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Microbial cell factories are the workhorses of industrial biotechnology and improving their performances can significantly optimize industrial bioprocesses. Microbial strain engineering is often employed for increasing the competitiveness of bio‐based product synthesis over more classical petroleum‐based synthesis. Recently, efforts for strain optimization have been standardized within the iterative concept of “design‐build‐test‐learn” (DBTL). This approach has been successfully employed for the improvement of traditional cell factories like Escherichia coli and Saccharomyces cerevisiae. Within the past decade, several new‐to‐industry microorganisms have been investigated as novel cell factories, including the versatile α‐proteobacterium Rhodobacter sphaeroides. Despite its history as a laboratory strain for fundamental studies, there is a growing interest in this bacterium for its ability to synthesize relevant compounds for the bioeconomy, such as isoprenoids, poly‐β‐hydroxybutyrate, and hydrogen. In this study, we reflect on the reasons for establishing R. sphaeroides as a cell factory from the perspective of the DBTL concept. Moreover, we discuss current and future opportunities for extending the use of this microorganism for the bio‐based economy. We believe that applying the DBTL pipeline for R. sphaeroides will further strengthen its relevance as a microbial cell factory. Moreover, the proposed use of strain engineering via the DBTL approach may be extended to other microorganisms that have not been critically investigated yet for industrial applications., Rhodobacter sphaeroides is a purple non‐sulfur bacteria which has served for decades as model laboratory strain for fundamental studies. Nevertheless, in the last decades this microorganism has matured into a versatile microbial cell factory for multiple biotechnological processes. In this work, authors describe this transition by means of the ‘design‐build‐test‐learn’ concept for strain engineering.

Background Microbial cell factories are usually engineered and employed for cultivations that combine product synthesis with growth. Such a strategy inevitably invests part of the substrate pool towards the generation of biomass and cellular maintenance. Hence, engineering strains for the formation of a specific product under non-growth conditions would allow to reach higher product yields. In this respect, isoprenoid biosynthesis represents an extensively studied example of growth-coupled synthesis with rather unexplored potential for growth-independent production. Rhodobacter sphaeroides is a model bacterium for isoprenoid biosynthesis, either via the native 2-methyl-d-erythritol 4-phosphate (MEP) pathway or the heterologous mevalonate (MVA) pathway, and for poly-β-hydroxybutyrate (PHB) biosynthesis. Results This study investigates the use of this bacterium for growth-independent production of isoprenoids, with amorpha-4,11-diene as reporter molecule. For this purpose, we employed the recently developed Cas9-based genome editing tool for R. sphaeroides to rapidly construct single and double deletion mutant strains of the MEP and PHB pathways, and we subsequently transformed the strains with the amorphadiene producing plasmid. Furthermore, we employed 13C-metabolic flux ratio analysis to monitor the changes in the isoprenoid metabolic fluxes under different cultivation conditions. We demonstrated that active flux via both isoprenoid pathways while inactivating PHB synthesis maximizes growth-coupled isoprenoid synthesis. On the other hand, the strain that showed the highest growth-independent isoprenoid yield and productivity, combined the plasmid-based heterologous expression of the orthogonal MVA pathway with the inactivation of the native MEP and PHB production pathways. Conclusions Apart from proposing a microbial cell factory for growth-independent isoprenoid synthesis, this work provides novel insights about the interaction of MEP and MVA pathways under different growth conditions.

