scholarly journals Combinatorial metabolic engineering of Saccharomyces cerevisiae for terminal alkene production

2015 ◽  
Author(s):  
Binbin Chen ◽  
Dong-Yup Lee ◽  
Matthew Wook Chang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acid ◽  
Industrial Applications ◽  
Yeast Saccharomyces Cerevisiae ◽  
Terminal Alkene ◽  
Terminal Alkenes ◽  
One Step ◽  
Combinatorial Engineering ◽  
Culturing Conditions ◽  
Alkene Production

Biological production of terminal alkenes has garnered a significant interest due to their industrial applications such as lubricants, detergents and fuels. Here, we engineered the yeast Saccharomyces cerevisiae to produce terminal alkenes via a one-step fatty acid decarboxylation pathway and improved the alkene production using combinatorial engineering strategies. In brief, we first characterized eight fatty acid decarboxylases to enable and enhance alkene production. We then increased the production titer 7-fold by improving the availability of the precursor fatty acids. We additionally increased the titer about 5-fold through genetic cofactor engineering and gene expression tuning in rich medium. Lastly, we further improved the titer 1.8-fold to 3.7 mg/L by optimizing the culturing conditions in bioreactors. This study represents the first report of terminal alkene biosynthesis in S. cerevisiae, and the abovementioned combinatorial engineering approaches collectively increased the titer 67.4-fold. We envision that these approaches could provide insights into devising engineering strategies to improve the production of fatty acid-derived biochemicals in S. cerevisiae.

scholarly journals Harnessing a Novel P450 Fatty Acid Decarboxylase from Macrococcus caseolyticus for Microbial Biosynthesis of Odd Chain Terminal Alkenes

2018 ◽  
Author(s):  
Jong-won Lee ◽  
Narayan P. Niraula ◽  
Cong T. Trinh
Keyword(s):  
Fatty Acid ◽  
Docking Simulation ◽  
Redox System ◽  
Acid Decarboxylase ◽  
Microbial Conversion ◽  
E Coli ◽  
Terminal Alkene ◽  
Terminal Alkenes ◽  
Alkene Production

ABSTRACTAlkenes are industrially important platform chemicals with broad applications. In this study, we report a microbial conversion route for direct biosynthesis of medium and long chain terminal alkenes from fermentable sugars by harnessing a novel P450 fatty acid (FA) decarboxylase from Macrococcus caseolyticus (OleTMC). We first characterized OleTMC and demonstrated its in vitro H2O2-independent activities towards linear and saturated C10:0-C18:0 FAs, with the highest activity for C16:0 and C18:0 FAs. Combining protein homology modeling, in silico residue mutation analysis, and docking simulation with direct experimental evidence, we elucidated the underlying mechanism for governing the observed substrate preference of OleTMC, which depends on the size of FA binding pocket, not the catalytic site. Next, we engineered the terminal alkene biosynthesis pathway, consisting of an engineered E. coli thioesterase (TesA*) and OleTMC, and introduced this pathway into E. coli for direct terminal alkene biosynthesis from glucose. The recombinant strain E. coli EcNN101 produced a total of 17.78 ± 0.63 mg/L odd-chain terminal alkenes, comprising of 0.9% ± 0.5% C11 alkene, 12.7% ± 2.2% C13 alkene, 82.7% ± 1.7% C15 alkene, and 3.7% ± 0.8% C17 alkene, and a yield of 0.87 ± 0.03 (mg/g) on glucose after 48 h in baffled shake flasks. To improve the terminal alkene production, we identified and overcame the electron transfer limitation in OleTMC, by introducing a two-component redox system, consisting of a putidaredoxin reductase CamA and a putidaredoxin CamB from Pseudomonas putida, into EcNN101, and demonstrated the terminal alkene production increased ∼2.8 fold after 48 h. Overall, this study provides a better understanding of the function of P450 FA decarboxylases and helps guide future protein and metabolic engineering for enhanced microbial production of target designer alkenes in a recombinant host.

scholarly journals The Spindle Checkpoint of the Yeast Saccharomyces cerevisiae Requires Kinetochore Function and Maps to the CBF3 Domain

Genetics ◽  
2001 ◽  
Vol 157 (4) ◽  
pp. 1493-1502
Author(s):  
Richard D Gardner ◽  
Atasi Poddar ◽  
Chris Yellman ◽  
Penny A Tavormina ◽  
M Cristina Monteagudo ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Critical Role ◽  
Spindle Checkpoint ◽  
Essential Genes ◽  
Gene Fusions ◽  
Yeast Saccharomyces Cerevisiae ◽  
Checkpoint Signaling ◽  
One Step ◽  
Diploid Cells ◽  
Kinetochore Function

