Glutaric Acid Production by Systems Metabolic Engineering of an L-Lysine–Overproducing Corynebacterium Glutamicum

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, pGA4 was used for further experiments. byproduct, indicating that there is a pathway for converting AVA to glutaric acid in C. glutamicum, and that further engineering of Rational Metabolic Engineering of Glutaric Acid Producers. To in- this strain might allow the efficient production of glutaric acid. crease the glutaric acid flux in the C. glutamicum BE strain, the In this work, we performed metabolic engineering of the C. target genes to be engineered were selected based on the results glutamicum BE strain to produce glutaric acid by applying mul- of previous studies performed on C. glutamicum wild-type (WT) tiple strategies (SI Appendix, Fig. S1). First, an optimal glutaric ATCC 13032 and ATCC 21831 strains (28, 29). Since 4 mol of acid synthetic pathway was established in C. glutamicum by fine- NADPH is required to biosynthesize 1 mol of L-lysine (22), tuning the gene expression levels. Next, additional metabolic en- sufficient NADPH level is a crucial factor in the synthesis of APPLIED BIOLOGICAL SCIENCES gineering, such as promoter change and introduction of additional L-lysine–derived products. Thus, the NADPH supply was en- copies of genes in the key L-lysine biosynthetic pathway, was per- hanced by increasing the pentose phosphate pathway (PPP) flux formed to increase L-lysine production and increase the precursor by changing the start codon of the pgi gene (encoding glucose-6- of glutaric acid. Multiomics analyses were performed to acquire a phosphate isomerase) from ATG to GTG and the start codon of comprehensive picture of the host strain and identify the target the zwf gene (encoding glucose-6-phosphate 1-dehydrogenase) genes to be manipulated for increasing glutaric acid-forming flux. from GTG to ATG (28, 29). In addition, the native promoter of Finally, the fed-batch fermentation condition was optimized for the the tkt operon was replaced with a stronger sod promoter. Next, high-level production of glutaric acid from glucose. the production of the key precursor, L-lysine, was enhanced by integrating the second copy of ddh encoding diaminopimelate Results and Discussion dehydrogenase next to the ddh gene in the genome. The ATG Design and Construction of the Glutaric Acid Biosynthetic Pathway. start codon of the icd gene encoding isocitrate dehydrogenase Among the three major pathways for glutaric acid production (SI was replaced by GTG. Furthermore, the lysE gene encoding an Appendix, Fig. S2), the L-lysine catabolic pathway via AVA from L-lysine exporter was deleted to reduce the excretion of L-lysine P. putida (Fig. 1A) was chosen because we wanted to take ad- into the medium and consequently secure more L-lysine for vantage of the L-lysine–overproducing C. glutamicum BE strain. subsequent conversion to glutaric acid. Six strains constructed This simple route does not involve highly reactive chemicals such (GA1 to GA6; SI Appendix, Table S1) were cultured in flasks to as hydrogen peroxide and was successfully used for glutaric acid examine L-lysine production. Unexpectedly, no significant in- production in E. coli in our previous study (15). For the con- crease in L-lysine production was observed (SI Appendix, Fig. version of L-lysine to AVA, pGA1 was constructed to express the S3). Moreover, the level of L-lysine produced by the GA6 strain, davAB operon from P. putida (Fig. 1B). The BE strain harboring in which the L-lysine exporter gene was deleted, was even slightly pGA1 converted almost all L-lysine to AVA, producing 8.1 g/L of higher than that of the BE strain (19.4 g/L vs. 18.5 g/L). None- AVA and 0.1 g/L of L-lysine (Fig. 1C). theless, the GA1 to GA6 strains were transformed with pGA4 Having developed an engineered strain efficiently converting and cultured in flasks to examine glutaric acid production. L-lysine to AVA, we then constructed the second synthetic As reflected in the L-lysine production results, all the strains pathway converting AVA to glutaric acid (Fig. 1A). Two candi- produced less glutaric acid than the BE strain (SI Appendix, Fig. date pathways are available for this: the P. putida DavT-DavD S3). These results suggest that rational metabolic engineering L-lysine catabolic pathway and C. glutamicum GabT-GabD strategies that worked for other C. glutamicum strains do not γ-aminobutyrate metabolic pathway. Homology modeling of C. work for the L-lysine–overproducing BE strain. Thus, compre- glutamicum GabT followed by molecular docking simulation hensive genomic, transcriptomic, and fluxomic analyses of the suggested that the nonnatural substrate AVA binds to the same BE strain were performed to better understand the metabolic pocket of GabT in an analogous manner to the natural substrate characteristics and identify engineering targets for enhanced γ-aminobutyrate (13). In addition, GabT shares high homology glutaric acid production. with P. putida DavT. Thus, both P. putida davTD and C. glutamicum gabTD operons were tested for the conversion of AVA to Identification of Engineering Targets by Multiomics Analyses of the C. glutaric acid. Using pGA1, three different plasmids—pGA2, glutamicum BE Strain. Although the BE strain is a known L-lysine pGA3, and pGA4 (Fig. 1B), harboring davTD, his-tagged gabTD, overproducer (13), its genomic and metabolic characteristics and gabTD operon, respectively—together with the davAB op- have not yet been assessed. Next-generation sequencing (NGS) eron were constructed and transformed to the BE strain. The of the BE strain was performed. The genome size of the BE flask cultivation of these three strains demonstrated that the BE strain was 3,343,309 bp, slightly larger than the 3,309,401 bp of

