Current Status of Biological Production Using C2 Feedstocks

Abstract

C2 feedstocks have emerged as promising carbon sources for the biological production of various value-added chemicals. Compared to the traditional C6/C5 sugars-contained/constituted feedstocks, C2 feedstocks have diverse and abundant sources, including non-food biomass, industrial by-products, and C1 gases. This diversification not only eliminates competition with human food demands but also aligns with environmental sustainability goals. Moreover, the metabolic route for C2 compounds to enter central carbon metabolism is more direct, which minimizes the carbon loss and enhances the efficiency of bio-based production processes. This review extensively analyzes three prominent C2 chemicals: ethylene glycol, ethanol, and acetate. After introducing the sources of those compounds, it details the metabolic pathways through which they are converted into acetyl-CoA in vivo. Several chemicals produced from these C2 feedstocks in fermentation are also exemplified. Furthermore, different perspectives are proposed to promote the efficient utilization of C2 feedstocks.

Keywords

C2 feedstocks / Ethylene glycol / Ethanol / Acetate / Carbon source

Author Contributions

D.-K.Y.: Writing—original draft. D.-Y.X.: Writing—editing. J.-F.L.: Writing—editing. P.X.: Writing—review & editing, Funding acquisition, Conceptualization. J.-Z.L.: Writing—review & editing, Supervision, Funding acquisition, Conceptualization.

Ethics Statement

Not applicable.

Informed Consent Statement

Not applicable.

Funding

This work was financially supported by National Key Research and Development Program of China (2024YFA0918000), Natural Science Foundation of Guangdong Province (305196996052), Guangdong S&T Program (2024B1111160007, 2024B1111130003), and Science and Technology Plan Project of Guangzhou (2024A04J3803).

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References

[1] Wendisch VF, Brito LF, Gil Lopez M, Hennig G, Pfeifenschneider J, Sgobba E, et al. The flexible feedstock concept in Industrial Biotechnology: Metabolic engineering of Escherichia coli, Corynebacterium glutamicum, Pseudomonas, Bacillus and yeast strains for access to alternative carbon sources. J. Biotechnol. 2016, 234, 139-157.

[2] Kiefer D, Merkel M, Lilge L, Henkel M, Hausmann R. From Acetate to Bio-Based Products: Underexploited Potential for Industrial Biotechnology. Trends Biotechnol. 2021, 39, 397-411.

[3] FAO IFAD, UNICEF WFP, WHO. The State of Food Security and Nutrition in the World 2024—Financing to End Hunger, Food Insecurity and Malnutrition in All Its Forms; FAO: Rome, Italy, 2024.

[4] Wiesfeld JJ, Peršolja P, Rollier FA, Elemans-Mehring AM, Hensen EJ. Cellulose conversion to ethylene glycol by tungsten oxide-based catalysts, Mol. Catal. 2019, 473, 110400.

[5] Puligundla P, Smogrovicova D, Mok C, Obulam VSR. A review of recent advances in high gravity ethanol fermentation, Renew. Energy 2019, 133, 1366-1379.

[6] Law PG, Sebran NH, Zawawi AZ, Hussain AS. Optimization Study of Biomass Hydrogenation to Ethylene Glycol Using Response Surface Methodology. Processes 2020, 8, 588.

[7] Tournier V, Topham CM, Gilles A, David B, Folgoas C, Moya-Leclair E, et al. An engineered PET depolymerase to break down and recycle plastic bottles. Nature 2020, 580, 216-219.

[8] Štrukil V. Highly Efficient Solid-State Hydrolysis of Waste Polyethylene Terephthalate by Mechanochemical Milling and Vapor-Assisted Aging. ChemSusChem 2021, 14, 330-338.

[9] Xu H, Rebollar D, He H, Chong L, Liu Y, Liu C, et al. Highly selective electrocatalytic CO₂ reduction to ethanol by metallic clusters dynamically formed from atomically dispersed copper. Nat. Energy 2020, 5, 623-632.

