scholarly journals A synthetic Calvin cycle enables autotrophic growth in yeast

2019 ◽  
Author(s):  
Thomas Gassler ◽  
Michael Sauer ◽  
Brigitte Gasser ◽  
Diethard Mattanovich ◽  
Matthias G. Steiger
Keyword(s):  
Metabolic Engineering ◽  
Carbon Source ◽  
Sole Carbon Source ◽  
Heterologous Protein ◽  
Calvin Cycle ◽  
Methylotrophic Yeast ◽  
Genes Encoding ◽  
Endogenous Genes ◽  
Multiple Cell ◽  
New Yeast

AbstractThe methylotrophic yeast Pichia pastoris is frequently used for heterologous protein production and it assimilates methanol efficiently via the xylulose-5-phosphate pathway. This pathway is entirely localized in the peroxisomes and has striking similarities to the Calvin-Benson-Bassham (CBB) cycle, which is used by a plethora of organisms like plants to assimilate CO2 and is likewise compartmentalized in chloroplasts. By metabolic engineering the methanol assimilation pathway of P. pastoris was re-wired to a CO2 fixation pathway resembling the CBB cycle. This new yeast strain efficiently assimilates CO2 into biomass and utilizes it as its sole carbon source, which changes the lifestyle from heterotrophic to autotrophic.In total eight genes, including genes encoding for RuBisCO and phosphoribulokinase, were integrated into the genome of P. pastoris, while three endogenous genes were deleted to block methanol assimilation. The enzymes necessary for the synthetic CBB cycle were targeted to the peroxisome. Methanol oxidation, which yields NADH, is employed for energy generation defining the lifestyle as chemoorganoautotrophic. This work demonstrates that the lifestyle of an organism can be changed from chemoorganoheterotrophic to chemoorganoautotrophic by metabolic engineering. The resulting strain can grow exponentially and perform multiple cell doublings on CO2 as sole carbon source with a µmax of 0.008 h−1.Graphical Abstract

scholarly journals Host engineering for improved valerolactam production in Pseudomonas putida

2019 ◽  
Author(s):  
Mitchell G. Thompson ◽  
Luis E. Valencia ◽  
Jacquelyn M. Blake-Hedges ◽  
Pablo Cruz-Morales ◽  
Alexandria E. Velasquez ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Carbon Source ◽  
Pseudomonas Putida ◽  
Aromatic Compounds ◽  
Sole Carbon Source ◽  
Carbon Sources ◽  
Shotgun Proteomics ◽  
Rich Media ◽  
Amide Hydrolase

ABSTRACTPseudomonas putida is a promising bacterial chassis for metabolic engineering given its ability to metabolize a wide array of carbon sources, especially aromatic compounds derived from lignin. However, this omnivorous metabolism can also be a hindrance when it can naturally metabolize products produced from engineered pathways. Herein we show that P. putida is able to use valerolactam as a sole carbon source, as well as degrade caprolactam. Lactams represent important nylon precursors, and are produced in quantities exceeding one million tons per year[1]. To better understand this metabolism we use a combination of Random Barcode Transposon Sequencing (RB-TnSeq) and shotgun proteomics to identify the oplBA locus as the likely responsible amide hydrolase that initiates valerolactam catabolism. Deletion of the oplBA genes prevented P. putida from growing on valerolactam, prevented the degradation of valerolactam in rich media, and dramatically reduced caprolactam degradation under the same conditions. Deletion of oplBA, as well as pathways that compete for precursors L-lysine or 5-aminovalerate, increased the titer of valerolactam from undetectable after 48 hours of production to ~90 mg/L. This work may serve as a template to rapidly eliminate undesirable metabolism in non-model hosts in future metabolic engineering efforts.

scholarly journals Hidden resources in the Escherichia coli genome restore PLP synthesis and robust growth after deletion of the essential gene pdxB

