Metabolic Engineering of Microorganisms Towards the Biomanufacturing of Non-Natural C5 and C6 Chemicals

2.1. 1-Pentanol 1-Pentanol is a straight-chain C5 alcohol commonly used as a coating solvent or in fragrances, while it also has potential as a fuel alternative [19]. It is considered a semi-natural alcohol, as only trace amounts are generated in yeast fermentations. Recent studies have reported the microbial production of 1-pentanol in E. coli through extending the 2-ketobutyrate (2-KB) pathway [58]. This pathway utilizes the chain elongation cycle mediated by the broad-substrate LeuABCD in the leucine biosynthesis and can be employed to produce C5-C7 alcohols that lack a natural biosynthetic pathway, such as 3-methyl-1-butanol and 3-methyl-1-pentanol [58]. However, the wild-type LeuABCD was unable to act on 2-KB for 1-pentanol production. To address this limitation, Zhang et al. introduced a leucine-feedback insensitive mutation into LeuA (G462D). Along with the action of 2-ketoisovalerate decarboxylase (KIVD) from Lactococcus lactis and alcohol dehydrogenase (ADH6) from Saccharomyces cerevisiae, they achieved the biosynthesis of 494 mg/L of 1-pentanol. Subsequent protein engineering of KIVD led to the identification of a more efficient mutant V461A/M538A, resulting in an increase in 1-pentanol to 750 mg/L [59]. Building on this foundation, Chen et al. attempted to further enhance the 1-pentanol production by specifically focusing on the effects of the residue V461 on KIVD activity. A saturated mutagenesis study of this residue revealed that the V461G mutant exhibited the highest activity and selectivity towards the 2-ketocaproate, the precursor of 1-pentanol. This improvement may be attributed to the extra space created within the binding pocket, allowing for a looser docking of the substrate. Notably, this particular mutant also showed a reduced catalytic efficiency for 2-KB and 2-ketovalerate, which decreased the production of the shorter-chain alcohols, 1-propanol, and 1-butanol, while increasing the production of 1-pentanol. Additional improvement was achieved by synergistically overexpressing the E. coli native threonine pathway alongside the CimA gene from Methanococcus jannaschii, whose catalytic activity was enhanced through directed evolution to bolster the citramalate pathway, ultimately resulting in an increased supply of 2-KB. Moreover, the elevated supply of acetyl-CoA, the iterative addition unit, by acetate feeding further directed the 2-ketoacid flux into the elongation cycle, thus enhancing the 1-pentanol productivity. As a result of these combined efforts, a final titer of 4.3 g/L was achieved [19]. 2.2. 1,5-Pentanediol (1,5-PDO) 1,5-PDO is a commodity chemical widely recognized for its industrial application as a plasticizer and its role in the production of polyester and other diol compounds [60]. Despite its high industrial value, the production and market size of 1,5-PDO has been constrained due to its complex manufacturing process and lack of readily available C5 feedstock in petroleum. 1,5-PDO is a non-natural chemical that lacks a natural biosynthesis pathway which is a great challenge for its microbial production [60]. However, metabolic engineering and synthetic biology allow the construction of non-natural pathways in microorganisms for their production (Figure 1).As of now, the microbial production of 1,5-PDO via metabolic engineering has only been studied in E. coli as the host microorganism. For example, Cen et al. created a lysine-overproducing E. coli strain by expressing lysC, dapA, and ddh, which are all genes that encode for rate-limiting enzymes in the biosynthesis pathway. Building upon the lysine-producing chassis, the davB and davA genes from Pseudomonas putida were introduced to allow for an efficient conversion from lysine to 5-AVA as well as an alcohol dehydrogenase encoded by yqhD. Furthermore, the performance of 4-aminobutyrate transaminases (ABAT) encoded by gabT genes from different organisms including E. coli, P. putida, and Pseudomonas stutzeri were evaluated, among which GabT from E. coli produced the highest amount of 5-hydroxyvalerate (5-HV). During the conversion from 5-AVA to 5-HV, a high accumulation of GA was observed. Therefore, Cen et al. included the use of two types of transporters, which are encoded by cgl0841 from C. glutamicum and gabP from E. coli into the plasmid construction to reduce the byproduct accumulation. As this article used the 5-HV-CoA-based pathway, Cen et al. screened different CoA-transferases from different sources, with the abfT gene from Clostridium aminobutyricum producing the highest titer. Ultimately, the final strain showed a considerable amount of 