Zongbao K. Zhao

Acid in ionic liquid: An efficient system for hydrolysis of lignocellulose

Acid in ionic liquid was demonstrated as an efficient system for hydrolysis of lignocellulosic materials with improved total reducing sugars (TRS) yield under mild conditions. TRS yields were up to 66%, 74%, 81% and 68% for hydrolysis of corn stalk, rice straw, pine wood and bagasse, respectively, in C4mimCl in the presence of 7 wt% hydrogen chloride at 100 °C under atmospheric pressure within 60 min. Different combinations between ionic liquids, such as C6mimCl, C4mimBr, AmimCl, C4mimHSO4, and SbmimHSO4, and acids, including sulfuric acid, nitric acid, phosphoric acid, as well as maleic acid, afforded similar results albeit longer reaction time was generally required comparing with the combination of C4mimCl and hydrochloric acid. FT-IR spectra and elemental analysis of the recovered residues indicated that modification of lignin occurred during sulfuric acid catalyzed hydrolysis. In addition, kinetic modeling based on experimental data suggested that the hydrolysis likely followed a consecutive first-order reaction sequence, where k1 and k2, the rate constants for TRS formation and TRS degradation, were determined as 0.068 min−1 and 0.007 min−1, respectively. This novel system may be valuable to facilitate cost-efficient conversion of biomass into biofuels and biobased products.

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.

Effects of biomass hydrolysis by-products on oleaginous yeast Rhodosporidium toruloides

Lignocellulosic biomass hydrolysis inevitably coproduces byproducts that may have various affects on downstream biotransformation. It is imperative to document the inhibitor tolerance ability of microbial strain in order to utilize biomass hydrolysate more effectively. To achieve better lipid production by Rhodosporidium toruloides Y4, we performed fermentation experiments in the presence of some representative inhibitors. We found that acetate, 5-hydroxymethylfurfural and syringaldehyde had slightly inhibitory effects; p-hydroxybenzaldehyde and vanillin were toxic at a concentration over 10 mM; and furfural and its derivatives furfuryl alcohol and furoic acid inhibited cell growth by 45% at around 1 mM. We further demonstrated that inhibition is generally additive, although strong synergistic inhibitions were also observed. Finally, lipid production afforded good results in the presence of six inhibitors at their respective concentrations usually found in biomass hydrolysates. Fatty acid compositional profile of lipid samples indicated that those inhibitors had little effects on lipid biosynthesis. Our work will be useful for optimization of biomass hydrolysis processes and lipid production using lignocellulosic materials.

Catalytic Conversion of Carbohydrates into 5‐Hydroxymethylfurfural by Germanium(IV) Chloride in Ionic Liquids

Direct conversion of carbohydrates into 5-hydroxymethylfurfural (HMF) catalyzed by germanium(IV) chloride in ionic liquids has been investigated in search of an efficient and environmentally friendly process. Monosaccharides D-fructose and D-glucose, disaccharides sucrose and maltose, and even the polysaccharide cellulose were successfully converted into HMF with good yields under mild conditions (yield up to 92 % in 5 min in the case of fructose). The structure of ionic liquids, catalyst loading, reaction temperature and water content had noticeable effects on this catalytic system. Addition of 5 Å molecular sieves during the dehydration of glucose resulted in an increase in HMF yield from 38.4 % to 48.4 %. A mechanism for glucose conversion to HMF catalyzed by germanium(IV) chloride was proposed according to ¹³C NMR spectra obtained in situ under different conditions using D-glucose-2-¹³C as the substrate.

Modular Pathway Engineering of Diterpenoid Synthases and the Mevalonic Acid Pathway for Miltiradiene Production

Microbial production can be advantageous over the extraction of phytoterpenoids from natural plant sources, but it remains challenging to rationally and rapidly access efficient pathway variants. Previous engineering attempts mainly focused on the mevalonic acid (MVA) or methyl-d-erythritol phosphate (MEP) pathways responsible for the generation of precursors for terpenoids biosynthesis, and potential interactions between diterpenoids synthases were unexplored. Miltiradiene, the product of the stepwise conversion of (E,E,E)-geranylgeranyl diphosphate (GGPP) catalyzed by diterpene synthases SmCPS and SmKSL, has recently been identified as the precursor to tanshionones, a group of abietane-type norditerpenoids rich in the Chinese medicinal herb Salvia miltiorrhiza . Here, we present the modular pathway engineering (MOPE) strategy and its application for rapid assembling synthetic miltiradiene pathways in the yeast Saccharomyces cerevisiae . We predicted and analyzed the molecular interactions between SmCPS and SmKSL, and engineered their active sites into close proximity for enhanced metabolic flux channeling to miltiradiene biosynthesis by constructing protein fusions. We show that the fusion of SmCPS and SmKSL, as well as the fusion of BTS1 (GGPP synthase) and ERG20 (farnesyl diphosphate synthase), led to significantly improved miltiradiene production and reduced byproduct accumulation. The MOPE strategy facilitated a comprehensive evaluation of pathway variants involving multiple genes, and, as a result, our best pathway with the diploid strain YJ2X reached miltiradiene titer of 365 mg/L in a 15-L bioreactor culture. These results suggest that terpenoids synthases and the precursor supplying enzymes should be engineered systematically to enable an efficient microbial production of phytoterpenoids.

