Single chamber electrochemical-biochemical synthesis of products from carbon dioxide

Abstract

The present invention provides for a vessel comprising: (a) electrodes, in electrical communication with a power source, and (b) a solution comprising a microbial strain capable of synthesizing a compound of interest, wherein the microbial strain obtains energy via water-splitting electrolysis through the electrodes and microbial hydrogen fermentation.

Description

Attorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory Single chamber electrochemical-biochemical synthesis of products from carbon dioxide Inventors: Eric Sundstrom, Changman Kim, Steven W. Singer, Justin Panich CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Ser. No. 63 / 641,823, filed May 2, 2024, which is incorporated by reference in its entirety. STATEMENT OF GOVERNMENTAL SUPPORT

[0002] The invention described and claimed herein was made utilizing funds supplied by the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. The government has certain rights in this invention. FIELD OF THE INVENTION

[0003] The present invention is in the field of electrochemical-biochemical synthesis of products. BACKGROUND OF THE INVENTION

[0004] Over the past few decades, decreasing CO2 emissions on a global scale to zero level and conversion to fuels is one of the most urgent challenges. Photosynthesis is a representative CO2removal process naturally, and it is used as an abundant energy source as sunlight, and simultaneously, it was used as the sole reagent with water and CO2. Photosynthesis is often described as a single-step reaction, but it is two steps involving photosynthetic transformation, which is described as a light reaction, and a dark reaction in nature. The light reaction used the sunlight for the endergonic splitting of the water into oxygen and hydrogen, stored as NADPH / H+reducing equivalent. The dark reaction led to use to power (i.e., ATP, which has translated from NADPH via ATP synthase) and the reduction of carbon dioxide by its combination with NADPH three-carbon products [1]. However, natural photosynthesis efficiency decreased in a diverse energy conversion bottleneck, which was reported mostly not to exceed 5-7% for microalgae in bubbled bioreactors, and 1% of most plant [2].

[0005] On the other hand, artificial photosynthesis (AP) had originated from the desire to mimic natures photosynthetic processes to improve efficiency, which deals with the understanding of the molecular aspects of mimicking the two steps to use sunlight in drivingAttorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory the conversion of the CO2 and water to carbohydrate and oxygen [3, 4]. Hence, various research groups have been numerous efforts that water splitting and CO2 reduction are multi- electron processes through developments of molecular catalysts for a few decades [5-7]. Herein, hydrogen is essential reagent of the photosynthesis that is a great energy carrier and easily convertible to electrical power without generating byproducts that are harmful to the environment. Moreover, the CO2reduction process has gained much attention that was derived from the photochemical reaction via accumulated electron charge transfer [4, 8]. Nonetheless, the device assembled from molecular catalysts is reported to be less stable and performs less efficiently. Recently, the biological autotrophic CO2conversion process was reported by using a biocatalyst as a whole-cell.

[0006] Last decade, microbial electrosynthesis (MES) is interested that has merited considerable interest in providing reducing equivalents for sustainable CO2 conversion based on the electrochemical device using renewable electricity [9, 10]. The applied potential induces MES progress as an electromotive force between electrodes for the water splitting or redox reaction. From the water electrolysis, the generated hydrogen carries the energy into a suspended electroactive microbe for fuel synthesis, that like the dark reaction of photosynthetic transformation [11, 12]. Hence, some research groups reported developing an integrated biological system that used H2-oxidizing autotrophic microbial as biocatalysts, producing hydrogen through electrochemical water splitting in an H-type reactor with a photovoltaics system

[0013] . Torella et al

[0014] ., achieved equivalent solar-to-biomass yields of up to 3.2% with Ralstonia eutropha when provided by an 18% efficient PV. Moreover, they reported that engineered R. eutropha obtained 216 mg / L of isopropanol yield. SUMMARY OF THE INVENTION

[0007] The present invention provides for a vessel comprising: (a) electrodes, in electrical communication with a power source, and (b) a solution comprising a microbial strain capable of synthesizing a compound of interest, wherein the microbial strain obtains energy via water-splitting electrolysis through the electrodes and microbial hydrogen fermentation.

[0008] In some embodiments, any toxicity generated by the electrolysis is overcome via use of a microbe strain which has natural resistance to heavy metal toxicity, a high affinity for hydrogen and carbon dioxide, and a high tolerance for oxygen. In some embodiments, the microbe strain has natural resistance to heavy metal toxicity, a high affinity for hydrogen andAttorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory carbon dioxide, and / or a high tolerance for oxygen. In some embodiments, the microbe strain has a higher affinity for hydrogen and carbon dioxide than that of Ralstonia eutropha.

[0009] In some embodiments, the microbe strain is a bacterial cell. In some embodiments, the bacterial cell is of the phylum Pseudomonadota, such as of the Class Betaproteobacteria, such as of the Order Burkholderiales, such as of the family Burkholderiaceae. In some embodiments, the microbe strain is of the genus Ralstonia, such as Ralstonia metallidurans, Ralstonia eutropha, Ralstonia insidiosa, Ralstonia mannitolilytica, Ralstonia pickettii, Ralstonia pseudosolanacearum, Ralstonia solanacearum, or Ralstonia syzygii. In some embodiments, the microbe strain is of the genus Cupriavidus, such as Cupriavidus metallidurans or Cupriavidus necator. In some embodiments, the microbe strain is chemolithoautotrophic, such as facultative chemolithoautotrophic. In some embodiments, the microbe strain is a facultative chemolithoautotrophic β-proteobacterium. In some embodiments, the microbe strain is able to assimilate CO2using the Calvin-Benson-Bassham (CBB) cycle. In some embodiments, the microbe strain is a thermophile and / or electrophile.

[0010] The microbe strain is of a more resilient strain allows use of standard low-cost electrode materials. Integrating electrolysis and fermentation simplifies the overall system and leverages gas dissolution directly from the electrodes, eliminating additional costs for gas dispersion, a major challenge for gas fermentation. In some embodiments, the microbial strain, such as R. metallidurans, has a flexible metabolism and can be engineered for production of one or more compounds of interest, including but not limited to complex, multi-carbon products, such as drop-in biofuels, biomaterials, and commodity chemicals.

[0011] In some embodiments, electrodes comprise at least one anode and at least one cathode. In some embodiments, the cathode is a reactive oxygen species (ROS)-resistant cathode. In some embodiments, one or more of the electrodes is a cobalt-phosphorous (Co-P) alloy electrode. In some embodiments, the cathode is a cobalt-phosphorous (Co-P) alloy cathode. In some embodiments, the anode is a self-healing Co-Pialloy anode. In some embodiments, the electrodes are not biocompatible electrodes, such as biocompatible with Ralstonia eutropha. In some embodiments, one or more of the electrodes are stainless electrodes, such as stainless steel, such as stainless steel with Cr. In some embodiments, one or more of the electrodes, or the solution, comprises a catalyst, such as Pt, Ir, or Pt-Ir catalyst. In some embodiments, the cathode comprises Pt. In some embodiments, the anode comprises Ir.Attorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory

[0012] This technology integrates (1) water-splitting electrolysis and (2) microbial fermentation into a single chamber by leveraging a microbial strain, such as a non-canonical microbial strain. Water-splitting electrolysis is used to split the aqueous culture medium into hydrogen and oxygen gas. CO2 is then supplemented as either a gas or as bicarbonate, thereby providing a carbon source (CO2), energy source (H2), and electron acceptor (O2) suitable for cultivation of hydrogen oxidizing bacteria. Both hydrogen and oxygen are supplied in microbubbles produced directly from the electrodes, eliminating the cost for gas dissolution into the aqueous phase as required for typical sparged bioreactors. No gas separation is required, eliminating cost and efficiency losses associated with gas separation membranes in conventional electrolyzers.

