shuang deng - Academia.edu (original) (raw)
Papers by shuang deng
Biotechnology for biofuels and bioproducts, Mar 29, 2023
Background Fuels and chemicals derived from non-fossil sources are needed to lessen human impacts... more Background Fuels and chemicals derived from non-fossil sources are needed to lessen human impacts on the environment while providing a healthy and growing economy. 3-hydroxypropionic acid (3-HP) is an important chemical building block that can be used for many products. Biosynthesis of 3-HP is possible; however, low production is typically observed in those natural systems. Biosynthetic pathways have been designed to produce 3-HP from a variety of feedstocks in different microorganisms. Results In this study, the 3-HP β-alanine pathway consisting of aspartate decarboxylase, β-alanine-pyruvate aminotransferase, and 3-hydroxypropionate dehydrogenase from selected microorganisms were codon optimized for Aspergillus species and placed under the control of constitutive promoters. The pathway was introduced into Aspergillus pseudoterreus and subsequently into Aspergillus niger, and 3-HP production was assessed in both hosts. A. niger produced higher initial 3-HP yields and fewer co-product contaminants and was selected as a suitable host for further engineering. Proteomic and metabolomic analysis of both Aspergillus species during 3-HP production identified genetic targets for improvement of flux toward 3-HP including pyruvate carboxylase, aspartate aminotransferase, malonate semialdehyde dehydrogenase, succinate semialdehyde dehydrogenase, oxaloacetate hydrolase, and a 3-HP transporter. Overexpression of pyruvate carboxylase improved yield in shake-flasks from 0.09 to 0.12 C-mol 3-HP C-mol −1 glucose in the base strain expressing 12 copies of the β-alanine pathway. Deletion or overexpression of individual target genes in the pyruvate carboxylase overexpression strain improved yield to 0.22 C-mol 3-HP C-mol −1 glucose after deletion of the major malonate semialdehyde dehydrogenase. Further incorporation of additional β-alanine pathway genes and optimization of culture conditions (sugars, temperature, nitrogen, phosphate, trace elements) for 3-HP production from deacetylated and mechanically refined corn stover hydrolysate improved yield to 0.48 C-mol 3-HP C-mol −1 sugars and resulted in a final titer of 36.0 g/L 3-HP.
Metabolic Engineering Communications
The global regulator LaeA controls secondary metabolism in diverse Aspergillus species. Here we e... more The global regulator LaeA controls secondary metabolism in diverse Aspergillus species. Here we explored its role in regulation of itaconic acid production in Aspergillus pseudoterreus. To understand its role in regulating metabolism, we deleted and overexpressed laeA, and assessed the transcriptome, proteome, and secreted metabolome prior to and during initiation of phosphate limitation induced itaconic acid production. We found that secondary metabolite clusters, including the itaconic acid biosynthetic gene cluster, are regulated by laeA and that laeA is required for high yield production of itaconic acid. Overexpression of LaeA improves itaconic acid yield at the expense of biomass by increasing the expression of key biosynthetic pathway enzymes and attenuating the expression of genes involved in phosphate acquisition and scavenging. Increased yield was observed in optimized conditions as well as conditions containing excess nutrients that may be present in inexpensive sugar containing feedstocks such as excess phosphate or complex nutrient sources. This suggests that global regulators of metabolism may be useful targets for engineering metabolic flux that is robust to environmental heterogeneity.