5-HV and lysine accumulation and a low yield of the final product, 1,5-PDO, of less than 1 g/L. This result suggests there is a huge metabolic imbalance present between all three modules, which could be due to the lack of NADPH available for the entire pathway [60]. The same group of researchers continued to optimize this reaction and found a new pathway using cadaverine, which was found to be more energy efficient. As a result, in addition to the gabT gene and alcohol dehydrogenase used to synthesize 5-HV, this study required the involvement of a putrescine transaminase (patA), 4-aminobutyraldehyde dehydrogenase (patD), and an endogenous lysine decarboxylase. In E. coli, there are two lysine decarboxylases, encoded by cadA and ldcC, while the former was chosen for its greater stability. CadA and patAD were co-expressed along with gabT and yqhD or yahK as the alcohol dehydrogenase. Unlike the previous study, where yqhD was used in the final strain, this study saw yahK producing a higher titer of 1,5-PDO. Cen et al. also made an adjustment in this study by using the CAR-based pathway from 5-HV to 1,5-PDO, therefore, different CAR enzymes were screened to test its efficiency for the production of 5-hydroxypentanal. Ultimately, the CAR from M. marinum produced the highest 1,5-PDO titer. Another addition is the knock-out of the gdhA and gabD genes, which are involved in a separate GA synthesis pathway, in an attempt to reduce its accumulation. The strain consisting of these changes resulted in an overall titer of 9.25 g/L of 1,5-PDO, which is significantly higher than the previous study but is still considered low on the industry scale. Furthermore, a high accumulation of byproducts still remained as well, therefore, this engineered pathway needs to be further optimized and tested [20]. In a separate study conducted by Wang et al., they explored the charged amino acid catabolic pathway to synthesize various diols, including 1,3-PDO, 1,4-BDO, and 1,5-PDO. However, for the purpose of this discussion, the focus will be primarily on the last compound. Starting with glucose as the carbon source to produce lysine, Wang et al. applied a similar strategy as discussed in prior studies. They inserted a synthetic AMV pathway by introducing davAB from P. putida, gabT, and yqhD from native E. coli. Additionally, they employed a CAR-based pathway for the conversion of ω-hydroxy acids (ω-HA) to diols. This pathway specifically consisted of the CAR enzyme from M. marinum and phosphopantetheine transferase (sfp) from B. subtilis. The study revealed that the titer of 1,5-PDO increased exponentially only when lysine was exogenously supplied to the cell culture, highlighting the bottleneck limitation imposed by lysine supply on the pathway. To address this issue, Wang et al. co-expressed lysC and dapA, to enhance the lysine degradation pathway. However, even after deleting the iclR gene in an effort to maintain the precursor supply of oxaloacetate, the highest titer reached only 0.97 g/L [61]. 2.3. Cadaverine Cadaverine, or 1,5-diaminopentane is a straight-chain diamine that has shown to have many commercial applications, notably in bio-based materials, and is seen in chelating agents [89]. Cadaverine is naturally formed by bacterial decarboxylation of lysine that occurs during putrefaction of animal tissues. There is a growing interest in enhancing cadaverine production using microbial engineering methods with a focus on renewable carbon sources (Figure 1) [11]. 2.3.1. Cadaverine Production in E. coli In E. coli, the majority of studies focused on the bioconversion of lysine to cadaverine instead of a direct fermentative approach from glucose in order to avoid limitations of precursor availability and cell growth [89]. There are two main lysine decarboxylases (LDC) that can achieve this conversion: CadA and LdcC, both of which originate from E. coli. When compared, CadA has a higher catalytic activity than LdcC, thus the former is more frequently used to produce cadaverine. Hong et al. worked to improve the enzymatic stability and activity of the CadA by inserting a combination of mutations: F14C, K44C, L7M, and N8G. This mutated strain produced an overall titer of 157 g/L of cadaverine [62]. Another study by Kou et al. focused on enhancing the thermal and pH stability of CadA by overexpressing a T88S mutant to achieve an increase in titer to 198 g/L [63]. Xi et al. also attempted to accomplish this by introducing additional mutations, K477R, E445Q, and F102V, along with T88S. However, this combinatorial mutagenesis approach led to a slight decrease in concentration of 160.7 g/L compared to the previous study [64]. Gao et al. discovered a combination of different