[0013] The primary challenge for integration of electrolysis and fermentation is harmonization of electrolysis and fermentation conditions, coupled with toxicity generated at the electrodes (heavy metals and reactive oxygen species). To overcome this toxicity using the canonical strain Ralstonia eutropha, previous demonstrations of similar integrated systems have relied on novel, biocompatible electrodes (see Liu et al., Science, 2016). In the present invention, one can use a more robust noncanonical host, such as Ralstonia metallidurans. This strain has high tolerance for heavy metal toxicity and high oxygen concentrations. In addition, it offers high CO2 and hydrogen affinity, enabling efficient single-pass gas uptake. Using this strain, one can achieve a high level of performance with a conventional stainless / Pt cathode and Ir anode, reducing system cost and enabling more flexibility with the electrolysis configuration. This strain is genetically tractable, and we have now demonstrated engineering of R. metallidurans to express a type III PKS and produce the pigment molecule flaviolin.

[0014] To date, we have demonstrated a proof of concept for the integrated system. This includes operation of water-splitting electrolysis in 200 mL bioreactors and in situ cultivation of R. metallidurans. We have integrated the electrolysis system with solar panels to enable growth and production directly from photons, and we have engineered the biocatalyst to produce the pigment molecule flaviolin, a type III PKS product. We have initiated process optimization, including electrode geometry, current / voltage conditions, growth medium optimization, and electrode surface area.

[0015] This invention can be used to synthesize complex products directly from renewable power and CO2. This is a compelling platform, that could be used by existing hydrogenAttorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory fermentation companies, as well as large-scale chemical producers in both the petrochemical and biochemical industries. Ultimately this type of technology could replace sugar with electricity / CO2as a biochemical feedstock, making it a platform production technology of significant interest in the biochemical industries. Likewise, this technology has the potential to replace petrochemical fuel and chemical production with production from electricity / CO2. As it requires few inputs, this process would allow distributed and flexible generation of chemicals, which could appeal to military and space flight end users.

[0016] For the present invention, there is integration of water-splitting electrolysis and fermentation leverages the electrodes for gas diffusion, saving substantial energy typically required to sparge these gases into an aqueous bioreactor. The integrated configuration also reduces system complexity by eliminating costs for electrode housings, gas separation membranes, and an electrolyzer stack used to produce hydrogen and oxygen in separate streams.

[0017] Previous researchers have demonstrated single chamber water-splitting electrolysis and fermentation in a similar configuration (Liu et al, “Water splitting–biosynthetic system with CO2reduction efficiencies exceeding photosynthesis,” Vol.352 (No.6290): pp.1210- 1213, 2016), which addressed toxicity via use of novel biocompatible electrodes. Because electrode assemblies represent a significant portion of the overall product cost, use of higher cost biocompatible electrodes may compromise overall economics. By contrast, our system uses a more chemically resistant strain to achieve the same level of performance with standard electrode assemblies. This additional flexibility should enable use of lower cost and higher efficiency electrodes, reducing capital cost and energy cost of the overall system.

[0018] Other objects, features, and advantages of the present invention will be apparent to one of skill in the art from the following detailed description and figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The foregoing aspects and others will be readily appreciated by the skilled artisan from the following description of illustrative embodiments when read in conjunction with the accompanying drawings.

[0020] Figure 1 shows the one pot electrolysis and fermentation apparatus / design of the present invention.Attorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory

[0021] Figure 2 shows the R. eutropha ceramic shielding of Li et al. (Science, 2012).

[0022] Figure 3 shows the R. eutropha biocompatible CoP catalyst design of Liu et al. (Science, 2016).

[0023] Figure 4 shows electrode and growth medium co-optimization.

[0024] Figure 5 shows the cell density of R. eutropha and R. metallidurans with and without bicarbonate after 2 or 4 days growth in different Ni2+concentration.

[0025] Figure 6 shows the cell density by days growth in low or high oxygen. The “low oxygen” culture is grown with “mix gas 1”: 2.5% H2, 1.5% O2, 1.5% CO2, N2 balance (94.5%). The “high oxygen” culture is grown with “mix gas 2”: 2.5% H2, 21.5% O2, 1.5% CO2, N2balance (74.5%).

[0026] Figure 7 shows the cell density versus time of growth from solar cells with low-cost stainless electrodes.

[0027] Figure 8 shows the cell density versus time of growth in carbon dioxide sparging (with or without electrolyzer) and bolus NaHCO3 (with or without electrolyzer).

[0028] Figure 9 shows the comparison of the autotrophic growth by (a) sparging CO2 or bolus NaHCO3, and (b) gas sparing like as aerobic / anaerobic atmosphere (gas composition: aerobic; 21% O2, anaerobic 1.5% O2 / 2.5 % H2, CO2, and N2balanced)

[0029] Figure 10 shows: (a) Representative diagram for flaviolin production pathway and comparison of the (b) autotrophic growth and absorbance for RppA expression in Cupriavidus metallidurans.

[0030] Figure 11 shows the comparison of cell growth in electrolyzer under different energy sources

[0031] Figure 12 shows artificial photosynthetic cultivation in RppA protein expressed C. metallidurans CH34 in ACE; (a) Cell density, (b) Flaviolin product. DETAILED DESCRIPTION OF THE INVENTION

[0032] Before the invention is described in detail, it is to be understood that, unless otherwise indicated, this invention is not limited to particular sequences, expression vectors, enzymes,Attorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory host cells, microorganisms, or processes, as such may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting.

[0033] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an "expression vector" includes a single expression vector as well as a plurality of expression vectors, either the same (e.g., the same operon) or different; reference to "cell" includes a single cell as well as a plurality of cells; and the like.

[0034] In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:

[0035] The terms "optional" or "optionally" as used herein mean that the subsequently described feature or structure may or may not be present, or that the subsequently described event or circumstance may or may not occur, and that the description includes instances where a particular feature or structure is present and instances where the feature or structure is absent, or instances where the event or circumstance occurs and instances where it does not.

[0036] The term "about" as used herein means a value that includes 10% less and 10% more than the value referred to.

[0037] The terms “host cell” and "host microorganism" are used interchangeably herein to refer to a living biological cell, such as a microorganism, that can be transformed via insertion of an expression vector. Thus, a host organism or cell as described herein may be a prokaryotic organism (e.g., an organism of the kingdom Eubacteria) or a eukaryotic cell. As will be appreciated by one of ordinary skill in the art, a prokaryotic cell lacks a membrane- bound nucleus, while a eukaryotic cell has a membrane-bound nucleus.

[0038] The term "heterologous DNA" as used herein refers to a polymer of nucleic acids wherein at least one of the following is true: (a) the sequence of nucleic acids is foreign to (i.e., not naturally found in) a given host cell; (b) the sequence may be naturally found in a given host cell, but in an unnatural (e.g., greater than expected) amount; or (c) the sequence of nucleic acids comprises two or more subsequences that are not found in the same relationship to each other in nature. The term "heterologous" as used herein refers to aAttorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory structure or molecule wherein at least one of the following is true: (a) the structure or molecule is foreign to (i.e., not naturally found in) a given host cell; or (b) the structure or molecule may be naturally found in a given host cell, but in an unnatural (e.g., greater than expected) amount. For example, regarding instance (c), a heterologous nucleic acid sequence that is recombinantly produced will have two or more sequences from unrelated genes arranged to make a new functional nucleic acid. Specifically, the present invention describes the introduction of an expression vector into a host cell, wherein the expression vector contains a nucleic acid sequence coding for an enzyme that is not normally found in a host cell. With reference to the host cell’s genome, then, the nucleic acid sequence that codes for the enzyme is heterologous.