Applied Microbiology and Biotechnology, 2020
The filamentous fungus Aspergillus terreus has been successfully used for industrial production o... more The filamentous fungus Aspergillus terreus has been successfully used for industrial production of itaconic acid (IA) for many years. The IA biosynthesis pathway has recently been characterized at a molecular genetic level as an IA gene cluster by a clonebased transcriptomic approach. The cluster consists of four genes, including genes for cis-aconitic acid decarboxylase (cadA), a predicted transcription factor (tf), a mitochondrial organic acid transporter (mttA) and an MFS (major facilitator superfamily) type transporter (mfsA). In this research, we performed expressed sequence tag (EST) analysis and systematic gene deletions to further investigate the role of those genes during IA biosynthesis in A. pseudoterreus ATCC32359. EST analysis showed a similar expression pattern among those four genes that were distinct from neighboring genes and further confirmed that they belong to the same biosynthesis cluster. Systematic gene deletion analysis demonstrated that tf, cadA, mttA and mfsA genes in the cluster are essential for IA production; deletion of any of them will either completely abolish the IA production or dramatically decrease the amount of IA produced. The tf gene plays a regulatory role in this cluster. Deletion of tf led to decreased expression levels of cadA, mttA and mfsA. More importantly, a significant amount of aconitic acid was detected in the cadA deletion strain but not in the other deletion strains. Therefore, by deleting only one gene, the cadA, we established a novel microbial host for the production of aconitic acid and other value-added chemicals from sugars in lignocellulosic biomass. Keywords Itaconic acid (IA). Aconitic acid (AA). Aspergillus pseudoterreus ATCC32359. cis-aconitic acid decarboxylase (cadA). Transporter
Fungal Genetics and Biology, 2014
Protein glycosylation, an important and complex post-translational modification (PTM), is involve... more Protein glycosylation, an important and complex post-translational modification (PTM), is involved in various biological processes, including the receptor-ligand and cell-cell interaction, and plays a crucial role in many biological functions. However, little is known about the glycan structures of important biological complex samples, and the conventional glycan enrichment strategy (i.e., size-exclusion column [SEC] separation) prior to nuclear magnetic resonance (NMR) detection is time-consuming and tedious. In this study, we developed a glycan enrichment strategy that couples Zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) with dialysis strategies to enrich the glycans from the pronase E digests of RNase B, followed by NMR analysis of the glycoconjugate. Our results suggest that the ZIC-HILIC enrichment coupled with dialysis is a simple, fast, and efficient sample preparation approach. The approach was thus applied to the analysis of a biological complex sample, the pronase E digest of the secreted proteins from the fungus Aspergillus niger. The NMR spectra revealed that the secreted proteins from A. niger contain both N-linked glycans with a high-mannose core similar to the structure of the glycan from RNase B, and O-linked glycans bearing mannose and glucose with 1->3 and 1->6 linkages. In all, our study provides compelling evidence that ZIC-HILIC separation coupled with dialysis is very effective and accessible in preparing glycans for the downstream NMR analysis, which could greatly facilitate the future NMR-based glycoproteomics research.
Journal of Bacteriology, 2008
MtrC and OmcA are cell surface-exposed lipoproteins important for reducing solid metal oxides. De... more MtrC and OmcA are cell surface-exposed lipoproteins important for reducing solid metal oxides. Deletions of type II secretion system (T2SS) genes reduced their extracellular release and their accessibility to the proteinase K treatment, demonstrating the direct involvement of T2SS in translocation of MtrC and OmcA to the bacterial cell surface.
Molecular Microbiology, 2005
The bacterial chromosome is organized into multiple independent domains, each capable of constrai... more The bacterial chromosome is organized into multiple independent domains, each capable of constraining the plectonemic negative supercoil energy introduced by DNA gyrase. Different experimental approaches have estimated the number of domains to be between 40 and 150. The sitespecific resolution systems of closely related transposons Tn3 and γδ are valuable tools for measuring supercoil diffusion and analysing bacterial chromosome dynamics in vivo. Once made, the wild-type resolvase persists in cells for time periods greater than the cell doubling time. To examine chromosome dynamics over shorter time frames that are more closely tuned to processes like inducible transcription, we constructed a set of resolvases with cellular half-lives ranging from less than 5 min to 30 min. Analysing chromosomes on different time scales shows domain structure to be dynamic. Rather than the 150 domains detected with the Tn3 resolvase, wild-type cells measured over a 10 min time span have more than 400 domains per genome equivalent, and some gyrase mutants exceed 1000.
Molecular Microbiology, 2005
During a normal cell cycle, chromosomes are exposed to many biochemical reactions that require sp... more During a normal cell cycle, chromosomes are exposed to many biochemical reactions that require specific types of DNA movement. Separation forces move replicated chromosomes into separate sister cell compartments during cell division, and the contemporaneous acts of DNA replication, RNA transcription and cotranscriptional translation of membrane proteins cause specific regions of DNA to twist, writhe and expand or contract. Recent experiments indicate that a dynamic and stochastic mechanism creates supercoil DNA domains soon after DNA replication. Domain structure is subsequently reorganized by RNA transcription. Examples of transcription-dependent chromosome remodelling are also emerging from eukaryotic cell systems.
Proceedings of the National Academy of Sciences, 2004
Transcription and replication both influence and are influenced by superhelical changes in DNA. E... more Transcription and replication both influence and are influenced by superhelical changes in DNA. Explaining how supercoil movement is channeled in living chromosomes has been a major problem for 30 years. Transcription of membrane-associated proteins leads to localized hypersupercoiling of plasmid DNA, and this behavior indicates the presence of aberrant supercoil diffusion. Using the lambda Red recombination system, we constructed model domains in the Salmonella typhimurium chromosome to analyze supercoiling dynamics of regions encoding membrane proteins. Regulation of Tn 10 -derived tetracycline resistance involves a repressor, TetR, and a membrane-bound export pump, TetA. Strains deficient in TetR activity had 60-fold higher transcription levels (from P A ) than TetR-positive strains. High tetA transcription caused a 10- to 80-fold decrease in the γδ resolution efficiency for the domain that includes the Tet module. Replacing tetA with genes encoding cytosolic proteins LacZ and Ka...