mutations, V12C and D41C, using rational engineering on the CadA. In addition to optimizing the pH conditions during fermentation, this strain achieved the highest cadaverine titer at 418 g/L [21]. Additionally, Ma et al. co-expressed the CadA gene with CadB, a lysine and cadaverine antiporter fused with a signal sequence PelB, leading to a production titer of 221 g/L [65]. Huang et al. further built upon this system by deleting the genes responsible for the accumulation of byproducts, including speE, speG, ygjC, and puuA. However, this knockout strain only produced 32.1 g/L of cadaverine [66]. An additional effort was made by Moon et al. on modification of the ATP regeneration system within the pathway. They overexpressed cadA, along with a polyphosphate kinase (ppk), and pyridoxal kinase (pdxY), to assist in the conversion of lysine. They further optimized the cell system by introducing hexadecyltrimethylammonium bromide (CTAB) to increase the permeability of the cell membrane. The implementation of both these strategies resulted in the production of 1 M of cadaverine, with a 100% conversion rate from lysine [67]. Xue et al. achieved a titer of 83.2 g/L by utilizing a similar strategy of co-expressing cadA with only pdxY [68]. In another study, Wang et al. introduced lysine decarboxylase (LDC) from Hafnia alvei with the mutation, E583G into E. coli. This heterologous expression saw a decent concentration of 63.9 g/L [69]. Noh et al. conducted an experiment involving the engineering of a synthetic sRNA scaffold. Using the cadaverine biosynthesis pathway to test their approach, they identified and used 67 genes to construct the strongest scaffold. To determine the effect of the modifications on carbon metabolism, this strategy utilized direct fermentation from glucose. As a result, 13.7 g/L of cadaverine was produced following fed-batch fermentation [70]. As for the study performed by Wei et al., their experiment focused on altering the fermentation process by using a CO₂ reconversion approach to control pH levels, which reduced cellular damage and led to a titer of 208.2 g/L [71]. Instead of using glucose as a carbon source, Kwak et al. utilized galactose as the carbon source to produce cadaverine in E. coli. To metabolize galactose, Kwak et al. engineered the Leloir pathway by overexpressing the genes involved, galE, galT, galK, galM, galP, and pgm. Additionally, they modified the carbon flux to increase the supply of lysine by inserting asd, dapA ^fbr, dapB, ddh, lysA, and lysC ^fbr into the chromosome. Disruption of the cadaverine degradation pathway was also attempted by knocking out speE, speG, ygjG, and puuPA, resulting in a titer of 8.80 g/L [22]. 2.3.2. Cadaverine Production in C. glutamicum Corynebacterium glutamicum has shown tremendous success in constructing engineered microbial cells in the past, making it an excellent candidate as the host organism for cadaverine production. Kind et al. utilized a previously engineered C. glutamicum strain, LYS-12, that hyper-produces lysine as a precursor. In this strain, Kind et al. opted to introduce the constitutive gene, ldcC from E. coli, as the lysine decarboxylase, along with a major facilitator permease, Cg2893, to enhance cellular transport. They additionally deleted the lysine exporter gene, lysE, to maintain the precursor supply, and removed NCgl1469, a gene involved in the production of by-product N-acetyl-diaminopentane. This modified strain resulted in a titer of 88 g/L following fed-batch fermentation using glucose as the starting substrate [72]. Similarly, the direct fermentation approach was utilized by Li et al., who employed a heterologous co-expression system involving cadB from E. coli and the LDC gene from Hafnia alvei, producing 2.75 g/L of cadaverine [73]. In another study, Kim et al. used a strong synthetic H30 promoter to express the ldcC gene from E. coli, where it significantly increased the activity of lysine decarboxylase. Coupled with the deletion of lysE, this approach achieved one of the highest titers from glycose in C. glutamicum, 103.78 g/L of cadaverine [23]. Kobayashi et al. utilized a different approach by constructing a system with an oxygen-responsive switch that could transition the carbon flux from glycolysis to the pentose phosphate pathway (PPP). This was accomplished through the replacement of the glucose 6-phosphate isomerase (pgi) promoter with the promoter of lactate dehydrogenase (ldhA). Furthermore, they deleted the lactate pathway through the disruption of ldhA, menaquinone oxidoreductase (pqo), acetate kinase (ack), and phosphotransacetylase (pta). However, to improve the supply of oxaloacetate, a precursor of lysine, they introduced a