Summary Advances in synthetic biology and metabolic engineering have proven the potential of introducing metabolic by‐passes within cell factories. These pathways can provide a more efficient alternative to endogenous counterparts due to their insensitivity to host's regulatory mechanisms. In this work, we replaced the endogenous essential 2‐C‐methyl‐D‐erythritol 4‐phosphate (MEP) pathway for isoprenoid biosynthesis in the industrially relevant bacterium Rhodobacter sphaeroides by an orthogonal metabolic route. The native 2‐C‐methyl‐D‐erythritol 4‐phosphate (MEP) pathway was successfully replaced by a heterologous mevalonate (MVA) pathway from a related bacterium. The functional replacement was confirmed by analysis of the reporter molecule amorpha‐4,11‐diene after cultivation with [4‐13C]glucose. The engineered R. sphaeroides strain relying exclusively on the MVA pathway was completely functional in conditions for sesquiterpene production and, upon increased expression of the MVA enzymes, it reached even higher sesquiterpene yields than the control strain coexpressing both MEP and MVA modules. This work represents an example where substitution of an essential biochemical pathway by an alternative, heterologous pathway leads to enhanced biosynthetic performance., This work describes the design and implementation of pathway replacement in isoprenoid metabolism in the bacterium Rhodobacter sphaeroides. Integration of a heterologous mevalonate pathway was followed by the inactivation of the endogenous MEP pathway via Cas9 counter‐selection. The resulting strain showed increased amorphadiene yields compared to the parental strain. Therefore replacement of endogenous pathways with non‐native counterparts is suggested for rational design of microbial cell factories.

Metabolic engineering for increased isoprenoid production often benefits from the simultaneous expression of the two naturally available isoprenoid metabolic routes, namely the 2-methyl-D-erythritol 4-phosphate (MEP) pathway and the mevalonate (MVA) pathway. Quantification of the contribution of these pathways to the overall isoprenoid production can help to obtain a better understanding of the metabolism within a microbial cell factory. Such type of investigation can benefit from 13C metabolic flux ratio studies. Here, we designed a method based on parallel labeling experiments (PLEs), using [1-13C]- and [4-13C]glucose as tracers to quantify the metabolic flux ratios in the glycolytic and isoprenoid pathways. By just analyzing a reporter isoprenoid molecule and employing only four equations, we could describe the metabolism involved from substrate catabolism to product formation. These equations infer 13C atom incorporation into the universal isoprenoid building blocks, isopentenyl-pyrophosphate (IPP) and dimethylallyl-pyrophosphate (DMAPP). Therefore, this renders the method applicable to the study of any of isoprenoid of interest. As proof of principle, we applied it to study amorpha-4,11-diene biosynthesis in the bacterium Rhodobacter sphaeroides. We confirmed that in this species the Entner-Doudoroff pathway is the major pathway for glucose catabolism, while the Embden-Meyerhof-Parnas pathway contributes to a lesser extent. Additionally, we demonstrated that co-expression of the MEP and MVA pathways caused a mutual enhancement of their metabolic flux capacity. Surprisingly, we also observed that the isoprenoid flux ratio remains constant under exponential growth conditions, independently from the expression level of the MVA pathway. Apart from proposing and applying a tool for studying isoprenoid biosynthesis within a microbial cell factory, our work reveals important insights from the co-expression of MEP and MVA pathways, including the existence of a yet unclear interaction between them.

Materials science and genetic engineering have joined forces over the last three decades in the development of so-called protein-based polymers. These are proteins, typically with repetitive amino acid sequences, that have such physical properties that they can be used as functional materials. Well-known natural examples are collagen, silk, and elastin, but also artificial sequences have been devised. These proteins can be produced in a suitable host via recombinant DNA technology, and it is this inherent control over monomer sequence and molecular size that renders this class of polymers of particular interest to the fields of nanomaterials and biomedical research. Traditionally, Escherichia coli has been the main workhorse for the production of these polymers, but the methylotrophic yeast Pichia pastoris is finding increased use in view of the often high yields and potential bioprocessing benefits. We here provide an overview of protein-based polymers produced in P. pastoris. We summarize their physicochemical properties, briefly note possible applications, and detail their biosynthesis. Some challenges that may be faced when using P. pastoris for polymer production are identified: (i) low yields and poor process control in shake flask cultures; i.e., the need for bioreactors, (ii) proteolytic degradation, and (iii) self-assembly in vivo. Strategies to overcome these challenges are discussed, which we anticipate will be of interest also to readers involved in protein expression in P. pastoris in general.