[0039] The terms "expression vector" or "vector" refer to a compound and / or composition that transduces, transforms, or infects a host cell, thereby causing the cell to express nucleic acids and / or proteins other than those native to the cell, or in a manner not native to the cell. An "expression vector" contains a sequence of nucleic acids (ordinarily RNA or DNA) to be expressed by the host cell. Optionally, the expression vector also comprises materials to aid in achieving entry of the nucleic acid into the host cell, such as a virus, liposome, protein coating, or the like. The expression vectors contemplated for use in the present invention include those into which a nucleic acid sequence can be inserted, along with any preferred or required operational elements. Further, the expression vector must be one that can be transferred into a host cell and replicated therein. Preferred expression vectors are plasmids, particularly those with restriction sites that have been well documented and that contain the operational elements preferred or required for transcription of the nucleic acid sequence. Such plasmids, as well as other expression vectors, are well known to those of ordinary skill in the art.

[0040] The term "transduce" as used herein refers to the transfer of a sequence of nucleic acids into a host cell or cell. Only when the sequence of nucleic acids becomes stably replicated by the cell does the host cell or cell become "transformed." As will be appreciated by those of ordinary skill in the art, "transformation" may take place either by incorporation of the sequence of nucleic acids into the cellular genome, i.e., chromosomal integration, or by extrachromosomal integration. In contrast, an expression vector, e.g., a virus, is "infective" when it transduces a host cell, replicates, and (without the benefit of any complementary virus or vector) spreads progeny expression vectors, e.g., viruses, of the same type as theAttorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory original transducing expression vector to other microorganisms, wherein the progeny expression vectors possess the same ability to reproduce.

[0041] As used herein, the terms "nucleic acid sequence," "sequence of nucleic acids," and variations thereof shall be generic to polydeoxyribonucleotides (containing 2-deoxy-D- ribose), to polyribonucleotides (containing D-ribose), to any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, and to other polymers containing nonnucleotidic backbones, provided that the polymers contain nucleobases in a configuration that allows for base pairing and base stacking, as found in DNA and RNA. Thus, these terms include known types of nucleic acid sequence modifications, for example, substitution of one or more of the naturally occurring nucleotides with an analog; intemucleotide modifications, such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., arninoalklyphosphoramidates, aminoalkylphosphotriesters); those containing pendant moieties, such as, for example, proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.); those with intercalators (e.g., acridine, psoralen, etc.); and those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.). As used herein, the symbols for nucleotides and polynucleotides are those recommended by the IUPAC-IUB Commission of Biochemical Nomenclature (Biochem.9:4022, 1970).

[0042] The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0043] In some embodiments, the microbial strain is a genetically modified host cell comprising one or more nucleic acids encoding one or more biosynthetic enzyme(s), operatively linked to one or more promoters, capable of expression in the microbial strain. In some embodiments, the biosynthetic enzyme(s) are capable of synthesizing, from one or more precursor(s) endogenously synthesized or able to be absorbed by the microbial strain in or into the cytoplasm of the microbial strain, one or more compound of interest. In some embodiments, the one or more biosynthetic enzyme(s) are encoded on one or more nucleicAttorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory acid which are transformed into the genetically modified host cell, or host cell prior to genetic modification. In some embodiments, each nucleic acid of the one or more nucleic acids is on a vector capable of stable introduction into the genome of the host cell and / or stable maintenance in cytoplasm of the host cell. In some embodiments, the genetically modified host cell is capable of producing one or more compounds, such as the compound of interest, in titers or yields equal to or more than the titers or yields described herein. In some embodiments, one or more of the biosynthetic enzyme(s) are heterologous to the microbial strain.

[0044] In some embodiments, the microbial strain is a genetically modified host cell or microorganism, or a genetically modified host cell or microorganism comprising the genetic modifications, disclosed by U.S. Patent Nos.9,752,163; 9,879,286; 10,273,506; and, 10,814,724; and, PCT International Patent Application Nos. PCT / EP2009 / 067784 and PCT / US2021 / 040757; and by Zheng, et al. “Metabolic engineering of Escherichia coli for high-specificity production of isoprenol and prenol as next generation of biofuels,” Biotechnology for Biofuels 6:57 (2013); George, et al. “Metabolic engineering for the high- yield production of isoprenoid-based C5alcohols in E. coli,” Scientific Reports 5:11128 (2015); Wang, et al. “Tolerance Characterization and Isoprenol Production of Adapted Escherichia coli in the Presence of Ionic Liquids,” ACS Sustainable Chem. Eng. 7(1):1457-1463 (2018); and, Kim et al.cerevisiae for isoprenol production,” Metabolic Engineering 64:154-166 (2021) (whereby all are incorporated by reference in their entireties).

[0045] In some embodiments, the compound of interest is a compound naturally produced by the microbial strain. In some embodiments, the compound of interest is a compound not naturally produced by the microbial strain. In some embodiments, the compound of interest is a biofuel or bioproduct, or any other organic compound, and the corresponding biosynthetic enzyme(s) for producing the compound of interest thereof, are described and taught in U.S. Patent Nos.7,985,567; 8,420,833; 8,852,902; 9,109,175; 9,200,298; 9,334,514; 9,376,691; 9,382,553; 9,631,210; 9,951,345; 10,167,488; 10,273,605; 10,814,724; and 11,660,961; and PCT International Patent Application Nos. PCT / US2014 / 48293, PCT / US2018 / 049609, PCT / US2017 / 036168, PCT / US2018 / 029668, PCT / US2008 / 068833, PCT / US2008 / 068756, PCT / US2008 / 068831, PCT / US2009 / 042132, PCT / US2010 / 033299, PCT / US2011 / 053787, PCT / US2011 / 058660, PCT / US2011 / 059784, PCT / US2011 / 061900, PCT / US2012 / 031025,Attorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory and PCT / US2013 / 074214 (all of which are incorporated in their entireties by reference). In some embodiments, the biosynthetic enzyme is a polyketide synthase (PKS). In some embodiments, the PKS is naturally occurring or non-naturally occurring PKS. In some embodiments, the non-naturally occurring PKS is a hybrid PKS. In some embodiments, the PKS is one described in one of the references incorporated herein. In some embodiments, the PKS is flaviolin PKS, and the compound of interest is flaviolin. In some embodiments, the compound of interest is a terpene, isoprenoid, carboxylic acid, lactone, trimethylpentanoic acid, 1-deoxyxylulose 5-phosphate, 1-deoxy-D-xylulose 5-phosphate (DXP), fatty acid, orderivatives thereof, alkyl lactone, lactam, isoprenyl alkanoate, 3- me t h y l - 2 - b u t e n - 1 - o l ,3 - me t h y l - 3 - b u t e n - 1 - o l , a n d 3 - me t h y l - b u t a n - 1 - o l , f a t t y a c i d e s t e r ,a l p h a - o l e f i n , d i a c i d , d i a mi n e , sesquiterpene, bisabolene, or oxidized aromaticamino acid. In some embodiments, the compound of interest is any product or intermediate in the mevalonate (MVA) pathway, including any compound from acetyl-CoA to mevalonate. In some embodiments, the biosynthetic enzyme(s) are phosphomevalonate decarboxylase (PMD), phosphatase, AtoB, hydroxymethylglutaryl-CoA synthase (HMGS), hydroxymethylglutaryl-CoA reductase (HMGR), and / or mevalonate kinase (MK).

