Enhanced production of maltobionic acid by a metabolically engineered Escherichia coli incapable of maltose utilization.

To produce maltobionic acid (MBA) from maltose in Escherichia coli, we recombinantly expressed a glucose dehydrogenase gene (gdh1) from Enterobacter cloacae and a pyrroloquinoline quinone (PQQ) synthesis gene cluster (pqqFABCDEMIH) from Pseudomonas taetrolens. Although the recombinant E. coli strain (E. coli [pKK-ECGDH1 + pACYC-PQQ]) successfully produced MBA from maltose, the yield of MBA was rather low, indicating that E. coli has other maltose utilization pathways. Amylomaltase (MalQ) is the first enzyme in the maltose utilization pathway in E. coli. To investigate the potential role of MalQ on MBA production, E. coli malQ was inactivated. The culturing of the recombinant E. coli strain (E. coli ∆malQ [pKK-ECGDH1 + pACYC-PQQ]) in a flask resulted in higher MBA production titer, yield, and productivity (209.3 g/L, 100%, and 1.1 g/L/h, respectively) than those of E. coli [pKK-ECGDH1 + pACYC-PQQ] (162.1 g/L, 77.4%, and 0.5 g/L/h, respectively), indicating that the MalQ inactivation was highly effective in improving the MBA production ability of E. coli. After fermentation using 5-L bioreactor, MBA production titer, yield, and productivity of the recombinant E. coli strain were 209.3 g/L, 100%, and 1.5 g/L/h, respectively, which were 1.3-, 1.3-, 2.3-fold higher than those of E. coli [pKK-ECGDH1 + pACYC-PQQ] (167.3 g/L, 79.9%, and 0.65 g/L/h), respectively. Thus, our results provide an important foundation for efficient MBA production using recombinant E. coli strain.

Conversion of waste cooked rice into maltose by a Bacillus subtilis-derived maltogenic amylase and production of maltobionic acid from the prepared maltose using genetically modified Pseudomonas taetrolens.

In this study, we attempted to produce maltobionic acid (MBA) from waste cooked rice (WCR) using maltose as an intermediate. In our previous study, we produced maltose from WCR using a commercial maltogenic amylase (Maltogenase L). However, in the present study, we used wild-type Bacillus subtilis, which inherently produces maltogenic amylase (AmyE), instead of Maltogenase L to produce maltose from WCR. During cultivation of B. subtilis with WCR, maltose was successfully produced by AmyE in the culture medium. To improve maltose production, we constructed a recombinant B. subtilis strain expressing AmyE and used it for maltose production. Following cultivation of the recombinant B. subtilis strain, the maltose production titer (34.6 g/L) increased approximately 3.6-fold that (9.6 g/L) obtained from the cultivation of wild-type B. subtilis. Using Pseudomonas taetrolens, an efficient MBA-producing bacterium, 28.8 g/L of MBA was produced from the prepared maltose (27.6 g/L). The above results indicated that MBA was successfully produced from WCR via a two-step process, which involved the conversion of WCR into maltose by maltogenic amylase-producing B. subtilis and the production of MBA from the WCR-derived maltose by P. taetrolens.