Bing-Zhi Li

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.

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.

“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

Bug mapping and fitness testing of chemically synthesized chromosome X

INTRODUCTION Design and construction of an extensively modified yeast genome is a direct means to interrogate the integrity, comprehensiveness, and accuracy of the knowledge amassed by the yeast community to date. The international synthetic yeast genome project (Sc2.0) aims to build an entirely designer, synthetic Saccharomyces cerevisiae genome. The synthetic genome is designed to increase genome stability and genetic flexibility while maintaining cell fitness near that of the wild type. A major challenge for a genome synthesis lies in identifying and eliminating fitness-reducing sequence variants referred to as “bugs.” RATIONALE Debugging is imperative for successfully building a fit strain encoding a synthetic genome. However, it is time-consuming and laborious to replace wild-type genes and measure strain fitness systematically. The Sc2.0 PCRTag system, which specifies recoded sequences within open reading frames (ORFs), is designed to distinguish synthetic from wild-type DNA in a simple polymerase chain reaction (PCR) assay. This system provides an opportunity to efficiently map bugs to the related genes by using a pooling strategy and subsequently correct them. Further, as we identify bugs in designer sequences, we will identify gaps in our knowledge and gain a deeper understanding of genome biology, allowing refinement of future design strategies. RESULTS We chemically synthesized yeast chromosome X, synX, designed to be 707,459 base pairs. A high-throughput mapping strategy called pooled PCRTag mapping (PoPM) was developed to identify unexpected bugs during chromosome assembly. With this method, the genotypes of pools of colonies with normal or defective fitness are assessed by PCRTag analysis. The PoPM method exploits the patchwork structure of synthetic and wild-type sequences observed in the majority of putative synthetic DNA integrants or meiotic progeny derived from synthetic/wild-type strain backcross. PCRTag analysis with both synthetic and wild-type specific primers, carried out with genomic DNA extracted from the two pools of clones (normal fitness versus a specific growth defect), can be used to identify regions of synthetic DNA missing from the normal fitness pool and, analogously, sections of wild-type DNA absent from the specific growth-defect pool. In this way, the defect can be efficiently mapped to a very small overlapping region, and subsequent systematic analysis of designed changes in that region can be used to identify the bug. Several bugs were identified and corrected, including a growth defect mapping to a specific synonymously recoded PCRTag sequence in the essential FIP1 ORF and the effect of introducing a loxPsym site that unexpectedly altered the the promoter function of a nearby gene, ATP2. In addition, meiotic crossover was employed to repair the massive duplications and rearrangements in the synthetic chromosome. The debugged synX strain exhibited high fitness under a variety of conditions tested and in competitive growth with the wild-type strain. CONCLUSION Synthetic yeast chromosome X was chemically synthesized from scratch, a rigorous, incremental step toward complete synthesis of the whole yeast genome. Thousands of designer modifications in synX revealed extensive flexibility of the yeast genome. We developed an efficient mapping method, PoPM, to identify bugs during genome synthesis, generalizable to any watermarked synthetic chromosome, and several details of yeast biology were uncovered by debugging. Considering the numerous gene-associated PCRTags available in the synthetic chromosomes, PoPM may represent a powerful tool to map interesting phenotypes of mutated synthetic strains or even mutated wild-type strains to the relevant genes. It may also be useful to study yeast genetic interactions when an unexpected phenotype is generated by alterations in two or more genes, substantially expanding understanding of yeast genomic and cellular functions. The PoPM method is also likely to be useful for mapping phenotype(s) resulting from the genome SCRaMbLE system. Characterization of synX and debugging by pooled PCRTag mapping. (Top) Design overview of synthetic chromosome X. (Bottom) Flow diagram of pooled PCRTag mapping (PoPM). Debugging a genome sequence is imperative for successfully building a synthetic genome. As part of the effort to build a designer eukaryotic genome, yeast synthetic chromosome X (synX), designed as 707,459 base pairs, was synthesized chemically. SynX exhibited good fitness under a wide variety of conditions. A highly efficient mapping strategy called pooled PCRTag mapping (PoPM), which can be generalized to any watermarked synthetic chromosome, was developed to identify genetic alterations that affect cell fitness (“bugs”). A series of bugs were corrected that included a large region bearing complex amplifications, a growth defect mapping to a recoded sequence in FIP1, and a loxPsym site affecting promoter function of ATP2. PoPM is a powerful tool for synthetic yeast genome debugging and an efficient strategy for phenotype-genotype mapping.

