Invited Oral Abstract Presentation
Effects of agricultural wastes and energy crops anaerobic co-digestion on anaerobic microbes and corresponding digestion performance
Mr. Yuan Zhong and Dr. Wei Liao, Michigan State University, East Lansing, MI, USA
2017 SIMB Annual Meeting and Exhibition
In the past decades, microbial communities of anaerobic digestion (AD) have been intensively investigated, with majority of these studies focusing on the correlation between microbial diversity and biogas production. However, currently there is a lack of comprehensive research on the relationship between microbial communities and compositional changes of the solid digestate (AD fiber). Therefore, a distinct understanding on the relationship between mixed feedstock, microbial communities, biogas production, and solid digestate quality should be concluded to promote AD technology for the next-generation biorefining. The objective of this study was to understand the responses of microbial communities to different feedstock combinations and ratios of anaerobic co-digestion and their influences on biogas production and solid digestate quality. Three feedstock combinations (dairy manure with corn stover, dairy manure with switchgrass and dairy manure with miscanthus) and two feedstock ratios (4 to1 and 3 to 2) were investigated with a completely randomized design. The 16S rRNA gene-based 454 pyrosequencing, Terminal Restriction Fragment Length Polymorphism (T-RFLP) and clone library were used to investigate the communities. Microbial communities were also statistically correlated with performance parameters such as total solids reduction, biogas production, and AD fiber quality (cellulose, xylan, and lignin).
Invited Oral Abstract Presentation
Engineering Yarrowia lipolytica for triacetic acid lactone production
Claire Palmer, Kelly Markham and Hal Alper, The University of Texas at Austin, Austin, TX, USA
2017 SIMB Annual Meeting and Exhibition
Yarrowia lipolytica, an industrially attractive, non-conventional yeast, boasts a high innate capacity to produce acyl-CoA derived molecules such as triacylglycerides. Here we demonstrate the potential of rewiring Y. lipolytica to divert this precursor pool away from lipids and into alternative molecules of interest. Specifically, we explored the production of the simple polyketide, triacetic acid lactone (TAL). TAL has been proposed as a biorenewable platform chemical that can be converted into many downstream products including sorbic acid. Previous efforts to produce TAL in hosts such as E. coli and S. cerevisiae have been limited by the availability of acyl-CoA precursors. We enabled TAL production in the precursor rich host, Y. lipolytica, through heterologous expression of 2-pyrone synthase, an enzyme from Gerbera hybrida that catalyzes the formation of TAL by condensation of acetyl-CoA and malonyl-CoA. We next performed a series of strain engineering approaches to boost TAL production by metabolically rewiring Y. lipolytica for enhanced precursor accumulation. Final strain characterization was conducted in bioreactors to further optimize production titer, rate, and yield. Ultimately, we established a strain that produced the highest titer of TAL reported to date in any host. Here we present the details of these genetic engineering efforts as well as the production characterization of the resulting strain.