Background Cutaneotrichosporon oleaginosus ATCC 20509 is a fast-growing oleaginous basidiomycete yeast that is able to grow in a wide range of low-cost carbon sources including crude glycerol, a byproduct of biodiesel production. When glycerol is used as a carbon source, this yeast can accumulate more than 50% lipids (w/w) with high concentrations of mono-unsaturated fatty acids. Results To increase our understanding of this yeast and to provide a knowledge base for further industrial use, a FAIR re-annotated genome was used to build a genome-scale, constraint-based metabolic model containing 1553 reactions involving 1373 metabolites in 11 compartments. A new description of the biomass synthesis reaction was introduced to account for massive lipid accumulation in conditions with high carbon-to-nitrogen (C/N) ratio in the media. This condition-specific biomass objective function is shown to better predict conditions with high lipid accumulation using glucose, fructose, sucrose, xylose, and glycerol as sole carbon source. Conclusion Contributing to the economic viability of biodiesel as renewable fuel, C. oleaginosus ATCC 20509 can effectively convert crude glycerol waste streams in lipids as a potential bioenergy source. Performance simulations are essential to identify optimal production conditions and to develop and fine tune a cost-effective production process. Our model suggests ATP-citrate lyase as a possible target to further improve lipid production.

Background Microbial cell factories are usually engineered and employed for cultivations that combine product synthesis with growth. Such a strategy inevitably invests part of the substrate pool towards the generation of biomass and cellular maintenance. Hence, engineering strains for the formation of a specific product under non-growth conditions would allow to reach higher product yields. In this respect, isoprenoid biosynthesis represents an extensively studied example of growth-coupled synthesis with rather unexplored potential for growth-independent production. Rhodobacter sphaeroides is a model bacterium for isoprenoid biosynthesis, either via the native 2-methyl-d-erythritol 4-phosphate (MEP) pathway or the heterologous mevalonate (MVA) pathway, and for poly-β-hydroxybutyrate (PHB) biosynthesis. Results This study investigates the use of this bacterium for growth-independent production of isoprenoids, with amorpha-4,11-diene as reporter molecule. For this purpose, we employed the recently developed Cas9-based genome editing tool for R. sphaeroides to rapidly construct single and double deletion mutant strains of the MEP and PHB pathways, and we subsequently transformed the strains with the amorphadiene producing plasmid. Furthermore, we employed 13C-metabolic flux ratio analysis to monitor the changes in the isoprenoid metabolic fluxes under different cultivation conditions. We demonstrated that active flux via both isoprenoid pathways while inactivating PHB synthesis maximizes growth-coupled isoprenoid synthesis. On the other hand, the strain that showed the highest growth-independent isoprenoid yield and productivity, combined the plasmid-based heterologous expression of the orthogonal MVA pathway with the inactivation of the native MEP and PHB production pathways. Conclusions Apart from proposing a microbial cell factory for growth-independent isoprenoid synthesis, this work provides novel insights about the interaction of MEP and MVA pathways under different growth conditions.

Genetic transformation of microalgae remains a challenge due to poor intracellular delivery of exogenous molecules. This limitation is caused by the structure and composition of the cell wall and cell membrane of each species. Moreover, successful delivery of proteins or nucleic acids cannot be assessed by determining transformability since their functionality is not always known in the studied microorganism. We propose a quick and effective screening tool for the prediction and optimization of electroporation settings by monitoring cell permeability and viability using Sytox Green and propidium iodide respectively. We determined voltage settings for the microalgae Chlamydomonas reinhardtii, Chlorella vulgaris, Neochloris oleoabundans and Acutodesmus obliquus. To evaluate the predicted settings, we delivered labelled DNA and proteins into the cells. We demonstrated that high transformation efficiencies can be accomplished when predicted values were applied with functional plasmids. Additionally, we increased transformation efficiencies by testing cell concentrations, light intensities and fragment sizes. This method can be used to determine suitable transformation conditions for non-transformed microalgae species and to increase the insight on established transformation protocols.

