Exploiting the Anaerobic Expression of Pyruvate Dehydrogenase for the Production of Biofuels

The Pyruvate dehydrogense complex (PDH) is a primarily aerobic enzyme which catalyses pyruvate to acetyl-CoA and carbon dioxide. Its counterpart in anaerobic metabolism is pyruvate formate lyase (Pfl) which converts pyruvate to acetyl-CoA and formate. A novel fermentation pathway involving PDH rather than Pfl (or equivalent), which retains the reducing equivalents from pyruvate oxidation, could provide a novel route for ethanol production, as well as changes in redox balance opening up opportunities for the production of higher alcohols such as butanol. Utilising PDH for the production of biofuels has been investigated in three microorganisms: Geobacillus thermodenitrificans, Bacillus subtilis, and E. coli. Geobacillus thermodenitrificans does express Pfl, thus PDH is always active in the G. thermodenitrificans regardless of whether the bacterium is growing in aerobic or anaerobic conditions. To utilise this PDH in the production of ethanol a bi-functional alcohol dehydrogenase (AdhE) was introduced to G. thermodenitrificans K1041. Further optimisation of ethanol production was achieved by knocking-out lactate dehydrogenase (Ldh), which would otherwise compete with ethanol for flux from acetyl-CoA, and activity of the PDH promoter verses potential alternative promoters to increase the expression of the native PDH was investigated. Like G. thermodenitrificans, Bacillus subtilis also does not have a PFL pathway, but does have a native Adh so can undergo fermentation, albeit poorly. To increase ethanol production competing fermentation pathways were knocked-out, however this resulted in strains which were unable to grow anaerobically. The activity of the native PDH promoter was investigated, and PDH subsequently upregulated. The production of 1-butanol from B. subtilis was also achieved using expression of Clostridial genes encoding a butanol synthetic pathway from a plasmid and from chromosomal integrations. PDH in Gram-negative bacteria such as E. coli are not active during anerobic growth due to fermentation resulting in elevated levels of intracellular NADH; which in turn triggers negative feedback inhibition of PDH. A consequence of this is E. coli strains which are engineered to produce increased titres of ethanol by knocking-out pfl are unable to grow anaerobically. To alleviate this problem a PDH from gram-positive bacteria was expressed in E. coli. The effect of these PDH was also used to assess their potential benefits on 1-butanol in E. coli, by introducing Clostridial genes encoding a butanol synthetic pathway via plasmids

A 1.5-million-year record of orbital and millennial climate variability in the North Atlantic

Climate during the last glacial period was marked by abrupt instability on millennial timescales that included large swings of temperature in and around Greenland (Daansgard-Oeschger events) and smaller, more gradual changes in Antarctica (AIM events). Less is known about the existence and nature of similar variability during older glacial periods, especially during the early Pleistocene when glacial cycles were dominantly occurring at 41 kyr intervals compared to the much longer and deeper glaciations of the more recent period. Here, we report a continuous millennially resolved record of stable isotopes of planktic and benthic foraminifera at IODP Site U1385 (the "Shackleton Site") from the southwestern Iberian margin for the last 1.5 million years, which includes the Middle Pleistocene Transition (MPT). Our results demonstrate that millennial climate variability (MCV) was a persistent feature of glacial climate, both before and after the MPT. Prior to 1.2 Ma in the early Pleistocene, the amplitude of MCV was modulated by the 41 kyr obliquity cycle and increased when axial tilt dropped below 23.5° and benthic δ18O exceeded ∼3.8 ‰ (corrected to Uvigerina), indicating a threshold response to orbital forcing. Afterwards, MCV became focused mainly on the transitions into and out of glacial states (i.e. inceptions and terminations) and during times of intermediate ice volume. After 1.2 Ma, obliquity continued to play a role in modulating the amplitude of MCV, especially during times of glacial inceptions, which are always associated with declining obliquity. A non-linear role for obliquity is also indicated by the appearance of multiples (82, 123 kyr) and combination tones (28 kyr) of the 41 kyr cycle. Near the end of the MPT (∼0.65 Ma), obliquity modulation of MCV amplitude wanes as quasi-periodic 100 kyr and precession power increase, coinciding with the growth of oversized ice sheets on North America and the appearance of Heinrich layers in North Atlantic sediments. Whereas the planktic δ18O of Site U1385 shows a strong resemblance to Greenland temperature and atmospheric methane (i.e. Northern Hemisphere climate), millennial changes in benthic δ18O closely follow the temperature history of Antarctica for the past 800 kyr. The phasing of millennial planktic and benthic δ18O variation is similar to that observed for MIS 3 throughout much of the record, which has been suggested to mimic the signature of the bipolar seesaw - i.e. an interhemispheric asymmetry between the timing of cooling in Antarctica and warming in Greenland. The Iberian margin isotopic record suggests that bipolar asymmetry was a robust feature of interhemispheric glacial climate variations for at least the past 1.5 Ma despite changing glacial boundary conditions. A strong correlation exists between millennial increases in planktic δ18O (cooling) and decreases in benthic δ13C, indicating that millennial variations in North Atlantic surface temperature are mirrored by changes in deep-water circulation and remineralization of carbon in the abyssal ocean. We find strong evidence that climate variability on millennial and orbital scales is coupled across different timescales and interacts in both directions, which may be important for linking internal climate dynamics and external astronomical forcing