[10] Tamura J, Ono A, Sugano Y, Huang C, Nishizawa H, Mikoshiba S. Electrochemical reduction of CO₂ to ethylene glycol on imidazolium ion-terminated self-assembly monolayer-modified Au electrodes in an aqueous solution. Phys. Chem. Chem. Phys. 2015, 17, 26072-26078.

[11] Yang X, Fugate E, Mueanngern Y, Baker L. Photo-Electrochemical CO₂ Reduction to Acetate on Iron-Copper Oxide Catalysts. ACS Catal. 2016, 7, 177-180.

[12] Yue H, Zhao Y, Ma X, Gong J. Ethylene glycol: properties, synthesis, and applications. Chem. Soc. Rev. 2012, 41, 4218-4244.

[13] Lum Y, Huang JE, Wang Z, Luo M, Nam D-H, Leow WR, et al. Tuning OH binding energy enables selective electrochemical oxidation of ethylene to ethylene glycol. Nat. Catal. 2020, 3, 14-22.

[14] Leow WR, Lum Y, Ozden A, Wang Y, Nam DH, Chen B, et al. Chloride-mediated selective electrosynthesis of ethylene and propylene oxides at high current density. Science 2020, 368, 1228-1233.

[15] Wang A, Zhang T. One-pot conversion of cellulose to ethylene glycol with multifunctional tungsten-based catalysts. Acc. Chem. Res. 2013, 46, 1377-1386.

[16] Rorrer NA, Nicholson S, Carpenter A, Biddy MJ, Grundl NJ, Beckham GT. Combining Reclaimed PET with Bio-based Monomers Enables Plastics Upcycling. Joule 2019, 3, 1006-1027.

[17] Werner AZ, Clare R, Mand TD, Pardo I, Ramirez KJ, Haugen SJ, et al. Tandem chemical deconstruction and biological upcycling of poly(ethylene terephthalate) to β-ketoadipic acid by Pseudomonas putida KT2440. Metab. Eng. 2021, 67, 250-261.

[18] Laopaiboon L, Nuanpeng S, Srinophakun P, Klanrit P, Laopaiboon P. Ethanol production from sweet sorghum juice using very high gravity technology: effects of carbon and nitrogen supplementations. Bioresour. Technol. 2009, 100, 4176-4182.

[19] Pradeep P, Reddy OV. High gravity fermentation of sugarcane molasses to produce ethanol: Effect of nutrients. Indian J. Microbiol. 2010, 50 (Suppl. 1), 82-87.

[20] Arshad M, Khan ZM, Khalil ur R, Shah FA, Rajoka MI. Optimization of process variables for minimization of byproduct formation during fermentation of blackstrap molasses to ethanol at industrial scale. Lett. Appl. Microbiol. 2008, 47, 410-414.

[21] Goswami M, Meena S, Navatha S, Prasanna Rani KN, Pandey A, Sukumaran RK, et al. Hydrolysis of biomass using a reusable solid carbon acid catalyst and fermentation of the catalytic hydrolysate to ethanol. Bioresour. Technol. 2015, 188, 99-102.

[22] Bai FW, Zhao XQ. High Gravity Ethanol Fermentations and Yeast Tolerance. In Microbial Stress Tolerance for Biofuels: Systems Biology; Liu ZL, Ed.; Springer: Berlin/Heidelberg, Germany, 2012; pp. 117-135.

[23] Thomas KC, Hynes SH, Ingledew WM. Practical and theoretical considerations in the production of high concentrations of alcohol by fermentation. Process Biochem. 1996, 31, 321-331.

[24] Pietrzak W, Kawa-Rygielska J. Simultaneous saccharification and ethanol fermentation of waste wheat-rye bread at very high solids loading: Effect of enzymatic liquefaction conditions. Fuel 2015, 147, 236-242.

[25] Koppram R, Tomás-Pejó E, Xiros C, Olsson L. Lignocellulosic ethanol production at high-gravity: challenges and perspectives. Trends Biotechnol. 2014, 32, 46-53.

