Ying-Jin Yuan

Process optimization to convert forage and sweet sorghum bagasse to ethanol based on ammonia fiber expansion (AFEX) pretreatment.

With growing demand for bio-based fuels and chemicals, there has been much attention given to the performance of different feedstocks. We have optimized the ammonia fiber expansion (AFEX) pretreatment and fermentation process to convert forage and sweet sorghum bagasse to ethanol. AFEX pretreatment was optimized for forage sorghum and sweet sorghum bagasse. Supplementing xylanase with cellulase during enzymatic hydrolysis increased both glucan and xylan conversion to 90% at 1% glucan loading. High solid loading hydrolyzates from the optimized AFEX conditions were fermented using Saccharomyces cerevisiae 424A (LNH-ST) without any external nutrient supplementation or detoxification. The strain was better able to utilize xylose at pH 6.0 than at pH 4.8, but glycerol production was higher for the former pH than the latter. The maximum final ethanol concentration in the fermentation broth was 30.9 g/L (forage sorghum) and 42.3 g/L (sweet sorghum bagasse). A complete mass balance for the process is given.

Mass balance and transformation of corn stover by pretreatment with different dilute organic acids

Previous studies indicated high xylose yield could be achieved after pretreatment using organic acids, but it is necessary to systematically investigate the effects of different parameters during organic acid pretreatments. Corn stover was pretreated with sulfuric, oxalic, citric, tartaric and acetic acid at 50 and 90 mM from 130 to 190°C. The xylan balance for each different acid was distinct, but all balances were very close to 100% by determining xylan recovery, xylooligomer yield, xylose yield and furfural yield. The effects of combined severity on the recovery or yields of these components were also studied. The acid pK(a) value affected the proportion of xylan degradation products. The maximum value of xylose and xylooligomer yield for specific acid pretreatment was also determined by pK(a) value. The maximum xylose yield was obtained after pretreatment with sulfuric and oxalic acid, but more xylooligomers were obtained after pretreatment with weaker acids.

Transcriptome shifts in response to furfural and acetic acid in Saccharomyces cerevisiae

Furfural and acetic acid are two prevalent inhibitors to microorganisms during cellulosic ethanol production, but molecular mechanisms of tolerance to these inhibitors are still unclear. In this study, genome-wide transcriptional responses to furfural and acetic acid were investigated in Saccharomyces cerevisiae using microarray analysis. We found that 103 and 227 genes were differentially expressed in the response to furfural and acetic acid, respectively. Furfural downregulated genes related to transcriptional control and translational control, while it upregulated stress-responsive genes. Furthermore, furfural also interrupted the transcription of genes involved in metabolism of essential chemicals, such as etrahydrofolate, spermidine, spermine, and riboflavin monophosphate. Acetic acid downregulated genes encoding mitochondrial ribosomal proteins and genes involved in carbohydrate metabolism and regulation and upregulated genes related to amino acid metabolism. The results revealed that furfural and acetic acid had effects on multiple aspects of cellular metabolism on the transcriptional level and that mitochondria might play important roles in response to both furfural and acetic acid. This research has provided insights into molecular response to furfural and acetic acid in S. cerevisiae, and it will be helpful to construct more resistant strains for cellulosic ethanol production.

Integrated proteomic and metabolomic analysis of an artificial microbial community for two-step production of vitamin C.

An artificial microbial community consisted of Ketogulonicigenium vulgare and Bacillus megaterium has been used in industry to produce 2-keto-gulonic acid (2-KGA), the precursor of vitamin C. During the mix culture fermentation process, sporulation and cell lysis of B. megaterium can be observed. In order to investigate how these phenomena correlate with 2-KGA production, and to explore how two species interact with each other during the fermentation process, an integrated time-series proteomic and metabolomic analysis was applied to the system. The study quantitatively identified approximate 100 metabolites and 258 proteins. Principal Component Analysis of all the metabolites identified showed that glutamic acid, 5-oxo-proline, L-sorbose, 2-KGA, 2, 6-dipicolinic acid and tyrosine were potential biomarkers to distinguish the different time-series samples. Interestingly, most of these metabolites were closely correlated with the sporulation process of B. megaterium. Together with several sporulation-relevant proteins identified, the results pointed to the possibility that Bacillus sporulation process might be important part of the microbial interaction. After sporulation, cell lysis of B. megaterium was observed in the co-culture system. The proteomic results showed that proteins combating against intracellular reactive oxygen stress (ROS), and proteins involved in pentose phosphate pathway, L-sorbose pathway, tricarboxylic acid cycle and amino acids metabolism were up-regulated when the cell lysis of B. megaterium occurred. The cell lysis might supply purine substrates needed for K. vulgare growth. These discoveries showed B. megaterium provided key elements necessary for K. vulgare to grow better and produce more 2-KGA. The study represents the first attempt to decipher 2-KGA-producing microbial communities using quantitative systems biology analysis.

