Browsing by Author "Sheng, Jiayuan"

Now showing 1 - 11 of 11
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    13C-Metabolic Flux Analysis: An Accurate Approach to Demystify Microbial Metabolism for Biochemical Production
    Guo, Weihua; Sheng, Jiayuan; Feng, Xueyang (MDPI, 2015-12-25)
    Metabolic engineering of various industrial microorganisms to produce chemicals, fuels, and drugs has raised interest since it is environmentally friendly, sustainable, and independent of nonrenewable resources. However, microbial metabolism is so complex that only a few metabolic engineering efforts have been able to achieve a satisfactory yield, titer or productivity of the target chemicals for industrial commercialization. In order to overcome this challenge, 13C Metabolic Flux Analysis (13C-MFA) has been continuously developed and widely applied to rigorously investigate cell metabolism and quantify the carbon flux distribution in central metabolic pathways. In the past decade, many 13C-MFA studies have been performed in academic labs and biotechnology industries to pinpoint key issues related to microbe-based chemical production. Insightful information about the metabolic rewiring has been provided to guide the development of the appropriate metabolic engineering strategies for improving the biochemical production. In this review, we will introduce the basics of 13C-MFA and illustrate how 13C-MFA has been applied via integration with metabolic engineering to identify and tackle the rate-limiting steps in biochemical production for various host microorganisms
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    Cell-free protein synthesis of norovirus virus-like particles
    Sheng, Jiayuan; Lei, Shaohua; Yuan, Lijuan; Feng, Xueyang (Royal Society of Chemistry, 2017-05-25)
    Norovirus vaccine development largely depends on recombinant virus-like-particles (VLPs). Norovirus VLPs have been produced in several cell-based expression systems with long production times. Here we report, for the first time, that norovirus VLPs can be expressed and assembled by using a cell-free protein expression system within four hours.
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    Metabolic Engineering for Production of Small Molecule Drugs: Challenges and Solutions
    Huttanus, Herbert M.; Sheng, Jiayuan; Feng, Xueyang (MDPI, 2016-02-19)
    Production of small molecule drugs in a recombinant host is becoming an increasingly popular alternative to chemical synthesis or production in natural hosts such as plants due to the ease of growing microorganisms with higher titers and less cost. While there are a wide variety of well-developed cloning techniques to produce small molecule drugs in a heterologous host, there are still many challenges towards efficient production. Therefore, this paper reviews some of these recently developed tools for metabolic engineering and categorizes them according to a chronological series of steps for a generalized method of drug production in a heterologous host, including 1) pathway discovery from a natural host, 2) pathway assembly in the recombinant host, and 3) pathway optimization to increase titers and yield.
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    Metabolic engineering of Saccharomyces cerevisiae to produce 1-hexadecanol from xylose
    Guo, Weihua; Sheng, Jiayuan; Zhao, Huimin; Feng, Xueyang (2016-02-01)
    Background An advantageous but challenging approach to overcome the limited supply of petroleum and relieve the greenhouse effect is to produce bulk chemicals from renewable materials. Fatty alcohols, with a billion-dollar global market, are important raw chemicals for detergents, emulsifiers, lubricants, and cosmetics production. Microbial production of fatty alcohols has been successfully achieved in several industrial microorganisms. However, most of the achievements were using glucose, an edible sugar, as the carbon source. To produce fatty alcohols in a renewable manner, non-edible sugars such as xylose will be a more appropriate feedstock. Results In this study, we aim to engineer a Saccharomyces cerevisiae strain that can efficiently convert xylose to fatty alcohols. To this end, we first introduced the fungal xylose utilization pathway consisting of xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulose kinase (XKS) into a fatty alcohol-producing S. cerevisiae strain (XF3) that was developed in our previous studies to achieve 1-hexadecanol production from xylose at 0.4 g/L. We next applied promoter engineering on the xylose utilization pathway to optimize the expression levels of XR, XDH, and XKS, and increased the 1-hexadecanol titer by 171 %. To further improve the xylose-based fatty alcohol production, two optimized S. cerevisiae strains from promoter engineering were evolved with the xylose as the sole carbon source. We found that the cell growth rate was improved at the expense of decreased fatty alcohol production, which indicated 1-hexadecanol was mainly produced as a non-growth associated product. Finally, through fed-batch fermentation, we successfully achieved 1-hexadecanol production at over 1.2 g/L using xylose as the sole carbon source, which represents the highest titer of xylose-based 1-hexadecanol reported in microbes to date. Conclusions A fatty alcohol-producing S. cerevisiae strain was engineered in this study to produce 1-hexadecanol from xylose. Although the xylose pathway we developed in this study could be further improved, this proof-of-concept study, for the first time to our best knowledge, demonstrated that the xylose-based fatty alcohol could be produced in S. cerevisiae with potential applications in developing consolidated bioprocessing for producing other fatty acid-derived chemicals.