[26] Saha BC, Iten LB, Cotta MA, Wu YV. Dilute acid pretreatment, enzymatic saccharification, and fermentation of rice hulls to ethanol. Biotechnol. Prog. 2005, 21, 816-822.

[27] Cara C, Ruiz E, Oliva JM, Sáez F, Castro E. Conversion of olive tree biomass into fermentable sugars by dilute acid pretreatment and enzymatic saccharification. Bioresour. Technol. 2008, 99, 1869-1876.

[28] Zhao X, Cheng K, Liu D. Organosolv pretreatment of lignocellulosic biomass for enzymatic hydrolysis. Appl. Microb. Biotechnol. 2009, 82, 815-827.

[29] Mutyala S, Kim JR. Recent advances and challenges in the bioconversion of acetate to value-added chemicals. Bioresour. Technol. 2022, 364, 128064.

[30] Budiman AW, Nam JS, Park JH, Mukti RI, Chang TS, Bae JW, et al. Review of Acetic Acid Synthesis from Various Feedstocks Through Different Catalytic Processes. Catal. Surv. Asia 2016, 20, 173-193.

[31] Nayak J, Pal P. Acetic Acid Production and Purification: Critical Review Towards Process Intensification. Sep. Purif. Rev. 2016, 46, 44-61.

[32] Krauch DC, Pier DM.Verfahren zur Darstellung von Kohlenwasserstoffen. German Patent DE 1935, 24 January 1935.

[33] Mullen A. Carbonylations Catalyzed by Metal Carbonyls—Reppe Reactions. ChemInform 1981, 12. doi:10.1002/chin.198131346.

[34] Reppe W, Kröper H, v. Kutepow N, Pistor HJ. Carbonylierung III. Umsetzung von Alkoholen und offenen Äthern mit Kohlenoxyd zu Carbonsäuren. Justus Liebigs Ann. Chem. 1953, 582, 72-86.

[35] Kim Y, Lama S, Agrawal D, Kumar V, Park S. Acetate as a potential feedstock for the production of value-added chemicals: Metabolism and applications. Biotechnol. Adv. 2021, 49, 107736.

[36] Raspor P, Goranovič D. Biotechnological Applications of Acetic Acid Bacteria. Crit. Rev. Biotechnol. 2008, 28, 101-124.

[37] Arnold S, Moss K, Henkel M, Hausmann R. Biotechnological Perspectives of Pyrolysis Oil for a Bio-Based Economy. Trends Biotechnol. 2017, 35, 925-936.

[38] Demirbas A. The influence of temperature on the yields of compounds existing in bio-oils obtained from biomass samples via pyrolysis. Fuel Process Technol. 2007, 88, 591-597.

[39] Lian J, Garcia-Perez M, Coates R, Wu H, Chen S. Yeast fermentation of carboxylic acids obtained from pyrolytic aqueous phases for lipid production. Bioresour. Technol. 2012, 118, 177-186.

[40] Hu P, Rismani-Yazdi H, Stephanopoulos G. Anaerobic CO₂ fixation by the acetogenic bacterium Moorella thermoacetica. AIChE J. 2013, 59, 3176-3183.

[41] Panda S, Fung VYK, Zhou JFJ, Liang H, Zhou K. Improving ethylene glycol utilization in Escherichia coli fermentation. Biochem. Eng. J. 2021, 168, 107957.

[42] Lu Z, Cabiscol E, Obradors N, Tamarit J, Ros J, Aguilar J, et al. Evolution of an Escherichia coli protein with increased resistance to oxidative stress. J. Biol. Chem. 1998, 273, 8308-8316.

[43] Mückschel B, Simon O, Klebensberger J, Graf N, Rosche B, Altenbuchner J, et al. Ethylene glycol metabolism by Pseudomonas putida. Appl. Environ. Microbiol. 2012, 78, 8531-8539.

[44] Shimizu T, Suzuki K, Inui M. A mycofactocin-associated dehydrogenase is essential for ethylene glycol metabolism by Rhodococcus jostii RHA1. Appl. Microbiol. Biotechnol. 2024, 108, 58.