The oleaginous microalga Ettlia oleoabundans accumulates both starch and lipids to high levels under stress conditions such as nitrogen starvation (N−). To steer biosynthesis towards starch or lipids only, it is important to understand the regulatory mechanisms involved. Here physiological and transcriptional changes under nitrogen starvation were analysed in controlled flat-panel photobioreactors at both short and long time-scales. Starch accumulation was transient and occurred rapidly within 24 h upon starvation, while lipid accumulation was gradual and reached a maximum after 4 days. The major fraction of accumulated lipids was composed of de novo synthesized neutral lipids - triacylglycerides (TAG) - and was characterized by a decreased composition of the polyunsaturated fatty acids (PUFAs) C18:3 and C16:3 and an increased composition of the mono-unsaturated (MUFAs) and saturated (SFAs) fatty acids C18:1/C16:1 and C18:0/C16:0, respectively. RNA-sequencing revealed that starch biosynthesis and degradation genes show different expression dynamics from lipid biosynthesis ones. An immediate rapid increase in starch synthetic transcripts was followed by an increase in starch degrading transcripts and a decrease in the starch synthetic ones. In contrast, increased gene expression for fatty acid and TAG synthesis was initiated later and occurred more gradually. Expression of several fatty acid desaturase (FAD) genes was decreased upon starvation, which corresponds to the observed changes to higher levels of MUFAs and SFAs. Moreover, several homologs of transcription regulators that were implicated in controlling starch and lipid metabolism in other microalgae showed differential gene expression and might be key regulators of starch and lipid metabolism in E. oleoabundans as well. Our data provide insights into the genetic foundation of starch and lipid metabolism in E. oleoabundans under nitrogen starvation and should facilitate metabolic engineering towards tailored strains with desired storage compound composition. Fil: Sturme, Mark H.J.. University of Agriculture Wageningen; Países Bajos Fil: Gong, Yanhai. Qingdao Institute Of Bioenergy And Bioprocess Technolog; China Fil: Heinrich, Josué Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Klok, Anne J.. University of Agriculture Wageningen; Países Bajos Fil: Eggink, Gerrit. University of Agriculture Wageningen; Países Bajos Fil: Wang, Dongmei. Qingdao Institute Of Bioenergy And Bioprocess Technolog; China Fil: Xu, Jian. Qingdao Institute Of Bioenergy And Bioprocess Technolog; China Fil: Wijffels, Rene H.. University of Agriculture Wageningen; Países Bajos

Thermostable enzymes are a promising alternative for chemical catalysts currently used for the production of N-acetylglucosamine (GlcNAc) from chitin. In this study, a novel thermostable β-N-acetylglucosaminidase MthNAG was cloned and purified from the thermophilic fungus Myceliophthora thermophila C1. MthNAG is a protein with a molecular weight of 71 kDa as determined with MALDI-TOF-MS. MthNAG has the highest activity at 50 °C and pH 4.5. The enzyme shows high thermostability above the optimum temperature: at 55 °C (144 h, 75% activity), 60 °C (48 h, 85% activity; half-life 82 h), and 70 °C (24 h, 33% activity; half-life 18 h). MthNAG releases GlcNAc from chitin oligosaccharides (GlcNAc)2–5, p-nitrophenol derivatives of chitin oligosaccharides (GlcNAc)1–3-pNP, and the polymeric substrates swollen chitin and soluble chitosan. The highest activity was detected towards (GlcNAc)2. MthNAG released GlcNAc from the non-reducing end of the substrate. We found that MthNAG and Chitinase Chi1 from M. thermophila C1 synergistically degraded swollen chitin and released GlcNAc in concentration of approximately 130 times higher than when only MthNAG was used. Therefore, chitinase Chi1 and MthNAG have great potential in the industrial production of GlcNAc.

Direct and selective terminal oxidation of medium-chain n-alkanes is a major challenge in chemistry. Efforts to achieve this have so far resulted in low specificity and overoxidized products. Biocatalytic oxidation of medium-chain n-alkanes – with for example the alkane monooxygenase AlkB from P. putida GPo1- on the other hand is highly selective. However, it also results in overoxidation. Moreover, diterminal oxidation of medium-chain n-alkanes is inefficient. Hence, α,ω-bifunctional monomers are mostly produced from olefins using energy intensive, multi-step processes. By combining biocatalytic oxidation with esterification we drastically increased diterminal oxidation upto 92 mol% and reduced overoxidation to 3% for n-hexane. This methodology allowed us to convert medium-chain n-alkanes into α,ω-diacetoxyalkanes and esterified α,ω-dicarboxylic acids. We achieved this in a one-pot reaction with resting-cell suspensions of genetically engineered Escherichia coli. The combination of terminal oxidation and esterification constitutes a versatile toolbox to produce α,ω-bifunctional monomers from n-alkanes.