[0046] The nucleic acid constructs of the present invention comprise nucleic acid sequences encoding one or more of the subject enzymes. The nucleic acid of the subject enzymes is operably linked to promoters and optionally control sequences such that the subject enzymes are expressed in a host cell cultured under suitable conditions. The promoters and control sequences are specific for each host cell species. In some embodiments, expression vectors comprise the nucleic acid constructs. Methods for designing and making nucleic acid constructs and expression vectors are well known to those skilled in the art.

[0047] Sequences of nucleic acids encoding the subject enzymes are prepared by any suitable method known to those of ordinary skill in the art, including, for example, direct chemical synthesis or cloning. For direct chemical synthesis, formation of a polymer of nucleic acids typically involves sequential addition of 3'-blocked and 5'-blocked nucleotide monomers to the terminal 5'-hydroxyl group of a growing nucleotide chain, wherein each addition is effected by nucleophilic attack of the terminal 5'-hydroxyl group of the growing chain on the 3'-position of the added monomer, which is typically a phosphorus derivative, such as a phosphotriester, phosphoramidite, or the like. Such methodology is known to those of ordinary skill in the art and is described in the pertinent texts and literature (e.g., in MatteuciAttorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory et al. (1980) Tet. Lett.521:719; U.S. Pat. Nos.4,500,707; 5,436,327; and 5,700,637). In addition, the desired sequences may be isolated from natural sources by splitting DNA using appropriate restriction enzymes, separating the fragments using gel electrophoresis, and thereafter, recovering the desired nucleic acid sequence from the gel via techniques known to those of ordinary skill in the art, such as utilization of polymerase chain reactions (PCR; e.g., U.S. Pat. No.4,683,195).

[0048] Each nucleic acid sequence encoding the desired subject enzyme can be incorporated into an expression vector. Incorporation of the individual nucleic acid sequences may be accomplished through known methods that include, for example, the use of restriction enzymes (such as BamHI, EcoRI, HhaI, Xhol, XmaI, and so forth) to cleave specific sites in the expression vector, e.g., plasmid. The restriction enzyme produces single stranded ends that may be annealed to a nucleic acid sequence having, or synthesized to have, a terminus with a sequence complementary to the ends of the cleaved expression vector. Annealing is performed using an appropriate enzyme, e.g., DNA ligase. As will be appreciated by those of ordinary skill in the art, both the expression vector and the desired nucleic acid sequence are often cleaved with the same restriction enzyme, thereby assuring that the ends of the expression vector and the ends of the nucleic acid sequence are complementary to each other. In addition, DNA linkers may be used to facilitate linking of nucleic acids sequences into an expression vector.

[0049] A series of individual nucleic acid sequences can also be combined by utilizing methods that are known to those having ordinary skill in the art (e.g., U.S. Pat. No. 4,683,195).

[0050] For example, each of the desired nucleic acid sequences can be initially generated in a separate PCR. Thereafter, specific primers are designed such that the ends of the PCR products contain complementary sequences. When the PCR products are mixed, denatured, and reannealed, the strands having the matching sequences at their 3' ends overlap and can act as primers for each other Extension of this overlap by DNA polymerase produces a molecule in which the original sequences are "spliced" together. In this way, a series of individual nucleic acid sequences may be "spliced" together and subsequently transduced into a host cell simultaneously. Thus, expression of each of the plurality of nucleic acid sequences is effected.Attorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory

[0051] Individual nucleic acid sequences, or "spliced" nucleic acid sequences, are then incorporated into an expression vector. The invention is not limited with respect to the process by which the nucleic acid sequence is incorporated into the expression vector. Those of ordinary skill in the art are familiar with the necessary steps for incorporating a nucleic acid sequence into an expression vector. A typical expression vector contains the desired nucleic acid sequence preceded by one or more regulatory regions, along with a ribosome binding site, e.g., a nucleotide sequence that is 3-9 nucleotides in length and located 3-11 nucleotides upstream of the initiation codon in E. coli. See Shine et al. (1975) Nature 254:34 and Steitz, in Biological Regulation and Development: Gene Expression (ed. R. F. Goldberger), vol.1, p.349, 1979, Plenum Publishing, N.Y.

[0052] Regulatory regions include, for example, those regions that contain a promoter and an operator. A promoter is operably linked to the desired nucleic acid sequence, thereby initiating transcription of the nucleic acid sequence via an RNA polymerase enzyme. An operator is a sequence of nucleic acids adjacent to the promoter, which contains a protein- binding domain where a repressor protein can bind. In the absence of a repressor protein, transcription initiates through the promoter. When present, the repressor protein specific to the protein-binding domain of the operator binds to the operator, thereby inhibiting transcription. In this way, control of transcription is accomplished, based upon the particular regulatory regions used and the presence or absence of the corresponding repressor protein. An example includes lactose promoters (LacI repressor protein changes conformation when contacted with lactose, thereby preventing the LacI repressor protein from binding to the operator). Another example is the tac promoter. (See deBoer et al. (1983) Proc. Natl. Acad. Sci. USA, 80:21-25.) As will be appreciated by those of ordinary skill in the art, these and other expression vectors may be used in the present invention, and the invention is not limited in this respect.

[0053] Although any suitable expression vector may be used to incorporate the desired sequences, readily available expression vectors include, without limitation: plasmids, such as pSC101, pBR322, pBBR1MCS-3, pUR, pEX, pMR100, pCR4, pBAD24, pUC19; bacteriophages, such as M13 phage and λ phage. Of course, such expression vectors may only be suitable for particular host cells. One of ordinary skill in the art, however, can readily determine through routine experimentation whether any particular expression vector is suited for any given host cell. For example, the expression vector can be introduced into the hostAttorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory cell, which is then monitored for viability and expression of the sequences contained in the vector. In addition, reference may be made to the relevant texts and literature, which describe expression vectors and their suitability to any particular host cell.

[0054] The expression vectors of the invention must be introduced or transferred into the host cell. Such methods for transferring the expression vectors into host cells are well known to those of ordinary skill in the art. For example, one method for transforming E. coli with an expression vector involves a calcium chloride treatment wherein the expression vector is introduced via a calcium precipitate. Other salts, e.g., calcium phosphate, may also be used following a similar procedure. In addition, electroporation (i.e., the application of current to increase the permeability of cells to nucleic acid sequences) may be used to transfect the host cell. Also, microinjection of the nucleic acid sequencers) provides the ability to transfect host cell. Other means, such as lipid complexes, liposomes, and dendrimers, may also be employed. Those of ordinary skill in the art can transfect a host cell with a desired sequence using these or other methods.

[0055] For identifying a transfected host cell, a variety of methods are available. For example, a culture of potentially transfected host cells may be separated, using a suitable dilution, into individual cells and thereafter individually grown and tested for expression of the desired nucleic acid sequence. In addition, when plasmids are used, an often-used practice involves the selection of cells based upon antimicrobial resistance that has been conferred by genes intentionally contained within the expression vector, such as the amp, gpt, neo, and hyg genes.

[0056] When the host cell is transformed with at least one expression vector. When only a single expression vector is used (without the addition of an intermediate), the vector will contain all of the nucleic acid sequences necessary.

[0057] Once the host cell has been transformed with the expression vector, the host cell is allowed to grow. For microbial hosts, this process entails culturing the cells in a suitable medium. It is important that the culture medium contain an excess carbon source, such as a sugar (e.g., glucose) when an intermediate is not introduced. In this way, cellular production of the isoprenol ensured. When added, any intermediate is present in an excess amount in the culture medium.