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    Metabolic Engineering of Yeast to Produce Fatty Acid-derived Biofuels: Bottlenecks and Solutions
    Sheng, Jiayuan; Feng, Xueyang (Frontiers, 2015-06-08)
    Fatty acid-derived biofuels can be a better solution than bioethanol to replace petroleum fuel, since they have similar energy content and combustion properties as current transportation fuels. The environmentally friendly microbial fermentation process has been used to synthesize advanced biofuels from renewable feedstock. Due to their robustness as well as the high tolerance to fermentation inhibitors and phage contamination, yeast strains such as Saccharomyces cerevisiae and Yarrowia lipolytica have attracted tremendous attention in recent studies regarding the production of fatty acid-derived biofuels, including fatty acids, fatty acid ethyl esters, fatty alcohols, and fatty alkanes. However, the native yeast strains cannot produce fatty acids and fatty acid-derived biofuels in large quantities. To this end, we have summarized recent publications in this review on metabolic engineering of yeast strains to improve the production of fatty acid-derived biofuels, identified the bottlenecks that limit the productivity of biofuels, and categorized the appropriate approaches to overcome these obstacles.
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    Mini-review: In vitro Metabolic Engineering for Biomanufacturing of High-value Products
    Guo, Weihua; Sheng, Jiayuan; Feng, Xueyang (Elsevier, 2017-01-19)
    With the breakthroughs in biomolecular engineering and synthetic biology, many valuable biologically active compound and commodity chemicals have been successfully manufactured using cell-based approaches in the past decade. However, because of the high complexity of cell metabolism, the identification and optimization of rate-limiting metabolic pathways for improving the product yield is often difficult, which represents a significant and unavoidable barrier of traditional in vivo metabolic engineering. Recently, some in vitro engineering approaches were proposed as alternative strategies to solve this problem. In brief, by reconstituting a biosynthetic pathway in a cell-free environment with the supplement of cofactors and substrates, the performance of each biosynthetic pathway could be evaluated and optimized systematically. Several value-added products, including chemicals, nutraceuticals, and drug precursors, have been biosynthesized as proof-of-concept demonstrations of in vitro metabolic engineering. This mini-review summarizes the recent progresses on the emerging topic of in vitro metabolic engineering and comments on the potential application of cell-free technology to speed up the “design-build-test” cycles of biomanufacturing.
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    Molecular basis of 5-hydroxytryptophan synthesis in Saccharomyces cerevisiae
    Zhang, Jiantao; Wu, Chaochen; Sheng, Jiayuan; Feng, Xueyang (Royal Society of Chemistry, 2016-03-18)
    We report for the first time that 5-hydroxytryptophan can be synthesized in Saccharomyces cerevisiae by heterologously expressing prokaryotic phenylalanine 4-hydroxylase or eukaryotic tryptophan 3/5-hydroxylase, together with enhanced synthesis of MH4 or BH4 cofactors. The innate DFR1 gene in the folate synthesis pathway was found to play pivotal roles in 5-hydroxytryptophan synthesis.
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    On-chip manufacturing of synthetic proteins for point-of-care therapeutics
    Murphy, Travis W.; Sheng, Jiayuan; Naler, Lynette B.; Feng, Xueyang; Lu, Chang (Nature, 2019)
    Therapeutic proteins have recently received increasing attention because of their clinical potential. Currently, most therapeutic proteins are produced on a large scale using various cell culture systems. However, storing and transporting these therapeutic proteins at low temperatures makes their distribution expensive and problematic, especially for applications in remote locations. To this end, an emerging solution is to use point-of-care technologies that enable immediate and accessible protein production at or near the patient’s bedside. Here we present the development of “Therapeutics-On-a-Chip (TOC)”, an integrated microfluidic platform that enables point-of-care synthesis and purification of therapeutic proteins. We used fresh and lyophilized materials for cell-free synthesis of therapeutic proteins on microfluidic chips and applied immunoprecipitation for highly efficient, on-chip protein purification. We first demonstrated this approach by expressing and purifying a reporter protein, green fluorescent protein. Next, we used TOC to produce cecropin B, an antimicrobial peptide that is widely used to control biofilmassociated diseases. We successfully synthesized and purified cecropin B at 63 ng/μl within 6 h with a 92% purity, followed by confirming its antimicrobial functionality using a growth inhibition assay. Our TOC technology provides a new platform for point-of-care production of therapeutic proteins at a clinically relevant quantity.