2019 ◽  
Vol 116 (48) ◽  
pp. 24164-24173 ◽  
Author(s):  
Juhan Kim ◽  
Jake J. Flood ◽  
Michael R. Kristofich ◽  
Cyrus Gidfar ◽  
Andrew B. Morgenthaler ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Sole Carbon Source ◽  
Essential Gene ◽  
Alternative Pathway ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Phosphoglycerate Dehydrogenase ◽  
Functional Pathway

PdxB (erythronate 4-phosphate dehydrogenase) is expected to be required for synthesis of the essential cofactor pyridoxal 5′-phosphate (PLP) in Escherichia coli. Surprisingly, incubation of the ∆pdxB strain in medium containing glucose as a sole carbon source for 10 d resulted in visible turbidity, suggesting that PLP is being produced by some alternative pathway. Continued evolution of parallel lineages for 110 to 150 generations produced several strains that grow robustly in glucose. We identified a 4-step bypass pathway patched together from promiscuous enzymes that restores PLP synthesis in strain JK1. None of the mutations in JK1 occurs in a gene encoding an enzyme in the new pathway. Two mutations indirectly enhance the ability of SerA (3-phosphoglycerate dehydrogenase) to perform a new function in the bypass pathway. Another disrupts a gene encoding a PLP phosphatase, thus preserving PLP levels. These results demonstrate that a functional pathway can be patched together from promiscuous enzymes in the proteome, even without mutations in the genes encoding those enzymes.

scholarly journals Endogenous Xylose Pathway in Saccharomyces cerevisiae

2004 ◽  
Vol 70 (6) ◽  
pp. 3681-3686 ◽  
Author(s):  
Mervi H. Toivari ◽  
Laura Salusj�rvi ◽  
Laura Ruohonen ◽  
Merja Penttil�
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Xylose Reductase ◽  
Pichia Stipitis ◽  
Dry Mass ◽  
Genes Encoding ◽  
Main Carbon Source ◽  
Endogenous Genes ◽  
Endogenous Enzymes ◽  
Main Carbon

ABSTRACT The baker's yeast Saccharomyces cerevisiae is generally classified as a non-xylose-utilizing organism. We found that S. cerevisiae can grow on d-xylose when only the endogenous genes GRE3 (YHR104w), coding for a nonspecific aldose reductase, and XYL2 (YLR070c, ScXYL2), coding for a xylitol dehydrogenase (XDH), are overexpressed under endogenous promoters. In nontransformed S. cerevisiae strains, XDH activity was significantly higher in the presence of xylose, but xylose reductase (XR) activity was not affected by the choice of carbon source. The expression of SOR1, encoding a sorbitol dehydrogenase, was elevated in the presence of xylose as were the genes encoding transketolase and transaldolase. An S. cerevisiae strain carrying the XR and XDH enzymes from the xylose-utilizing yeast Pichia stipitis grew more quickly and accumulated less xylitol than did the strain overexpressing the endogenous enzymes. Overexpression of the GRE3 and ScXYL2 genes in the S. cerevisiae CEN.PK2 strain resulted in a growth rate of 0.01 g of cell dry mass liter−1 h−1 and a xylitol yield of 55% when xylose was the main carbon source.

scholarly journals Biodegradation of the Organic Disulfide 4,4′-Dithiodibutyric Acid by Rhodococcus spp.

2015 ◽  
Vol 81 (24) ◽  
pp. 8294-8306 ◽  
Author(s):  
Heba Khairy ◽  
Jan Hendrik Wübbeler ◽  
Alexander Steinbüchel
Keyword(s):  
Carbon Source ◽  
Sole Carbon Source ◽  
Rhodococcus Erythropolis ◽  
Model Organism ◽  
Hypothetical Protein ◽  
Gene Encoding ◽  
Content Type ◽  
Gene Coding ◽  
Initial Cleavage ◽  
Genes Encoding