[0058] The present invention provides for a method for constructing genetically modifiedAttorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory yeast host cell of the present invention comprising: (a) introducing one or more nucleic acid comprising open reading frames (ORF) encoding the enzymes described herein wherein each is operatively linked to a promoter capable of transcribing each ORF to which it is operatively linked, and / or (b) optionally knocking out one or more of the enzymes described herein such that the modified host cell does not express the one or more knocked out enzymes.

[0059] In some embodiments, the host cells are genetically modified in that heterologous nucleic acid have been introduced into the host cells, and as such the genetically modified host cells do not occur in nature. The suitable host cell is one capable of expressing a nucleic acid construct encoding one or more enzymes described herein. The gene(s) encoding the enzyme(s) may be heterologous to the host cell or the gene may be native to the host cell but is operatively linked to a heterologous promoter and one or more control regions which result in a higher expression of the gene in the host cell.

[0060] The enzyme can be native or heterologous to the host cell. Where the enzyme is native to the host cell, the host cell is genetically modified to modulate expression of the enzyme. This modification can involve the modification of the chromosomal gene encoding the enzyme in the host cell or a nucleic acid construct encoding the gene of the enzyme is introduced into the host cell. One of the effects of the modification is the expression of the enzyme is modulated in the host cell, such as the increased expression of the enzyme in the host cell as compared to the expression of the enzyme in an unmodified host cell.

[0061] References cited herein: [1] D.K. Dogutan, D.G. Nocera, Artificial photosynthesis at efficiencies greatly exceeding that of natural photosynthesis, Accounts of Chemical Research 52 (2019) 3143-3148. [2] R.E. Blankenship, D.M. Tiede, J. Barber, G.W. Brudvig, G. Fleming, M. Ghirardi, M. Gunner, W. Junge, D.M. Kramer, A. Melis, Comparing photosynthetic and photovoltaic efficiencies and recognizing the potential for improvement, science 332 (2011) 805-809. [3] S. Berhanu, T. Ueda, Y. Kuruma, Artificial photosynthetic cell producing energy for protein synthesis, Nature communications 10 (2019) 1325. [4] D. Kim, K.K. Sakimoto, D. Hong, P. Yang, Artificial photosynthesis for sustainable fuel and chemical production, Angewandte Chemie International Edition 54 (2015) 3259-3266. [5] S. Perathoner, G. Centi, Chapter 21 - Artificial leaves using sunlight to produce fuels, in: A. Basile, G. Centi, M.D. Falco, G. Iaquaniello (Eds.) Studies in Surface Science and Catalysis, Elsevier2020, pp.415-430.Attorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory [6] Y. Wang, H. Suzuki, J. Xie, O. Tomita, D.J. Martin, M. Higashi, D. Kong, R. Abe, J. Tang, Mimicking natural photosynthesis: solar to renewable H2 fuel synthesis by Z-scheme water splitting systems, Chemical reviews 118 (2018) 5201-5241. [7] M. Đokić, H.S. Soo, Artificial photosynthesis by light absorption, charge separation, and multielectron catalysis, Chemical Communications 54 (2018) 6554-6572. [8] D.R. Whang, D.H. Apaydin, Artificial photosynthesis: Learning from nature, ChemPhotoChem 2 (2018) 148-160. [9] A. Prévoteau, J.M. Carvajal-Arroyo, R. Ganigué, K. Rabaey, Microbial electrosynthesis from CO2: forever a promise?, Current opinion in biotechnology 62 (2020) 48-57.

[0010] B.S. Thapa, T. Kim, S. Pandit, Y.E. Song, Y.P. Afsharian, M. Rahimnejad, J.R. Kim, S.- E. Oh, Overview of electroactive microorganisms and electron transfer mechanisms in microbial electrochemistry, Bioresource Technology 347 (2022) 126579.

[0011] E.V. LaBelle, C.W. Marshall, H.D. May, Microbiome for the Electrosynthesis of Chemicals from Carbon Dioxide, Accounts of Chemical Research 53 (2020) 62-71.

[0012] Y.E. Song, A. Mohamed, C. Kim, M. Kim, S. Li, E. Sundstrom, H. Beyenal, J.R. Kim, Biofilm matrix and artificial mediator for efficient electron transport in CO2 microbial electrosynthesis, Chemical Engineering Journal 427 (2022) 131885.

[0013] L. Lin, H. Huang, X. Zhang, L. Dong, Y. Chen, Hydrogen-oxidizing bacteria and their applications in resource recovery and pollutant removal, Science of The Total Environment 835 (2022) 155559.

[0014] J.P. Torella, C.J. Gagliardi, J.S. Chen, D.K. Bediako, B. Colón, J.C. Way, P.A. Silver, D.G. Nocera, Efficient solar-to-fuels production from a hybrid microbial–water-splitting catalyst system, Proceedings of the National Academy of Sciences 112 (2015) 2337-2342.

[0015] J. Panich, B. Fong, S.W. Singer, Metabolic engineering of Cupriavidus necator H16 for sustainable biofuels from CO2, Trends in biotechnology 39 (2021) 412-424.

[0016] K. Tanaka, A. Ishizaki, T. Kanamaru, T. Kawano, Production of poly (D‐3‐hydroxybutyrate) from CO2, H2, and O2 by high cell density autotrophic cultivation of Alcaligenes eutrophus, Biotechnology and bioengineering 45 (1995) 268-275.

[0017] D. Yang, W.J. Kim, S.M. Yoo, J.H. Choi, S.H. Ha, M.H. Lee, S.Y. Lee, Repurposing type III polyketide synthase as a malonyl-CoA biosensor for metabolic engineering in bacteria, Proceedings of the National Academy of Sciences 115 (2018) 9835-9844.

[0018] E.S. Boyd, M.J. Amenabar, S. Poudel, A.S. Templeton, Bioenergetic constraints on the origin of autotrophic metabolism, Philosophical Transactions of the Royal Society A 378 (2020) 20190151.

[0019] J. Marc, E. Grousseau, E. Lombard, A.J. Sinskey, N. Gorret, S.E. Guillouet, Over expression of GroESL in Cupriavidus necator for heterotrophic and autotrophic isopropanol production, Metabolic Engineering 42 (2017) 74-84.

[0020] P.J. Janssen, R. Van Houdt, H. Moors, P. Monsieurs, N. Morin, A. Michaux, M.A. Benotmane, N. Leys, T. Vallaeys, A. Lapidus, The complete genome sequence of Cupriavidus metallidurans strain CH34, a master survivalist in harsh and anthropogenic environments, PLoS One 5 (2010) e10433.Attorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory

[0021] H. Li, P.H. Opgenorth, D.G. Wernick, S. Rogers, T.-Y. Wu, W. Higashide, P. Malati, Y.-X. Huo, K.M. Cho, J.C. Liao, Integrated Electromicrobial Conversion of CO2 to Higher Alcohols, Science 335 (2012) 1596-1596.

[0022] E. Rasten, G. Hagen, R. Tunold, Electrocatalysis in water electrolysis with solid polymer electrolyte, Electrochimica acta 48 (2003) 3945-3952.

[0023] H.M. Woo, Solar-to-chemical and solar-to-fuel production from CO2 by metabolically engineered microorganisms, Current Opinion in Biotechnology 45 (2017) 1-7.

[0062] It is to be understood that, while the invention has been described in conjunction with the preferred specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

[0063] All patents, patent applications, and publications mentioned herein are hereby incorporated by reference in their entireties.