mutation in isocitrate dehydrogenase to reduce its activity. They also introduced a P328S mutation into pyruvate carboxylase (pyc) to increase its enzymatic activity. Finally, a lysC-T311I mutant was introduced into the strain to increase lysine by eliminating the feedback inhibition. The final strain produced 0.74 g/L of cadaverine [74]. In the studies by Buschke et al., xylose was used as the carbon source. They constructed a mutant strain by expressing xylA and xylB genes from E. coli, that converts xylose into xylulose 5-phosphate to be used as a metabolic intermediate [24]. Using this recombinant strain, Buschke et al. overexpressed icd, along with fructose bisphosphatase (fbp), and transketolase (tkt), enzymes involved in the production of lysine from a carbon source. This strain consumed xylose to achieve 103 g/L of cadaverine, a high titer seen in C. glutamicum [25]. Mao et al. also employed xylose as their substrate, but their method centered on the introduction of Beta-xylosidase BSU17580 from B. subtilis into the strain, facilitated by a PorH anchor protein. This enzyme played a crucial role in generating the xylose supply through the conversion of xylo-oligosaccharides. Along with the overexpression of the xylA and LDC genes, Imao et al. achieved a yield of 11.6 mM of cadaverine [75]. 2.4. δ-Valerolactam δ-valerolactam plays a pivotal role in the synthesis of bioplastics, which can subsequently be utilized in the manufacturing of various items and goods [76]. This class of products has garnered increased attention due to its reliance on non-petroleum-based sources and its exceptional durability, with predictions suggesting that bioplastics production may exceed 2.43 million tons in the near future [26]. While there are various other compounds capable of synthesizing biopolyamides, δ-valerolactam has been shown to be a potential candidate for large-scale bioplastics production. However, currently, there are limited methods for δ-valerolactam production, achieved either through plant extraction or the chemical conversion of 5-AVA. Therefore, engineered microorganisms have become a focal point, although there have only been a handful of studies reporting on this compound (Figure 1). While there have been relatively few studies on the synthesis of δ-valerolactam via metabolic engineering, Xu et al. proposed a novel pathway that included L-pipecolic acid (L-PA) as the intermediate. In this study, Xu et al. uses E. coli as the host organism to first introduce the DavB genes from P. putida to convert L-PA to δ-valerolactam, which produced an initial titer of 90.3 mg/L. Further modifications involved co-expressing DavB with multiple proteins including an apoptosis-inducing protein (rAIP) from Scomber japonicus, Bacillus subtilis glucose dehydrogenase (GDH), P. putida Δ-piperideine-2-carboxylae reductase (DpkA), and Oryza sativa lysine permease (LysP). These changes allowed for δ-valerolactam to be produced directly from L-lysine, resulting in a final titer of 242 mg/L of δ-valerolactam [76]. In a separate study, Cheng et al. built upon the information from this previous experiment and further optimized the pathway to co-produce δ-valerolactam and 5-AVA. While Cheng et al. also introduced the RaiP from S. japonicus, they also expressed a catalase gene, katE. They supplemented the process with H₂O₂ and adjusted the pH level to the optimal value of 9.0. With these modifications, this strain obtained a significantly improved titer of 6.88 g/L [26]. 2.5. Glutaric Acid (GA) GA, also known as 1,5-pentanedioic acid, is a dicarboxylic acid that is an important component for the production of polyester and polyamides, which are essential for the manufacturing of fabric and textiles, amongst other materials [87]. Typically, GA is synthesized via a chemical process using potassium cyanide to catalyze the ring-opening reaction of butyrolactone or using other toxic petroleum-based materials [30,90]. Apparently, some of the main issues associated with this approach are the high costs as well as the environmental burdens. Therefore, there is a preference for transitioning to efficient biobased approaches over traditional chemical methods. 