Biotechnology for biofuels and bioproducts, Mar 29, 2023
Background Fuels and chemicals derived from non-fossil sources are needed to lessen human impacts... more Background Fuels and chemicals derived from non-fossil sources are needed to lessen human impacts on the environment while providing a healthy and growing economy. 3-hydroxypropionic acid (3-HP) is an important chemical building block that can be used for many products. Biosynthesis of 3-HP is possible; however, low production is typically observed in those natural systems. Biosynthetic pathways have been designed to produce 3-HP from a variety of feedstocks in different microorganisms. Results In this study, the 3-HP β-alanine pathway consisting of aspartate decarboxylase, β-alanine-pyruvate aminotransferase, and 3-hydroxypropionate dehydrogenase from selected microorganisms were codon optimized for Aspergillus species and placed under the control of constitutive promoters. The pathway was introduced into Aspergillus pseudoterreus and subsequently into Aspergillus niger, and 3-HP production was assessed in both hosts. A. niger produced higher initial 3-HP yields and fewer co-product contaminants and was selected as a suitable host for further engineering. Proteomic and metabolomic analysis of both Aspergillus species during 3-HP production identified genetic targets for improvement of flux toward 3-HP including pyruvate carboxylase, aspartate aminotransferase, malonate semialdehyde dehydrogenase, succinate semialdehyde dehydrogenase, oxaloacetate hydrolase, and a 3-HP transporter. Overexpression of pyruvate carboxylase improved yield in shake-flasks from 0.09 to 0.12 C-mol 3-HP C-mol −1 glucose in the base strain expressing 12 copies of the β-alanine pathway. Deletion or overexpression of individual target genes in the pyruvate carboxylase overexpression strain improved yield to 0.22 C-mol 3-HP C-mol −1 glucose after deletion of the major malonate semialdehyde dehydrogenase. Further incorporation of additional β-alanine pathway genes and optimization of culture conditions (sugars, temperature, nitrogen, phosphate, trace elements) for 3-HP production from deacetylated and mechanically refined corn stover hydrolysate improved yield to 0.48 C-mol 3-HP C-mol −1 sugars and resulted in a final titer of 36.0 g/L 3-HP.
Metabolic Engineering Communications
The global regulator LaeA controls secondary metabolism in diverse Aspergillus species. Here we e... more The global regulator LaeA controls secondary metabolism in diverse Aspergillus species. Here we explored its role in regulation of itaconic acid production in Aspergillus pseudoterreus. To understand its role in regulating metabolism, we deleted and overexpressed laeA, and assessed the transcriptome, proteome, and secreted metabolome prior to and during initiation of phosphate limitation induced itaconic acid production. We found that secondary metabolite clusters, including the itaconic acid biosynthetic gene cluster, are regulated by laeA and that laeA is required for high yield production of itaconic acid. Overexpression of LaeA improves itaconic acid yield at the expense of biomass by increasing the expression of key biosynthetic pathway enzymes and attenuating the expression of genes involved in phosphate acquisition and scavenging. Increased yield was observed in optimized conditions as well as conditions containing excess nutrients that may be present in inexpensive sugar containing feedstocks such as excess phosphate or complex nutrient sources. This suggests that global regulators of metabolism may be useful targets for engineering metabolic flux that is robust to environmental heterogeneity.
Applied Microbiology and Biotechnology, 2020
The filamentous fungus Aspergillus terreus has been successfully used for industrial production o... more The filamentous fungus Aspergillus terreus has been successfully used for industrial production of itaconic acid (IA) for many years. The IA biosynthesis pathway has recently been characterized at a molecular genetic level as an IA gene cluster by a clonebased transcriptomic approach. The cluster consists of four genes, including genes for cis-aconitic acid decarboxylase (cadA), a predicted transcription factor (tf), a mitochondrial organic acid transporter (mttA) and an MFS (major facilitator superfamily) type transporter (mfsA). In this research, we performed expressed sequence tag (EST) analysis and systematic gene deletions to further investigate the role of those genes during IA biosynthesis in A. pseudoterreus ATCC32359. EST analysis showed a similar expression pattern among those four genes that were distinct from neighboring genes and further confirmed that they belong to the same biosynthesis cluster. Systematic gene deletion analysis demonstrated that tf, cadA, mttA and mfsA genes in the cluster are essential for IA production; deletion of any of them will either completely abolish the IA production or dramatically decrease the amount of IA produced. The tf gene plays a regulatory role in this cluster. Deletion of tf led to decreased expression levels of cadA, mttA and mfsA. More importantly, a significant amount of aconitic acid was detected in the cadA deletion strain but not in the other deletion strains. Therefore, by deleting only one gene, the cadA, we established a novel microbial host for the production of aconitic acid and other value-added chemicals from sugars in lignocellulosic biomass. Keywords Itaconic acid (IA). Aconitic acid (AA). Aspergillus pseudoterreus ATCC32359. cis-aconitic acid decarboxylase (cadA). Transporter
