A cell engineering approach to enzyme-based fed-batch fermentation

BACKGROUND: A fundamental problem associated with E. coli fermentations is the difficulty in achieving high cell densities in batch cultures, attributed in large part to the production and accumulation of acetate through a phenomenon known as overflow metabolism when supplying enough glucose for the cell density desired. Although a fed-batch configuration is the standard method for reducing such issues, traditional fed-batch systems require components which become problematic when applying them at smaller scale. One alternative has been the development of a system whereby the enzymatic degradation of starch is used to release glucose at a controlled rate. However, to date, amylolytic enzymes have only been applied to the culture exogenously, whereas our goal is to design and construct a self-secreting amylolytic chassis capable of self-regulated enzyme-based fed-batch fermentation. RESULTS: A putative glucoamylase from C. violaceum has been cloned and expressed in E. coli BL21(DE3) and W3110, which exhibits significant glucose releasing amylolytic activity. Extracellular amylolytic activity was enhanced following a replacement of the enzymes native signal peptide with the DsbA signal sequence, contributing to a glucoamylase secreting strain capable of utilising starch as a sole carbon source in defined media. Introduction of PcstA, a glucose sensitive K12 compatible promoter, and the incorporation of this alongside C. violaceum glucoamylase in E. coli W3110, gave rise to increased cell densities in cultures grown on starch (OD600 ∼ 30) compared to those grown on an equivalent amount of glucose (OD600 ∼ 15). Lastly, a novel self-secreting enzyme-based fed-batch fermentation system was demonstrated via the simultaneous expression of the C. violaceum glucoamylase and a recombinant protein of interest (eGFP), resulting in a fourfold increase in yield when grown in media containing starch compared with the glucose equivalent. CONCLUSIONS: This study has developed, through the secretion of a previously uncharacterised bacterial glucoamylase, a novel amylolytic E. coli strain capable of direct starch to glucose conversion. The ability of this strain to achieve increased cell densities as well as an associated increase in recombinant protein yield when grown on starch compared with an equivalent amount of glucose, demonstrates for the first time a cell engineering approach to enzyme-based fed-batch fermentation

Patient-specific prediction of long-term outcomes will change stroke rehabilitation for the better.

Stroke is the most common cause of neurological disability and yet our ability to predict long-term outcomes remains poor. The paper by Selles et al1 used upper limb outcomes from 450 patients with first-time ischaemic stroke to take a refreshingly different approach to the prediction problem. First, allowing repeated clinical measures to contribute to the prediction acknowledges what most clinicians already know—that rate of clinical change is helpful in prognostication. Second, rather than predict outcome at a single future time point, they have created likely recovery trajectories (with CIs) for individual patients. Third, access to this predictive model is freely available online, so that stroke services around the world can more accurately begin to make predictions of individual recovery. Although the current approach concerns upper limb recovery, the principle should apply to all domains

Novel transaminases from thermophiles: from discovery to application

Transaminases (TAs) are promising biocatalysts for chiral amine synthesis; however, only few thermophilic TAs have been described to date. In this work, a genome mining approach was taken to seek novel TAs from nine thermophilic microorganisms. TA sequences were identified from their respective genome sequences and their Pfam were predicted confirming that TAs class I–II are the most abundant (50%), followed by class III (26%), V (16%), IV (8%) and VI (1%). The percentage of open reading frames (ORFs) that are TAs ranges from 0.689% in Thermococcus litoralis to 0.424% in Sulfolobus solfataricus. A total of 94 putative TAs were successfully cloned and expressed into E. coli, showing mostly good expression levels when using a chemical chaperone media containing d-sorbitol. Kinetic and end-point colorimetric assays with different amino donors–acceptors confirmed TAs activity allowing for initial exploration of the substrate scope. Stereoselective and non-stereoselective serine-TAs were selected for the synthesis of hydroxypyruvate (HPA). Low HPA reaction yields were observed with four non-stereoselective serine-TAs, whilst two stereoselective serine-TAs showed significantly higher yields. Coupling serine-TA reactions to a transketolase to yield l-erythrulose (Ery) substantially increased serine conversion into HPA. Combining both stereoselective serine-TAs and transketolase using the inexpensive racemic D/L-serine led to high Ery yield (82%). Thermal characterization of stereoselective serine-TAs confirmed they have excellent thermostability up to 60°C and high optimum temperatures

Optimizing the Performance of Aerosol Photoacoustic Cells using a Finite Element Model. Part 2: Application to a Two-Resonator Cell

This is the author accepted manuscript. The final version is available from Taylor & Francis via the DOI in this recordData availability: For data related to this paper, please contact Michael I. Cotterell ([email protected]).Photoacoustic spectroscopy (PAS) measures aerosol absorption in a non-contact manner, providing accurate absorption measurements that are needed to improve aerosol optical property representations in climate models. Central to PAS is resonant amplification of the acoustic pressure wave generated from laser-heated aerosol transferring heat to surrounding gas by a photoacoustic cell. Although this cell amplifies pressure sources from aerosol absorption (signal), it also amplifies noise and background sources. It is important to maximise the cell signal-to-background ratio (SBR) for sensitive absorption measurements. Many researchers have adopted the two-resonator cell design described by Lack et al. (2006). We show that the uncertainty in PAS measurements of aerosol absorption using this two-resonator cell is significantly degraded by its large sensitivity to background contributions from laser scattering and absorption at the cell windows. In Part 1, we described the use of a finite element method (FEM) to predict cell acoustic properties, validated this framework by comparing model predictions to measurements, and used FEM to test various strategies applied commonly to single-resonator cell optimisation. In this second part, we apply FEM to understand the excitation of resonant modes of the two-resonator cell, with comparison measurements demonstrating accurate predictions of acoustic response. We perform geometry optimisation studies to maximise the SBR and demonstrate that the laser-window interaction background is reduced to undetectable levels for an optimal cell. This optimised two-resonator cell will improve the sensitivity and accuracy of future aerosol absorption measurements.Defence Science and Technology Laboratory (DSTL)Royal Society of ChemistryAnalytical Chemistry Trust FundNatural Environment Research Council (NERC