Abstract We have measured the activity of the spindle checkpoint in null mutants lacking kinetochore activity in the yeast Saccharomyces cerevisiae. We constructed deletion mutants for nonessential genes by one-step gene replacements. We constructed heterozygous deletions of one copy of essential genes in diploid cells and purified spores containing the deletion allele. In addition, we made gene fusions for three essential genes to target the encoded proteins for proteolysis (degron alleles). We determined that Ndc10p, Ctf13p, and Cep3p are required for checkpoint activity. In contrast, cells lacking Cbf1p, Ctf19p, Mcm21p, Slk19p, Cse4p, Mif2p, Mck1p, and Kar3p are checkpoint proficient. We conclude that the kinetochore plays a critical role in checkpoint signaling in S. cerevisiae. Spindle checkpoint activity maps to a discreet domain within the kinetochore and depends on the CBF3 protein complex.

scholarly journals Screening and directed evolution of transporters to improve xylodextrin utilization in the yeast Saccharomyces cerevisiae

2017 ◽  
Author(s):  
Chenlu Zhang ◽  
Ligia Acosta-Sampson ◽  
Vivian Yaci Yu ◽  
Jamie H. D. Cate
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Directed Evolution ◽  
Plant Biomass ◽  
Carbon Sources ◽  
Industrial Applications ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cellulosic Biofuel ◽  
Economic Production ◽  
Report Analysis ◽  
Selection Medium

AbstractThe economic production of cellulosic biofuel requires efficient and full utilization of all abundant carbohydrates naturally released from plant biomass by enzyme cocktails. Recently, we reconstituted the Neurospora crassa xylodextrin transport and consumption system in Saccharomyces cerevisiae, enabling growth of yeast on xylodextrins aerobically. However, the consumption rate of xylodextrin requires improvement for industrial applications, including consumption in anaerobic conditions. As a first step in this improvement, we report analysis of orthologues of the N. crassa transporters CDT-1 and CDT-2. Transporter ST16 from Trichoderma virens enables faster aerobic growth of S. cerevisiae on xylodextrins compared to CDT-2. ST16 is a xylodextrin-specific transporter, and the xylobiose transport activity of ST16 is not inhibited by cellobiose. Other transporters identified in the screen also enable growth on xylodextrins including xylotriose. Taken together, these results indicate that multiple transporters might prove useful to improve xylodextrin utilization in S. cerevisiae. Efforts to use directed evolution to improve ST16 from a chromosomally-integrated copy were not successful, due to background growth of yeast on other carbon sources present in the selection medium. Future experiments will require increasing the baseline growth rate of the yeast population on xylodextrins, to ensure that the selective pressure exerted on xylodextrin transport can lead to isolation of improved xylodextrin transporters.

scholarly journals Identification of a Caenorhabditis elegans Δ6-fatty-acid-desaturase by heterologous expression in Saccharomyces cerevisiae

1998 ◽  
Vol 330 (2) ◽  
pp. 611-614 ◽  
Author(s):  
A. Johnathan NAPIER ◽  
J. Sandra HEY ◽  
J. Dominic LACEY ◽  
R. Peter SHEWRY
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acid ◽  
Caenorhabditis Elegans ◽  
Expressed Sequence Tag ◽  
Cytochrome B5 ◽  
Fatty Acid Desaturase ◽  
Higher Plant ◽  
Yeast Saccharomyces Cerevisiae ◽  
C Elegans ◽  
Δ6 Desaturase

We identified a cDNA expressed sequence tag from an animal (the nematode worm Caenorhabditis elegans) that showed weak similarity to a higher-plant microsomal Δ6-desaturase. A full-length cDNA clone was isolated and expressed in the yeast Saccharomyces cerevisiae. This demonstrated that the protein encoded by the C. elegans cDNA was that of a fatty acid Δ6-desaturase, as determined by the accumulation of γ-linolenic acid. The C. elegans Δ6-desaturase contained an N-terminalcytochrome b5 domain, indicating that it had a similar structure to that of the higher-plant Δ6-desaturase. The C. elegans Δ6-desaturase mapped to cosmid W08D2, a region of chromosome III. This is the first example of a Δ6-desaturase isolated from an animal and also the first example of an animal desaturase containing a cytochrome b5 domain.

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