Invited Oral Abstract Presentation
Chemical and bioactivity screening of secondary metabolites from Oregonian bacterial strains
Chenxi Zhu, Cassandra Lew and Prof. Sandra Loesgen, Oregon State University, Corvallis, OR, USA
2017 SIMB Annual Meeting and Exhibition
Soil-dwelling bacteria have given us a remarkable chemical diversity and an unprecedented amount of bioactive small molecules, such as the clinical used approved anticancer drugs doxorubicin, mitomycin, and many antibiotics, such as erythromycin and kanamycin. Here we present the chemical and bioactivity screening of seventeen bacterial strains isolated from soils collected near Bend Oregon from an arid, high desert area. Species diversity was assessed using 16S rRNA gene sequencing, while chemical diversity was explored via untargeted comparative metabolomic analysis based on liquid-chromatography coupled mass spectrometry (LCMS) derived data. Organic extracts were prepared from each bacterium cultivated in malt-based liquid media and tested for their respective anti-bacterial and cytotoxic activities. Antibacterial screening was carried out against four human pathogens, Enterococcus faecium, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, by broth dilution method. Cell viability was evaluated against a panel of human cell lines to assess their potential for the treatment of cancer. Five model systems were used to determine the ability of extracts to inhibit the growth of human colon cancer adenocarcinoma (HCT-116), breast adenocarcinoma (MCF-7), lung carcinoma (A549), malignant melanoma (SK-Mel-5), and prostate adenocarcinoma (PC-3) via MTT cell proliferation procedures. Noteworthy, six extracts (35%) showed strong inhibition with less than 50% cell proliferation at 10 μg/ml in all cell lines. Active compounds are purified and characterized by HPLC and NMR. Principle component analysis of organic extract LCMS data revealed drastic differences in the metabolic profiles found in these panel of high desert derived bacteria
Invited Oral Abstract Presentation
Developing the thermotolerant yeast Kluyveromyces marxianus as a microbial host for volatile ester biosynthesis
Ann-Kathrin Loebs, Ronja Engel, Cory Schwartz and Ian Wheeldon, University of California Riverside, Riverside, CA, USA
2017 SIMB Annual Meeting and Exhibition
The yeast Kluyveromyces marxianus is a promising candidate for chemicals biosynthesis. It has rapid growth kinetics at temperatures upwards of 45 °C and can produce short and medium chain volatile esters and ethanol at high rates. Of particular interest is its capacity to produce ethyl acetate at rates upwards of 2 g/L/hr in aerated cultures. Both ethanol and ethyl acetate production rely on the activity of alcohol dehydrogenases to oxidize acetaldehyde to ethanol, which serves a substrate for ethyl acetate production. Little is known about the ester production pathways in K. marxianus. Prior findings suggest the presence of alcohol acetyltransferase (Atf) activity and the absence of significant reverse esterase activity. Our studies suggest the presence of one ATF and seven different alcohol dehydrogenase (ADH) genes in K. marxianus. In this work, we design a hybrid-synthetic RNA polymerase III promoter to create a CRISPR-Cas9 genome editing systems for K. marxianus. This system was used to construct a disruption library of ADH and ATF genes to study their function in volatile metabolite production. ATF disruption and overexpression analyses suggest a limited role of Atf in overall ethyl acetate biosynthesis in K. marxianus. ADH2 disruption resulted in reduced ethanol production along with accumulation of acetaldehyde suggesting an importance of Adh2 in ethanol production. The terminal step of ethyl acetate synthesis remains puzzling but overexpression of ADH7 shows promiscuous activity towards ethyl acetate production from hemiacetal. These findings serve as starting point for metabolic engineering approaches towards ethyl acetate and longer chain esters production.
Invited Oral Abstract Presentation
Break
2017 SIMB Annual Meeting and Exhibition
Invited Oral Abstract Presentation
Measuring dynamic interactions and metabolic fluxes in microbial communities
Mr. Nikodimos Gebreselassie and Maciek Antoniewicz, University of Delaware, Newark, DE, USA
2017 SIMB Annual Meeting and Exhibition
In nature, microorganisms don’t exist in isolation, rather they are part of complex interacting systems. Recent efforts in metabolic engineering have also taken advantage of multi-microorganism systems to enhance product yields and productivities. Despite these advances, quantitative characterization tools to elucidate microbial metabolism and drive engineering efforts are still lacking for multi-microorganism systems. For example, metabolic flux analysis, a widely used approach for metabolic engineering of pure cultures, has not been developed and applied towards multi-microorganism systems.
In this work, we present a novel approach for performing 13C-metabolic flux analysis (13C-MFA) in co-cultures. We demonstrate for the first time that it is possible to determine population dynamics, metabolic interactions (e.g. cross-feeding of molecules between cells), and metabolic flux distributions in multiple species simultaneously without the need for physical separation of cells or proteins, or overexpression of species-specific products. We have applied this methodology to study an interacting synthetic co-culture consisting of a glucose consuming wild-type E. coli strain and a glucose non-consuming ΔptsI Δglk E. coli strain. Here, the ΔptsI Δglk strain relies on acetate produced by wild-type E. coli for cell growth. We have performed a complete characterization of this interacting system using our dynamic co-culture 13C-MFA methodology. In addition to measuring intracellular metabolic fluxes for individual strains in the co-culture, for the first time, population dynamics and acetate cross-feeding was fully characterized. This work lays the foundation for more detailed studies of complex interacting microbial communities including systems consisting of more than two species.