Metabolic engineering for increased isoprenoid production often benefits from the simultaneous expression of the two naturally available isoprenoid metabolic routes, namely the 2-methyl-D-erythritol 4-phosphate (MEP) pathway and the mevalonate (MVA) pathway. Quantification of the contribution of these pathways to the overall isoprenoid production can help to obtain a better understanding of the metabolism within a microbial cell factory. Such type of investigation can benefit from

Rhodobacter sphaeroides is a metabolically versatile bacterium capable of producing terpenes natively. Surprisingly, terpene biosynthesis in this species has always been investigated in complex media, with unknown compounds possibly acting as carbon and nitrogen sources. Here, a defined medium was adapted for R. sphaeroides dark heterotrophic growth, and was used to investigate the conversion of different organic substrates into the reporter terpene amorphadiene. The amorphadiene synthase was cloned in R. sphaeroides, allowing its biosynthesis via the native 2-methyl-d-erythritol-4-phosphate (MEP) pathway and, additionally, via a heterologous mevalonate one. The latter condition increased titers up to eightfold. Consequently, better yields and productivities to previously reported complex media cultivations were achieved. Productivity was further investigated under different cultivation conditions, including nitrogen and oxygen availability. This novel cultivation setup provided useful insight into the understanding of terpene biosynthesis in R. sphaeroides, allowing to better comprehend its dynamics and regulation during chemoheterotrophic cultivation.

Sugars obtained from pretreated lignocellulose are interesting as substrate for the production of lactic acid in fermentation processes. However, by-products formed during pretreatment of lignocellulose can inhibit microbial growth. In this study, a small-scale rapid screening method was used to identify inhibitory effects of single and combined by-products on growth of lactic acid producing micro-organisms. The small-scale screening was performed in 48-well plates using 5 bacterial species and 12 by-products. Large differences were observed in inhibitory effects of by-products between different species. Predictions can be made for growth behaviour of different micro-organisms on acid pretreated or alkaline pretreated bagasse substrates using data from the small-scale screening. Both individual and combined inhibition effects were shown to be important parameters to predict growth. Synergy between coumaric acid, formic acid and acetic acid is a key inhibitory parameter in alkaline pretreated lignocellulose, while furfural is a key inhibitor in acid pretreated lignocellulose.

A thermostable Chitinase Chi1 from Myceliophthora thermophila C1 was homologously produced and characterized. Chitinase Chi1 shows high thermostability at 40 °C (>140 h 90% activity), 50 °C (>168 h 90% activity), and 55 °C (half-life 48 h). Chitinase Chi1 has broad substrate specificity and converts chitin, chitosan, modified chitosan, and chitin oligosaccharides. The activity of Chitinase Chi1 is strongly affected by the degree of deacetylation (DDA), molecular weight (Mw), and side chain modification of chitosan. Chitinase Chi1 releases mainly (GlcNAc)2 from insoluble chitin and chito-oligosaccharides with a polymerization degree (DP) ranging from 2 to 12 from chitosan, in a processive way. Chitinase Chi1 shows higher activity toward chitin oligosaccharides (GlcNAc)4–6 than toward (GlcNAc)3 and is inactive for (GlcNAc)2. During hydrolysis, oligosaccharides bind at subsites −2 to +2 in the enzyme’s active site. Chitinase Chi1 can be used for chitin valorisation and for production of chitin- and chito-oligosaccharides at industrial scale.

