Hun-Suk Song

Increase in furfural tolerance by combinatorial overexpression of NAD salvage pathway enzymes in engineered isobutanol-producing E. coli

To reduce the furfural toxicity for biochemical production in E. coli, a new strategy was successfully applied by supplying NAD(P)H through the nicotine amide salvage pathway. To alleviate the toxicity, nicotinamide salvage pathway genes were overexpressed in recombinant, isobutanol-producing E. coli. Gene expression of pncB and nadE respectively showed increased tolerance to furfural among these pathways. The combined expression of pncB and nadE was the most effective in increasing the tolerance of the cells to toxic aldehydes. By comparing noxE- and fdh-harbouring strains, the form of NADH, rather than NAD+, was the major effector of furfural tolerance. Overall, this study is the application of the salvage pathway to isobutanol production in the presence of furfural, and this system seems to be applicable to alleviate furfural toxicity in the production of other biochemical.

Production and characterization of medium-chain-length polyhydroxyalkanoate copolymer from Arctic psychrotrophic bacterium Pseudomonas sp. PAMC 28620

Arctic psychrotrophic bacterium Pseudomonas sp. PAMC 28620 was found to produce a distinctive medium-chain-length polyhydroxyalkanoate (MCL-PHA) copolymer when grown on structurally unrelated carbon sources including glycerol. The maximum MCL-PHA copolymer yield was obtained about 52.18±4.12% from 7.95±0.66g/L of biomass at 144h of fermentation when 3% glycerol was used as sole carbon and energy source during the laboratory-scale bioreactor process. Characterization of the copolymer was carried out using fourier transform infrared spectroscopy (FTIR), gas chromatography-mass spectrometry (GC-MS), proton (1H) and carbon (13C) nuclear magnetic resonance spectroscopy (NMR), gel permeation chromatography (GPC), differential scanning calorimeter (DSC) and thermo-gravimetric analysis (TGA). The copolymer produced by Pseudomonas sp. PAMC 28620 consisting of four PHA monomers and identified as 3-hydroxyoctanoate (3HO), 3-hydroxydecanoate (3HD), 3-hydroxydodecanoate (3HDD) and 3-hydroxytetradecanoate (3HTD). An average molecular weight of the copolymer was found approximately 30.244kDa with polydispersity index (PDI) value of 2.05. Thermal analysis showed the produced MCL-PHA copolymer to be low-crystalline (43.73%) polymer with great thermal stability, having the thermal decomposition temperature of 230°C-280°C, endothermic melting temperature (Tm) of 172.84°C, glass transition (Tg) temperature of 3.99°C, and apparent melting enthalpy fusion (ΔHm) about 63.85Jg-1.

Enhanced isobutanol production from acetate by combinatorial overexpression of acetyl-CoA synthetase and anaplerotic enzymes in engineered Escherichia coli

Acetic acid is an abundant material that can be used as a carbon source by microorganisms. Despite its abundance, its toxicity and low energy content make it hard to utilize as a sole carbon source for biochemical production. To increase acetate utilization and isobutanol production with engineered Escherichia coli, the feasibility of utilizing acetate and metabolic engineering was investigated. The expression of acs, pckA, and maeB increased isobutanol production by up to 26%, and the addition of TCA cycle intermediates indicated that the intermediates can enhance isobutanol production. For isobutanol production from acetate, acetate uptake rates and the NADPH pool were not limiting factors compared to glucose as a carbon source. This work represents the first approach to produce isobutanol from acetate with pyruvate flux optimization to extend the applicability of acetate. This technique suggests a strategy for biochemical production utilizing acetate as the sole carbon source. This article is protected by copyright. All rights reserved.

Bioconversion of plant biomass hydrolysate into bioplastic (polyhydroxyalkanoates) using Ralstonia eutropha 5119

Pretreatment of lignocellulosic biomass results in the formation of byproducts (furfural, hydroxymethylfurfural [HMF], vanillin, acetate etc.), which affect microbial growth and productivity. Furfural (0.02%), HMF (0.04%), and acetate (0.6%) showed positive effects on Ralstonia eutropha 5119 growth and polyhydroxyalkanoate (PHA) production, while vanillin exhibited negative effects. Response optimization and interaction studies between the variables glucose, ammonium chloride, furfural, HMF, and acetate using the response surface methodology resulted in maximum PHA production (2.1 g/L) at optimal variable values of 15.3 g/L, 0.43 g/L, 0.04 g/L, 0.05 g/L, and 2.34 g/L, respectively. Different lignocellulosic biomass hydrolysates (LBHs), including barley biomass hydrolysate (BBH), Miscanthus biomass hydrolysate (MBH), and pine biomass hydrolysate (PBH), were evaluated as potential carbon sources for R. eutropha 5119 and resulted in 1.8, 2.0, and 1.7 g/L PHA production, respectively. MBH proved the best carbon source, resulted in higher biomass (Yx/s, 0.31 g/g) and PHA (Yp/s, 0.14 g/g) yield.

L-Glycine Alleviates Furfural-Induced Growth Inhibition during Isobutanol Production in Escherichia coli

Lignocellulose is now a promising raw material for biofuel production. However, the lignin complex and crystalline cellulose require pretreatment steps for breakdown of the crystalline structure of cellulose for the generation of fermentable sugars. Moreover, several fermentation inhibitors are generated with sugar compounds, majorly furfural. The mitigation of these inhibitors is required for the further fermentation steps to proceed. Amino acids were investigated on furfural-induced growth inhibition in E. coli producing isobutanol. Glycine and serine were the most effective compounds against furfural. In minimal media, glycine conferred tolerance against furfural. From the IC₅₀ value for inhibitors in the production media, only glycine could alleviate growth arrest for furfural, where 6 mM glycine addition led to a slight increase in growth rate and isobutanol production from 2.6 to 2.8 g/l under furfural stress. Overexpression of glycine pathway genes did not lead to alleviation. However, addition of glycine to engineered strains blocked the growth arrest and increased the isobutanol production about 2.3-fold.

The role of NdgR in glycerol metabolism in Streptomyces coelicolor

Streptomyces, which produces many pharmaceutical antibiotics and anticancer agents, is a genus of soil-dwelling bacteria with numerous regulators that control both primary and secondary metabolism. NdgR is highly conserved in Streptomyces spp. and is known to be involved in antibiotic production, tolerance against shock and physical stress, nitrogen metabolism, leucine metabolism, and N-acetylglucosamine metabolism. As another function of NdgR, we report the involvement of NdgR in glycerol metabolism in S. coelicolor. Initially, a glycerol utilization operon containing gylCABX was found to be up-regulated in an ndgR deletion mutant (BG11) grown in N-acetylglucosamine solid minimal media compared with wild-type strain (M145). BG11 produced more antibiotics with a small amount of glycerol and increased glycerol utilization, yielding higher concentrations of lactate and acetate per cell. Moreover, fatty acid production was also changed in BG11 to produce longer chain fatty acids, phenolic compounds, alkanes, and fatty alcohols. Using a gel retardation assay, NdgR was found to bind the upstream region of gylC, working as a repressor. NdgR is a second regulator of a glycerol utilization operon, for which only one regulator, GylR was previously known.