skip to main content


Title: Porting the synthetic D‐glucaric acid pathway from Escherichia coli to Saccharomyces cerevisiae
Abstract

D‐Glucaric acid can be produced as a value‐added chemical from biomass through a de novo pathway inEscherichia coli. However, previous studies have identified pH‐mediated toxicity at product concentrations of 5 g/L and have also found the eukaryotic myo‐inositol oxygenase (MIOX) enzyme to be rate‐limiting. We ported this pathway toSaccaromyces cerevisiae, which is naturally acid‐tolerant and evaluate a codon‐optimized MIOX homologue. We constructed two engineered yeast strains that were distinguished solely by theirMIOXgene – either the previous version fromMus musculusor a homologue fromArabidopsis thalianacodon‐optimized for expression inS. cerevisiae– in order to identify the rate‐limiting steps for D‐glucaric acid production both from a fermentative and non‐fermentative carbon source. myo‐Inositol availability was found to be rate‐limiting from glucose in both strains and demonstrated to be dependent on growth rate, whereas the previously usedM. musculusMIOX activity was found to be rate‐limiting from glycerol. Maximum titers were 0.56 g/L from glucose in batch mode, 0.98 g/L from glucose in fed‐batch mode, and 1.6 g/L from glucose supplemented with myo‐inositol. Future work focusing on the MIOX enzyme, the interplay between growth and production modes, and promoting aerobic respiration should further improve this pathway.

 
more » « less
NSF-PAR ID:
10235785
Author(s) / Creator(s):
 ;  ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Biotechnology Journal
Volume:
11
Issue:
9
ISSN:
1860-6768
Page Range / eLocation ID:
p. 1201-1208
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ABSTRACT  
    more » « less
  2. Abstract

    Several chromosomally expressed AceE variants were constructed inEscherichia coli ΔldhA ΔpoxB ΔppsAand compared using glucose as the sole carbon source. These variants were examined in shake flask cultures for growth rate, pyruvate accumulation, and acetoin production via heterologous expression of thebudAandbudBgenes fromEnterobacter cloacae ssp. dissolvens. The best acetoin‐producing strains were subsequently studied in controlled batch culture at the one‐liter scale. PDH variant strains attained up to four‐fold greater acetoin than the strain expressing the wild‐type PDH. In a repeated batch process, the H106V PDH variant strain attained over 43 g/L of pyruvate‐derived products, acetoin (38.5 g/L) and 2R,3R‐butanediol (5.0 g/L), corresponding to an effective concentration of 59 g/L considering the dilution. The acetoin yield from glucose was 0.29 g/g with a volumetric productivity of 0.9 g/L·h (0.34 g/g and 1.0 g/L·h total products). The results demonstrate a new tool in pathway engineering, the modification of a key metabolic enzyme to improve the formation of a product via a kinetically slow, introduced pathway. Direct modification of the pathway enzyme offers an alternative to promoter engineering in cases where the promoter is involved in a complex regulatory network.

     
    more » « less
  3. Abstract

    Kluyveromyces marxianusis a promising nonconventional yeast for biobased chemical production due to its rapid growth rate, high TCA cycle flux, and tolerance to low pH and high temperature. UnlikeSaccharomyces cerevisiae, K. marxianusgrows on low‐cost substrates to cell densities that equal or surpass densities in glucose, which can be beneficial for utilization of lignocellulosic biomass (xylose), biofuel production waste (glycerol), and whey (lactose). We have evaluatedK. marxianusfor the synthesis of polyketides, using triacetic acid lactone (TAL) as the product. The 2‐pyrone synthase (2‐PS) was expressed on a CEN/ARS plasmid in three different strains, and the effects of temperature, carbon source, and cultivation strategy on TAL levels were determined. The highest titer was obtained in defined 1% xylose medium at 37°C, with substantial titers at 41 and 43°C. The introduction of a high‐stability 2‐PS mutant and a promoter substitution increased titer four‐fold. 2‐PS expression from a multi‐copy pKD1‐based plasmid improved TAL titers a further five‐fold. Combining the best plasmid, promoter, and strain resulted in a TAL titer of 1.24 g/L and a yield of 0.0295 mol TAL/mol carbon for this otherwise unengineered strain in 3 ml tube culture. This is an excellent titer and yield (on xylose) before metabolic engineering or fed‐batch culture relative to other hosts (on glucose), and demonstrates the promise of this rapidly growing and thermotolerant yeast species for polyketide production.