Invited Oral Abstract Presentation
Uncovering the role of branched-chain amino acid transaminases in Saccharomyces cerevisiae isobutanol biosynthesis
Sarah Hammer and Prof. José Avalos, Princeton University, Princeton, NJ, USA
2017 SIMB Annual Meeting and Exhibition
Engineering metabolic pathways in yeast is a promising strategy for producing commodity and high-value chemicals from renewable sources. Yeast are particularly attractive hosts for the production of branched-chain higher alcohols (BCHAs), which they naturally synthesize in trace amounts during ethanol fermentation. BCHA production is complicated by separation of the upstream and downstream biosynthetic pathways between the mitochondria and the cytosol, as well as the presence of branched-chain amino acid transaminases (BCATs) in both compartments. In the past, researchers have deleted or overexpressed the yeast mitochondrial (BAT1) or cytosolic (BAT2) BCAT genes in order to reduce BCHA production and improve the flavor and aroma profiles of fermented alcoholic beverages. However, these studies have yielded inconsistent results regarding the effects of BCATs on isobutanol production. Recently, there has been a renewed interest in understanding the role of these transaminases in the production of BCHAs – specifically isobutanol – as advanced biofuels. This work aimed to resolve previous conflicting results and conclusively determine the role of BCATs in the biosynthetic route from glucose to isobutanol. Manipulation of transamination activity alone enabled us to increase isobutanol production by more than 15-fold over wild type strains. Addressing the intracellular transport bottleneck implicated in our transamination activity experiments led to an additional 92% increase in isobutanol production. This work answers the longstanding question of the role of transamination activity in BCHA biosynthesis and develops valuable strains for future optimization of isobutanol production.
Invited Oral Abstract Presentation
Bioproduction of 1-piperideine using engineered Escherichia coli strains
Mr. Valentine Anyanwu, Ms. Juliana Lebeau, Mr. Aidan Grimsley, Dr. Stephen Hall, Dr. Anca Pordea and Gill Stephens, University of Nottingham, Nottingham, United Kingdom
2017 SIMB Annual Meeting and Exhibition
Abstract
Although the
bioproduction of complex, functionalised N-heterocycles has been
reported, bioproduction of unsubstituted platform N-heterocycles has not
yet been achieved. Therefore, the suitability of putrescine oxidase from Rhodococcus
erythropolis (PuORH) for bioproduction of Δ1-piperideine
was studied. PuORH catalyses the oxidation of
cadaverine to 5-aminopentanal. Although this product is known to cyclise
spontaneously into Δ1-piperideine, direct formation of this
product catalysed by PuORH has not been demonstrated,
except by using o-aminobenzaldehyde as a reagent to trap Δ1-piperideine
and shift the equilibrium for cyclisation. The PuORH
gene was cloned and expressed in E. coli BL21 (DE3) using the pET20b
vector, and the His-tagged enzyme was purified. Steady state kinetics of PuORH
were determined by monitoring oxygen consumption; the KM
and kcat values were 0.24 ± 0.05 mM and 26.6 ± 0.08 s-1
for cadaverine, and 0.17 ± 0.03 mM and 147.4 ± 0.4 s-1 for
putrescine, respectively. Whereas the kcat/KM values are
lower than those reported using peroxidase-coupled assays, this result should
represent the true kinetics of PuORH. Using the
purified enzyme, the conversion of cadaverine to Δ1-piperideine
was demonstrated qualitatively using LC-ESI-MS and 1H NMR;
5-aminopentanal could not be detected. Δ1-Piperideine also
formed the corresponding dimer and trimers, a known spontaneous reaction, and
the product ratio could be adjusted by varying the pH. This preliminary study
indicates that PuORH is suitable for bioproduction of Δ1-piperideine.
The next steps are to optimise the reaction conditions, quantify, extract and
purify the products, and develop whole cell bioproduction of N-heterocycles
from renewable feedstocks.