A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants

Most published genome sequences are drafts, and most are dominated by computational gene prediction. Draft genomes typically incorporate considerable sequence data that are not assigned to chromosomes, and predicted genes without quality confidence measures. The current Actinidia chinensis (kiwifruit) 'Hongyang' draft genome has 164\ua0Mb of sequences unassigned to pseudo-chromosomes, and omissions have been identified in the gene models

The first continuous Late Glacial - Holocene peat bog multi-proxy record from the Dolomites (NE Italian Alps)

Paleoclimate and paleoenvironmental studies in the north-eastern Italian Alps have been hampered by the rarity of well-preserved high-altitude deposits and the lack of high-resolution multi-proxy records with adequate chronological control. This paper presents the first complete Late Glacial to Holocene peat succession from the Dolomites (Danta di Cadore, Belluno, Italian Alps). A 7 m core was used to evaluate the potential of the ombrotrophic Val di Ciampo peat deposit (1400 m a.s.l.) as an archive of environmental and climate change. The depth age scale is based upon independent C-14 and Pb-210 dates and combined with peat stratigraphy demonstrates that the peat core covers more than 13,200 cal BP, extending to the end of the last part of the Late Glacial. Bulk density, inorganic matter content, pore water pH, conductivity, Ca/Mg ratios, and Ca, Sr and Ti trends were used to identify changes in trophic conditions through the bog. The boundary between ombrotrophic and minerotrophic conditions occurs at approximately 400 cm below the surface and demonstrates that this core is the longest Eastern Alpine ombrotrophic record yet obtained, corresponding to 7000 cal BP. The high-resolution chemical data of this peat archive improves our understanding of European Alpine Holocene climate variability and the relationship between natural climate fluctuations and anthropogenic climate change during the present interglacial in the Dolomites

High quality genome annotation and expression visualisation of a mupirocin-producing bacterium

Pseudomonas strain NCIMB10586, in the P. fluorescens subgroup, produces the polyketide antibiotic mupirocin, and has potential as a host for industrial production of a range of valuable products. To underpin further studies on its genetics and physiology, we have used a combination of standard and atypical approaches to achieve a quality of the genome sequence and annotation, above current standards for automated pathways. Assembly of Illumina reads to a PacBio genome sequence created a retrospectively hybrid assembly, identifying and fixing 415 sequencing errors which would otherwise affect almost 5% of annotated coding regions. Our annotation pipeline combined automation based on related well-annotated genomes and stringent, partially manual, tests for functional features. The strain was close to P. synxantha and P. libaniensis and was found to be highly similar to a strain being developed as a weed-pest control agent in Canada. Since mupirocin is a secondary metabolite whose production is switched on late in exponential phase, we carried out RNAseq analysis over an 18 h growth period and have developed a method to normalise RNAseq samples as a group, rather than pair-wise. To review such data we have developed an easily interpreted way to present the expression profiles across a region, or the whole genome at a glance. At the 2-hour granularity of our time-course, the mupirocin cluster increases in expression as an essentially uniform bloc, although the mupirocin resistance gene stands out as being expressed at all the time points

Genome editing reveals that pSCL4 is required for chromosome linearity in Streptomyces clavuligerus

Streptomyces clavuligerus is an industrially important actinomycete whose genetic manipulation is limited by low transformation and conjugation efficiencies, low levels of recombination of introduced DNA, and difficulty in obtaining consistent sporulation. We describe the construction and application of versatile vectors for Cas9-mediated genome editing of this strain. To design spacer sequences with confidence, we derived a highly accurate genome assembly for an isolate of the type strain (ATCC 27064). This yielded a chromosome assembly (6.75 Mb) plus assemblies for pSCL4 (1795 kb) and pSCL2 (149 kb). The strain also carries pSCL1 (12 kb), but its small size resulted in only partial sequence coverage. The previously described pSCL3 (444 kb) is not present in this isolate. Using our Cas9 vectors, we cured pSCL4 with high efficiency by targeting the plasmid's parB gene. Five of the resulting pSCL4-cured isolates were characterized and all showed impaired sporulation. Shotgun genome sequencing of each of these derivatives revealed large deletions at the ends of the chromosomes in all of them, and for two clones sufficient sequence data was obtained to show that the chromosome had circularized. Taken together, these data indicate that pSCL4 is essential for the structural stability of the linear chromosome