High temperature aqueous ammonia pretreatment and post-washing enhance the high solids enzymatic hydrolysis of corn stover

Aqueous ammonia pretreatment was optimized and the limiting factors in high solids enzymatic hydrolysis were assessed. The recommended pretreatment condition to achieve high enzymatic yield was: 180 °C, 20% (w/w) ammonia, 30 min, and 20% solids content. FT-IR and GC-MS results indicated that most of the lignin was degraded to soluble fragments after pretreatment. The pretreated solids after post-washing showed higher enzymatic digestibility at high solids loading than that without washing. The washed solids required lower cellulase and xylanase dosage than unwashed solids to achieve high sugar yield. Enzymatic conversions were declined with the increased solids loading of pretreated solids, pretreated-washed solids, and filter papers. The results indicated that solids loading in enzymatic hydrolysis was an important factor affecting sugar yield. The increasing concentration of glucose and ligno-phenolics mainly inhibited the enzymatic hydrolysis of aqueous ammonia pretreated corn stover.

Comparative metabolic profiling revealed limitations in xylose-fermenting yeast during co-fermentation of glucose and xylose in the presence of inhibitors.

During lignocellulosic ethanol fermentation, yeasts are exposed to various lignocellulose‐derived inhibitors, which disrupt the efficiency of hexose and pentose co‐fermentation. To understand the metabolic response of fermentation microbes to these inhibitors, a comparative metabolomic investigation was performed on a xylose‐fermenting Saccharomyces cerevisiae 424A (LNH‐ST) and its parental strain 4124 with and without three typical inhibitors (furfural, acetic acid, and phenol). Three traits were uncovered according to fermentation results. First, the growth of strain 424A (LNH‐ST) was more sensitive to inhibitors than strain 4124. Through metabolomic analysis, the variance of trehalose, cadaverine, glutamate and γ‐aminobutyric acid (GABA) suggested that strain 424A (LNH‐ST) had a lower capability to buffer redox changes caused by inhibitors. Second, lower ethanol yield in glucose and xylose co‐fermentation than glucose fermentation was observed in strain 424A (LNH‐ST), which was considered to be correlated with the generation of xylitol, as well as the reduced levels of lysine, glutamate, glycine and isoleucine in strain 424A (LNH‐ST). Accumulation of glycerol, galactinol and mannitol was also observed in strain 424A (LNH‐ST) during xylose fermentation. Third, xylose utilization of strain 424A (LNH‐ST) was more significantly disturbed by inhibitors than glucose utilization. Through the analysis of fermentation and metabolomic results, it was suggested that xylose catabolism and energy supply, rather than xylose uptake, were the limiting steps in xylose utilization in the presence of inhibitors. Biotechnol. Bioeng. 2014;111: 152–164.

Simultaneous saccharification and co-fermentation of aqueous ammonia pretreated corn stover with an engineered Saccharomyces cerevisiae syBE005.

Co-fermentation of glucose and xylose from lignocelluloses is an efficient approach to increasing ethanol production. Simultaneous saccharification and co-fermentation (SSCF) of corn stover pretreated with aqueous ammonia was performed using engineered yeast with xylose utilization pathway. Thus far, the effect of the several key factors on SSCF was investigated, including temperature, inoculation size, pre-hydrolysis and pH. Ethanol concentration was achieved to 36.5 g/L during SSCF process with 6% glucan loading. The addition of Tween 20 reduced enzyme loading, i.e., from 15 to 7.5 FPU/gglucan with the same final ethanol concentration. The ethanol concentration was achieved to 70.1g/L at 12% glucan loading. Yeast feeding, combined with substrate and enzyme feeding, was proved to be an efficient approach for SSCF with high solid loading.

Transcriptome analysis of differential responses of diploid and haploid yeast to ethanol stress

Diploid and haploid strains often exhibit different tolerances to variety of stresses, which facilitates the comparative studies to understand the mechanism of the tolerances to stresses. Gene expression profiles of a diploid strain and two homologous haploid strains in the presence of ethanol at different concentrations were investigated by microarray and the data were validated with quantitative real-time PCR. In all the three strains, upregulated genes in the presence of ethanol were involved in regulation of lipid synthesis and ribosomal biogenesis, while downregulated genes in the presence of ethanol were involved in synthesis of several amino acids, metabolism of biotin and folic acid. In addition, differentially expressed genes in different ploidy strains, which showed less responsive to ethanol, were involved in pheromone response or mating, energy, stress response, metal transport and cell wall. Furthermore, our data also revealed significant differences between transcriptome shift after ethanol acclimation and transcriptome response to short-term ethanol shock. Taken together, these results provide molecular insights into tolerance difference of haploid and diploid yeast strains and molecular information to further understand ethanol tolerance mechanism in yeast.