ABSTRACTFourRhodococcusspp. exhibited the ability to use 4,4′-dithiodibutyric acid (DTDB) as a sole carbon source for growth. The most important step for the production of a novel polythioester (PTE) using DTDB as a precursor substrate is the initial cleavage of DTDB. Thus, identification of the enzyme responsible for this step was mandatory. BecauseRhodococcus erythropolisstrain MI2 serves as a model organism for elucidation of the biodegradation of DTDB, it was used to identify the genes encoding the enzymes involved in DTDB utilization. To identify these genes, transposon mutagenesis ofR. erythropolisMI2 was carried out using transposon pTNR-TA. Among 3,261 mutants screened, 8 showed no growth with DTDB as the sole carbon source. In five mutants, the insertion locus was mapped either within a gene coding for a polysaccharide deacetyltransferase, a putative ATPase, or an acetyl coenzyme A transferase, 1 bp upstream of a gene coding for a putative methylase, or 176 bp downstream of a gene coding for a putative kinase. In another mutant, the insertion was localized between genes encoding a putative transcriptional regulator of the TetR family (noxR) and an NADH:flavin oxidoreductase (nox). Moreover, in two other mutants, the insertion loci were mapped within a gene encoding a hypothetical protein in the vicinity ofnoxRandnox. The interruption mutant generated,R. erythropolisMI2noxΩtsr, was unable to grow with DTDB as the sole carbon source. Subsequently,noxwas overexpressed and purified, and its activity with DTDB was measured. The specific enzyme activity of Nox amounted to 1.2 ± 0.15 U/mg. Therefore, we propose that Nox is responsible for the initial cleavage of DTDB into 2 molecules of 4-mercaptobutyric acid (4MB).

scholarly journals Contribution of Gene Loss to the Pathogenic Evolution of Burkholderia pseudomallei and Burkholderia mallei

2004 ◽  
Vol 72 (7) ◽  
pp. 4172-4187 ◽  
Author(s):  
Richard A. Moore ◽  
Shauna Reckseidler-Zenteno ◽  
Heenam Kim ◽  
William Nierman ◽  
Yan Yu ◽  
...  
Keyword(s):  
Carbon Source ◽  
Burkholderia Pseudomallei ◽  
Sole Carbon Source ◽  
Lethal Dose ◽  
Selective Advantage ◽  
Burkholderia Mallei ◽  
Closely Related Species ◽  
Transposon Insertion ◽  
Genes Encoding ◽  
Pathogenic Evolution

ABSTRACT Burkholderia pseudomallei is the causative agent of melioidosis. Burkholderia thailandensis is a closely related species that can readily utilize l-arabinose as a sole carbon source, whereas B. pseudomallei cannot. We used Tn5-OT182 mutagenesis to isolate an arabinose-negative mutant of B. thailandensis. Sequence analysis of regions flanking the transposon insertion revealed the presence of an arabinose assimilation operon consisting of nine genes. Analysis of the B. pseudomallei chromosome showed a deletion of the operon from this organism. This deletion was detected in all B. pseudomallei and Burkholderia mallei strains investigated. We cloned the B. thailandensis E264 arabinose assimilation operon and introduced the entire operon into the chromosome of B. pseudomallei 406e via homologous recombination. The resultant strain, B. pseudomallei SZ5028, was able to utilize l-arabinose as a sole carbon source. Strain SZ5028 had a significantly higher 50% lethal dose for Syrian hamsters compared to the parent strain 406e. Microarray analysis revealed that a number of genes in a type III secretion system were down-regulated in strain SZ5028 when cells were grown in l-arabinose, suggesting a regulatory role for l-arabinose or a metabolite of l-arabinose. These results suggest that the ability to metabolize l-arabinose reduces the virulence of B. pseudomallei and that the genes encoding arabinose assimilation may be considered antivirulence genes. The increase in virulence associated with the loss of these genes may have provided a selective advantage for B. pseudomallei as these organisms adapted to survival in animal hosts.

