Design of galactose utilization and n-butanol production modules in E. coli
The galactose metabolizing pathway, also known as the “Leloir pathway”, involves several enzymes that catalyze galactose catabolism in addition to glucose metabolism. As shown in Fig. 1, galactose is imported by galactose transporters (encoded by galP, and mglBAC) and is subsequently converted into glucose-1-phosphate through multiple enzymes encoded by the galactose operon (galETKM). Finally, phosphoglucomutase (encoded by pgm) converts glucose-1-phosphate to glucose-6-phosphate to enter glycolysis. In the presence of glucose, however, galactose metabolism is regulated by several factors, including a Gal repressor (GalR), Gal iso-repressor (GalS), and intracellular cyclic AMP, which induces CCR, enabling the preferential utilization of glucose. Although multiple enzymatic steps and the regulatory hierarchy in the pathway limit the rate of galactose utilization and the simultaneous fermentation of many carbohydrates [21], the reconstruction of galactose pathway on chromosome enhanced the galactose utilization rate and the simultaneous assimilation of galactose and glucose [10].
The assembly of both modules for synthetic galactose utilization and n-butanol production was rationally designed to enhance n-butanol production from increased galactose utilization. As the platform strain to introduce each module, we used previously reported E. coli strain JHL59 whose by-product pathways (acetate, lactate, ethanol, succinate) were blocked to amplify flux toward acetoacetyl-CoA [18]. For the galactose utilization module, the native galactose metabolism was engineered by deleting the Gal repressor (encoded by galR) and replacing the nascent regulatory elements on the “Leloir pathway”, including the enzymes encoded by galP, galETKM, and pgm, with predictable regulatory elements such as a synthetic constitutive strong promoter (BBa_J23100 from the Registry of Standard Biological Parts, http://partsregistry.org), and an optimized 5′-UTR designed by UTR Designer (http://sbi.postech.ac.kr/utr_designer) to amplify gene expression and avoid the host regulatory system [22]. Consequently, optimized galactose utilization module could be constructed (E. coli GAL_059).
In the case of the n-butanol production module, n-butanol, non-native metabolite in E. coli, is mainly produced by the introduction of four heterologous enzymes from Clostridium acetobutylicum and Treponema denticola with endogenous acetoacetyl-CoA as a starting material. Acetoacetyl-CoA (catalyzed by AtoB in E. coli) is firstly converted to 3-hydroxybutyryl-CoA by 3-hydroxybutyryl-CoA dehydrogenase (encoded by hbd from C. acetobutylicum). The crotonyl-CoA is then produced by crotonase (encoded by crt from C. acetobutylicum) from 3-hydroxybutyryl-CoA. Crotonyl-CoA is further modified to butyryl-CoA by trans-enoyl-CoA reductase (encoded by ter from T. denticola). Subsequently, n-butanol is finally produced with bifunctional aldehyde/alcohol dehydrogenase (encoded by adhE2 from C. acetobutylicum) (Fig. 1). Accompanying deletion of by-product forming pathway, the overexpression of these heterologous enzymes with synthetic promoter and UTRs designed in the previous study [18] was utilized. Accordingly, the strain with both optimized modules could be obtained by transforming the pCDF-BuOH harboring maximized n-butanol production module [18] into E. coli GAL_059 (E. coli GAL_061).
Enhanced n-butanol production of engineered strain with galactose
The E. coli GAL_059 strain was aerobically cultivated to evaluate its metabolic capacity when grown in M9 minimal medium containing galactose as a sole carbon source (Fig. 2a). As expected, the engineered strain showed a 37.2 % higher specific growth rate and a 66.4 % improvement in specific sugar uptake rate (Fig. 2b, c). The higher sugar uptake rate by the engineered strain resulted in the depletion of the galactose supplement in the medium within 14 h. In contrast, the parental strain JHL61 consumed about 60 % of the initial galactose.
Furthermore, the n-butanol production capacity of our engineered strain GAL_061 was evaluated after anaerobic fermentation in galactose-supplemented medium for 48 h. After fermentation, this strain showed a dramatically increased final n-butanol titer of 4.5 g/L, about 2.5-fold higher than that produced by the parental strain. Moreover, the amount of galactose consumed was notably greater for this strain (19 g/L) than for the parental strain JHL61 (7.4 g/L), indicating that intracellular carbon flux was enhanced in the engineered strain (Fig. 3).
