4. Discussion
The conversion between aldose and ketose forms of sugars can be catalyzed by isomerase in one step or by oxidoreductase in two steps. Until 2018, many studies had focused on the isomerase reaction usingL-arabinose isomerase to produce tagatose from galactose. Several studies attempted to increase the conversion rate using immobilized enzymes,[16] recombinant cell expression,[17-20] immobilized recombinant cells,[8,21] cell encapsulation,[22] or cell surface display.[23,24] However, the isomerase reaction had a lower conversion rate because of the unfavorable thermodynamic equilibria.[8,9] Therefore, an oxidoreductive pathway was selected for tagatose synthesis in this study.
Oxidoreductases are usually cofactor-dependent, and the imbalance of cofactors in the cascade reaction may limit the conversion of the substrate. Alleviating the redox imbalance has been reported to be beneficial for improving the yield of the target production using cofactor engineering strategies.[25-28] Cahn et al. developed a general tool named CSR-SALAD to help reverse the cofactor preference of oxidoreductases due to the large demand for balancing cofactor availability.[27] Cofactor reversal has now been realized and applied to many enzymes.[29,30] For example, alcohol dehydrogenase in Clostridium autoethanogenum was modified to NADH dependence, resulting in improved growth and ethanol production.[31] Xylitol dehydrogenase was modified to be NADPH-dependent to reduce cofactor imbalance in xylose assimilation, and the engineered S. cerevisiae exhibited high xylose utilization and ethanol production capabilities.[13,32-33] To alleviate the cofactor imbalance in the biosynthesis of branched-chain amino acids, the coenzyme preference of ketol-acid reductoisomerase was altered from NADPH to NADH.[34,35] However, cofactor engineering in the redox pathway of tagatose synthesis has not yet been reported. Therefore, we designed and demonstrated the oxidoreductive Pathway II and the conversion rate was greatly improved (Figure 3).
Basic amino acid residues in the cofactor binding region are believed to favor the binding of NADPH, whereas acidic amino acids facilitate the affinity of NADH.[36,37] Based on this principle, we designed 100 mutants of Pd PDH (Table S5). Complete reversal of the coenzyme specificity was achieved with those containing D36A(G/R/H/K/I/F)/I37R (Table 2), which agrees with the results of a previous study.13 We investigated the binding mechanism between the mutants or the wild-type Pd PDH and the NAD+ or NADP+ cofactors. Interactions between protein and ligand, such as strong hydrogen interactions, are essential for stable binding, and steric hindrance and electrostatic repulsion may hinder their affinity.[38,39] In the wild-type Pd PDH, the adenosine portion of NAD+ could be stabilized by strong hydrogen interactions between the 2′-and 3′-hydroxyl groups and the negatively charged amino acid Asp36, as shown in Figure S5A. In the D36A/I37R mutant, the 2′-and 3′-hydroxyl groups of NAD+ were pushed apart by the amino group of Arg37 due to spatial site resistance and electrostatic repulsion, and strong hydrogen interaction was lacking between the 2′- and 3′-hydroxyl groups of NAD+ and Ala36, resulting in less interaction with D36A/I37R than that in the wild-type Pd PDH. For the NADP+ cofactor, mutation of the Ile37 side chain to Arg37 resulted in a stable and strong hydrogen interaction with the 2′-phosphate group of NADP+, as shown in Figure S5. Therefore, the Km values of NAD+ and NADP+ of Pd PDH were 0.7 mM and infinity, respectively, whereas those of thePd PDHD36A/I37R were infinity and 0.942 mM, respectively.
Enzyme assembly could improve the efficiency of the reaction. Various strategies for constructing artificial multienzyme complexes have been widely reported and successfully applied, including fusion protein technology with a linker,[40,41]SpyTag003/SpyCatcher003,[42] and self-assembling scaffold-EutM.43 We constructed the GS-linker (Figure S4B), SpyTag003/SpyCatcher003 (Figures S4A and B), and EutM-scaffold systems (Figures S4C and D) and found that the GS-linker system played the best-facilitating role. We speculated that this linkage maintained a suitable distance between Ps XR andPd PDHD36A/I37R, allowing a faster reaction transfer. The SpyTag003/Catcher003 linkage system may form a longer distance compared with that of the GS-linker system because the SpyTag003 and SpyCatcher003 were all fused to the N-terminus or C-terminus of the protein with the same GS-linker (Figure 3A). Therefore, the tagatose yield only increased by 19% at 24 h compared with that of the free enzyme system. With the EutM-scaffold system, we speculated that this linkage may not have brought the enzymes spatially closer together as the activity was not considerably improved compared with that of the free enzyme system.
The reuse of industrial waste is important for saving energy and reducing emissions, while the conversion of waste into high-value products is an important method for establishing a sustainable bioeconomy. Whey, a by-product of the dairy industry, is an environmental pollutant.[12] However, whey has lactose and abundant nitrogen sources. Thus, whey is a suitable material for microbial fermentation. Engineered E. coli strain was used to produce tagatose from cheese whey.[18,28] However, nongenetically modified organism (non-GMO) tagatose production technology is being explored to obtain an increased tagatose yield from lactose. Herein, we demonstrated an enzyme cascade pathway for producingD-tagatose from 1 kg of WP, including 81% lactose, and the tagatose yield from lactose and WP reached 0.32 and 0.27 g/g, respectively. In this reaction, we found that galactose was entirely consumed; by contrast, there was no change in glucose concentration (Figure 5). These conversion efficiencies are similar to those of microbial cell factories.[11,28] However, this study constructed a more robust non-GMO technology, which avoids consumer anxiety about genetically modified technology. Furthermore, yeast fermentation in the enzyme reaction solution was performed without tagatose separation. We compared the ethanol production with and without adding nitrogen source after tagatose production, and no significant change in ethanol yields was observed (Figure 5). This suggested the presence of an abundant nitrogen source and trace elements in whey for yeast growth and ethanol production under anaerobic conditions. Moreover, no change in tagatose concentration in the broth was observed, suggesting that natural S. cerevisiae cannot utilize tagatose. Finally, we obtained 371.3 g of ethanol and 215.4 g of dry yeast cells from 1 kg of WP, and the protein content in the dry yeast was approximately 38% (w/w) (Table S9). Bioethanol is an important fuel for mitigating global warming and conserving fossil fuels.[43]Microbial protein, or single-cell protein, has been regarded as an important protein reservoir for future nutritional needs.[44] Microbial proteins have been used as a feed or feed supplement.[45] Microbial proteins have good environmental benefits as an alternative to ruminant meat.[46] S. cerevisiae is considered a generally recognized as safe (GRAS) strain and is therefore a good candidate for microbial protein.[14,47,48]