets and is environmentally unsustainable (Ritala et al. 2017). In recent years, the microbial conversion of point source CO 2 and CO emissions have received great attention as an alternative to sugar fermentations (Nisar et al. 2021;Naresh Kumar et al. 2022). The availability of these gases is practically unlimited, flue gases are practically free, and the valorization of CO 2 and CO acts as an atmospheric pollution and climate change mitigation strategy (Metz et al. 2018). However, despite the potential for sustainable production of added-value compounds from CO 2 and CO, current physico-chemical and biological technologies are seldomly cost-effective. Physico-chemical transformation of CO 2 into industrial compounds of interest is difficult because it requires high energy input as a result of the oxidized low-energy carbon of the CO 2 molecule (Gabrielli et al. 2020). For biological CO 2 transformation, photobioreactors are the most studied and developed biological technologies (Anand et al. 2020). However, photosynthesis has a relatively low energy efficiency (less than 3%). Open systems have limitations such as uneven light intensity, evaporative losses, diffusion of CO 2 to the atmosphere, undesired contamination and a larger footprint. Closed photobioreactors are expensive and often require nitrogen dosing to improve productivity (Zeng 2019;Bose et al. 2019). CO, instead, is a high-energy molecule and can be converted using chemical synthesis. Nevertheless, chemical catalysis usually requires energyintensive conditions (e.g. Fischer-Tropsch process), a fixed H 2 to CO ratio, and costly removal of syngas impurities to avoid catalyst poisoning (Cordero et al. 2019).
CO 2 and CO can be converted using chemolithoautotrophic microorganisms in a process independent from light, known as dark carbon fixation. Chemolithoautotrophic organisms obtain their energy from redox reactions and their carbon from CO 2 fixation or/and CO. Technologically, dark carbon fixation can be a cost-effective option to transform CO 2 and CO emissions into chemical building blocks (Claassens et al. 2018). Promising cell factories are researched for the production of plastics, diesels, single cell protein and extracellular polysaccharides. Table 1 shows an overview of chemicals produced from CO 2 and CO (complete review of microbial compounds produced from CO 2 and CO, yields and references in supplementary information 1, Table S1). However, current processes apply a restricted number of model microorganisms, such as acetogens, with a small portfolio of products, mainly alcohols and organic acids (Zeng 2019). Consequently, the development of new biotechnologies that expand microbial catalysts used for transforming CO 2 and CO and their product portfolio is of great interest for the transition pathway to a fossil-free circular economy.

High-value compounds produced by microbes at high salinities: ectoine and hydroxyectoine
Microorganisms growing in high-salinity environments produce organic osmolytes (also known as extremolytes) to protect cell integrity and ensure survival (Czech et al. 2018a). Industry has spotlighted extremolytes formed at high salinity as an 'unexploited gold-mine' with opportunities for the cosmetic, medical, and food industries (Becker and Wittmann 2020). Out of all the extremolytes, ectoines (ectoine and its hydroxyectoine derivative) are highly interesting as they provide protection against an array of stress factors (e.g. salinity, desiccation, oxygen radicals, temperature, UV light) which makes of them multi-functional agents of great market value (Becker and Wittmann 2020;Liu et al. 2021). Ectoine is a cyclic imino-acid (2-methyl-1,4,5,6tetrahydropyrimidine-4-carboxylic acid) which molecular structure enhances hydrogen bonding in aqueous solution (Czech et al. 2018a). This allows ectoine to bind water and form an ectoine-hydrocomplex that acts as a protective hydro-layer on cell surfaces and macromolecules. This makes ectoine a highly effective stabilizer of proteins, DNA-protein complexes, nucleic acids, cell membranes and tissues (Fig. 1). Current ectoine retail value is of approximately 1000 € kg −1 and market trends indicate that ectoine's importance in the chemical industry will continue to grow inferred from its novel commercial products (Liu et al. 2021). Bitop AG manufactures 90% of the current market needs (data 2016), but other companies such as Bloomage Biotechnology Corporation are starting to produce ectoine industrially (Becker and Wittmann 2020). The primary application is the cosmetic industry: mainly for skin and hair care products (industrial producers: Bitop AG, DADO-cosmed GmbH, Börlind Gesellschaft für, Jan Dekker). There is also expansion towards the medical industry due to ectoines anti-allergenic and anti-desiccant properties. Companies like Johnson & Johnson, Pari GmbH, and Bitop AG are already incorporating ectoine in nasal sprays, eye drops, mouth and throat spray, anti-dermatitis creams and lung inhalation fluids (Becker and Wittmann 2020).