2.5.1. GA Production in E. coli E. coli is a widely used host microorganism for metabolic engineering due to its fast growth and capacity to express heterologous genes, enabling the rapid construction of efficient microbial systems for producing desired compounds. GA is a compound derived from lysine and 5-AVA, whose pathways require the introduction of heterologous enzymes from different organisms. Two separate studies conducted by Park et al. and Adkins et al. first focused on the production of 5-AVA and then the conversion of 5-AVA to GA in E. coli (Figure 1). Both studies synthesized 5-AVA from L-lysine by introducing the davAB genes from P. putida into an E. coli, which encodes for the δ-aminovaleramidase and lysine 2-monooxygenase, respectively. The gabTD genes from P. putida, which encode for 5-AVA aminotransferase and GA semialdehyde dehydrogenase, were also co-expressed to catalyze the conversion of 5-AVA to GA. Adkins et al. further deleted the cadA and ldcC genes, which were involved in a separate lysine degradation pathway to reduce the formation of the byproduct, cadaverine. With similar modifications, Adkins et al. and Park et al. obtained a final titer of 0.82 and 1.7 g/L, respectively [77,78]. In a study conducted by Li et al., their approach closely resembled the biosynthesis of 1,5-PDO via the cadaverine pathway, as previously explored by Cen et al [59]. In this approach, E. coli native genes cadA, patAD, and gabTD were incorporated to facilitate the synthesis of GA. Furthermore, to reduce the accumulation of cadaverine, Li et al. overexpressed PotE, a bi-directional cadaverine transporter. The study also focused on boosting the supply of lysine by enhancing the precursor oxaloacetate, achieved through the deletion of iclR. These modifications resulted in minimal byproduct formation, culminating in a final GA titer of 54.5 g/L [27]. Wang et al. also utilized the cadaverine pathway in their study, albeit with slight variations. They introduced the patAD genes from Klebsiella pneumoniae and gabTD from Pseudomonas fluorescens into their pathway. Wang et al. also discovered the patA gene was a rate-limiting enzyme in the 5-AVA synthesis reaction, to which they improved its efficiency via a hydrophobic scanning strategy, where certain hydrophilic residues within the catalytic binding pocket were replaced with a hydrophobic residue. The introduction of a T332A/E120G patA mutant into the strain resulted in an enhancement of 5-AVA production. The transporter gene, gabP, was also overexpressed to reduce the accumulation of 5-AVA. This particular strain achieved an impressive titer of GA, reaching 77.62 g/L [28]. Hong et al. improved the production of GA via the 5-AVA pathway by coupling the overexpression of the gabTD genes with the Nox gene from Lactobacillus sanfranciscensis that encodes for an NADPH oxidase. In doing so, it reduced the need for NAD⁺ as a cofactor and allowed the overall production of GA to reach 282 mM [79]. In another study, they inserted the gabTD genes from B. subtilis into E. coli, where they obtained 191 μmol of GA after five cycles of whole-cell bioconversion [80]. Yang et al. attempted to improve this experiment by using polyvinyl alcohol and polyethylene glycol (PVA-PEG) hydrogel to entrap and immobilize the entire system. With this addition, the production of GA from 5-AVA increased to 995.2 mM [81]. The same group published another study focusing on the involvement of α-ketoglutaric acid, where they introduced glutamate oxidase (GOX) from Streptomyces mobaraensis to lower the demand of α-KG by regenerating its supply, with the addition of a catalase to further enhance the system. Despite these changes, the titer of GA lowered to 468.5 mM [82]. The whole-cell biocatalysts approach was also explored by Wang et al., who used E. coli to build a synthetic microbial consortium. In this study, they used two engineered strains to express the davAB and gabTD genes separatly, as they realized that these two genes had a negative interaction when placed in a single cell strain. This approach resulted in the production of 43.8 g/L [83]. In a separate study, Wang et al. established a new pathway by using a ketoacid chain elongation strategy in E. coli. They extend the TCA cycle intermediate α-ketoglutarate to form α-ketoadipate (α-KA) by introducing three enzymes from S. cerevisiae: homocitrate synthase, homoaconitase, and homoisocitrate dehydrogenase. After the formation of α-KA, they expressed a α-ketoacid decarboxylase, KivD from L. lactis, and a semialdehyde dehydrogenase, gabD from P. putida, to catalyze the conversion to GA. To optimize this alternative pathway, Wang et al. used CRISPR to silence sucAB genes from the TCA cycle, in order to increase the supply of the precursor, α-ketoglutarate. This novel pathway resulted in a GA titer of 420 mg/L [76]. The same research group explored the GA biosynthesis using xylose as a carbon source instead of glucose. They also investigated different metabolic pathways to produce GA and found that a combination of the xylose isomerase and Weimberg pathways yielded the highest titer of 602 mg/L [29]. Yu et al. proposed an alternative strategy to produce GA in E. coli, by utilizing