Glutaric acid production by systems metabolic engineering of an L-lysine–overproducing Corynebacterium glutamicum Taehee Hana, Gi Bae Kima, and Sang Yup Leea,b,c,1 aMetabolic and Biomolecular Engineering National Research Laboratory, Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Yuseong-gu, 34141 Daejeon, Republic of Korea; bBioInformatics Research Center, Korea Advanced Institute of Science and Technology, Yuseong-gu, 34141 Daejeon, Republic of Korea; and cBioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology, Yuseong-gu, 34141, Daejeon, Republic of Korea Contributed by Sang Yup Lee, October 6, 2020 (sent for review August 18, 2020; reviewed by Tae Seok Moon and Blake A. Simmons) There is increasing industrial demand for five-carbon platform processes rely on nonrenewable and toxic starting materials, chemicals, particularly glutaric acid, a widely used building block however. Thus, various approaches have been taken to biologi- chemical for the synthesis of polyesters and polyamides. Here we cally produce glutaric acid from renewable resources (13–19). report the development of an efficient glutaric acid microbial pro- Naturally, glutaric acid is a metabolite of L-lysine catabolism in ducer by systems metabolic engineering of an L-lysine–overproducing Pseudomonas species, in which L-lysine is converted to glutaric Corynebacterium glutamicum BE strain. Based on our previous study, acid by the 5-aminovaleric acid (AVA) pathway (20, 21). We an optimal synthetic metabolic pathway comprising Pseudomonas previously reported the development of the first glutaric acid- putida L-lysine monooxygenase (davB) and 5-aminovaleramide amido- producing Escherichia coli by introducing this pathway compris- hydrolase (davA) genes and C. glutamicum 4-aminobutyrate amino- ing Pseudomonas putida davB, davA, davT, and davD genes transferase (gabT) and succinate-semialdehyde dehydrogenase (gabD) encoding L-lysine 2-monooxygenase (DavB), 5-aminovaleramide genes, was introduced into the C. glutamicum BE strain. Through amidohydrolase (DavA), 5-aminovalerate aminotransferase system-wide analyses including genome-scale metabolic simulation, (DavT), and glutarate semialdehyde dehydrogenase (DavD), comparative transcriptome analysis, and flux response analysis, 11 tar- respectively (15). Another pathway through condensation of get genes to be manipulated were identified and expressed at desired α-ketoglutarate with acetyl-CoA was also attempted in E. coli, levels to increase the supply of direct precursor L-lysine and reduce but the titer achieved by flask cultivation was only 0.42 g/L (16). precursor loss. A glutaric acid exporter encoded by ynfM was discov- Glutaric acid production by metabolically engineered Coryne- ered and overexpressed to further enhance glutaric acid production. bacterium glutamicum has also been reported in several studies Fermentation conditions, including oxygen transfer rate, batch-phase (13, 17, 19). Recently, Wittmann and colleagues developed a glu- glucose level, and nutrient feeding strategy, were optimized for the taric acid-overproducing C. glutamicum strain (17) by engineering efficient production of glutaric acid. Fed-batch culture of the final an AVA-producing strain that they had previously developed (22). engineered strain produced 105.3 g/L of glutaric acid in 69 h without They overexpressed the genes encoding 5-aminovalerate amino- any byproduct. The strategies of metabolic engineering and fer- transferase (GabT encoded by gabT), succinate-semialdehyde de- mentation optimization described here will be useful for devel- hydrogenase (GabD encoded by gabD), and a newly discovered oping engineered microorganisms for the high-level bio-based AVA importer (Ncgl0464) to create a C. glutamicum strain that production of other chemicals of interest to industry. metabolic engineering | Corynebacterium glutamicum | glutaric acid | Significance multiomics Glutaric acid is an important dicarboxylic acid used in the ue to the increasing concerns around climate change and synthesis of commercial polymers. Here we report metabolic – Dour heavy dependence on fossil resources, substantial effort engineering of an L-lysine overproducing Corynebacterium has been exerted for the bio-based production of chemicals, glutamicum strain for the high-level production of glutaric acid. fuels, and materials from renewable resources. Metabolic engi- The glutaric acid biosynthesis pathway was established in C. glutamicum by optimally expressing the genes encoding key neering, a key enabling technology for developing microbial cell enzymes from Pseudomonas putida and C. glutamicum for the factories, has