Although the pathway for ectoine production can vary slightly from one organism to another, it involves three specific enzymes encoded by the conserved gene cluster ectABC ( Fig. 1): l-2,4-diaminobutyric acid (DABA) transaminase (EctB), DABA acetyltransferase (EctA) and ectoine synthase (EctC). EctC is considered a marker protein for ectoine producers (Czech et al. 2019). Sometimes an additional gene of

Sesquiterpene (E)-α-bisabolene
Hydrogenophaga pseudoflava CO, CO 2 H 2 , CO aspartokinase (ask_ect) and/or the gene (ectR) for a MarR-type regulator are observed within the ectABC cluster (Czech et al. 2018a). Members of Halomonas, Brevibacterium and Chromohalobacter genera can naturally accumulate high concentrations of ectoine (15% cdw) when cultured at high salinity with simple sugars and yeast extract. Halomonas elongata is the most widely used strain for industrial ectoine production. Bitop AG uses non-genetically modified 'superleaky' mutants of H. elongata to produce tons of ectoines yearly (Kunte et al. 2014;Becker and Wittmann 2020). These mutants excrete ectoine to the medium avoiding the need for hiper and hipo osmotic shocks to recover intra-cellular ectoine from the biomass (bio-milking process). Gene cluster for ectoine and hydroxyectoine production in Acidiphilum cryptum and general microbial biosynthetic pathway for ectoine and hydroxyectoine production in the cell. ectR encodes a MarR-type regulator; ectA, the protein diaminobutyric acid (DABA) acetyltransferase (EctA); ectB, DABA aminotransferase (EctB); ectC, ectoine synthase (EctC); ectD, ectoine hydroxylase (EctD); ask_ect, a specialized aspartokinase (Ask); Asd stands for l-aspartate-semialdehyde-dehydrogenase. Dotted box with possible mechanisms for ectoine protection in the cells. Figure created with biorender Besides this advantage, the process with 'superleaky' strains is still costly due the high amounts of glucose and oxygen required, and the corrosive characteristics of the high salinity medium (Kunte et al. 2014;Liu et al. 2021). Recent research and commercial attempts have focused on cost-effective biotechnology aimed at synthesizing ectoine at lower salinities using engineered microorganisms, however, the preference of using natural non-GMO extremolytes by the cosmetic market calls for other alternative processes (Becker and Wittmann 2020). Table 2 shows some of the high-yield ectoine producers (complete overview of ectoine producers can be found in supplementary information 1, Table S2).
A compatible solute derived from ectoine that has recently received commercial attention is hydroxyectoine (5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) (Czech et al. 2019). Hydroxyectoine is produced by a substantial amount of ectoine producers through a positionand stereo-specific hydroxylation catalyzed by an ectoine hydroxylase (EctD) (Czech et al. 2018a). Compared to ectoine, hydroxyectoine confers additional protective properties due to its hydroxylated nature and has a higher price (1200 € kg −1 ) (Becker and Wittmann 2020). Hydroxyectoine plays an important role in heat stress protection, increasing the melting temperature of DNA, and confers stronger desiccation resistance due to its  (Czech et al. 2018b) ability to form glasses (Liu et al. 2019). With the exception of some Marinococcus strains and Pseudomonas stuzeri (Schiraldi et al. 2006;Seip et al. 2011), hydroxyectoine and ectoine are naturally coproduced. Commercial strains for ectoine production, such as H. elongata, can be manipulated to increase its hydroxyectoine content (to a maximum of approximately 50%) via desiccation at extreme salinity and elevated temperature. However, growth under extreme conditions and subsequent chromatographic separation procedures increase production costs. Thus, industrial ectoines are usually sold as mixtures of ectoine and hydroxyectoine (Liu et al. 2021). As hydroxyectoine is in high demand due to its superior protective properties, current research has focused on the singular production of this analogue. The highest hydroxyectoine production yields have been obtained in cultivations with natural Halomonas species at high salinity. Although some recent research has focused on the genetic modification of non-natural producers with the aim of synthesizing hydroxyectoine as the only osmolite (Table 3; a whole overview of hydroxyectoine producers, conditions, and yields can be found in supplementary information 1, Table S3).