α-ketoglutarate in a reported glutaconate biosynthesis pathway. They first formed trans-glutaconyl-CoA by expressing hgdH, gctAB, and hgdC from A. fermentans, and hgdAB from C. symbiosum. This compound can then be converted to form GA using either trans-enoyl-CoA reductase from E. gracilis (egTer) or T. denticola (tdTer), along with the E. coli thioesterase enzymes (TesB). Using this pathway, the best strain obtained 3.8 mg/L of GA with egTer, although it accumulated a higher amount of glutaconate with 27.7 mg/L [88]. Another study by Zhao et al. explored the production of GA in E. coli through the Thermobifida fusca native reverse adipate degradation pathway (RADP), with the overexpression of five enzymes: Tfu_0875, Tfu_2399, Tfu_0067, Tfu_1647, and Tfu_2576-7. Using this pathway, Zhao et al. attempted to use both acetyl-CoA and malonyl-CoA as precursors for GA. They increased the supply of malonyl-CoA by using the antibiotic, cerulenin, to inhibit a fatty acid synthesis pathway. Additionally, to increase acetyl-CoA, genes encoding for L-lactate dehydrogenase (ldhA), acetyl-CoA acetyltransferase (atoB), and formate C-acetyltransferase I (pflB) were deleted to disrupt competitive pathways and reduce byproduct formation. The resulting yield of GA from this engineered E. coli strain was approximately 4.8 g/L [91]. Years later, another study was published extending upon this established pathway. As the previous experiment found a limitation with the supply of malonyl-CoA, Sui et al. introduced the matB and matC genes from Clover rhizobia to convert malonic acid to malonyl-CoA. The addition of malonic acid into this pathway increased the overall titer to 6.3 g/L [92]. 2.5.2. GA Production in C. glutamicum C. glutamicum has shown to be an excellent microorganism for the synthesis of amino acids such as lysine and glutamate via fermentation [87]. As GA biosynthesis requires the catabolism of lysine, the engineering of lysine producing C. glutamicum was thought to be a promising approach. Rohles et al. conducted a study in C. glutamicum using strategies similar to those of Park et al. Deletion of the lysE gene that encodes for the lysine exporter reduced the amount of lysine excretion, resulting in the production of a total 7 g/L of GA [84]. In a subsequent study, Rohles et al. further refined their approach by using native gabTD genes and overexpressing a 5-AVA importer, NCg10464, which would re-import 5-AVA back into the cell to maintain the supply for GA production. Amplifying the transport protein significantly improved the final titer of GA after fed-batch fermentation to 90 g/L, with little to no presence of any known byproduct [85]. Kim et al. adopted a different strategy, where they used a synthetic promoter H30 to express the davAB and gabTD genes to enhance the enzyme activity. Additionally, Kim et al. fused the davB gene with an N-terminal His6-tag, with the intention of improving its solubility and expression within the cell. As a result, this incorporation led to 24.5 g/L of GA to be produced [86]. Han et al. continued to optimize the engineered C. glutamicum strain by overexpressing or deleting 11 different genes (icd, ddh, dapB, pyc, dapA, ppc, lysA, lysI, lysE, lysC, and pck) that were found to increase the supply of lysine as the precursor. Additionally, they introduced the gene ynfM, which encodes for a GA exporter, into the strain, producing a final titer of 105.3 g/L of GA [30]. Perez-Garcia et al. developed a new pathway that does not require oxygen to produce GA. For this, they employed the use of the transaminase-oxidoreductase pathway to form 5-AVA by heterologous expression of the patAD and ldcC genes from E. coli. To reduce byproduct formation and increase the production of 5-AVA as a precursor, they deleted the transcriptional repressor (sugR), lactose dehydrogenase (ldh), N-acetyltransferase (snA), and diamine exporter (cgmA). In this study, Perez-Garcia examined the gabTD genes from three different Pseudomonas species to produce GA, with the gene from P. stutzeri performing the best. Additionally, the glutamate biosynthesis pathway was eliminated by deleting the gdh gene. The final modified C. glutamicum strain produced 25.2 g/L of GA through fed-batch fermentation [87]. 