scholarly journals Identification of genes encoding a novel ABC transporter in Lactobacillus delbrueckii for inulin polymers uptake

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuji Tsujikawa ◽  
Shu Ishikawa ◽  
Iwao Sakane ◽  
Ken-ichi Yoshida ◽  
Ro Osawa
Keyword(s):  
Bacillus Subtilis ◽  
Carbon Source ◽  
Heterologous Expression ◽  
Gene Cluster ◽  
Abc Transporter ◽  
Transcriptome Analysis ◽  
Sole Carbon Source ◽  
Cultured Cells ◽  
Lactobacillus Delbrueckii ◽  
Genes Encoding

AbstractLactobacillus delbrueckii JCM 1002T grows on highly polymerized inulin-type fructans as its sole carbon source. When it was grown on inulin, a > 10 kb long gene cluster inuABCDEF (Ldb1381-1386) encoding a plausible ABC transporter was suggested to be induced, since a transcriptome analysis revealed that the fourth gene inuD (Ldb1384) was up-regulated most prominently. Although Bacillus subtilis 168 is originally unable to utilize inulin, it became to grow on inulin upon heterologous expression of inuABCDEF. When freshly cultured cells of the recombinant B. subtilis were then densely suspended in buffer containing inulin polymers and incubated, inulin gradually disappeared from the buffer and accumulated in the cells without being degraded, whereas levan-type fructans did not disappear. The results imply that inuABCDEF might encode a novel ABC transporter in L. delbrueckii to “monopolize” inulin polymers selectively, thereby, providing a possible advantage in competition with other concomitant inulin-utilizing bacteria.

scholarly journals Bioconversion of biphenyl to a polyhydroxyalkanoate copolymer by Alcaligenes denitrificans A41

2020 ◽  
Author(s):  
Taito Yajima ◽  
Mizuki Nagatomo ◽  
Aiko Wakabayashi ◽  
Michio Sato ◽  
Seiichi Taguchi ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Carbon Source ◽  
Sole Carbon Source ◽  
Average Molecular Weight ◽  
Gel Permeation Chromatography ◽  
Nitrogen Sources ◽  
Dry Weight ◽  
Environmental Pollutant ◽  
Genes Encoding ◽  
Copolymer Poly

Abstract A polyhydroxyalkanoate (PHA) copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)], was biosynthesized from biphenyl as the sole carbon source using Alcaligenes (currently Achromobacter) denitrificans A41. This strain is capable of degrading polychlorinated biphenyls (PCBs) and biphenyl. This proof-of-concept of the conversion of aromatic chemicals such as the environmental pollutant PCBs/biphenyl to eco-friendly products such as biodegradable polyester PHA was inspired by the uncovering of two genes encoding PHA synthases in the A. denitrificans A41 genome. When the carbon/nitrogen (C/N) ratio was set at 21, the cellular P(3HB-co-3HV) content in strain A41 reached its highest value of 10.1% of the cell dry weight (CDW). A two-step cultivation protocol improved the accumulation of P(3HB-co-3HV) by up to 26.2% of the CDW, consisting of 13.0 mol% 3HV when grown on minimum salt medium without nitrogen sources. The highest cellular content of P(3HB-co-3HV) (47.6% of the CDW) was obtained through the two-step cultivation of strain A41 on biphenyl as the sole carbon source. The purified copolymer had ultra-high molecular weight (weight-average molecular weight of 3.5 × 106), as revealed through gel-permeation chromatography. Based on the genomic information related to both polymer synthesis and biphenyl degradation, we finally proposed a metabolic pathway for the production of P(3HB-co-3HV) associated with the degradation of biphenyl by strain A41.

scholarly journals Identification of Genes Required for Recycling Reducing Power during Photosynthetic Growth

