Production of dihydroxyacetone from glycerol by engineered Escherichia coli cells co-expressing gldA and nox genes

Glycerol can be converted into more valuable compound dihydroxyacetone by the nicotinamide adenine dinucleotide (NAD+)-dependent glycerol dehydrogenase. However, it is economically prohibitive to produce dihydroxyacetone using purified glycerol dehydrogenase at the expense of a stoichiometric amount of the cofactor NAD+. In this study, Escherichia coli was engineered for dihydroxyacetone production by enhancing its glycerol dehydrogenase activity and introducing NADH oxidase activity. Under optimized conditions, dihydroxyacetone productivity reached 0.13 g/h/g wet cell mass by recombinant E. coli D4 (pET-24b-gldA+nox) cells co-expressing gldA gene from E. coli and nox gene from Enterococcus faecalis. It was interesting to note that exogenous NAD+ greatly improved dihydroxyacetone production for the whole-cell biotransformation process. These results should be useful for the development of advanced bioprocess in terms of glycerol utilization.Keywords: Dihydroxyacetone, Glycerol dehydrogenase, NAD+, whole-cell biotransformation, Escherichia coliAfrican Journal of Biotechnology Vol. 12(27), pp. 4387-439

Engineering NAD+ availability for Escherichia coli whole-cell biocatalysis: A case study for dihydroxyacetone production

Background: Whole-cell redox biocatalysis has been intensively explored for the production of valuable compounds because excellent selectivity is routinely achieved. Although the cellular cofactor level, redox state and the corresponding enzymatic activity are expected to have major effects on the performance of the biocatalysts, our ability remains limited to predict the outcome upon variation of those factors as well as the relationship among them. Results: In order to investigate the effects of cofactor availability on whole-cell redox biocatalysis, we devised recombinant Escherichia coli strains for the production of dihydroxyacetone (DHA) catalyzed by the NAD + -dependent glycerol dehydrogenase (GldA). In this model system, a water-forming NAD + oxidase (NOX) and a NAD + transporter (NTT4) were also co-expressed for cofactor regeneration and extracellular NAD + uptake, respectively. We found that cellular cofactor level, NAD + /NADH ratio and NOX activity were not only strain-dependent, but also growth condition-dependent, leading to significant differences in specific DHA titer among different whole-cell biocatalysts. The host E. coli DH5α had the highest DHA specific titer of 0.81\ua0g/g DCW with the highest NAD + /NADH ratio of 6.7 and NOX activity of 3900 U. The biocatalyst had a higher activity when induced with IPTG at 37\ub0C for 8\ua0h compared with those at 30\ub0C for 8\ua0h and 18\ua0h. When cells were transformed with the ntt4 gene, feeding NAD + during the cell culture stage increased cellular NAD(H) level by 1.44 fold and DHA specific titer by 1.58 fold to 2.13\ua0g/g DCW . Supplementing NAD + during the biotransformation stage was also beneficial to cellular NAD(H) level and DHA production, and the highest DHA productivity reached 0.76\ua0g/g DCW /h. Cellular NAD(H) level, NAD + /NADH ratio, and NOX and GldA activity dropped over time during the biotransformation process.Conclusions: High NAD + /NADH ratio driving by NOX was very important for DHA production. Once cofactor was efficiently cycled, high cellular NAD(H) level was also beneficial for whole-cell redox biocatalysis. Our results indicated that NAD + transporter could be applied to manipulate redox cofactor level for biocatalysis. Moreover, we suggested that genetically designed redox transformation should be carefully profiled for further optimizing whole-cell biocatalysis. \ua9 2013 Zhou et al.; licensee BioMed Central Ltd

Simultaneous utilization of glucose and xylose for lipid production by Trichosporon cutaneum

<p>Abstract</p> <p>Background</p> <p>Biochemical conversion of lignocellulose hydrolysates remains challenging, largely because most microbial processes have markedly reduced efficiency in the presence of both hexoses and pentoses. Thus, identification of microorganisms capable of efficient and simultaneous utilization of both glucose and xylose is pivotal to improving this process.</p> <p>Results</p> <p>In this study, we found that the oleaginous yeast strain <it>Trichosporon cutaneum </it>AS 2.571 assimilated glucose and xylose simultaneously, and accumulated intracellular lipid up to 59 wt% with a lipid coefficient up to 0.17 g/g sugar, upon cultivation on a 2:1 glucose/xylose mixture in a 3-liter stirred-tank bioreactor. In addition, no classic pattern of diauxic growth behavior was seen; the microbial cell mass increased during the whole culture process without any lag periods. In shake-flask cultures with different initial glucose:xylose ratios, glucose and xylose were consumed simultaneously at rates roughly proportional to their individual concentrations in the medium, leading to complete utilization of both sugars at the same time. Simultaneous utilization of glucose and xylose was also seen during fermentation of corn-stover hydrolysate with a lipid content and coefficient of 39.2% and 0.15 g/g sugar, respectively. The lipid produced had a fatty-acid compositional profile similar to those of conventional vegetable oil, indicating that it could have potential as a raw material for biodiesel production.</p> <p>Conclusion</p> <p>Efficient lipid production with simultaneous consumption of glucose and xylose was achieved in this study. This process provides an exciting opportunity to transform lignocellulosic materials into biofuel molecules, and should also encourage further study to elucidate this unique sugar-assimilation mechanism.</p