Han et al. PNAS | December 1, 2020 | vol. 117 | no. 48 | 30329 Downloaded by guest on September 30, 2021 Fig. 1. Overview for construction of the glutaric acid biosynthesis pathway in the C. glutamicum BE strain. (A) Schematic of the glutaric acid biosynthesis pathway from glucose. The genes in light green are obtained from P. putida, and the genes in blue are obtained from C. glutamicum. Each gene encodes the following: davA, 5-aminovaleramide amidohydrolase; davB, L-lysine 2-monooxygenase; davT, 5-aminovalerate aminotransferase; davD, glutarate semialdehyde dehydrogenase; gabT, 4-aminobutyrate aminotransferase; and gabD, succinate-semialdehyde dehydrogenase. (B) Schematic of plasmids used for glutaric acid biosynthesis. (C) Flask cultivation results of glutaric acid-producing strains. Error bars represent SD, and the white circles represent individual data points. Experiments were performed in triplicate. Cells were cultured for 48 h. (D) Fed-batch fermentation profile of the BE strain harboring pGA4. ASP, L-aspartate; AVA, 5-aminovaleric acid; GLC, glucose; GLT, glutaric acid; LYS, L-lysine; OAA, oxaloacetate; PYR, pyruvate.

the C. glutamicum ATCC 13032 WT strain (hereinafter the WT Next, comparative transcriptome analysis between the BE and strain). The GC content of 54.1% was also higher than the 53.8% WT strains was performed. The endogenous genes with signifi- of the WT strain (SI Appendix, Fig. S4). cantly different expression levels between the two strains are Using the genomic data, the genome-scale metabolic model summarized in Fig. 3 and SI Appendix, Table S4. The fold change (GEM) of the BE strain was reconstructed. The heterologous value (BE/WT) indicates the expression level of each gene in the metabolic reactions comprising the glutaric acid biosynthetic BE strain with respect to that in the WT strain. Here the positive pathway were added for subsequent fluxome analyses. To iden- and negative fold change values mean that the mRNA level of tify the target genes to be overexpressed for enhanced glutaric the corresponding gene in the BE strain is higher and lower, acid production, flux variability scanning based on enforced ob- respectively, than that of the WT strain. As expected, dapA, jective flux (FVSEOF) simulation (30) was performed using the dapC (encoding succinyl-aminoketopimelate transaminase), reconstructed GEM. The Pearson correlation value of each re- dapE (encoding succinyl-diaminopimelate desuccinylase), dapF action was obtained to examine the correlation between the (encoding diaminopimelate epimerase), lysE, and lysI (encoding average of the flux variability of the reaction and the glutaric acid L-lysine small transporter protein), which are responsible for production flux (SI Appendix, Table S3). The positive Pearson L-lysine production, showed high positive fold change values, correlation value indicates that glutaric acid production is more indicating strong L-lysine flux in the BE strain. In contrast, gabT, likely to be increased by stronger flux of the corresponding re- gabD2, and gabD3, the genes involved in L-lysine degradation, action. If a reaction showed a Pearson correlation value >0.9, showed negative fold change values of −16.54, −4.40, and −5.14, then the corresponding gene