[0064] The invention having been described, the following examples are offered to illustrate the subject invention by way of illustration, not by way of limitation. EXAMPLE 1 One pot electrolysis and fermentation to produce flaviolin

[0065] ELECTRONS TO PRODUCTS: CHALLENGES AND OPPORTUNITIES: Electrons are rapidly becoming cost-competitive with alternative electron donors. Product specificity and flexibility: (1) Challenges thermochemical, electrochemical, and anaerobic biological conversion. (2) Conversion efficiency and intensity. (3) Must couple biological and electrochemical conversion efficiency. (4) High flux rate of electron shuttle required. (5) Attached growth challenged by surface area requirement. Gas transfer efficiency: (1) High transfer rates of low-solubility H2 required. (2) Must also consider CO2 and O2 transfer if necessary. Interface of biology and electrochemistry: (1) Low-solubility or toxic electron shuttles favor in situ generation. (2) Biological and electrochemical conditions are not always compatible.Attorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory

[0066] INTEGRATING ELECTROLYSIS AND FERMENTATION: One pot electrolysis and fermentation have one or more of the following benefits: (1) Leverages electrolyzer for gas transfer. (2) No CAPEX or efficiency loss from gas separation membranes. (3) Direct production of long-chain hydrocarbons. See Figure 1.

[0067] OVERCOMING ELECTROLYSIS TOXICITY VIA ELECTROCHEMISTRY: Prior art includes Cerami R. eutropha, Ceramic shielding. Li et al., Science, 2012. See Figure 2. R. eutropha, Biocompatible CoP catalyst design. Liu et al. Science, 2016. See Figure 3.

[0068] OVERCOMING ELECTROLYSIS TOXICITY VIA STRAIN SELECTION: Electrode and growth medium co-optimization. See Figure 4. Strain selection: Ralstonia Metallidurans. Broad heavy metal resistance. Higher CO2 and H2 affinity than R. eutropha. Genetic tools available. See Figure 5. Mixed gas 1: 2.5% H2, 1.5% O2, 1.5% CO2, N2 balance (94.5%). Mixed gas 2: 2.5% H2, 21.5% O2, 1.5% CO2, N2 balance (74.5%). See Figure 6.

[0069] SYSTEM INTEGRATION: Electrode biocompatibility. Growth achieved directly from solar cells with low cost stainless electrodes. Figure 7. Carbon supply is optimized. OD >1 is achieved in 4 days with carbon supply optimization. Stainless electrodes with Pt-Ir catalyst. Figure 8. Future opportunities include: (1) Engineering for bioproduct formation. (2) Process intensification - H2supply and media supplementation. (3) Exploring additional strains – thermophiles and acidophiles.

[0070] STRAIN ENGINEERING: Strain engineered for flaviolin production: (1) Single gene type III PKS insert. (2) Reddish pigment (electrochemically converts to yellow in our system). (3) POC for one pot conversion of photons or electrons to complex metabolites. Control: no electricity. Engineered R. metallidurans is used in the electrochemical reactors. EXAMPLE 2 Single-chamber biosynthesis of complex organic molecules from CO2and renewable electrons

[0071] Aerobic biological conversion of electrolytically generated hydrogen and carbon dioxide is a compelling route for high-specificity production of organic chemicals from waste feedstocks. This chemistry requires a gaseous electron donor, acceptor, and carbon source, and minimizing gas transfer cost for CO2, H2, and O2is therefore a primary limitation for commercial deployment. Directly integrating fermentation and water-splitting electrolysisAttorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory could potentially address energetic costs for gas dispersion by leveraging direct formation of gaseous microbubbles from the electrodes, while concurrently decreasing infrastructure costs via consolidation of unit operations into a single reaction vessel. In practice, fully consolidating electrolysis and fermentation requires careful consideration, to avoid exposing the biocatalyst to reactive oxygen species and heavy metal toxicity, and to avoid crossover of gases between cathode and anode. In this study, we investigate the potential of Cupriavidus metallidurans CH34 as a multi-resistant chemolithotrophic host suitable for aerobic- autotrophic growth in a single chamber water-splitting electrolyzer. To address cultivation under electrolysis conditions, we developed a prototype single-chamber electrolyzer and demonstrated autotrophic growth of C. metallidurans to an OD600 of 1.2 when powered directly from a solar cell, with H2 and O2 supplied directly from the electrodes and no intermediate shielding. We then engineered C. metallidurans CH34 strains for inducible heterologous expression of the heterologous polyketide flaviolin, achieving titers of 696 ± 66 mg / L within the electrochemical cell. This result confirms that this one pot reactor configuration, when paired with a genetically tractable and high-tolerance host, is a feasible geometry for single-chamber synthesis of complex products from CO2and renewable electrons.

[0072] In this study, Cupriavidus metallidurans CH34 strain was screened, which is the facultative chemolithoautotrophic β-proteobacterium. It was reported to assimilate CO2using the Calvin-Benson-Bassham (CBB) cycle like C. necator H16 (formerly Ralstonia eutropha H16), and it also has a high concentration tolerance of heavy metal ions [15, 16]. Moreover, it has a malonyl-CoA pathway, which can convert into various manufactured products as fuels. However, quantifying intracellular malonyl-CoA concentration requires a highly accurate but time-consuming analytical method such as LC-MS, due to the presence of many different intracellular metabolisms. Moreover, it has to require a complex sample preparation process due to the dynamic intermediate with rapid turnover rate, and sensitivity to environmental conditions, such as pH and temperature

[0017] .

[0073] Therefore, the engineered C. metallidurans expressed the RppA gene, and it directly showed the malonyl-CoA concentration measurement via colorimetric indicator as 2,5,7- trihydroxy-1,4-naphthoquinone (i.e., Flaviolin). Moreover, we demonstrated and maximized electro-fermentation by mimicking artificial photosynthesis based on an electrolyzer in which we developed a scalable all-in-one single electrolyzer.Attorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory MATERIAL AND METHODS

[0074] Strains and culture media: Cupriavidus metallidurans CH34 was cultivated in minimal medium at 30 °C. The minimal medium was contained (per 1L); 2.895g Na2HPO4, 2.32g NaH2PO4, 0.117g Na2SO4, 0.943 g (NH4)2SO4, 0.8g MgSO4▪7H2O, 0.097g CaSO4▪2H2O. Moreover, the engineered strain was cultivated with modified media, adding 0.1 g yeast extract, 0.1g arabinose, and 0.4g chloramphenicol.

[0075] Reactor configuration: The reactor was designed as a double layer All-in-one Cylinder type Electrolyzer (ACE), which consisted of the working chamber and water jacket for temperature control. The total chamber volume is 320ml, but the actual working volume is 280ml. The electrodes can insert both sides of the reactor bottom caps, and the glass separator was located middle of the reactor bottom. The electrodes used Iridium oxide (IrO2) and Platinum (Pt) coated on three pairs 2X2 cm2stainless steel mash electrode layers at the anode and cathode, respectively. Moreover, the reference electrode used the customized Ag / AgCl 3M KCl.

[0076] Electrolyzer operation: Cupriavidus metallidurans CH34 was inoculated at a final optical density of 0.1 at 600 mn in minimal media. For CO2supplement, most electrolyzer operation added bolus NaHCO3 (0.2g / L), but some test was feed 10ml / min CO2 gas directly. For the applied power source, all electrolyzer was induced 4.5V by chronoamperometry (vs. Ag / AgCl 3M KCl, potentiostat, Interface 1010e, Gamry instruments, USA), or solar panel array Photovoltaic system (PV). The media preparation was sparged 50 ml / min N2 gas for 2 hours for eliminated dissolved oxygen. The temperature was controlled at 30 ± 1 °C by recirculating chillers (VWR, USA).

[0077] Analyses: The electrochemical characteristics of the catalytic electrodes were examined by Liner Sweep voltammetry (LSV) using a potentiostat (Interface 1010e, Gamry instruments, USA) with Gamry Framework software. The polarization curve was investigated by LSV at a scan rate of 10 mV / s from 0 to 5V versus Ag / AgCl 3M KCl, respectively.