Fungal Genetics and Biology, 2014
Protein glycosylation, an important and complex post-translational modification (PTM), is involve... more Protein glycosylation, an important and complex post-translational modification (PTM), is involved in various biological processes, including the receptor-ligand and cell-cell interaction, and plays a crucial role in many biological functions. However, little is known about the glycan structures of important biological complex samples, and the conventional glycan enrichment strategy (i.e., size-exclusion column [SEC] separation) prior to nuclear magnetic resonance (NMR) detection is time-consuming and tedious. In this study, we developed a glycan enrichment strategy that couples Zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) with dialysis strategies to enrich the glycans from the pronase E digests of RNase B, followed by NMR analysis of the glycoconjugate. Our results suggest that the ZIC-HILIC enrichment coupled with dialysis is a simple, fast, and efficient sample preparation approach. The approach was thus applied to the analysis of a biological complex sample, the pronase E digest of the secreted proteins from the fungus Aspergillus niger. The NMR spectra revealed that the secreted proteins from A. niger contain both N-linked glycans with a high-mannose core similar to the structure of the glycan from RNase B, and O-linked glycans bearing mannose and glucose with 1->3 and 1->6 linkages. In all, our study provides compelling evidence that ZIC-HILIC separation coupled with dialysis is very effective and accessible in preparing glycans for the downstream NMR analysis, which could greatly facilitate the future NMR-based glycoproteomics research.
Journal of Bacteriology, 2008
MtrC and OmcA are cell surface-exposed lipoproteins important for reducing solid metal oxides. De... more MtrC and OmcA are cell surface-exposed lipoproteins important for reducing solid metal oxides. Deletions of type II secretion system (T2SS) genes reduced their extracellular release and their accessibility to the proteinase K treatment, demonstrating the direct involvement of T2SS in translocation of MtrC and OmcA to the bacterial cell surface.
Molecular Microbiology, 2005
The bacterial chromosome is organized into multiple independent domains, each capable of constrai... more The bacterial chromosome is organized into multiple independent domains, each capable of constraining the plectonemic negative supercoil energy introduced by DNA gyrase. Different experimental approaches have estimated the number of domains to be between 40 and 150. The sitespecific resolution systems of closely related transposons Tn3 and γδ are valuable tools for measuring supercoil diffusion and analysing bacterial chromosome dynamics in vivo. Once made, the wild-type resolvase persists in cells for time periods greater than the cell doubling time. To examine chromosome dynamics over shorter time frames that are more closely tuned to processes like inducible transcription, we constructed a set of resolvases with cellular half-lives ranging from less than 5 min to 30 min. Analysing chromosomes on different time scales shows domain structure to be dynamic. Rather than the 150 domains detected with the Tn3 resolvase, wild-type cells measured over a 10 min time span have more than 400 domains per genome equivalent, and some gyrase mutants exceed 1000.
Molecular Microbiology, 2005
During a normal cell cycle, chromosomes are exposed to many biochemical reactions that require sp... more During a normal cell cycle, chromosomes are exposed to many biochemical reactions that require specific types of DNA movement. Separation forces move replicated chromosomes into separate sister cell compartments during cell division, and the contemporaneous acts of DNA replication, RNA transcription and cotranscriptional translation of membrane proteins cause specific regions of DNA to twist, writhe and expand or contract. Recent experiments indicate that a dynamic and stochastic mechanism creates supercoil DNA domains soon after DNA replication. Domain structure is subsequently reorganized by RNA transcription. Examples of transcription-dependent chromosome remodelling are also emerging from eukaryotic cell systems.
Proceedings of the National Academy of Sciences, 2004
Transcription and replication both influence and are influenced by superhelical changes in DNA. E... more Transcription and replication both influence and are influenced by superhelical changes in DNA. Explaining how supercoil movement is channeled in living chromosomes has been a major problem for 30 years. Transcription of membrane-associated proteins leads to localized hypersupercoiling of plasmid DNA, and this behavior indicates the presence of aberrant supercoil diffusion. Using the lambda Red recombination system, we constructed model domains in the Salmonella typhimurium chromosome to analyze supercoiling dynamics of regions encoding membrane proteins. Regulation of Tn 10 -derived tetracycline resistance involves a repressor, TetR, and a membrane-bound export pump, TetA. Strains deficient in TetR activity had 60-fold higher transcription levels (from P A ) than TetR-positive strains. High tetA transcription caused a 10- to 80-fold decrease in the γδ resolution efficiency for the domain that includes the Tet module. Replacing tetA with genes encoding cytosolic proteins LacZ and Ka...