was selected as an overexpression respectively, which also explains the high level of L-lysine pro- target. Flux response analysis was also performed to analyze the duction in the BE strain. Unexpectedly, five genes—lysA, dapB¸ correlation between glutaric acid production flux and the flux of lysC, ddh,andppc (encoding phosphoenolpyruvate carboxylase)— each reaction in glycolysis, PPP, and the tricarboxylic acid (TCA) showed negative fold changes in the BE strain, even though they cycle (Fig. 2); those genes responsible for the reactions that showed are involved in L-lysine biosynthesis. Thus, these five genes were positive correlations with the glutaric acid production flux were selected as overexpression targets. Among these five genes, lysA, selected as the candidate genes to be amplified. Based on the re- dapB, dapA¸andlysC overlapped with those amplification targets sults of FVSEOF analysis and flux response analysis, the amplifi- identified by fluxome analysis. Thus, a total of six overexpression cation targets were narrowed down to five genes: lysA (encoding target genes—lysA, dapB, dapA¸ lysC, ddh,andppc—were selected. diaminopimelate decarboxylase), dapB (encoding dihydropicolinate In addition, the lysE and lysI genes encoding proteins exporting reductase), dapA (encoding 4-hydroxy-tetrahydrodipicolinate syn- L-lysine were selected as deletion targets to increase the intracel- thase), lysC (encoding aspartokinase), and ddh. lular L-lysine available for glutaric acid production. Furthermore,

30330 | www.pnas.org/cgi/doi/10.1073/pnas.2017483117 Han et al. Downloaded by guest on September 30, 2021 Fig. 2. Flux response analysis results for glutaric acid production. Each box represents the response of glutaric acid production flux to varying flux of reactions of genes in glycolysis, PPP, the TCA cycle, and the glutaric acid biosynthetic pathway. Each reaction of the genes in glycolysis, PPP, the TCA cycle, and the glutaric acid biosynthetic pathway was gradually increased from the minimum flux to the maximum flux while maximizing glutaric acid production flux. The negative flux of each reaction indicates the reverse reaction. Genes shown here are listed in SI Appendix, Note S1. APPLIED BIOLOGICAL SCIENCES

well-known strategies, such as amplification of pyc (encoding py- Construction of a Glutaric Acid-Producing Platform Strain. To con- ruvate carboxylase), deletion of pck (encoding phosphoenolpyr- struct a C. glutamicum strain with higher glutaric acid flux, 11 uvate carboxykinase), and reduced expression of icd (28, 29), target genes identified above were engineered one by one into were used. the BE strain (Fig. 4A). First, the GA7 strain was constructed by

Fig. 3. Comparative transcriptome analysis between the BE and WT strains. The fold change value (BE/WT) of each gene indicating the comparative gene expression level between the BE and WT strains is represented in color. ACT, acetate; AKB, α-ketobutyrate; ASP, L-aspartate; FA, fatty acid; GLC, glucose; GLU, L-glutamate; KIV, ketoisovalerate; LA, lactate; LEU, L-leucine; LYS, L-lysine; MET, L-methionine; THR, L-threonine; OAA, oxaloacetate; PAN, pantoate; PRM, pyrimidine; PYR, pyruvate; VAL, L-valine. The genes shown here are listed in SI Appendix, Note S2.