Neochloris oleoabundans is an oleaginous microalgal species that can be cultivated in fresh water as well as salt water. Using salt water gives the opportunity to reduce production costs and the fresh water footprint for large scale cultivation. Production of triacylglycerols (TAG) usually includes a biomass growth phase in nitrogen-replete conditions followed by a TAG accumulation phase under nitrogen-deplete conditions. This is the first report that provides insight in the saline resistance mechanism of a fresh water oleaginous microalgae. To better understand the osmoregulatory mechanism of N. oleoabundans during growth and TAG accumulating conditions, the transcriptome was sequenced under four different conditions: fresh water nitrogen-replete and -deplete conditions, and salt water (525 mM dissolved salts, 448mM extra NaCl) nitrogen-replete and -deplete conditions. In this study, several pathways are identified to be responsible for salt water adaptation of N. oleoabundans under both nitrogen-replete and -deplete conditions. Proline and the ascorbate-glutathione cycle seem to be of importance for successful osmoregulation in N. oleoabundans. Genes involved in Proline biosynthesis were found to be upregulated in salt water. This was supported by Nuclear magnetic resonance (NMR) spectroscopy, which indicated an increase in proline content in the salt water nitrogen-replete condition. Additionally, the lipid accumulation pathway was studied to gain insight in the gene regulation in the first 24 hours after nitrogen was depleted. Oil accumulation is increased under nitrogen-deplete conditions in a comparable way in both fresh and salt water. The mechanism behind the biosynthesis of compatible osmolytes can be used to improve N. oleoabundans and other industrially relevant microalgal strains to create a more robust and sustainable production platform for microalgae derived products in the future.

Bernard Witholt was appointed a professor of biochemistry at the University of Groningen at the age of 30 years. He was born in 1941 in The Hague (the Netherlands), spent his early life in Brazil and moved to the USA during his teenage years. After studying biology at Amherst College (Massachusetts), he undertook a PhD at Johns Hopkins University in Baltimore. Before returning to the Netherlands, he did his post-doc and became a staff member at the Department of Biology and Chemistry at the University of California, San Diego.

Background Medium chain length (C6–C12) α,ω-dicarboxylic acids (DCAs) and corresponding esters are important building blocks for the polymer industry. For DCAs of 12 carbon atoms and longer, a sustainable process based on monooxygenase catalyzed ω-oxidation of fatty-acids has been realized. For medium-chain DCAs with a shorter chain length however, such a process has not been developed yet, since monooxygenases poorly ω-oxidize medium-chain fatty acids (MCFAs). On the contrary, esterified MCFAs are ω-oxidized well by the AlkBGTHJ proteins from Pseudomonas putida GPo1. Results We show that MCFAs can be efficiently esterified and subsequently ω-oxidized in vivo. We combined ethyl ester synthesis and ω-oxidation in one-pot, whole-cell biocatalysis in Escherichia coli. Ethyl ester production was achieved by applying acyl-CoA ligase AlkK and an alcohol acyltransferase, either AtfA or Eeb1. E. coli expressing these proteins in combination with the ω-oxidation pathway consisting of AlkBGTHJ, produced mono-ethyl DCAs directly from C6, C8 and C9 fatty acids. The highest molar yield was 0.75, for mono-ethyl azelate production from nonanoic acid. Furthermore, di-ethyl esters were produced. Diethyl suberate was produced most among the di-ethyl esters, with a molar yield of 0.24 from octanoic acid. Conclusion The results indicate that esterification of MCFAs and subsequent ω-oxidation to mono-ethyl DCAs via whole-cell biocatalysis is possible. This process could be the first step towards sustainable production of medium-chain DCAs and medium-chain di-ethyl esters. Electronic supplementary material The online version of this article (10.1186/s12934-017-0803-9) contains supplementary material, which is available to authorized users.

In this study, stearoyl-ACP desaturase (SAD), the enzyme that converts stearic acid into oleic acid, is silenced by artificial microRNA in the green microalga Chlamydomonas reinhardtii. Two different constructs, which target different positions on the mRNA of stearoyl-ACP desaturase, were tested. The mRNA levels for SAD were reduced after the silencing construct was induced. In one of the strains, the reduction in SAD mRNA resulted in a doubling of the stearic acid content in triacylglycerol molecules, which shows that stearic acid production in microalgae is possible.