2005 ◽  
Vol 187 (15) ◽  
pp. 5249-5258 ◽  
Author(s):  
Christine L. Tavano ◽  
Angela M. Podevels ◽  
Timothy J. Donohue
Keyword(s):  
Carbon Source ◽  
Calvin Cycle ◽  
Photosynthetic Apparatus ◽  
Reducing Power ◽  
Global Gene Expression ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Glycolate Metabolism ◽  
Global Gene Expression Analysis ◽  
Photoheterotrophic Growth

ABSTRACT Photosynthetic organisms have the unique ability to transform light energy into reducing power. We study the requirements for photosynthesis in the α-proteobacterium Rhodobacter sphaeroides. Global gene expression analysis found that ∼50 uncharacterized genes were regulated by changes in light intensity and O2 tension, similar to the expression of genes known to be required for photosynthetic growth of this bacterium. These uncharacterized genes included RSP4157 to -4159, which appeared to be cotranscribed and map to plasmid P004. A mutant containing a polar insertion in RSP4157, CT01, was able to grow via photosynthesis under autotrophic conditions using H2 as an electron donor and CO2 as a carbon source. However, CT01 was unable to grow photoheterotrophically in a succinate-based medium unless compounds that could be used to recycle reducing power (the external electron acceptor dimethyl sulfoxide (DMSO) or CO2) were provided. This suggests that the insertion in RSP4157 caused a defect in recycling reducing power during photosynthetic growth when a fixed carbon source was present. CT01 had decreased levels of RNA for genes encoding putative glycolate degradation functions. We found that exogenous glycolate also rescued photoheterotrophic growth of CT01, leading us to propose that CO2 produced from glycolate metabolism can be used by the Calvin cycle to recycle reducing power generated in the photosynthetic apparatus. The ability of glycolate, CO2, or DMSO to support photoheterotrophic growth of CT01 suggests that one or more products of RSP4157 to -4159 serve a previously unknown role in recycling reducing power under photosynthetic conditions.

Metabolic Engineering of E. coli for Xylose Production from Glucose as the Sole Carbon Source

2021 ◽  
Vol 10 (9) ◽  
pp. 2266-2275
Author(s):  
Wencheng Yin ◽  
Yujin Cao ◽  
Miaomiao Jin ◽  
Mo Xian ◽  
Wei Liu
Keyword(s):  
Metabolic Engineering ◽  
Carbon Source ◽  
Sole Carbon Source ◽  
E Coli ◽  
Xylose Production

scholarly journals Metabolic Engineering of Zymomonas moblis for Ethylene Production

2019 ◽  
Author(s):  
Yan He ◽  
Bo Wu ◽  
Wei Xia ◽  
Kun-Yang Zhao ◽  
Qiong Tan ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Carbon Source ◽  
Ethylene Production ◽  
Sole Carbon Source ◽  
High Stress ◽  
Cellulosic Biomass ◽  
Corn Straw ◽  
Enzymatic Hydrolysate ◽  
Alternative Approach ◽  
Engineering Work

Abstract Background: Biological ethylene production via the ethylene-forming enzyme (EFE) can offer a promising sustainable alternative approach for fossil-based ethylene production. The high stress tolerance of Z. mobilis make it as promising bio-ethylene producer.Results: In this study, Heterologous expression of the efe gene in Z. mobilis successfully converted the non-ethylene producing strain into an ethylene producer. What’s more, we systematically performed the effect of knocking out the competitive metabolic pathway of pyruvate and the addition of nutrients to the medium to improve the ethylene production in Z. mobilis. These optimization pathways and different substrate supplies resulted in higher ethylene productivity (from 1.36 to 12.83 nmol/OD600/ml), which may guide future engineering work on ethylene production in other organisms to further improve ethylene productivity. Meanwhile, we obtained ethylene production of 5.8 nmol/OD600/ml in strain ZM532-efe by using enzymatic hydrolysate of corn straw as the sole carbon source. This is also the first report on the production of ethylene from cellulosic biomass.Conclusions: These results indicate that the engineered Z. mobilis show great potential for production of ethylene from cellulosic biomass in the future.

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