2.6. Glutaconic Acid Glutaconic acid is an unsaturated dicarboxylic acid that can be reduced to synthesize GA. As such, it has similar applications, being a precursor to polyamides and other biodegradable polymers [31]. Currently, the only natural biosynthetic pathway is found in anaerobic organisms such as Acidaminococcus fermentans and Clostridium symbiosum, which are capable of removing the ammonia from glutamate to convert into glutaconic acid [9]. However, since these microbes cannot produce glutaconic acid on an industrial scale, alternative microbial production methods have been developed (Figure 1). In prior studies, it has been shown that anaerobic bacteria are capable of converting glutamate to glutaconate in fermentation conditions. From this information, Djurdjevic et al. attempted to modify E. coli to produce glutaconate by introducing enzymes from those bacteria. Specifically, they heterologously introduced six genes: 2-hydroxyglutarate dehydrogenase (hgdH), glutaconate CoA-transferase (gctAB), 2-hydroxyglutaryl-CoA dehydratase (hgdC) from A. fermentans, and 2-hydroxyglutaryl-CoA dehydratase (hgdAB) from C. symbiosum. When this modified strain was cultivated in an anaerobic medium to replicate the conditions of an anaerobic bacteria, it yielded a glutaconate concentration of approximately 2.7 mM [31]. Sun et al. conducted another study in which they developed a novel pathway in E. coli using the estradiol ring catechol degradation pathway. Through this pathway, catechol can be catabolized to ketoacid, 4-oxalocrotonate, which can then be converted to glutaconic acid. Sun et al. first started this experiment by introducing the genes directly involved in this reaction: C23O and DmpI from P. putida, and DmpC from R. eutropha. Once 4-oxalocrotonate was reached, KivD from L. lactis and gabD from P. putida are capable of converting it into glutaconic acid. To synthesize catechol, Sun et al. used salicylic acid as the precursor, which required the expression of nahG to catalyze this bioconversion. Furthermore, the entC and pchB genes were also expressed in E. coli to help produce salicylic acid from glucose. This modified strain resulted in a titer of 35.3 mg/L of glutaconic acid [93]. 2.7. 5-Hydroxyvaleric Acid 5-hydroxyvaleric acid (5-HV), also referred to as 5-hydroxypentanoic acid, is a versatile compound with a wide range of applications. It is used in the production of various materials, such polyurethanes and polyhydroxyalkanoates (PHA) [32]. In addition, 5-HV serves as a precursor to 1,5-pentanediol. Similar to GA, the current chemical approach primarily relies on furfural derivatives, involving costly substrates and catalysts, and is a highly resource-intensive process [33]. As of recently, the interest in PHAs have been rising due to its applicability in packaging and in the biomedical industry. Given that 5-HV is a major building block for the material, the interest in its production has risen significantly as well. In a previous study by Rohles et al., they established an innovative pathway for synthesizing 5-AVA, a valuable precursor to 5-HV, by incorporating the davAB and davTD genes from P. putida into C. glutamicum. In this study, Sohn et al. attempts to increase the production of 5-HV by building onto this established 5-AVA pathway, while making slight modifications such as introducing an appropriate aldehyde reductase that would efficiently convert glutarate semialdehyde to 5-HV (Figure 1). In the pathway found by Rohles et al., the absence of 5-HV demonstrated a clear preference in the system's metabolic flux toward GA. To address this issue, Sohn et al. deleted the gabD gene to eliminate the GA pathway entirely. Despite this deletion, the overall titer of 5-HV did not improve, indicating that further investigation is needed to understand this metabolic flux. Additionally, yahK gene from E. coli was used in the final strain as the aldehyde reductase. The culmination of these modifications resulted in an overall titer of 52.1 g/L of 5-HV following fermentation, however, a significant amount of GA remained present [32]. In contrast, instead of using an engineered C. glutamicum strain, Chen et al. utilized E. coli to produce 5-HV. The main pathway is very similar to Sohl et al. with the implementation of the davAB gene, however, a key difference is the use of a native enzyme, aminobutyrate aminotransferase encoded by gabT to convert 5-AVA to glutarate semialdehyde. Additionally, Chen et al. screened yqhD and yahK genes from E. coli and yajO gene from P. putida for the best aldehyde reductase activity. Although it was found that the yqhD gene performed the best, a dual vector system was employed, constructing a secondary promoter with yajO to overexpress the enzyme activity. Chen et al. also introduced genes that encoded for a 5-aminovalerate exporter and a lysine-specific permease, gabP and lysP, respectively, to increase the transport rate of key intermediates into the cell. Furthermore, a hok/sok system was introduced to reduce plasmid instability. Despite these changes differing from Sohl et al., the outcome of this study yielded a lower titer of 5-HV at 35.6 g/L [33].