Microbial Biomanufacturing Using Chemically Synthesized Non-Natural Sugars as the Substrate.

The bioproduction of valuable materials using biomass sugars is attracting attention as an environmentally friendly technology. However, its ability to fulfil the enormous demand to produce fuels and chemical products is limited. With a view towards the future development of a novel bioproduction process that addresses these concerns, this study investigated the feasibility of bioproduction of valuable substances using Corynebacterium glutamicum (C. glutamicum) with a chemically synthesized non-natural sugar solution. Cells were grown using the synthesized sugar solution as the sole carbon source and they produced lactate under oxygen-limited conditions. It was also found that some of the sugars produced by the series of chemical reactions inhibited cell growth since prior removal of these sugars increased the cell growth rate. The results obtained in this study indicate that chemically synthesized sugars have the potential to resolve the concerns regarding future biomass sugar supply in microbial biomanufacturing.

Construction of an autocatalytic reaction cycle in neutral medium for synthesis of life-sustaining sugars.

Autocatalytic mechanisms in carbon metabolism, such as the Calvin cycle, are responsible for the biological assimilation of CO2 to form organic compounds with complex structures, including sugars. Compounds that form C-C bonds with CO2 are regenerated in these autocatalytic reaction cycles, and the products are concurrently released. The formose reaction in basic aqueous solution has attracted attention as a nonbiological reaction involving an autocatalytic reaction cycle that non-enzymatically synthesizes sugars from the C1 compound formaldehyde. However, formaldehyde and sugars, which are the substrate and products of the formose reaction, respectively, are consumed in Cannizzaro reactions, particularly under basic aqueous conditions, which makes the formose reaction a fragile sugar-production system. Here, we constructed an autocatalytic reaction cycle for sugar synthesis under neutral conditions. We focused on the weak Brønsted basicity of oxometalate anions such as tungstates and molybdates as catalysts, thereby enabling the aldol reaction, retro-aldol reaction, and aldose-ketose transformation, which collectively constitute the autocatalytic reaction cycle. These bases acted on sugar molecules of substrates together with sodium ions of a Lewis acid to promote deprotonation under neutral conditions, which is the initiation step of the reactions forming an autocatalytic cycle, whereas the Cannizzaro reaction was inhibited. The autocatalytic reaction cycle established using this abiotic approach is a robust sugar production system. Furthermore, we found that the synthesized sugars work as energy storage substances that sustain microbial growth despite their absence in nature.

Quantification of NAD(P)H in cyanobacterial cells by a phenol extraction method.

In photosynthetic organisms, it is recognized that the intracellular redox ratio of NADPH is regulated within an appropriate range for the cooperative function of a wide variety of physiological processes. However, despite its importance, there is large variability in the values of the NADPH fraction [NADPH/(NADPH + NADP+)] quantitatively estimated to date. In the present study, the light response of the NADPH fraction was investigated by applying a novel NADP(H) extraction method using phenol / chloroform / isoamyl alcohol (PCI) in the cyanobacterium Synechocystis sp. PCC 6803. The light response of NADP(H) observed using PCI extraction was qualitatively consistent with the NAD(P)H fluorescence time course measured in vivo. Moreover, the results obtained by PCI extraction and the fluorescence-based methods were also consistent in a mutant lacking the ability to oxidize NAD(P)H in the respiratory chain, and exhibiting a unique NADPH light response. These observations indicate that the PCI extraction method allowed quantitative determination of NADP(H) redox. Notably, the PCI extraction method showed that not all NADP(H) was oxidized or reduced by light-dark transition. Specifically, the fraction of NADPH was 42% in the dark-adapted cell, and saturated at 68% in light conditions.