Engineering yeast for high-level production of diterpenoid sclareol

The diterpenoid sclareol is an industrially important precursor for alternative sustainable supply of ambergris. However, its current production from plant extraction is neither economical nor environmental-friendly, since it requires laborious and cost-intensive purification procedures and plants cultivation is susceptible to environmental factors. Engineering cell factories for bio-manufacturing can enable sustainable production of natural products. However, stringent metabolic regulation poses challenges to rewire cellular metabolism for overproduction of compounds of interest. Here we used a modular approach to globally rewire the cellular metabolism for improving sclareol production to 11.4 g/L in budding yeast Saccharomyces cerevisiae, the highest reported diterpenoid titer in microbes. Metabolic flux analysis showed that modular balanced metabolism drove the metabolic flux toward the biosynthesis of targeted molecules, and transcriptomic analysis revealed that the expression of central metabolism genes was shaped for a new balanced metabolism, which laid a foundation in extensive metabolic engineering of other microbial species for sustainable bio-production

Production of lipid from N-acetylglucosamine by Cryptococcus curvatus

N-Acetylglucosamine (GlcNAc), the monomeric constituent of chitin, is rarely used as a carbon source for fermentation technology. In this study, we demonstrate that the oleaginous yeast Cryptococcus curvatus ATCC 20509 can produce intracellular lipid during the cultivation process and total lipid content can reach 54% on a GlcNAc-based medium. Culture of C. curvatus under various conditions indicated that lipid accumulation also occurred at a relatively broad range of temperatures as well as relatively high initial GlcNAc concentrations. Fatty acid analysis indicated that the product was rich in palmitic acid, stearic acid, and oleic acid, closely resembling the composition of palm oil. More importantly, the lipid sample produced at 22 degrees C had a total saturated fatty acid content of 54.2 wt%, suggesting that it may be explored as cocoa-butter equivalent. Our data suggested that GlcNAc could be used as a feedstock for industrial biotechnology and that C. curvatus ATCC 20509 is a strain capable of accumulating high intracellular lipid using this nitrogen-rich renewable material

Microwave-assisted conversion of lignocellulosic biomass into furans in ionic liquid

Production of 5-hydroxymethylfurfural (HMF) and furfural from lignocellulosic biomass was studied in ionic liquid in the presence of CrCl(3) under microwave irradiation. Corn stalk, rice straw and pine wood treated under typical reaction conditions produced HMF and furfural in yields of 45-52% and 23-31%, respectively, within 3 min. This method should be valuable to facilitate energy-efficient and cost-effective conversion of biomass into biofuels and platform chemicals. (C) 2009 Elsevier Ltd. All rights reserved

Mechanistic studies of IspH in the deoxyxylulose phosphate pathway: Heterolytic C-O bond cleavage at C(4) position

Isoprenoids are one of the largest and most structurally diverse groups of metabolites in nature. Their biosyntheses require two precursors, isopentenyl diphosphate (IPP) and its isomer, dimethylallyl diphosphate (DMAPP). There are two different pathways for the synthesis of IPP and DMAPP: the deoxyxylulose phosphate (DXP) pathway and the mevalonic acid (MVA) pathway. More importantly, these two pathways have a well-defined distribution among different kingdoms. Most pathogenic bacteria and protozoan parasites utilize the DXP pathway, while animals synthesize their isoprenoid precursors from acety-CoA via the MVA pathway. Plants have both DXP and MVA pathways. Thus, mechanistic studies on the DXP pathway enzymes may lead to the development of mechanism-based inhibitors as herbicides, broad-spectrum antibiotics, and antimalaria drugs. IspH in the DXP pathway catalyzes the reductive dehydration of (E)-4-hydroxy-3-metho-2-butenyl diphosphate (HMBPP) to form IPP and DMAPP, the last step in the DXP pathway. Recent EPR studies reported in literature suggest that IspH is a unique iron-site-containing [4Fe-4S] protein. In this study, we studied the IspH-catalyzed reductive dehydration mechanism using two substrate analogues. Our data reported herein provide evidence to not only support the integrity of the C1 position C-O bond during reaction, they also suggest a heterolytic C-O bond cleavage at the C4 position for IspH-catatyzed reductive dehydration reaction. Our kinetic studies also suggest that the C4 hydroxyl group is involved in substrate binding. Because the IspH-catalyzed reductive dehydration reaction does not fall into the two known classes of unique iron-site-containing [4Fe-4S] proteins, aconitase-type and radical SAM-type enzymes, IspH may represent a new class of iron-sulfur-containing proteins

Efficient acid-catalyzed hydrolysis of cellulose in ionic liquid

A novel method for cellulose hydrolysis catalyzed by mineral acids in the ionic liquid 1-butyl-3-methylimidazolium chloride ([C(4)mim]Cl) has been developed that facilitates the hydrolysis of cellulose with dramatically accelerated reaction rates at 100 degrees C under atmospheric pressure and without pretreatment