Han et al. PNAS | December 1, 2020 | vol. 117 | no. 48 | 30331 Downloaded by guest on September 30, 2021 changing the start codon of the icd gene from ATG to GTG to glutaric acid production. The glutaric acid yields of the three decrease the flux of TCA cycle and thereby increase glutaric acid strains were the same as 0.13 g glutaric acid per 1 g of glucose. flux. To enhance L-lysine biosynthesis, an additional copy of the Next, to prevent L-lysine export and consequently increase the ddh gene was inserted next to the ddh gene in the genome of intracellular L-lysine pool for glutaric acid production, the GA14 GA7 to make the GA8 strain. Then the native promoter of the and GA15 strains were constructed by deleting lysI and lysE, dapB gene was changed to a stronger H36 promoter in GA8 to respectively, in the GA13 strain. While the GA14 (pGA4) strain make the GA9 strain. Flask cultures of the GA7, GA8, and GA9 showed no improved glutaric acid production (4.5 g/L; SI Ap- strains harboring pGA4 produced 2.7, 3.1, and 3.4 g/L of glutaric pendix, Fig. S5), the GA15 (pGA4) strain produced 5.3 g/L of acid with respective yields of 0.11, 0.12, and 0.12 g of glutaric glutaric acid, which is 18% higher than that obtained with GA13 B acid per 1 g of glucose (Fig. 4 ). Thus, the yields of the three (pGA4) (Fig. 4B). Thus, as expected and reported previously constructed strains were all higher than that (0.10) of the BE (17), deletion of the L-lysine exporter gene was beneficial for (pGA4) strain, but the titer was higher only in the GA9 enhanced glutaric acid production. (pGA4) strain. Finally, the GA16 strain was constructed by integrating an The GA9 strain was further engineered by replacing the pro- additional copy of lysC into the middle of pck. This lysC knock-in moter of the pyc gene with the H36 promoter to make the GA10 strain. However, the GA10 (pGA4) strain produced less glutaric strategy allows the reduction of oxaloacetate conversion to py- acid (3.2 g/L) than the GA9 (pGA4) strain (SI Appendix, Fig. ruvate and enhanced conversion of aspartate to L-aspartate- S5). Thus, we returned to the GA9 strain and proceeded by phosphate. The GA16 (pGA4) produced 6.1 g/L of glutaric acid replacing the promoters of dapA, ppc, and lysA with the H36 (Fig. 4B), 1.9-fold greater than the amount produced by the BE promoter to construct the GA11, GA12, and GA13 strains, re- (pGA4) strain. The glutaric acid yield (0.15 g/g) obtained with spectively. The GA11, GA12, and GA13 strains harboring pGA4 the GA16 (pGA4) strain was also the highest among the produced 3.4, 3.7, and 4.5 g/L, respectively, all showing increased different strains.

Fig. 4. Construction of the glutaric acid-producing platform strain. (A) Overview of the engineering strategies to enhance glutaric acid flux. The blue and dotted arrows represent up-regulated and down-regulated flux, respectively. The strategies that brought no significant increase in glutaric acid production are shown in gray boxes. (B) Comparison of glutaric acid titers, cell growth, and yields obtained by flask cultivation of engineered C. glutamicum strains. Each strain harboring pGA4 was tested. The P values of the data showing increases compared with BE (pGA4) are *P < 0.05, **P < 0.01, and ***P < 0.001. (C)The effect of putative glutaric acid exporter gene amplification on glutaric acid production. Each plasmid was transformed to the GA16 strain together with pGA4, and the generated strains were cultured in flasks. The P values of the data showing increases compared with GA16 (pGA4, pEKEx1) are *P < 0.05, **P <

0.01, and ***P < 0.001. The blue and gray bars indicate glutaric acid titer (g/L) and OD600, respectively, while the white circles indicate yield. The error bars represent SD. Experiments were performed in triplicate. Cells were cultured for 48 h. AKG, α-ketoglutarate; ASA, L-aspartyl-semialdehyde; ASP, L-aspartate; ASPP, L-aspartate-phosphate; DAP, diaminopimelate; DHDP, L-2,3-dihydropicolinate; GLC, glucose; GLT, glutaric acid; ICT, isocitrate; LYS, L-lysine; OAA, oxaloacetate; PDC, L-piperidine-2,6-discarboxylate; PEP, phosphoenolpyruvate; PYR, pyruvate.