rapidly advanced to the stage of systems-level efficient conversion of L-lysine to glutaric acid. Further meta- metabolic engineering for the development of engineered mi- – bolic engineering and overexpression of a newly discovered croorganisms capable of efficiently producing them (1 4). Var- glutaric acid exporter gene resulted in the production of ious microorganisms capable of producing diverse natural and 105.3 g/L of glutaric acid without byproducts from glucose. The nonnatural chemicals and materials have been developed over metabolic engineering strategies described here will be useful the last decade (5). In particular, bio-based polymers and their for developing microbial strains for the bio-based production building blocks have been receiving much attention as environ- of other chemicals in addition to glutaric acid. mentally friendly alternatives to the petroleum-derived plastics (6, 7). Author contributions: S.Y.L. and T.H. designed research; T.H. performed research; G.B.K. Glutaric acid, also known as pentanedioic acid, is a widely contributed in silico simulations; T.H. analyzed data; and T.H. and S.Y.L. wrote the paper. used chemical for various applications, including production of Reviewers: T.S.M., Washington University in St. Louis; and B.A.S., Joint BioEnergy Institute. polyamides, polyurethanes, glutaric anhydride, 1,5-pentanediol, The authors declare no competing interest. and 5-hydroxyvaleric acid (8), which are used in consumer goods, Published under the PNAS license. textile, and footwear industries (9). Glutaric acid has been pro- 1To whom correspondence may be addressed. Email: [email protected]. duced by various petroleum-based chemical methods, including This article contains supporting information online at https://www.pnas.org/lookup/suppl/ oxidation of 2-cyanocylopentanone catalyzed by nitric acid and doi:10.1073/pnas.2017483117/-/DCSupplemental. condensation of acrylonitrile with ethyl malonate (10–12). These First published November 16, 2020. 30328–30334 | PNAS | December 1, 2020 | vol. 117 | no. 48 www.pnas.org/cgi/doi/10.1073/pnas.2017483117 Downloaded by guest on September 30, 2021 produced 90 g/L glutaric acid with a productivity of 1.8 g/L/h and strain harboring pGA4 showed the highest glutaric acid titer yield of 0.7 mol glutaric acid per mol (glucose + molasses) from compared with those of others, producing 6.4 g/L of glutaric acid glucose and molasses by fed-batch fermentation. However, con- (Fig. 1C). Next, pGA5 was constructed by changing the back- sidering that C. glutamicum has the capability of producing >130 g/ bone plasmid from pCES208s to pEKEx1 with a higher copy L(andeven>200 g/L in industry) of L-lysine (23), further im- number and a tac promoter. provement of glutaric acid production seems possible. Further construction of pGA6 harboring the gabTD operon C. glutamicum is a widely used organism for global amino acid followed, to test the two-vector system. Both plasmids were production (24). It has been engineered for the annual produc- transformed to the BE strain to identify the best combination for tion of several million tons of L-glutamate and L-lysine (25). glutaric acid production. pGA6 was transformed to the BE strain Beyond amino acids, many other commodity chemicals, diols, together with pGA1. Consistent with the previous results, the BE diamines, carotenoids, and terpenes have been produced by strain harboring pGA4 produced the most glutaric acid metabolically engineered C. glutamicum (25, 26). In addition, C. (Fig. 1C), indicating that the combination of codon-optimized glutamicum can utilize various carbon sources, such as glucose, davAB operon and native gabTD operon under the control of sucrose, fructose, and xylose (27), which provides the additional H36 promoter is the best for efficient glutaric acid production in advantage of substrate flexibility for industrial use. Compared C. glutamicum. To further validate the synthetic pathway, fed- with other microorganisms, such as E. coli and P. putida,no batch fermentation of the BE strain harboring pGA4 was con- L-lysine degradation pathway exists in C. glutamicum. We have ducted. As shown in Fig. 1D, 54.4 g/L of glutaric acid (yield of −1 previously demonstrated the conversion of L-lysine to AVA by 0.19 g/g and productivity of 0.35 g/L h ) was produced without introduction of the davAB genes from P. putida into the C. glu- any L-lysine, and AVA remained, indicating the efficient con- tamicum BE strain, an L-lysine overproducer (13). The engi- version of these precursors to glutaric acid by the synthetic neered strain produced not only AVA, but also glutaric acid as a pathways. Therefore,