A multi-omic map of the lipid-producing yeast Rhodosporidium toruloides

Triacylglycerols are among the most attractive alternative raw materials for biofuel development. Current oil plant-based technologies are limited in terms of triacylglycerol production capacity and rate. These limitations may be circumvented by biotransformation of carbohydrates into lipids; however, our understanding of microbial oleaginicity remains limited. Here we present the results of a multi-omic analysis of Rhodosporidium toruloides, a robust triacylglycerol-producing fungus. The assembly of genome and transcriptome sequencing data reveals a genome of 20.2 Mb containing 8,171 protein-coding genes, the majority of which have multiple introns. Genes including a novel fatty acid synthase are predicted to participate in metabolic pathways absent in non-oleaginous yeasts. Transcriptomic and proteomic data suggest that lipid accumulation under nitrogen-limited conditions correlates with the induction of lipogenesis, nitrogenous compound recycling, macromolecule metabolism and autophagy. The multi-omic map of R. toruloides therefore provides a valuable resource for efforts to rationally engineer lipid-production pathways.

Solid acid and microwave-assisted hydrolysis of cellulose in ionic liquid

Solid acid-catalyzed hydrolysis of cellulose in ionic liquid was greatly promoted by microwave heating. H-form zeolites with a lower Si/Al molar ratio and a larger surface area showed a relatively higher catalytic activity. These solid catalysts exhibited better performance than the sulfated ion-exchanging resin NKC-9. Compared with conventional oil bath heating mode, microwave irradiation at an appropriate power significantly reduced the reaction time and increased the yields of reducing sugars. A typical hydrolysis reaction with Avicel cellulose produced glucose in around 37% yield within 8min.

Phosphate-limitation mediated lipid production by Rhodosporidium toruloides

Nitrogen-limited conditions have been routinely prepared for efficient lipid production by oleaginous microorganisms. However, it is difficult to attain high cellular lipid contents with natural nitrogen-rich substrates. In the present study, we showed that lipid accumulation by Rhodosporidium toruloides Y4 was directly linked to the carbon to phosphorus (C/P) molar ratios of the culture media. Moreover, such lipid accumulation phenomena were effective regardless of the presence of high amounts of nitrogen sources. Thus, cellular lipid content and lipid yield were 62.2% and 0.205 g/g glucose, respectively, using a medium with a carbon to nitrogen (C/N) molar ratio of 6.1 and a C/P molar ratio of 9552. This work suggested that phosphorus limitation can be equally effective and efficient to mediate lipid accumulation, which in turn, provides opportunities to produce microbial lipid more economically using natural or waste materials with high nitrogen content.

Microbial lipid production by Rhodosporidium toruloides under sulfate-limited conditions.

Novel biochemical approaches remain to be developed to improve microbial lipid technology. This study demonstrated that sulfate limitation was effective to promote accumulating substantial amounts of intracellular lipid by the oleaginous yeast Rhodosporidium toruloides Y4. When it was cultivated using a medium with an initial carbon-to-sulfur (C/S) molar ratio of 46,750, cellular lipid content reached up to 58.3%. The time courses of cell growth, lipid accumulation and nutrient depletion were analyzed and discussed in terms of lipid biosynthesis. Moreover, lipid accumulation under sulfate-limited conditions was effective regardless of the presence of a high concentration of nitrogen sources. Thus, lipid contents almost held constant at near 57% in the media with an initial C/S molar ratio of 11,380 although the carbon-to-nitrogen molar ratio ranged from 28.3 to 5.7. Taken together, our results established the sulfate-limitation approach to control lipid biosynthesis, which should be valuable to explore nitrogen-rich raw materials as the feedstock for lipid production.

CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts

Cytochrome P450 enzymes (CYPs) play major roles in generating highly functionalized terpenoids, but identifying the exact biotransformation step(s) catalyzed by plant CYP in terpenoid biosynthesis is extremely challenging. Tanshinones are abietane-type norditerpenoid naphthoquinones that are the main lipophilic bioactive components of the Chinese medicinal herb danshen (Salvia miltiorrhiza). Whereas the diterpene synthases responsible for the conversion of (E,E,E)-geranylgeranyl diphosphate into the abietane miltiradiene, a potential precursor to tanshinones, have been recently described, molecular characterization of further transformation of miltiradiene remains unavailable. Here we report stable-isotope labeling results that demonstrate the intermediacy of miltiradiene in tanshinone biosynthesis. We further use a next-generation sequencing approach to identify six candidate CYP genes being coregulated with the diterpene synthase genes in both the rhizome and danshen hairy roots, and demonstrate that one of these, CYP76AH1, catalyzes a unique four-electron oxidation cascade on miltiradiene to produce ferruginol both in vitro and in vivo. We then build upon the previous establishment of miltiradiene production in Saccharomyces cerevisiae, with incorporation of CYP76AH1 and phyto-CYP reductase genes leading to heterologous production of ferruginol at 10.5 mg/L. As ferruginol has been found in many plants including danshen, the results and the approaches that were described here provide a solid foundation to further elucidate the biosynthesis of tanshinones and related diterpenoids. Moreover, these results should facilitate the construction of microbial cell factories for the production of phytoterpenoids.