30332 | www.pnas.org/cgi/doi/10.1073/pnas.2017483117 Han et al. Downloaded by guest on September 30, 2021 Fed-Batch Fermentation for Glutaric Acid Production. To enhance from a plasmid, 4.6 g/L of glutaric acid was produced. These glutaric acid production, fermentation conditions, including results suggest that ynfM encodes a glutaric acid exporter. Given carbon source, oxygen transfer rate, nutrient feeding strategy, that ynfM has previously been suggested to encode a succinic and the batch-phase glucose level, were optimized. First, simul- acid exporter (33), the gene product of ynfM seems to be a good taneous utilization of glucose and sucrose was examined, because dicarboxylic acid exporter. this strategy improved L-arginine production in C. glutamicum in Having constructed an efficient glutaric acid producer, GA16 our previous study (29). Four different mass ratios of glucose to (pGA4, pEK_GAex5), we performed fed-batch fermentation. sucrose (1:0, 1:1, 3:1, and 0:1) were examined in flask cultivation Surprisingly, only 45.5 g/L of glutaric acid was produced (SI (SI Appendix, Fig. S6). However, glutaric acid titers obtained Appendix, Fig. S10), which was even lower than the 54.4 g/L were similar to the titer obtained with glucose only, and thus obtained by fed-batch culture of the BE (pGA4) strain. It was glucose was used as a carbon source in further experiments. reasoned that the use of two-plasmid system caused a metabolic Second, the effect of oxygen supply on glutaric acid was ex- burden, resulting in lower glutaric acid production. Thus, instead amined. Generally, C. glutamicum requires enough oxygen for of plasmid-based overexpression of ynfM, its native promoter was rapid growth, and a sufficient oxygen level is beneficial for the replaced with a stronger promoter in the genome. The GA17 and production of chemicals. Moreover, DavB, which converts L-ly- GA18 strains were constructed by replacing the native promoter sine to 5-aminovaleramide, requires oxygen to catalyze the re- of ynfM with H30 and H36 promoters, respectively. Flask cultures action (13). Thus, it was hypothesized that a higher oxygen of the GA17 (pGA4) and GA18 (pGA4) strains produced 9.4 transfer rate during fermentation would benefit glutaric acid and 9.2 g/L, respectively (SI Appendix, Fig. S11), more than that production. For the fed-batch fermentation of the GA16 (pGA4) produced using the two-plasmid system. producing 61.0 g/L of glutaric acid (SI Appendix, Fig. S7A), the Fed-batch culture of the GA17 (pGA4) strain produced 105.3 agitation speed was set at 600 rpm, and no additional oxygen was g/L of glutaric acid with a yield and productivity of 0.54 g/g and − supplied. To increase the oxygen transfer rate, the agitation speed 1.53 g/L h 1, respectively (Fig. 5). The titer, yield, and produc- was automatically controlled by dissolved oxygen (DO) level from tivity were all significantly higher than those (54.4 g/L, 0.13 g/g, − 600 to 1,000 rpm. Pure oxygen was also automatically added when and 0.35 g/L h 1) obtained with the starting strain, BE (pGA4). needed. The fed-batch culture of the GA16 (pGA4) under this This is the highest glutaric acid titer achieved to date. It is also high oxygen transfer condition produced 75.1 g/L of glutaric acid notable that no byproduct was produced in fermentation, which (SI Appendix,Fig.S7B), suggesting that higher oxygen transfer is a big advantage in the purification process. To sum up, the rate increased both cell growth and glutaric acid production. metabolically engineered C. glutamicum strain has proven po- Third, the nutrient feeding method was examined. In the tential as a competitive microbial host for sustainable glutaric APPLIED BIOLOGICAL SCIENCES aforementioned fed-batch cultures, glucose was manually fed acid production. when the residual glucose concentration became low. Instead of manual feeding, the pH-stat feeding strategy, which automati- Conclusions cally adds a feeding solution when the pH rises higher than the In this study, a highly efficient glutaric acid producer was de- set point, was examined. The pH-stat fed-batch culture of GA16 veloped by metabolic engineering of an L-lysine–overproducing (pGA4) under high oxygen transfer conditions produced 90.5 g/L C. glutamicum BE strain. After construction of a synthetic of glutaric acid (SI Appendix, Fig. S8), significantly higher than pathway comprising the davA and davB genes from P. putida and that obtained with the manual feeding. In addition, the glutaric the gabT and gabD genes from C. glutamicum, their expression − acid productivity obtained by pH-stat feeding (0.81 g/L h 1) was levels were optimized. Then system-wide analyses were per- − much higher than that (0.55 g/L h 1) obtained by manual feed- formed by genome sequencing, followed by transcriptome and ing. In summary, the optimal fed-batch culture condition was fluxome analyses to better understand the metabolic character- achieved by using the pH-stat feeding strategy under high oxygen istics and identify target genes to be engineered to enhance transfer conditions using glucose as a carbon source. This con- glutaric acid production. Through engineering of 11 target dition was used in subsequent fed-batch cultures. genes by promoter exchange, gene deletion, and integration of