[0078] The SEM images of the catalytic electrode were taken with a Zeiss Gemini Ultra-55 analytical scanning electron microscope at an accelerating voltage of 3 keV. Energy- dispersive X-ray spectroscopy (EDS) was taken on a Zeiss Gemini Ultra-55 analytical scanning electron microscope (accelerating voltage 7 keV) with an EDAX EDS detector with Genesis software. A range of spot sizes and locations on film were measured beforeAttorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory concluding compositional analysis and to confirm uniformity.

[0079] The turbidity of the suspension was determined from the optical density using a UV- visible spectrophotometer (Thermo Fisher Scientific™, Evolution™ 60S UV-Visible Spectrophotometer, Waltham, MA) at 600 nm. The absorbance of the flaviolin was determined at a scan range from 200 to 800 nm, and specific products was indicated at 290 nm using plate reader (Synergy™ Mx, U.S. BioTek Laboratories, Shoreline, WA). RESULTS AND DISCUSSION

[0080] Characterization of the C. metallidurans CH34: Most of the autotrophic microbes are anaerobic and use CO2as both an oxidant and carbon source, but H2-dependent autotrophic, ferredoxin reduction is accomplished using one of several recently involved enzymatic mechanisms

[0018] . Cupriavidus metallidurans CH34 is reported for their facultative chemolithotrophic microbes, but they can autotrophic growth under sufficient hydrogen and CO2 supply by hydrogenase

[0019] . To investigate the proton energy carrier effect for autotrophic growth, C. metallidurans CH34 was cultured, added bolus NaHCO3, or fed CO2 gas directly. In Fig.9 (Panel a), the CO2-fed cultivation was indicated to decrease cell density gradually over time, whereas the bolus NaHCO3supplied cultivation showed slightly increased cell density. This phenomenon means that the C. metallidurans can be utilized soluble hydrogen as a proton via autotrophic growth. In addition, to demonstrate autotrophic growth in aerobic conditions, the strains were cultivated under mixed gas supplements (i.e., variable O2, 2.5% H2, CO2, and N2 balance) in which differently fed as high oxygen (i.e., ambient air: 21% O2), and low oxygen (i.e., like anaerobic: 1.5% O2). As a result, autotrophic growth was shown only as anaerobic condition, and a highly aerobic condition hardly grows as similar to CO2-fed fermentation (Fig.9 (Panel b)). Herein, the anaerobic condition contains less amount of oxygen at 1.5% which phenomenon means that oxygen is a critical inhibit factor for H2-dependent autotrophic growth, but sufficiently supplied hydrogen and CO2might grow in aerobic fermentation.

[0081] Therefore, C. metallidurans CH34 seems to have high-affinity hydrogenase activity which may help to strongly indicate that leads to facultative aerobically autotrophic fermentation. However, Janssen et al.,

[0020] reported that it is worked aerobic energy metabolism that the several specific gene clusters located on chromosomes encoding a life membrane-bound complex of NADH dehydrogenase, but simultaneously anaerobicAttorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory metabolism may resort to alternate modes of energy generation by using electron acceptors other than oxygen.

[0082] Expression of RppA expressed to the production of the Flaviolin: C. metallidurans CH34 strain can produce malonyl-CoA by autotrophic synthesis, and that malonyl-CoA precursor molecule, its major building block, benefits many value-added chemicals. However, quantifying intracellular malonyl-CoA concentration requires a highly accurate but time-consuming analytical method such as LC-MS, due to the presence of many different intracellular metabolisms. Moreover, it is known that has to require a complex sample preparation process due to the dynamic intermediate with rapid turnover rate, and sensitivity to environmental conditions, such as pH and temperature

[0017] . To overcome this limitation, the C. metallidurans strain was engineered with the colorimetric biosensor enzyme by encoding 1,3,6,8-Tetrahydroxynaphthalene synthetase (THNS, referred to as RppA) (Fig.10 (Panel a)).

[0083] RppA is synthesized from the five molecules of the malonyl-CoA to 1,3,6,8- Tetrahydroxynaphthalene (THN), in which the Flaviolin is spontaneously converted from THN, and it displayed red color by malonyl-CoA level. To confirm the colorimetric change, the engineered C. metallidurans, and wild-type strains were cultivated by feeding 10ml / min hydrogen with bolus NaHCO3 for 2 days. In Fig.10 (Panel b) the cell growth showed similarly around 0.5 OD in both cultivations, but only the mutant culture indicated a red color. In addition, the wild-type strain was not indicated as any peak when comparing the absorption wavelength spectra from 200 to 400 nm as different from mutant strains (Fig.10 (Panel c)). Meanwhiles, in the absorption scan wavelengths, the flaviolin compounds obtained attain their maximal value at approximately 280 to 290 nanometers, exceedingly elevated reactions at UV wavelengths, demonstrating a similar profile to the mean absorption spectra.

[0084] Optimization of the all-in-one cylinder electrolyzer (ACE) for electrochemical artificial autotrophic fermentation: Electrochemically artificial autotrophic cultivation needs integrated water splitting and CO2 biomass conversion progress. Hence, the electrolyzer fermentation has required the potential for water splitting over the thermodynamic potential of E=1.23V, but indeed, the considerable system resistance required a higher thermodynamic potential. Besides, thermodynamic ROS evolution potential was reported to be favored over hydrogen production (beyond 1.6V) and occurs below 2.7V

[0022] . Moreover, Torella et al.,Attorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory

[0014] reported that bacterial growth revealed the quizzical dependence that biological viability was observed only for applied potentials of over 2.7V vs. NHE. as support this insisted, the previous bioelectrochemical cells studied reported that the 4.0 to 5.5V vs. NHE applied potential is well-maintained cell viability

[0021] .

[0085] The developed all-in-one cylinder-type electrolyzer (ACE) consists of two sections such as water splitting part and fluidized bed fermentation. To improve water splitting performance, the catalytic electrode had to be considered for optimization, and the pair of the iridium oxide and platinum-coated on the stainless-steel electrode obtained the highest electric performance. Moreover, the electrode surface area was installed by triple pairs to provide sufficient hydrogen evolution through water splitting at 4.5V applied potential. In Fig.11, the autotrophic cell growth was enhanced up to 1.5 times higher as 1.24 OD for 140 hours than the single pair electrode, and simultaneously the consumption current was increased to record from 75 mA to 90 mA.

[0086] In addition, for the electrolyzer fermentation, the water splitting process is possibly used for occurring potential beyond 1.23 V via any type of electric source, but the potentiostat energy supply is favored in the small-size electro-fermentation for more sensitive electrochemical reaction analysis or control. Herein, to mimic the artificial autotrophic fermentation, the electrolyzer worked the water-splitting process from chronoamperometry to photovoltaic cells from solar flex energy. The potentiostat was recorded to consume a current of around 90mA during the water-splitting process, but the photovoltaic cell was supplied up to 150mA at the same applied potential (data not shown). However, the autotrophic cell growth level was similar around 1.25 OD in both power supplies (Fig.11).

[0087] This phenomenon seems that the increased supplied current directly affected hydrogen production and cell growth, but increased microbubble hydrogen production by increased electrode surface area seems to carry out the synergic effect with the hydrogenase of the C. metallidurans. However, the chronoamperometry current supply has a demand current level for the requirement of the redox reaction, whereas the photovoltaic cell is supplied by assigned power by solar panel. Thus, the highly supplied potential may be affected to lead the electrode load.