[45] Yan W, Qi X, Cao Z, Yao M, Ding M, Yuan Y. Biotransformation of ethylene glycol by engineered Escherichia coli. Synth. Syst. Biotechnol. 2024, 9, 531-539.

[46] Wagner N, Bade F, Straube E, Rabe K, Frazão CJR, Walther T. In vivo implementation of a synthetic metabolic pathway for the carbon-conserving conversion of glycolaldehyde to acetyl-CoA. Front. Bioeng. Biotechnol. 2023, 11, 1125544.

[47] Lu X, Liu Y, Yang Y, Wang S, Wang Q, Wang X, et al. Constructing a synthetic pathway for acetyl-coenzyme A from one-carbon through enzyme design. Nat. Commun. 2019, 10, 1378.

[48] Suzuki R, Katayama T, Kim BJ, Wakagi T, Shoun H, Ashida H, et al. Crystal structures of phosphoketolase: Thiamine diphosphate-dependent dehydration mechanism. J. Biol. Chem. 2010, 285, 34279-34287.

[49] Pandit AV, Harrison E, Mahadevan R. Engineering Escherichia coli for the utilization of ethylene glycol. Microb. Cell Fact. 2021, 20, 22.

[50] Panda S, Zhou JFJ, Feigis M, Harrison E, Ma X, Fung Kin Yuen V, et al. Engineering Escherichia coli to produce aromatic chemicals from ethylene glycol. Metab. Eng. 2023, 79, 38-48.

[51] de Jong BW, Shi S, Valle-Rodríguez JO, Siewers V, Nielsen J. Metabolic pathway engineering for fatty acid ethyl ester production in Saccharomyces cerevisiae using stable chromosomal integration. J. Ind. Microbiol. Biotechnol. 2015, 42, 477-486.

[52] Gao Q, Cao X, Huang YY, Yang JL, Chen J, Wei LJ, et al. Overproduction of Fatty Acid Ethyl Esters by the Oleaginous Yeast Yarrowia lipolytica through Metabolic Engineering and Process Optimization. ACS Synth. Biol. 2018, 7, 1371-1380.

[53] Liu Y, Bai C, Liu Q, Xu Q, Qian Z, Peng Q, et al. Engineered ethanol-driven biosynthetic system for improving production of acetyl-CoA derived drugs in Crabtree-negative yeast. Metab. Eng. 2019, 54, 275-284.

[54] Li T, Liu GS, Zhou W, Jiang M, Ren YH, Tao XY, et al. Metabolic Engineering of Saccharomyces cerevisiae To Overproduce Squalene. J. Agric. Food Chem. 2020, 68, 2132-2138.

[55] Kessler D, Leibrecht I, Knappe J. Pyruvate-formate-lyase-deactivase and acetyl-CoA reductase activities of Escherichia coli reside on a polymeric protein particle encoded by adhE. FEBS Lett. 1991, 281, 59-63.

[56] Membrillo-Hernandez J, Echave P, Cabiscol E, Tamarit J, Ros J, Lin EC. Evolution of the adhE gene product of Escherichia coli from a functional reductase to a dehydrogenase. Genetic and biochemical studies of the mutant proteins. J. Biol. Chem. 2000, 275, 33869-33875.

[57] Wenk S, Schann K, He H, Rainaldi V, Kim S, Lindner SN, et al. An “energy-auxotroph” Escherichia coli provides an in vivo platform for assessing NADH regeneration systems. Biotechnol. Bioeng. 2020, 117, 3422-3434.

[58] Lu J, Wang Y, Xu M, Fei Q, Gu Y, Luo Y, et al. Efficient biosynthesis of 3-hydroxypropionic acid from ethanol in metabolically engineered Escherichia coli. Bioresour. Technol. 2022, 363, 127907.

[59] Wang Y, Lu J, Xu M, Qian C, Zhao F, Luo Y, et al. Efficient Biosynthesis of Isopropanol from Ethanol by Metabolically Engineered Escherichia coli. ACS Sustain. Chem. Eng. 2022, 10, 13857-13864.