pET expression vector customized for efficient seamless cloning

Here we present a modification of the widely used pET29 expression vector for use in rapid and straightforward parallel cloning via a gene replacement and Golden Gate strategy. The modification can be applied to other expression vectors for Gram-negative bacteria. We have used the modified vectors to clone large numbers of bacterial natural enzyme variants from genomic and metagenomic sources for applications in biocatalysis

Integral field spectroscopy of massive young stellar objects in the N113 H II region in the Large Magellanic Cloud

The Spitzer Surveying the Agents of Galaxy Evolution (SAGE) survey has allowed the identification and analysis of significant samples of Young Stellar Object (YSO) candidates in the Large Magellanic Cloud (LMC). However, the angular resolution of Spitzer is relatively poor meaning that at the distance of the LMC, it is likely that many of the Spitzer YSO candidates in fact contain multiple components. We present high-resolution K-band integral field spectroscopic observations of the three most prominent massive YSO candidates in the N113 H II region using Very Large Telescope/Spectrograph for INtegral Field Observations in the Near Infrared (VLT/SINFONI). We have identified six K-band continuum sources within the three Spitzer sources and we have mapped the morphology and velocity fields of extended line emission around these sources. Br γ, He I and H2 emission is found at the position of all six K-band sources; we discuss whether the emission is associated with the continuum sources or whether it is ambient emission. H2 emission appears to be mostly ambient emission and no evidence of CO emission arising in the discs of YSOs has been found. We have mapped the centroid velocities of extended Br γ emission and He I emission and found evidence of two expanding compact H II regions. One source shows compact and strong H2 emission suggestive of a molecular outflow. The diversity of spectroscopic properties observed is interpreted in the context of a range of evolutionary stages associated with massive star formation

Pleistocene uplift and palaeoenvironments of Macquarie Island: evidence from palaeobeaches and sedimentary deposits

Macquarie Island (54°30'S, 159°00'E) is an emergent part of the Macquarie Ridge Complex composed of ocean-floor rocks of Miocene age now 4000 m above the ocean floor. A number of landforms, including palaeobeaches now above sea level (a.s.l.)on Macquarie Island, were formed by marine erosion during uplift of the island. During the last Pleistocene period of low sea level (c. 20 ka) the island was three times larger than now. Thermoluminescence (TL) dating of two palaeobeaches indicates Pleistocene ages: 172 ± 40 ka for one at 100 m a.s.l. and 340 ± 80 ka for another at 263 m a.s.l. Matching the altitude sequence of palaeobeaches on Macquarie Island with the pattern of peaks in world sea level determined from deep sea cores allows an independent estimate of beach ages. Comparison of the altitude and sea level sequences most plausibly places the 100 m palaeobeach in Oxygen Isotope Stage 5e (130-125 ka) and the 263 m palaeobeach in Stage 9 (340-330 ka), matching reasonably with the TL dates. Other palaeobeaches at about 50 m and 170-190 m a.s.l. then correlate with high sea levels. We calculate an average rate of uplift forthe island of 0.8 mma-I . At this rate, 4000 m of Macquarie Ridge uplift would have taken about five million years and the top of the island may first have emerged some 700 to 600 ka. During the six Pleistocene glacial-interglacial cycles since then, there has been periglacial rather than glacial activity on cold uplands, but conditions suitable for vegetation of the present type persisted close to sea level

A rapid, sensitive colorimetric assay for the high-throughput screening of transaminases in liquid or solid-phase

A new colorimetric method has been developed to screen transaminases using an inexpensive amine donor. The assay is sensitive, has a low level of background coloration, and can be used to identify and profile transaminase activities against aldehyde and ketone substrates in a high-throughput format. Significantly it is also amendable to solid phase colony screening

Furfurylamines from biomass: Transaminase catalysed upgrading of furfurals

Furfural is recognised as an attractive platform molecule for the production of solvents, plastics, resins and fuel additives. Furfurylamines have many applications as monomers in biopolymer synthesis and for the preparation of pharmacologically active compounds, although preparation via traditional synthetic routes is not straightforward due to by-product formation and sensitivity of the furan ring to reductive conditions. In this work transaminases (TAms) have been investigated as a mild sustainable method for the amination of furfural and derivatives to access furfurylamines. Preliminary screening with a recently reported colorimetric assay highlighted that a range of furfurals were readily accepted by several transaminases and the use of different amine donors was then investigated. Multistep synthetic routes were required to synthesise furfurylamine derivatives for use as analytical standards, highlighting the benefits of using a one step biocatalytic route. To demonstrate the potential of using TAms for the production of furfurals, the amination of selected compounds was then investigated on a preparative scale