Signal transduction pathway for oxidative burst and taxol production in suspension cultures of Taxus chinensis var. mairei induced by oligosaccharide from Fusarium oxysprum

Signal transduction pathway for oxidative burst and taxol production was studied in suspension cultures of Taxus chinensis var. mairei induced by oligosaccharide from Fusarium oxysprum. Suspension cells exhibited an oxidative burst approximately 4h after challenge with oligosaccharide. The secondary metabolism including accumulation of extracellular phenolics and taxol production were enhanced, while the alkalization of the outer medium and activity of phenylalanine ammonia lyase (PAL) was promoted significantly. Pretreatment with the tyrosine (Tyr) kinase inhibitor herbimycin A abolished the oxidative burst and the subsequent induction of taxol production within 15 min after addition of elicitor. Suramin, a G-protein inhibitor, inhibited the oxidative burst if added up to 30 min after oligosaccharide treatment. The oxidative burst was also induced by the ion channel generator amphotericin B. In contrast, the ion channel blocker anthracene-9-carboxylate acid and Ca2+ channel blocker nifedipine inhibited the oxidative burst within 45 min after treatment. Oligosaccharide rapidly induced Tyr phosphrylation of receptor, which was inhibited by herbimycin A and anthracene-9-carboxylate acid. These responses were also inhibited by phospholipase C-neomycin. The response time of oligosaccharide to phospholipase C pathway was ca at 60 min. These data suggested that the activation of ion channels and phospholipase C follow Tyr kinase and G-protein in the signal pathway leading to the oxidative burst.

Metabolome Profiling Reveals Metabolic Cooperation between Bacillus megaterium and Ketogulonicigenium vulgare during Induced Swarm Motility

ABSTRACT The metabolic cooperation in the ecosystem of Bacillus megaterium and Ketogulonicigenium vulgare was investigated by cultivating them spatially on a soft agar plate. We found that B. megaterium swarmed in a direction along the trace of K. vulgare on the agar plate. Metabolomics based on gas chromatography coupled with time-of-flight mass spectrometry (GC-TOF-MS) was employed to analyze the interaction mechanism between the two microorganisms. We found that the microorganisms interact by exchanging a number of metabolites. Both intracellular metabolism and cell-cell communication via metabolic cooperation were essential in determining the population dynamics of the ecosystem. The contents of amino acids and other nutritional compounds in K. vulgare were rather low in comparison to those in B. megaterium, but the levels of these compounds in the medium surrounding K. vulgare were fairly high, even higher than in fresh medium. Erythrose, erythritol, guanine, and inositol accumulated around B. megaterium were consumed by K. vulgare upon its migration. The oxidization products of K. vulgare, including 2-keto-gulonic acids (2KGA), were sharply increased. Upon coculturing of B. megaterium and K. vulgare, 2,6-dipicolinic acid (the biomarker of sporulation of B. megaterium), was remarkably increased compared with those in the monocultures. Therefore, the interactions between B. megaterium and K. vulgare were a synergistic combination of mutualism and antagonism. This paper is the first to systematically identify a symbiotic interaction mechanism via metabolites in the ecosystem established by two isolated colonies of B. megaterium and K. vulgare.

Fungal elicitor-induced cell apoptosis in suspension cultures of Taxus chinensis var. mairei for taxol production

Abstract A crude extract from Fusarium oxysprum caused significant cell apoptosis in suspension cultures of Taxus chinensis var. mairei as determined by transmission electron microscopy, DNA gel electrophoresis and terminal deoxynucleotidyl transferase-mediated dUTP nick labeling (TUNEL) analysis. Cells of T. chinensis var. mairei responded to the fungal elicitors by altering the general phenylpropanoid pathway and affecting the biosynthesis of taxol. The activity of l -phenylalanine ammonia lyase (PAL) increased appreciably within the first 36 h of elicitor treatment, and then decreased rapidly. A large quantity of phenolic compounds accumulated in the culture medium. The maximum concentration of taxol was three times higher than that of the control. Cell apoptosis and taxol production are closely relevant in elicitor-treated culture of T. chinensis var. mairei .

Evaluation of storage methods for the conversion of corn stover biomass to sugars based on steam explosion pretreatment

Effects of dry and wet storage methods without or with shredding on the conversion of corn stover biomass were investigated using steam explosion pretreatment and enzymatic hydrolysis. Sugar conversions and yields for wet stored biomass were obviously higher than those for dry stored biomass. Shredding reduced sugar conversions compared with non-shredding, but increased sugar yields. Glucan conversion and glucose yield for non-shredded wet stored biomass reached 91.5% and 87.6% after 3-month storage, respectively. Data of micro-structure and crystallinity of biomass indicated that corn stover biomass maintained the flexible and porous structure after wet storage, and hence led to the high permeability of corn stover biomass and the high efficiency of pretreatment and hydrolysis. Therefore, the wet storage methods would be desirable for the conversion of corn stover biomass to fermentable sugars based on steam explosion pretreatment and enzymatic hydrolysis.