[60] Liang H, Ma X, Ning W, Liu Y, Sinskey AJ, Stephanopoulos G, et al. Constructing an ethanol utilization pathway in Escherichia coli to produce acetyl-CoA derived compounds. Metab. Eng. 2021, 65, 223-231.

[61] Huang B, Yang H, Fang G, Zhang X, Wu H, Li Z, et al. Central pathway engineering for enhanced succinate biosynthesis from acetate in Escherichia coli. Biotechnol. Bioeng. 2018, 115, 943-954.

[62] Young EM, Zhao Z, Gielesen BEM, Wu L, Benjamin Gordon D, Roubos JA, et al. Iterative algorithm-guided design of massive strain libraries, applied to itaconic acid production in yeast. Metab. Eng. 2018, 48, 33-43.

[63] Xu Y, Li Z. Utilization of ethanol for itaconic acid biosynthesis by engineered Saccharomyces cerevisiae. FEMS Yeast Res. 2021, 21, foab043.

[64] Arndt A, Auchter M, Ishige T, Wendisch VF, Eikmanns BJ. Ethanol catabolism in Corynebacterium glutamicum. J. Mol. Microbiol. Biotechnol. 2008, 15, 222-233.

[65] Eggert MW, Byrne ME, Chambers RP. Kinetic involvement of acetaldehyde substrate inhibition on the rate equation of yeast aldehyde dehydrogenase. Appl. Biochem. Biotechnol. 2012, 168, 824-833.

[66] Felenbok B, Flipphi M, Nikolaev I. Ethanol catabolism in Aspergillus nidulans: a model system for studying gene regulation. Prog. Nucleic Acid. Res. Mol. Biol. 2001, 69, 149-204.

[67] Schobert M, Görisch H. Cytochrome c550 is an essential component of the quinoprotein ethanol oxidation system in Pseudomonas aeruginosa: Cloning and sequencing of the genes encoding cytochrome c550 and an adjacent acetaldehyde dehydrogenase. Microbiol. SGM 1999, 145 Pt. 2, 471-481.

[68] Kumari S, Tishel R, Eisenbach M, Wolfe AJ. Cloning, characterization, and functional expression of acs, the gene which encodes acetyl coenzyme A synthetase in Escherichia coli. J. Bacteriol. 1995, 177, 2878-2886.

[69] Kumari S, Beatty CM, Browning DF, Busby SJ, Simel EJ, Hovel-Miner G, et al. Regulation of acetyl coenzyme A synthetase in Escherichia coli. J. Bacteriol. 2000, 182, 4173-4179.

[70] Fox DK, Roseman S. Isolation and characterization of homogeneous acetate kinase from Salmonella typhimurium and Escherichia coli. J. Biol. Chem. 1986, 261, 13487-13497.

[71] Noh MH, Lim HG, Woo SH, Song J, Jung GY.Production of itaconic acid from acetate by engineering acid-tolerant Escherichia coli W. Biotechnol. Bioeng. 2018, 115, 729-738.

[72] Kutscha R, Tomin T, Birner-Gruenberger R, Bekiaris PS, Klamt S, Pflügl S. Efficiency of acetate-based isopropanol synthesis in Escherichia coli W is controlled by ATP demand. Biotechnol. Biofuels Bioprod. 2024, 17, 110.

[73] Beck SW, Ye DY, Hwang HG, Jung GY.Stepwise Flux Optimization for Enhanced GABA Production from Acetate in Escherichia coli. J. Agric. Food Chem. 2024, 72, 10420-10427.

[74] Archer CT, Kim JF, Jeong H, Park JH, Vickers CE, Lee SY, et al. The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli. BMC Genom. 2011, 12, 9.

[75] Alterthum F, Ingram LO. Efficient ethanol production from glucose, lactose, and xylose by recombinant Escherichia coli. Appl. Environ. Microbiol. 1989, 55, 1943-1948.