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    Pathway Compartmentalization in Peroxisome of Saccharomyces cerevisiae to Produce Versatile Medium Chain Fatty Alcohols
    Stevens, Joseph; Sheng, Jiayuan; Feng, Xueyang (Nature, 2016-05-27)
    Fatty alcohols are value-added chemicals and important components of a variety of industries, which have a >3 billion-dollar global market annually. Long chain fatty alcohols (>C12) are mainly used in surfactants, lubricants, detergents, pharmaceuticals and cosmetics while medium chain fatty alcohols (C6–C12) could be used as diesel-like biofuels. Microbial production of fatty alcohols from renewable feedstock stands as a promising strategy to enable sustainable supply of fatty alcohols. In this study, we report, for the first time, that medium chain fatty alcohols could be produced in yeast via targeted expression of a fatty acyl-CoA reductase (TaFAR) in the peroxisome of Saccharomyces cerevisiae. By tagging TaFAR enzyme with peroxisomal targeting signal peptides, the TaFAR could be compartmentalized into the matrix of the peroxisome to hijack the medium chain fatty acyl-CoA generated from the beta-oxidation pathway and convert them to versatile medium chain fatty alcohols (C10 & C12). The overexpression of genes encoding PEX7 and acetyl-CoA carboxylase further improved fatty alcohol production by 1.4-fold. After medium optimization in fed-batch fermentation using glucose as the sole carbon source, fatty alcohols were produced at 1.3 g/L, including 6.9% 1-decanol, 27.5% 1-dodecanol, 2.9% 1-tetradecanol and 62.7% 1-hexadecanol. This work revealed that peroxisome could be engineered as a compartmentalized organelle for producing fatty acid-derived chemicals in S. cerevisiae.
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    Systematic Optimization of Protein Secretory Pathways in Saccharomyces cerevisiae to Increase Expression of Hepatitis B Small Antigen
    Sheng, Jiayuan; Flick, Hunter; Feng, Xueyang (Frontiers, 2017-05-16)
    Hepatitis B is a major disease that chronically infects millions of people in the world, especially in developing countries. Currently, one of the effective vaccines to prevent Hepatitis B is the Hepatitis B Small Antigen (HBsAg), which is mainly produced by the recombinant yeast Saccharomyces cerevisiae. In order to bring down the price, which is still too high for people in developing countries to afford, it is important to understand key cellular processes that limit protein expression. In this study, we took advantage of yeast knockout collection (YKO) and screened 194 S. cerevisiae strains with single gene knocked out in four major steps of the protein secretory pathway, i.e., endoplasmic-reticulum (ER)-associated protein degradation, protein folding, unfolded protein response (UPR), and translocation and exocytosis. The screening showed that the single deletion of YPT32, SBH1, and HSP42 led to the most significant increase of HBsAg expression over the wild type while the deletion of IRE1 led to a profound decrease of HBsAg expression. The synergistic effects of gene knockout and gene overexpression were next tested. We found that simultaneously deleting YPT32 and overexpressing IRE1 led to a 2.12-fold increase in HBsAg expression over the wild type strain. The results of this study revealed novel genetic targets of protein secretory pathways that could potentially improve the manufacturing of broad scope vaccines in a cost-effective way using recombinant S. cerevisiae.
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    Transcriptional profiling reveals molecular basis and novel genetic targets for improved resistance to multiple fermentation inhibitors in Saccharomyces cerevisiae
    Chen, Yingying; Sheng, Jiayuan; Jiang, Tao; Stevens, Joseph; Feng, Xueyang; Wei, Na (2016-01-13)
    Background Lignocellulosic biomass is a promising source of renewable biofuels. However, pretreatment of lignocellulosic biomass generates fermentation inhibitors that adversely affect the growth of industrial microorganisms such as Saccharomyces cerevisiae and prevent economic production of lignocellulosic biofuels. A critical challenge on developing S. cerevisiae with improved inhibitor resistance lies in incomplete understanding of molecular basis for inhibitor stress response and limited information on effective genetic targets for increasing yeast resistance to mixed fermentation inhibitors. In this study, we applied comparative transcriptomic analysis to determine the molecular basis for acetic acid and/or furfural resistance in S. cerevisiae. Results We recently developed a yeast strain YC1 with superior resistance to acetic acid, furfural, and their mixture through inverse metabolic engineering. In this study, we first determined transcriptional changes through RNA sequencing in YC1 versus the wild-type strain S-C1 under three different inhibitor conditions, including acetic acid alone, furfural alone, and mixture of acetic acid and furfural. The genes associated with stress responses of S. cerevisiae to single and mixed inhibitors were revealed. Specifically, we identified 184 consensus genes that were differentially regulated in response to the distinct inhibitor resistance between YC1 and S-C1. Bioinformatic analysis next revealed key transcription factors (TFs) that regulate these consensus genes. The top TFs identified, Sfp1p and Ace2p, were experimentally tested as overexpression targets for strain optimization. Overexpression of the SFP1 gene improved specific ethanol productivity by nearly four times, while overexpression of the ACE2 gene enhanced the rate by three times in the presence of acetic acid and furfural. Overexpression of SFP1 gene in the resistant strain YC1 further resulted in 42 % increase in ethanol productivity in the presence of acetic acid and furfural, suggesting the effect of Sfp1p in optimizing the yeast strain for improved tolerance to mixed fermentation inhibitor. Conclusions Transcriptional regulation underlying yeast resistance to acetic acid and furfural was determined. Two transcription factors, Sfp1p and Ace2p, were uncovered for the first time for their functions in improving yeast resistance to mixed fermentation inhibitors. The study demonstrated an omics-guided metabolic engineering framework, which could be developed as a promising strategy to improve complex microbial phenotypes.