Identification of a Glutaric Acid Exporter. When overproducing a desired chemical, it is essential to efficiently excrete the product so that the product can be continuously synthesized inside the cell. A number of studies have already shown that overexpressing the exporters of target product can debottleneck biological production of target molecules (17, 31, 32). However, no glutaric acid exporter of C. glutamicum has been reported. Thus, we searched for exporters of other dicarboxylic acids from C. glutamicum, E. coli, and Pseudomonas aeruginosa (33–35). Then, from the genome sequence of the BE strain, their orthologous genes, including dctA1, dctX, dctM, ykuT, ynfM, dctA2, dauA1, and dauA2, were identified (SI Appendix, Table S5). These genes were cloned into pEKEx1, which is compatible with pGA4, to make pEK_GAex1-8. The GA16 strain was transformed with pGA4 and each of pEK_GAex1-8. Flask culture of the GA16 (pGA4, pEK_GAex5) strain expressing ynfM produced 7.6 g/L of glutaric acid (Fig. 4C). To investigate whether the ynfM gene is a true glutaric acid exporter, we constructed the GA16 ΔynfM strain by disrupting ΔynfM in the GA16 strain. After transforming pGA4 into the Δ GA16 ynfM strain, flask culture was performed. The mutant Fig. 5. Fed-batch fermentation profile of the GA17 strain harboring pGA4.

strain produced no glutaric acid, while the parent strain, GA16 The black triangle represents OD600; the white diamond, glucose (g/L); blue (pGA4), produced 6.1 g/L of glutaric acid (SI Appendix, Fig. S9). circles, glutaric acid (g/L). For reproducibility, the results of another fed- When the ynfM gene was expressed in the GA16 ΔynfM strain batch culture performed independently are shown in SI Appendix, Fig. S13.

Han et al. PNAS | December 1, 2020 | vol. 117 | no. 48 | 30333 Downloaded by guest on September 30, 2021 additional gene copy, glutaric acid production could be in- Materials and Methods creased to 6.1 g/L in flask culture. Then an optimal fed-batch All of the materials and methods used in this study are detailed in SI Ap- culture condition was found using the pH-stat feeding strategy pendix, Materials and Methods, including strains and plasmids, construction under high oxygen transfer conditions using glucose as a carbon of plasmids, culture conditions, fed-batch fermentation, genome manipu- source. In addition, a glutaric acid exporter gene, ynfM,was lation, analytical methods, GC/MS analysis, reconstruction of the C. gluta- discovered. When the expression level of this exporter gene was micum BE strain GEM and in silico simulations, and comparative increased from the chromosome, glutaric acid production could transcriptome analysis. be further enhanced. Fed-batch culture of the final strain produced 105.3 g/L of glutaric acid without any byproduct in 69 Data Availability. All study data are included in the main text and SI Appendix. h, the highest titer achieved to date. The strategies of systems metabolic engineering (1–3) and fermentation optimization ACKNOWLEDGMENTS. We thank Jae Sung Cho for helpful discussions. This research was supported by the Bio & Medical Technology Development Pro- described here (summarized in SI Appendix,TableS7)willbe gram of the National Research Foundation (Grant 2020M3A9I503788311) useful for the development of microbial cell factories capable of and funded by the Ministry of Science and Information Communication producing chemicals of interest with high efficiency. Technology, Republic of Korea.

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