Plant triterpenoids constitute a diverse class of organic compounds that play a major role in development, plant defence and environmental interaction. Several triterpenes have demonstrated potential as pharmaceuticals. One example is betulin, which has shown promise as a pharmaceutical precursor for the treatment of certain cancers and HIV. Major challenges for triterpenoid commercialization include their low production levels and their cost-effective purification from the complex mixtures present in their natural hosts. Therefore, attempts to produce these compounds in industrially relevant microbial systems such as bacteria and yeasts have attracted great interest. Here, we report the production of the triterpenes betulin and its precursor lupeol in the photosynthetic diatom Phaeodactylum tricornutum, a unicellular eukaryotic alga. This was achieved by introducing three plant enzymes in the microalga: a Lotus japonicus oxidosqualene cyclase and a Medicago truncatula cytochrome P450 along with its native reductase. The introduction of the L. japonicus oxidosqualene cyclase perturbed the mRNA expression levels of the native mevalonate and sterol biosynthesis pathway. The best performing strains were selected and grown in a 550-L pilot-scale photobioreactor facility. To our knowledge, this is the most extensive pathway engineering undertaken in a diatom and the first time that a sapogenin has been artificially produced in a microalga, demonstrating the feasibility of the photo-bio-production of more complex high-value, metabolites in microalgae.

The use of microorganisms as cell factories frequently requires extensive molecular manipulation. Therefore, the identification of genomic neutral sites for the stable integration of ectopic DNA is required to ensure a successful outcome. Here we describe the genome mapping and validation of five neutral sites in the chromosome of Synechocystis sp. PCC 6803, foreseeing the use of this cyanobacterium as a photoautotrophic chassis. To evaluate the neutrality of these loci, insertion/deletion mutants were produced, and to assess their functionality, a synthetic green fluorescent reporter module was introduced. The constructed integrative vectors include a BioBrick-compatible multiple cloning site insulated by transcription terminators, constituting robust cloning interfaces for synthetic biology approaches. Moreover, Synechocystis mutants (chassis) ready to receive purpose-built synthetic modules/circuits are also available. This work presents a systematic approach to map and validate chromosomal neutral sites in cyanobacteria, and that can be extended to other organisms.

There are few studies defining CHO host cell proteins (HCPs) and the flux of these throughout a downstream purification process. Here we have applied quantitative iTRAQ proteomics to follow the HCP profile of an antibody (mAb) producing CHO‐S cell line throughout a standard downstream purification procedure consisting of a Protein A, cation and anion exchange process. We used both 6 sample iTRAQ experiment to analyze technical replicates of three samples, which were culture harvest (HCCF), Protein A flow through and Protein A eluate and an 8 sample format to analyze technical replicates of four sample types; HCCF compared to Protein A eluate and subsequent cation and anion exchange purification. In the 6 sample iTRAQ experiment, 8781 spectra were confidently matched to peptides from 819 proteins (including the mAb chains). Across both the 6 and 8 sample experiments 936 proteins were identified. In the 8 sample comparison, 4187 spectra were confidently matched to peptides from 219 proteins. We then used the iTRAQ data to enable estimation of the relative change of individual proteins across the purification steps. These data provide the basis for application of iTRAQ for process development based upon knowledge of critical HCPs.

Post-translational modifications (PTMs), mass spectrometry (MS), liquid chromatography (LC), tandem MS (MS/MS), phosphopeptide (p-peptide), label-free quantification (LFQ), stable isotope labelling by amino acids in cell culture (SILAC), Tandem Mass Tags (TMT) isobaric tags for relative and absolute quantification (iTRAQ), phospho-serine (pSer), threonine (pThr), tyrosine (pTyr), immobilized metal ion affinity chromatography (IMAC), metal oxide affinity chromatography (MOAC), acetonitrile (ACN), sodium dodecyl sulfate -polyacrylamide gel electrophoresis (SDS-PAGE), filter assisted sample preparation (FASP), solid phase extraction (SPE), high performance liquid chromatography (HPLC), reverse phase (RP), strong cation exchange (SCX), electrostatic repulsion hydrophilic interaction chromatography (ERLIC), solution isoelectric focusing (sIEF), strong anion exchange (SAX), histidine (pHis), arginine (Arg),

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Seaweeds contain many varied and commercially valuable components, from individual pigments and metabolites through to whole biomass, and yet they remain an under cultivated and underutilised commodity. Currently, commercial exploitation of seaweeds is predominantly limited to whole biomass consumption or single product extracts for the food industry. The development of a seaweed biorefinery, based around multiple products and services, could provide an important opportunity to exploit new and currently underexplored markets. Here, we assessed the native and invasive seaweeds on the South West coast of the UK to determine their characteristics and potential for exploitation through a biorefinery pipeline, looking at multiple components including pigments, carbohydrates, lipids, proteins and other metabolites.

Electro-coagulation floatation (ECF) is a foam-floatation dewatering method that has been shown to be a highly effective, rapid, and scalable separation methodology. In this manuscript, an in-depth analysis of the gas and flocculant levels observed during the process is provided, with microbubbles observed in the 5–80 μm size range at a concentration of 10 2 –10 3 bubbles mL −1 . Electrolysis of microalgae culture was then observed, demonstrating both effective separation using aluminium electrodes (nine microalgal species tested, 1–40 μm size range, motile and non-motile, marine and freshwater), and sterilisation of culture through bleaching with inert titanium electrodes. Atomic force microscopy was used to visualise floc formation in the presence and absence of algae, showing nanoscale structures on the magnitude of 40–400 nm and entrapped microalgal cells. Improvements to aid industrial biotechnology processing were investigated: protein-doping was found to improve foam stability without inducing cell lysis, and an oxalate buffer wash regime was found to dissolve the flocculant whilst producing no observable difference in the final algal lipid or pigment profiles, leaving the cells viable at the end of the process. ECF separated microalgal culture had an algal biomass loading of 13% and as such was ideal for direct down-stream processing through hydrothermal liquefaction. High bio-crude yields were achieved, though this was reduced slightly on addition of the Al(OH) 3 after ECF, with carbon being distributed away to the aqueous and solid residue phases. The amenability and compatibility of ECF to integration with, or replacement of, existing centrifugation and settling processes suggests this process may be of significant interest to the biotechnology industry.

Highlights Proteomics is widely used in production strain analysis  The value of specific strategies is discussed with reference to case studies  Methodologies often based on prior application to eukaryotic systems  New developments target quantitative accuracy and proteome coverage AbstractProteomics is the large-scale study and analysis of proteins, directed to analysing protein function in a cellular context. Since the vast majority of the processes occurring in a living cell rely on protein activity, proteomics offer a unique vantage point from which researchers can dissect, characterise, understand and manipulate biological systems. When developing a production strain, proteomics offers a versatile toolkit of analytical techniques. In this commentary, we highlight a number of recent developments in this field using three industrially relevant case studies: targeted proteomic analysis of heterologous pathways in E. coli, biofuel production in Synechocystis PCC6803 and proteomic investigations of lignocellulose degradation. We conclude by discussing future developments in proteomics that will impact upon metabolic engineering and process monitoring of bio-producer strains.