[0088] Meanwhile, the ACE may occur ROS while the electrolyzer is, and it might affect the suspended microbe in the fluidized bed fermentation area because it is a differentAttorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory configuration from the fundamental H-type reactors. Therefore, the ROS effect was investigated on C. metallidurans CH34 strains, and it was cultivated to inoculate at 0.05 OD for 4 days under different ROS concentrations from 0 to 900uM. C. metallidurans CH34 strains showed well growth for 4 days and obtained less effect of the ROS cytotoxicity. These results addressed that the C. metallidurans CH34 demonstrated high affinity of hydrogenase activity and low ROS damage, which is more suitable for the electrolyzer fermentation strains. Moreover, High tolerance for cytotoxic ROS acts as the advantage of the high potential electro-fermentation process due to preventing the inhibition of cell growth in the evolution of the redox signaling and oxidative stress while water splitting.

[0089] Artificial phototrophic growth in all-in-one cylinder type electrolyzer: The ACE was coupled with a PV system, which supplied 4.5V and 150 mA for the electrochemical artificial photosynthesis system. The engineered C. metallidurans were initially inoculated to adjust at 0.05 OD and induced with 0.5g / L, then allowed to express autotrophic for 96 hours. Fig.12 (Panel A) compared with the open circuit operation, the mutant strain in the electrolyzer was grown up to 0.96 OD for 100 hours. However, the open circuit electrolyzer showed slight growth than inoculation cell density. Flaviolin is known to indicate the red color as accumulated. Indeed, the flask cultivation was expressed red in Fig.10, but when the cultivation in the ACE, the flaviolin accumulated yellow instead of the red colorimetric. To demonstrate the colorimetric change, the autotrophic cultured flaviolin was used for electrolyzer fermentation for 24 hours. The colorimetric flaviolin was changed into yellow after electrolysis progressed. Moreover, in the absorption scan wavelengths, the flaviolin and electrolysis flaviolin indicated different wavelength spectra. Nonetheless, those compounds obtained a maximum value at approximately 290 nanometers, exceedingly elevated reactions at absorption wavelengths, demonstrating a similar profile to the mean absorption spectra. Thus, to determine the electrolysis flaviolin concentration, the flaviolin was abiotically electrolysis process, and the electrolysis flaviolin concentration was estimated by absorption level. In Fig.12 (Panel b), the electrolysis flaviolin obtained that estimated at up to 695.9 ± 66.1 mg / L for 96 hours.

[0090] On the other hand, the photovoltaic cell reported that it could supply current at around 150mA, so it would be possible to damage the electrode. Therefore, the electrode catalyst was analyzed for degradation phenomena by long-term operation. The SEM image and EDS atomic mapping were captured and analyzed on the surface of the iridium oxide and platinumAttorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory catalyst-coated electrode. The iridium oxide-coated electrode as an anode electrode indicated a relatively faster degradation than the cathode electrode as coated with platinum on the surface after 4 weeks. Thus, a higher current supply seems to have damaged the catalytic stability and decreased the performance of the electrolyzer, which showed similar electrochemcial activity with bare stainless-steel electrodes (data not shown).

[0091] Implication: Artificial photosynthesis is interesting in that can be mimicking the natural cycle, which can remove CO2 and simultaneously generate biomass and fuels. However, biological artificial photosynthesis was studied less because of the less growth rate of the autotrophic microbial and the limitation of the cell growth conditions as mostly anaerobic cultivation. C. metallidurans CH34 strain overcomes the less cell growth and can facultative autotrophic growth in both aerobic and anaerobic. In addition, the developed all- in-one cylinder-type electrolyzer made it possible to scalable design the system for the application of the infrastructure. Thus, the diversly engineered C. metallidurans strains can give the chance to various malonyl-CoA derived chemicals and fuels as mimicking electrochemically artificial photosynthesis. CONCLUSION

[0092] C. metallidurans CH34 is addressed suitably for the electrochemical artificial photosynthesis process due to its facultative chemolithotrophic microbe, which has a high tolerance for ROS and high-affinity hydrogenase activity. Therefore, the all-in-one cylinder- type electrolyzer (ACE) with applied potential at 4.5 V by photovoltaic cells was investigated, mimicking artificial photosynthesis to convert CO2to biomass or fuels. The accumulated malonyl-CoA production is challenging to analyze, but the Raap protein expression on C. metallidurans was able to be simply indicated by colorimetric ways. Hence, the optimized ACE configuration as electrode and carbon fed led to enhance cell growth, and concomitantly, malonyl-CoA production has risen up to 695.9 ± 66.1 mg / L as estimated by flaviolin.

[0093] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit andAttorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

Attorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory WE CLAIM:

1. A vessel comprising: (a) electrodes, in electrical communication with a power source, and (b) a solution comprising a microbial strain capable of synthesizing a compound of interest, wherein the microbial strain obtains energy via water-splitting electrolysis through the electrodes and microbial hydrogen fermentation.

2. The vessel of claim 1, wherein the microbe strain which has natural resistance to heavy metal toxicity, a high affinity for hydrogen and carbon dioxide, and a high tolerance for oxygen.

3. The vessel of claim 1, wherein the microbe strain has a higher affinity for hydrogen and carbon dioxide than that of Ralstonia eutropha.

4. The vessel of claim 1, wherein the microbe strain is a bacterial cell.

5. The vessel of claim 4, wherein the bacterial cell is of the genus Ralstonia,.

6. The vessel of claim 5, wherein the bacterial cell is Ralstonia metallidurans, Ralstonia eutropha, Ralstonia insidiosa, Ralstonia mannitolilytica, Ralstonia pickettii, Ralstonia pseudosolanacearum, Ralstonia solanacearum, or Ralstonia syzygii.

7. The vessel of claim 6, wherein the bacterial cell is Ralstonia metallidurans.

8. The vessel of claim 4, wherein the microbe strain is of the genus Cupriavidus.

9. The vessel of claim 8, wherein the microbe strain is Cupriavidus metallidurans or Cupriavidus necator.

10. The vessel of claim 4, wherein the microbe strain is chemolithoautotrophic, 11. The vessel of claim 10, wherein the microbe strain is a facultative chemolithoautotrophic.

12. The vessel of claim 11, wherein the microbe strain is a facultative chemolithoautotrophic β-proteobacterium.

13. The vessel of claim 1, wherein the microbe strain is able to assimilate CO2 using the Calvin-Benson-Bassham (CBB) cycle.Attorney Docket: 2021-094-02 Lawrence Berkeley National Laboratory 14. The vessel of claim 13, wherein the microbe strain is a thermophile and / or electrophile.

15. The vessel of claim 1, wherein the microbe strain is of a resilient strain which allows use of standard low-cost electrode materials.

16. The vessel of claim 1, wherein the vessel is configured for integrating electrolysis and fermentation such that gas dissolution is directly from the electrodes.

17. The vessel of claim 1, wherein the microbial strain is engineered for production of one or more compounds of interest.

18. The vessel of claim 1, wherein the electrodes comprise at least one anode and at least one cathode.

19. The vessel of claim 18, wherein the cathode is a reactive oxygen species (ROS)- resistant cathode.

20. The vessel of claim 18, wherein one or more of the electrodes is a cobalt-phosphorous (Co-P) alloy electrode.

21. The vessel of claim 18, wherein the cathode is a cobalt-phosphorous (Co-P) alloy cathode.

22. The vessel of claim 18, wherein the anode is a self-healing Co-Pialloy anode.

23. The vessel of claim 18, wherein one or more of the electrodes, or the solution, comprises a catalyst.

24. The vessel of claim 23, wherein the catalyst is a Pt, Ir, or Pt-Ir catalyst.

25. The vessel of claim 23, wherein the cathode comprises Pt, and the anode comprises Ir.

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