Metabolic profiling reveals growth related FAME productivity and quality of Chlorella sorokiniana with different inoculum sizes

Inoculum size strongly affects cell growth and lipid accumulation of microalgae, one of the most potential biodiesel feedstock, however, the metabolic mechanism(s) of the lipid biosynthesis upon inoculum size has not been fully explored yet. The effects of inoculum size on cell growth, lipid accumulation, and metabolic changes of a green microalga Chlorella sorokiniana were investigated. In our experimental range of inoculum size, the productivity and the cetane number (CN) of fatty acid methyl esters (FAME) increased with increasing initial cell density, and the inoculum of 1 × 107 cells mL−1 processed much higher productivity (up to 2.02‐fold) and CN (up to 1.19‐fold) of the FAME than the others. A significant correlation between the metabolic profile and quantity and quality of lipid production was revealed by partial least‐squares to latent structures (PLS) analysis, and 15 key metabolites were identified. Most of those metabolites were involved in the photosynthetically fixed carbon metabolism. Furthermore, light intensity as one of the vital limitation factors for the high inoculum size cultivation was evaluated by illumination assay and the results revealed that increasing light intensity could improve the polyunsaturated fatty acids composition and lipid accumulation of C. sorokiniana. The lipid productivity of the culture was improved by 71.21% with the light intensity of 110 µmol m−2 s−1, compared to that under the irradiance of 65 µmol m−2 s−1. Biotechnol. Bioeng. 2012; 109:1651–1662.

“Perfect” designer chromosome V and behavior of a ring derivative

INTRODUCTION The Saccharomyces cerevisiae 2.0 project (Sc2.0) aims to modify the yeast genome with a series of densely spaced designer changes. Both a synthetic yeast chromosome arm (synIXR) and the entirely synthetic chromosome (synIII) function with high fitness in yeast. For designer genome synthesis projects, precise engineering of the physical sequence to match the specified design is important for the systematic evaluation of underlying design principles. Yeast can maintain nuclear chromosomes as rings, occurring by chance at repeated sequences, although the cyclized format is unfavorable in meiosis given the possibility of dicentric chromosome formation from meiotic recombination. Here, we describe the de novo synthesis of synthetic yeast chromosome V (synV) in the “Build-A-Genome China” course, perfectly matching the designer sequence and bearing loxPsym sites, distinguishable watermarks, and all the other features of the synthetic genome. We generated a ring synV derivative with user-specified cyclization coordinates and characterized its performance in mitosis and meiosis. RATIONALE Systematic evaluation of underlying Sc2.0 design principles requires that the final assembled synthetic genome perfectly match the designed sequence. Given the size of yeast chromosomes, synthetic chromosome construction is performed iteratively, and new mutations and unpredictable events may occur during synthesis; even a very small number of unintentional nucleotide changes across the genome could have substantial effects on phenotype. Therefore, precisely matching the physical sequence to the designed sequence is crucial for verification of the design principles in genome synthesis. Ring chromosomes can extend those design principles to provide a model for genomic rearrangement, ring chromosome evolution, and human ring chromosome disorders. RESULTS We chemically synthesized, assembled, and incorporated designer chromosome synV (536,024 base pairs) of S. cerevisiae according to Sc2.0 principles, based on the complete nucleotide sequence of native yeast chromosome V (576,874 base pairs). This work was performed as part of the “Build-A-Genome China” course in Tianjin University. We corrected all mutations found—including duplications, substitutions, and indels—in the initial synV strain by using integrative cotransformation of the precise desired changes and by means of a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)–based method. Altogether, 3331 corrected base pairs were required to match to the designed sequence. We generated a strain that exactly matches all designer sequence changes that displays high fitness under a variety of culture conditions. All corrections were verified with whole-genome sequencing; RNA sequencing revealed only minor changes in gene expression—most notably, decreases in expression of genes relocated near synthetic telomeres as a result of design. We constructed a functional circular synV (ring_synV) derivative in yeast by precisely joining both chromosome ends (telomeres) at specified coordinates. The ring chromosome showed restoration of subtelomeric gene expression levels. The ring_synV strain exhibited fitness comparable with that of the linear synV strain, revealed no change in sporulation frequency, but notably reduced spore viability. In meiosis, heterozygous or homozygous diploid ring_wtV and ring_synV chromosomes behaved similarly, exhibiting substantially higher frequency of the formation of zero-spore tetrads, a type that was not seen in the rod chromosome diploids. Rod synV chromosomes went through meiosis with high spore viability, despite no effort having been made to preserve meiotic competency in the design of synV. CONCLUSION The perfect designer-matched synthetic chromosome V provides strategies to edit sequence variants and correct unpredictable events, such as off-target integration of extra copies of synthetic DNA elsewhere in the genome. We also constructed a ring synthetic chromosome derivative and evaluated its fitness and stability in yeast. Both synV and synVI can be circularized and can power yeast cell growth without affecting fitness when gene content is maintained. These fitness and stability phenotypes of the ring synthetic chromosome in yeast provide a model system with which to probe the mechanism of human ring chromosome disorders. Synthesis, cyclization, and characterization of synV. (A) Synthetic chromosome V (synV, 536,024 base pairs) was designed in silico from native chromosome V (wtV, 576,874 base pairs), with extensive genotype modification designed to be phenotypically neutral. (B) CRISPR/Cas9 strategy for multiplex repair