[76] Nagata S. Growth of Escherichia coli ATCC 9637 through the uptake of compatible solutes at high osmolarity. J. Biosci. Bioeng. 2001, 92, 324-329.

[77] Kiefer D, Merkel M, Lilge L, Hausmann R, Henkel M. High cell density cultivation of Corynebacterium glutamicum on bio-based lignocellulosic acetate using pH-coupled online feeding control. Bioresour. Technol. 2021, 340, 125666.

[78] Schmollack M, Werner F, Huber J, Kiefer D, Merkel M, Hausmann R, et al. Metabolic engineering of Corynebacterium glutamicum for acetate-based itaconic acid production. Biotechnol. Biofuels Bioprod. 2022, 15, 139.

[79] Gu P, Zhao S, Niu H, Li C, Jiang S, Zhou H, et al. Synthesis of isobutanol using acetate as sole carbon source in Escherichia coli. Microb. Cell Fact. 2023, 22, 196.

[80] Sauer U, Eikmanns BJ. The PEP-pyruvate-oxaloacetate node as the switch point for carbon flux distribution in bacteria. FEMS Microbiol. Rev. 2005, 29, 765-794.

[81] Jo M, Noh MH, Lim HG, Kang CW, Im DK, Oh MK, et al. Precise tuning of the glyoxylate cycle in Escherichia coli for efficient tyrosine production from acetate. Microb. Cell Fact. 2019, 18, 57.

[82] Kim DH, Hwang HG, Jung GY.Optimum flux rerouting for efficient production of naringenin from acetate in engineered Escherichia coli. Biotechnol. Biofuels Bioprod. 2022, 15, 90.

[83] Zhou Y, Huang H, Zhou P, Xie J. Molecular mechanisms underlying the function diversity of transcriptional factor IclR family. Cell Signal 2012, 24, 1270-1275.

[84] Liu Z, Zhou L, Hua Q. Metabolic regulation of isocitrate lyase regulator in Escherichia coli based on metabolic flux information]. Sheng Wu Gong Cheng Xue Bao Chin. J. Biotechnol. 2012, 28, 565-576.

[85] Noh MH, Lim HG, Park S, Seo SW, Jung GY. Precise flux redistribution to glyoxylate cycle for 5-aminolevulinic acid production in Escherichia coli Escherichia coli. Metab. Eng. 2017, 43, 1-8.

[86] Zhang L, Li Y, Wang Z, Xia Y, Chen W, Tang K. Recent developments and future prospects of Vitreoscilla hemoglobin application in metabolic engineering. Biotechnol. Adv. 2007, 25, 123-136.

[87] Chong H, Yeow J, Wang I, Song H, Jiang R. Improving Acetate Tolerance of Escherichia coli by Rewiring Its Global Regulator cAMP Receptor Protein (CRP). PLoS ONE 2013, 8, e77422.

[88] Sun M, Gao AX, Liu X, Bai Z, Wang P, Ledesma-Amaro R. Microbial conversion of ethanol to high-value products: Progress and challenges. Biotechnol. Biofuels Bioprod. 2024, 17, 115.

[89] Zhang X, Zhang X-F, Li H-P, Wang L-Y, Zhang C, Xing X-H, et al. Atmospheric and room temperature plasma (ARTP) as a new powerful mutagenesis tool. Appl. Microbiol. Biotechnol. 2014, 98, 5387-5396.

[90] Yi X, Khey J, Kazlauskas RJ, Travisano M. Plasmid hypermutation using a targeted artificial DNA replisome. Sci. Adv. 2021, 7, eabg8712.

[91] Seong W, Han GH, Lim HS, Baek JI, Kim S-J, Kim D, et al. Adaptive laboratory evolution of Escherichia coli lacking cellular byproduct formation for enhanced acetate utilization through compensatory ATP consumption. Metab. Eng. 2020, 62, 249-259.

[92] Sun S, Ding Y, Liu M, Xian M, Zhao G. Comparison of Glucose, Acetate and Ethanol as Carbon Resource for Production of Poly(3-Hydroxybutyrate) and Other Acetyl-CoA Derivatives. Front. Bioeng. Biotechnol. 2020, 8, 833.