     
    more » « less
  4. Abstract

    Myo‐inositol oxygenase (MIOX) is the first enzyme in the inositol route to ascorbate (L‐ascorbic acid, AsA, vitamin C). We have previously shown that Arabidopsis plants constitutively expressingMIOXhave elevated foliar AsA content and displayed enhanced growth rate, biomass accumulation, and increased tolerance to multiple abiotic stresses. In this work, we used a combination of transcriptomics, chromatography, microscopy, and physiological measurements to gain a deeper understanding of the underlying mechanisms mediating the phenotype of theAtMIOX4 line. Transcriptomic analysis revealed increased expression of genes involved in auxin synthesis, hydrolysis, transport, and metabolism, which are supported by elevated auxin levels both in vitro and in vivo, and confirmed by assays demonstrating their effect on epidermal cell elongation in theAtMIOX4 over‐expressers. Additionally, we detected up‐regulation of transcripts involved in photosynthesis and this was validated by increased efficiency of the photosystem II and proton motive force. We also found increased expression of amylase leading to higher intracellular glucose levels. Multiple gene families conferring plants tolerance/expressed in response to cold, water limitation, and heat stresses were found to be elevated in theAtMIOX4 line. Interestingly, the high AsA plants also displayed up‐regulation of transcripts and hormones involved in defense including jasmonates, defensin, glucosinolates, and transcription factors that are known to be important for biotic stress tolerance. These results overall indicate that elevated levels of auxin and glucose, and enhanced photosynthetic efficiency in combination with up‐regulation of abiotic stresses response genes underly the higher growth rate and abiotic stresses tolerance phenotype of theAtMIOX4 over‐expressers.

     
    more » « less
  5. ABSTRACT Zymomonas mobilis produces ethanol from glucose near the theoretical maximum yield, making it a potential alternative to the yeast Saccharomyces cerevisiae for industrial ethanol production. A potentially useful industrial feature is the ability to form multicellular aggregates called flocs, which can settle quickly and exhibit higher resistance to harmful chemicals than single cells. While spontaneous floc-forming Z. mobilis mutants have been described, little is known about the natural conditions that induce Z. mobilis floc formation or about the genetic factors involved. Here we found that wild-type Z. mobilis forms flocs in response to aerobic growth conditions but only in a minimal medium. We identified a cellulose synthase gene cluster and a single diguanylate cyclase that are essential for both floc formation and survival in a minimal aerobic medium. We also found that NADH dehydrogenase 2, a key component of the aerobic respiratory chain, is important for survival in a minimal aerobic medium, providing a physiological role for this enzyme, which has previously been found to be disadvantageous in a rich aerobic medium. Supplementation of the minimal medium with vitamins also promoted survival but did not inhibit floc formation. IMPORTANCE The bacterium Zymomonas mobilis is best known for its anaerobic fermentative lifestyle, in which it converts glucose into ethanol at a yield surpassing that of yeast. However, Z. mobilis also has an aerobic lifestyle, which has confounded researchers with its attributes of poor growth, accumulation of toxic acetic acid and acetaldehyde, and respiratory enzymes that are detrimental for aerobic growth. Here we show that a major Z. mobilis respiratory enzyme and the ability to form multicellular aggregates, called flocs, are important for survival, but only during aerobic growth in a medium containing a minimum set of nutrients required for growth. Supplements, such as vitamins or yeast extract, promote aerobic growth and, in some cases, inhibit floc formation. We propose that Z. mobilis likely requires aerobic respiration and floc formation in order to survive in natural environments that lack protective factors found in supplements such as yeast extract. 
    more » « less