C 1 compounds as alternative feedstock for ectoine and hydroxyectoine production
Although there has been tremendous progress in ectoine and hydroxyectoine biosynthesis, there is little integration of sustainable feedstock and a lack of studies about the use of renewable carbon and energy sources for ectoine production. In this regard, new bioproduction systems using waste C 1 compounds as carbon supply could lower production costs, while mitigating greenhouse gas emissions. Different halotolerant aerobic methanotrophs have been identified as able to accumulate ectoine; most of them belong to the genus Methylomicrobium, e.g. M. alcaliphilum, M. buryatense, M. kenyense or M. japanense, although other bacteria such as, Methylobacter marinus and Methylohalobius cremeensis are also ectoine producers (Kalyuzhnaya et al. 2008). Out of all, M. alcaliphilum is considered the most efficient methanotrophic ectoine producer (achieving ectoine specific yields of 230 ± 20 mg g dcw −1 ) (Khmelenina et al. 2015). In fact, the process feasibility of M. alcaliphilum in bio-reactors has been demonstrated in aerobic fed-batch fermentation, reaching extracellular concentrations of 253.4 ± 55.1 mg L −1 ectoine and recoveries of ~ 70% of the total intra-cellular ectoine  (Cantera et al. 2017). Further on, the use of consortia has been preferred for the production of ectoine from CH 4 in continuous, due to their higher resilience and two times higher ectoine productivities (Cantera et al. 2020). In fact, a recent techno-Economic and Sensitivity Analysis of the production of ectoine from biogas in waste treatment facilities has demonstrated that the production of ectoine from biogas has high profitability with a net present value evaluated at 20 years (NPV 20 ) of 33.6 M€ (Pérez et al. 2021). Nevertheless, despite the enormous potential of biogas as feedstock for ectoine production, its large-scale production is still constrained by the limitation of CH 4 solubility in liquid medium and gas-liquid mass transfer, eventually resulting in low product titers. In view of overcoming these rate-limiting factor, a novel approach of using different C 1 gases with greater mass transfer, such as CO 2 , and processes that integrate aerobic and anaerobic microbes able to use CO 2 and CO as carbon source could broaden and make more sustainable this bioconversion platform.
Different approaches could be taken for the bioconversion of CO 2 and CO into ectoines. Synthetic co-cultures formed by mesophilic microorganisms able to produce organic acids or ethanol from CO 2 or CO, such as acetogens, coupled to heterotrophic ectoine producers, such as Halomonas species, could be used to enhance the valorization process of these C 1 gases. Nevertheless, two step fermentations would be required for the implementation of this technology by virtue of operational restrictions. These restrictions include the high salinity needed for ectoine production, the changes in the pH concomitant to the production of organic acids and alcohols, and the anaerobic restrictive conditions characteristic of acetogens. The use of mixotrophs also appears as a solution for ectoine production from CO 2 /CO. In fact, recently, it was observed that H. elongata anaplerotic flux can support an increase in ectoine production when using glucose as the main carbon source (Hobmeier et al. 2020). In other study, the addition of low concentrations of glucose (0.5 g L −1 ) to Halomonas stevensii cultures promoted ectoine contents of 15% and 22% at salinities of 6% and 12% NaCl, and supported an abatement of 37.1 mg CO 2 L −1 h −1 (Cantera et al. 2022). Besides glucose, other organic compounds, such as acetate and alcohols, could be considered for mixotrophic growth, though they have not yet been experimentally tested.
Nevertheless, a chemoautotrophic biotechnological platform that allows to produce ectoines purely with CO 2 as the sole carbon source has a great potential. Although, dark fixation provides less energy than aerobic respiration of sugars, this bioconversion process could use CO 2 from industrial flue gases, and waste gases (CO, H 2 ) or liquid waste (sulfur compounds, metals, ammonium) as energy source enhancing the feasibility and expansion of this residue-valorisation technology. Moreover, it could be implemented in both, aerobic and anaerobic conditions. However, CO 2 and CO have been disregarded for the production of ectoines, fact that is probably related to the current lack of fundamental studies proving that chemolithoautotrophs can produce ectoines.