[93] Vo TM, Park JY, Kim D, Park S. Use of acetate as substrate for sustainable production of homoserine and threonine by Escherichia coli W3110: A modular metabolic engineering approach. Metab. Eng. 2024, 84, 13-22.

[94] Kim DH, Hwang HG, Ye DY, Jung GY.Transcriptional and translational flux optimization at the key regulatory node for enhanced production of naringenin using acetate in engineered Escherichia coli. J. Ind. Microbiol. Biotechnol. 2024, 51, kuae006.

[95] Frazão CJR, Wagner N, Rabe K, Walther T. Construction of a synthetic metabolic pathway for biosynthesis of 2,4-dihydroxybutyric acid from ethylene glycol. Nat. Commun. 2023, 14, 1931.

[96] Kataoka M, Sasaki M, Hidalgo A-RG, Nakano M, Shimizu S. Glycolic Acid Production Using Ethylene Glycol-Oxidizing Microorganisms. Biosci. Biotechnol. Biochem. 2001, 65, 2265-2270.

[97] Hua X, Cao R, Zhou X, Xu Y. Integrated process for scalable bioproduction of glycolic acid from cell catalysis of ethylene glycol. Bioresour. Technol. 2018, 268, 402-407.

[98] Nguyen LT, Mai DHA, Sarwar A, Lee EY. Reconstructing ethanol oxidation pathway in Pseudomonas putida KT2440 for bio-upgrading of ethanol to biodegradable polyhydroxybutanoates. Int. J. Biol. Macromol. 2022, 222, 902-914.

[99] Kamzolova SV, Shamin RV, Stepanova NN, Morgunov GI, Lunina JN, Allayarov RK, et al. Fermentation conditions and media optimization for isocitric acid production from ethanol by Yarrowia lipolytica. Biomed. Res. Int. 2018, 2018, 2543210.

[100] Xu J, Liu N, Qiao K, Vogg S, Stephanopoulos G. Application of metabolic controls for the maximization of lipid production in semicontinuous fermentation. Proc. Nati. Acad. Sci. USA 2017, 114, E5308-E5316.

[101] Oswald F, Dörsam S, Veith N, Zwick M, Neumann A, Ochsenreither K, et al. Sequential Mixed Cultures: From Syngas to Malic Acid. Front. Microbiol. 2016, 7, 891.

[102] Garcia-Gonzalez L, De Wever H. Acetic acid as an indirect sink of CO₂ for the synthesis of polyhydroxyalkanoates (PHA): Comparison with PHA production processes directly using CO₂ as feedstock. Appl. Sci. 2018, 8, 1416.

[103] Tong T, Chen X, Tang K, Ma W, Gao C, Song W, et al. A new-to-nature photosynthesis system enhances utilization of one-carbon substrates in Escherichia coli. Nat. Commun. 2025, 16, 145.

[104] Yu T, Cui H, Li JC, Luo Y, Jiang G, Zhao H.Enzyme function prediction using contrastive learning. Science 2023, 379, 1358-1363.

[105] Yeh AH-W, Norn C, Kipnis Y, Tischer D, Pellock SJ, Evans D, et al. De novo design of luciferases using deep learning. Nature 2023, 614, 774-780.

[106] Yang KK, Wu Z, Arnold FH. Machine-learning-guided directed evolution for protein engineering. Nat. Methods 2019, 16, 687-694.

[107] Jian X, Guo X, Wang J, Tan ZL, Xing X-h, Wang L, et al. Microbial microdroplet culture system (MMC): An integrated platform for automated, high-throughput microbial cultivation and adaptive evolution. Biotechnol. Bioeng. 2020, 117, 1724-1737.

[108] Moon JH, Nam S, Jeung K, Noh MH, Jung GY.Biosensor-assisted engineering for diverse microbial cellular physiologies. J. Agric. Food Chem. 2024, 72, 18321-18334.