[Recent advances in the bioproduction of mannitol].

D-mannitol is a six-carbon sugar alcohol and one of the most abundant polyols in the nature. With antioxidant and osmotic pressure-regulating effects and non-metabolism by the human body, D-mannitol has been widely used in functional food and pharmaceutical industries. At present, a major way for industrial production of D-mannitol is chemical hydrogenation. In addition, D-Mannitol can be produced by microbial metabolism or catalysis. Compared with the chemical hydrogenation, the microbial methods for synthesizing mannitol do not produce sorbitol as a by-product and have the advantages of mild reaction conditions, strong specificity, and high conversion rate. Microbial fermentation is praised for easy access of strains and raw materials and simple separation of the product. Microbial catalysis usually adopts a multi-enzyme coupling strategy, which uses enzymes produced by engineered bacteria for whole-cell catalysis, and the cofactor recycling pathway is introduced to replenish expensive cofactor. This method can achieve high yields with cheap substrates under mild conditions without the formation of by-products. However, the application of microbial methods in the industrial production of D-mannitol is limited by the high costs of fermentation media and substrates and the long reaction time. This article reviews the reported microbial methods for producing D-mannitol, including the use of high-yielding strains and their fermentation processes, the utilization of low-cost substrates, whole-cell catalytic strategies, and the process control for high productivity. The biosynthesis of mannitol is not only of great significance for promoting industrial upgrading and realizing green manufacturing, but also provides strong support for the development of new bio-based products to meet the growing market demand. With the continuous improvement of technological innovation and industrial chain, it is expected to become one of the main ways of mannitol production in the future.

Improving the production of carbamoyltobramycin by an industrial Streptoalloteichus tenebrarius through metabolic engineering.

Tobramycin is an essential and extensively used broad-spectrum aminoglycoside antibiotic obtained through alkaline hydrolysis of carbamoyltobramycin, one of the fermentation products of Streptoalloteichus tenebrarius. To simplify the composition of fermentation products from industrial strain, the main byproduct apramycin was blocked by gene disruption and constructed a mutant mainly producing carbamoyltobramycin. The generation of antibiotics is significantly affected by the secondary metabolism of actinomycetes which could be controlled by modifying the pathway-specific regulatory proteins within the cluster. Within the tobramycin biosynthesis cluster, a transcriptional regulatory factor TobR belonging to the Lrp/AsnC family was identified. Based on the sequence and structural characteristics, tobR might encode a pathway-specific transcriptional regulatory factor during biosynthesis. Knockout and overexpression strains of tobR were constructed to investigate its role in carbamoyltobramycin production. Results showed that knockout of TobR increased carbamoyltobramycin biosynthesis by 22.35%, whereas its overexpression decreased carbamoyltobramycin production by 10.23%. In vitro electrophoretic mobility shift assay (EMSA) experiments confirmed that TobR interacts with DNA at the adjacent tobO promoter position. Strains overexpressing tobO with ermEp* promoter exhibited 36.36% increase, and tobO with kasOp* promoter exhibited 22.84% increase in carbamoyltobramycin titer. When the overexpressing of tobO and the knockout of tobR were combined, the production of carbamoyltobramycin was further enhanced. In the shake-flask fermentation, the titer reached 3.76 g/L, which was 42.42% higher than that of starting strain. Understanding the role of Lrp/AsnC family transcription regulators would be useful for other antibiotic biosynthesis in other actinomycetes. KEY POINTS: • The transcriptional regulator TobR belonging to the Lrp/AsnC family was identified.  • An oxygenase TobO was identified within the tobramycin biosynthesis cluster. • TobO and TobR have significant effects on the synthesis of carbamoyltobramycin.

Highly efficient biosynthesis of salidroside by a UDP-glucosyltransferase-catalyzed cascade reaction.

OBJECTIVE: Salidroside is an important plant-derived aromatic compound with diverse biological properties. The main objective of this study was to synthesize salidroside from tyrosol using UDP-glucosyltransferase (UGT) with in situ regeneration of UDP-glucose (UDPG). RESULTS: The UDP-glucosyltransferase 85A1 (UGT85A1) from Arabidopsis thaliana, which showed high activity and regioselectivity towards tyrosol, was selected for the production of salidroside. Then, an in vitro cascade reaction for in situ regeneration of UDPG was constructed by coupling UGT85A1 to sucrose synthase from Glycine max (GmSuSy). The optimal UGT85A1-GmSuSy activity ratio of 1:2 was determined to balance the efficiency of salidroside production and UDP-glucose regeneration. Different cascade reaction conditions for salidroside production were also determined. Under the optimized condition, salidroside was produced at a titer of 6.0 g/L with a corresponding molar conversion of 99.6% and a specific productivity of 199.1 mg/L/h in a continuous feeding reactor. CONCLUSION: This is the highest salidroside titer ever reported so far using biocatalytic approach.