In addition, butyrate, which can be catalyzed by endogenous tesB (encoding acyl-CoA thioesterases) [19], was produced abnormally as a by-product in the engineered strain. This can be accounted for accumulation of butyryl-CoA (an intermediate of both n-butanol and butyrate) with increased carbon flux using galactose, probably due to insufficient activity of AdhE2 or the stoichiometric imbalance of reducing equivalent, NADH [23].
Redox re-balancing by tuning the NADH supplementation module
Generally, increasing the availability of intracellular NADH is important to produce reduced metabolites, such as ethanol [24], succinate [25], and n-butanol [26, 27] and may be attainable by expressing yeast NAD+-dependent formate dehydrogenase (FDH1), an enzyme that converts formate to NADH and CO2 to supply additional NADH [25–27]. Our previous work elucidated that the optimal supply of NADH determined the efficiency of n-butanol production and was significantly different in galactose and glucose supplemented medium depending on the assimilation rate of sugars [18]. Moreover, the optimization of redox state was successfully accomplished through fine-tuning of FDH1 based on a predictive method of controlling translation using a biophysical thermodynamic model of the translation initiation process (UTR Designer) [22].
In this manner, the commitment of expression cassettes for various levels of FDH1, termed as NADH supplementation module, was expected to effectively coordinate galactose-utilizing and n-butanol producing modules by optimization of intracellular redox state. To find the optimal redox state for n-butanol production equipped with galactose module, fdh1 variants showing different levels of expression [18] were used to transform the GAL_061 strain, finally resulting in the GAL_080, 081, 082, 083, and 084 strains (Additional file 1: Table S1). After fermentation of the variants for 48 h, the relationship between n-butanol and fdh1 expression level titer exhibited a concave curve (Fig. 4a). The carbon flux towards n-butanol was enhanced up to 22 % depending on the enzymatic activity of FDH1, indicating that the GAL_083 strain was redox balanced.
Optimizing the production of n-butanol increased the consumption of galactose and decreased the production of oxidized metabolites, such as pyruvate and butyrate, consistent with previous findings (Additional file 1: Table S3). Moreover, the optimal redox state in galactose-supplemented medium was markedly higher than that of the parental strain, indicating that increased carbon flux elevated the availability of NADH in our engineered strain [18]. Ultimately, the redox balanced strain GAL_083 produced 6.2 g/L of n-butanol after 48 h of fermentation (Fig. 4b). Collectively, the redox balancing module successfully optimized the catalytic amounts of reducing equivalents depending on carbon flux, resulting in maximum production of n-butanol from galactose.
Simultaneous utilization of galactose and glucose for n-butanol production
Typically, biomass consists of a variety of polysaccharides with the composition depending on species, cultivation conditions, and methods of pretreatment [28–31]. Also, galactose is always obtainable as a sugar mixture of glucose from hydrolysate of seaweed, dairy waste [8, 9]. Therefore, the efficient conversion of cost-effective feedstock into valuable products requires the optimal, robust, and simultaneous consumption of galactose and glucose, regardless of their composition [11]. However, most microorganisms have evolved to utilize a preferred sugar in the presence of other sugars, enabling them to grow quickly under nutrient limited conditions [32]. The galactose metabolic pathway is repressed, both at the transcriptional and translational levels by glucose metabolism [33, 34].
We previously observed that deregulation and constitutive expression of transcripts whose 5′-UTR were designed for maximized expression allowed galactose to be utilized in the presence of glucose [10]. Accordingly, our engineered strain GAL_083 was expected to produce n-butanol through co-fermentation of galactose and glucose. To confirm this, GAL_083 and its parental strain were cultivated in simulated synthetic medium containing the same amounts of galactose and glucose that are generally present in seaweed and lactose [35]. Additionally, the ratios of these two sugars were varied from 2:1 to 1:2, reflecting the possible changes in the medium [29, 30, 36].
Under these conditions, the engineered strain utilized galactose and glucose simultaneously, while the parental strain showed no galactose consumption until glucose was depleted (Fig. 5). Moreover, n-butanol production by the engineered strain was consistently above 5 g/L regardless of the composition of the medium, while the yield of metabolites produced by the parental strain varied. Taken together, these results indicate that this strain would be promising as a platform strain for the optimal and robust production of valuable bio-chemicals from various galactose–glucose mixtures without regard to possible changes in the composition.