Potential chemolithoautotrophs able to produce ectoines
Although heat or cold stress can affect ectoine accumulation, salinity is the main trigger for the expression of genes involved in ectoine biosynthesis pathway (Argandoña et al. 2021). Thus, ectoine and hydroxyectoine production is usually observed in halophilic or halotolerant organisms. Several halophiles have the ability to grow on CO 2 and/or CO, but their potential to produce ectoines while using these gases is unexplored. Based on literature and genomic databases (supplementary information 2), we gathered a total of 143 species of halophilic chemolithotrophs, carboxydotrophs and carboxydovores. All the identified species that were able to use CO 2 and CO aerobically and anaerobically, together with their required energy sources and their optimum salinity for growth are summarized in supplementary information 2, Table S4 and Table S5. Genomes of the identified species were screened for genes encoding for enzymes involved in the ectoine and hydroxyectoine synthesis pathways: specifically ectA, ectB, ectC and ectD (supplementary information 2, Tables S6  and S7). Moreover, the presence of ectR, an important transcriptional regulator of this pathway, and two genes involved in the production of the precursor L-2,4-diaminobutyrate (aspartate kinase: ask and aspartate semi-aldehyde dehydrogenase: asd), were assessed. In total, 31 microbial genomes were identified with the ectABC gene cluster (Fig. 2) 2+ and reduced sulfur compounds (Acidihalobacter aeolianus, Acidihalobacter ferrooxidans, Acidihalobacter prosperus) as electron donors. This diversity indicates a broad range of possibilities for application in biotechnological processes. Some anaerobic chemolithoautotrophs also contained the genes for ectoine synthesis. These included Achromobacter ruhlandii and Alkalilimnicola erlichii (CO 2 fixation with H 2 as electron donor and NO 3 − as electron Fig. 2 Presence/absence of ectoine biosynthesis genes in halophilic chemolithotrophs. All genomes found with ectC and ectB were interrogated for other relevant genes in the ectoine biosynthesis pathway. All detected ectA and ectD were found within 10 kb of ectB and ectC. All surveyed genomes contained aspartate kinase (ask) genes. Additionally, ask copies found within 10 kb of the ect gene cluster are considered to represent ask_ect orthologs (orange). While most genomes contained genes belonging to the ectR gene family (COG1846; gray), only those found within 10 kb of the ect gene cluster were considered ectR orthologs (red). The phylogeny displayed at the left reflects the relationships between organisms based on their NCBI taxonomy acceptor), Desulfatibabacillum aliphaticivorans (CO 2 fixation with H 2 as electron donor and SO 4 2− as electron acceptor), Desulfocapsa sulfexigens (CO 2 fixation with H 2 as electron donor and S 0 , SO 3 2− , S 2 O 3 2− as electron acceptors), and the methanogen Methanobacterium subterraneum (CO 2 fixation with H 2 as electron donor). Theoretically, these could be potential candidates for the anaerobic conversion of CO 2 into ectoine.
As less CO-utilizing microbes are known, and therefore screened, their metabolic potential may be less well characterised. However, some aerobic carboxydotrophs such as Hydrogenibacillus schlegelii, and Hahella chejuensis, aerobic carboxydovores, such as Alkalispirillum mobile and Mycobacterium smegmatis, and the facultative anaerobe Alkalilimnicola erlichii were detected as putatively able to consume CO and produce ectoine (Hoeft et al. 2007).
The formation of hydroxyectoine depends on the initial synthesis of ectoine and its subsequent hydroxylation by the enzyme coded by the gene ectD. The ectD gene was found in 14 of the genomes that contained ectABC. Ten of those genomes belonged to aerobic CO 2 -utilizing microbes (Fig. 2), one to an anaerobic chemolithoautotrophic CO 2 consumer (Achromobacter ruhlandii), and three to aerobic CO-utilizing microbes (Mycobacterium smegmatis, Hydrogenibacillus schlegelii, and Hahella chejuensis).

Perspectives
This work sheds light in a new direction looking at yet overlooked microbes and strategies that can help in the development of CO 2 and CO valorization. The information provided has the potential to propel new research in this topic for different industrial scenarios, using H 2 , nitrogen and sulfur compounds or metals as the energy source. Moreover, the approaches and results from this review are applicable for the valorization of any CO 2 -containing gases, including emissions from industrial sites (steel mills, cement producers, electricity plants, etc.); flue gas emissions (natural gas-fired power plants, coal boilers) and syngas. Besides, biotransformations carried out at high salinity can be adapted to reduce cost (use of plastic-based reactors, sea water as medium) and are usually more robust processes due to the low risk of contamination. Therefore, this review offers a completely new strategy that can pave the way for a more cost-effective elimination of GHG and waste gasses, as well as more sustainable and circular chemical production systems.

Conflict of interest
The authors have no relevant financial or non-financial interests to disclose.
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