Hideo Kawaguchi

To synthesize a high-performance biopolyimide bioplastic from lignocellulosic feedstock, Escherichia coli was metabolically engineered to produce 4-amino-L-phenylalanine (4APhe) as a diamine monomer. A high-biomass sorghum cultivar was used as the model lignocellulosic feedstock, and the enzymatic hydrolysate was used as a substrate for 4APhe production in fed-batch culture. When using the ldh mutant strain, HKE6027, 4APhe production from glucose was increased by over three-fold compared to the parent strain. This increase was due to the disruption of biosynthetic pathways that produce either acetate or lactate as by-products. Comparative metabolomic analysis revealed increased flux in both the pentose phosphate and shikimate pathways in HKE6027, resulting in the highest yields. However, 5.7 g/L of 4APhe was produced from enzymatic hydrolysis by HKE6027, which was 24% lower than that obtained from glucose. The concentrations of 14 potential fermentation inhibitors present in the enzymatic hydrolysate were determined, and their inhibitory effects on both cell growth and 4APhe production were examined. The addition of groups of potential inhibitors present in the enzymatic hydrolysate of sorghum bagasse showed that benzaldehyde- and cinnamic acid derivatives inhibited 4APhe fermentation at low concentrations (half-maximum inhibitor concentration IC₅₀ = 16 mg/L), whereas furfural and 5-hydroxymethylfurfural, which are well-known fermentation inhibitors, inhibited 4APhe fermentation at relatively high concentrations (IC₅₀ = 640 mg/L). These results provide insight into the design of metabolic pathways tailored for the utilization of lignocellulosic biomass to produce aromatic compounds through the shikimate pathway.

The aromatic compound 3-amino-4-hydroxybenzoic acid (3,4-AHBA) can be employed as a raw material for high-performance industrial plastics. The aim of this study is to produce 3,4-AHBA via a recombinant Streptomyces lividans strain containing griI and griH genes derived from Streptomyces griseus using culture medium with glucose and/or xylose, which are the main components in lignocellulosic biomass. Production of 3,4-AHBA by the recombinant S. lividans strain was successful, and the productivity was affected by the kind of sugar used as an additional carbon source. Metabolic profiles revealed that L aspartate-4-semialdehyde (ASA), a precursor of 3,4-AHBA, and coenzyme NADPH were supplied in greater amounts in xylose medium than in glucose medium. Moreover, cultivation in TSB medium with a mixed sugar (glucose/xylose) was found to be effective for 3,4-AHBA production, and optimal conditions for efficient production were designed by changing the ratio of glucose to xylose. The best productivity of 2.70 g/L was achieved using a sugar mixture of 25 g/L glucose and 25 g/L xylose, which was 1.5 times higher than the result using 50 g/L glucose alone. These results suggest that Streptomyces is a suitable candidate platform for 3,4-AHBA production from lignocellulosic biomass-derived sugars under appropriate culture conditions.

Production of bioplastics from renewable biological resources is a prerequisite for the development of a circular and sustainable society. Current bioplastics are mostly heat‐sensitive aliphatic polymers, requiring thermoresistant aromatic bioplastics. Herein, 3‐amino‐4‐hydroxybenzoic acid (AHBA) and 4‐aminobenzoic acid (ABA) are produced from kraft pulp, an inedible cellulosic feedstock, using metabolically engineered bacteria. AHBA is chemically converted to 3,4‐diaminobenzoic acid (DABA); subsequently, poly(2,5‐benzimidazole) is obtained by the polycondensation of DABA and processed into an ultrahigh thermoresistant film. The copolymerization of DABA with a small amount of ABA dramatically increases the degradation temperatures of the resulting films (over 740 °C) to yield the most thermoresistant plastic on record. Density functional theory calculations indicate that the incorporation of ABA strengthens the interchain hydrogen bonds between aromatic imidazole rings. Thus, an alternative organic molecular design is proposed for thermoresistant plastics without using heavy inorganics, although continuous aromatic heterocycles are widely considered ideal for polymer thermoresistance. This innovative macromolecular design increases thermoresistance and can be widely applied to well‐processable plastics for the production of lightweight materials and is expected to contribute to the development of a more sustainable society.

We determined the complete and draft genome sequences of two strains of Corynebacterium glutamicum and revealed their genomic islands (GEIs). The two strains, ATCC 21799 and ATCC 31831, were found to have 3,079 and 3,109 coding sequences, respectively, with 13 GEIs each not present in the reference strain, ATCC 13032.

Controlling the carbon flux into a desired pathway is important for improving product yield in metabolic engineering. After entering a cell, glucose is channeled into glycolysis and the pentose phosphate pathway (PPP), which decreases the yield of target products whose synthesis relies on NADPH as a cofactor. Here, we demonstrate redirection of carbon flux into PPP under aerobic conditions in Corynebacterium glutamicum, achieved by replacing the promoter of glucose 6-phosphate isomerase gene (pgi) with an anaerobic-specific promoter of the lactate dehydrogenase gene (ldhA). The promoter replacement increased the split ratio of carbon flux into PPP from 39 to 83% under aerobic conditions. The titer, yield, and production rate of 1,5-diaminopentane, whose synthesis requires NADPH as a cofactor, were increased by 4.6-, 4.4-, and 2.6-fold, respectively. This is the largest improvement in the production of 1,5-diaminopentane or its precursor, lysine, reported to date. After aerobic cell growth, pgi expression was automatically induced under anaerobic conditions, altering the carbon flux from PPP to glycolysis, to produce succinate in a single metabolically engineered strain. Such an automatic redirection of metabolic pathway using an oxygen-responsive switch enables two-stage fermentation for efficient production of two different compounds by a single strain, potentially reducing the production costs and time for practical applications.