A Monascus ruber strain was isolated that was able to grow on mineral medium at high sugar concentrations and 175 g/l lactic acid at pH 2.8. Its genome and transcriptomes were sequenced and annotated. Genes encoding lactate dehydrogenase (LDH) were introduced to accomplish lactic acid production and two genes encoding pyruvate decarboxylase (PDC) were knocked out to subdue ethanol formation. The strain preferred lactic acid to glucose as carbon source, which hampered glucose consumption and therefore also lactic acid production. Lactic acid consumption was stopped by knocking out 4 cytochrome-dependent LDH (CLDH) genes, and evolutionary engineering was used to increase the glucose consumption rate. Application of this strain in a fed-batch fermentation resulted in a maximum lactic acid titer of 190 g/l at pH 3.8 and 129 g/l at pH 2.8, respectively 1.7 and 2.2 times higher than reported in literature before. Yield and productivity were on par with the best strains described in literature for lactic acid production at low pH.

Summary The AlkBGTL proteins coded on the alk operon from Pseudomonas putida GPo1 can selectively ω‐oxidize ethyl esters of C6 to C10 fatty acids in whole‐cell conversions with Escherichia coli. The major product in these conversions is the ω‐alcohol. However, AlkB also has the capacity to overoxidize the substrate to the ω‐aldehyde and ω‐acid. In this study, we show that alcohol dehydrogenase AlkJ and aldehyde dehydrogenase AlkH are able to oxidize ω‐alcohols and ω‐aldehydes of esterified fatty acids respectively. Resting E. coli expressing AlkBGTHJL enabled exclusive mono‐ethyl azelate production from ethyl nonanoate, with an initial specific activity of 61 U gcdw −1. Within 2 h, this strain produced 3.53 mM mono‐ethyl azelate, with a yield of 0.68 mol mol−1. This strain also produced mono‐ethyl dicarboxylic acids from ethyl esters of C6 to C10 fatty acids and mono‐methyl azelate from methyl nonanoate. Adding ethyl nonanoate dissolved in carrier solvent bis‐(2‐ethylhexyl) phthalate enabled an increase in product titres to 15.55 mM in two‐liquid phase conversions. These findings indicate that E. coli expressing AlkBGTHJL is an effective producer of mono‐esterified dicarboxylic acids from fatty acid esters.

The microalgae Tetradesmus obliquus is able to maintain a high photosynthetic efficiency under nitrogen limitation and is considered a promising green microalgae for sustainable production of diverse compounds, including biofuels. Here, we report the first draft whole-genome shotgun sequencing of T. obliquus . The final assembly comprises 108,715,903 bp with over 1,368 scaffolds.

Interest in sustainable development has led to efforts to replace petrochemical-based monomers with biomass-based ones. Itaconic acid, a C5-dicarboxylic acid, is a potential monomer for the chemical industry with many prospective applications. cis-aconitate decarboxylase (CadA) is the key enzyme of itaconate production, converting the citric acid cycle intermediate cis-aconitate into itaconate. Heterologous expression of cadA from Aspergillus terreus in Escherichia coli resulted in low CadA activities and production of trace amounts of itaconate on Luria-Bertani (LB) medium (

Lignocellulose might become an important feedstock for the future development of the biobased economy. Although up to 75 % of the lignocellulose dry weight consists of sugar, it is present in a polymerized state and cannot be used directly in most fermentation processes for the production of chemicals and fuels. Several methods have been developed to depolymerize the sugars present in lignocellulose, making the sugars available for fermentation. In this review, we describe five different pretreatment methods and their effect on the sugar and non-sugar fraction of lignocellulose. For several pretreatment methods and different types of lignocellulosic biomass, an overview is given of by-products formed. Most unwanted by-products present after pretreatment are dehydrated sugar monomers (furans), degraded lignin polymers (phenols) and small organic acids. Qualitative and quantitative effects of these by-products on fermentation processes have been studied. We conclude this review by giving an overview of techniques and methods to decrease inhibitory effects of unwanted by-products.

Background: Sugars derived from lignocellulose-rich sugarcane bagasse can be used as feedstock for production of L(+)-lactic acid, a precursor for renewable bioplastics. In our research, acid-pretreated bagasse was hydrolysed with the enzyme cocktail GC220 and fermented by the moderate thermophilic bacterium Bacillus coagulans DSM2314. Saccharification and fermentation were performed simultaneously (SSF), adding acid-pretreated bagasse either in one batch or in two stages. SSF was performed at low enzyme dosages of 10.5-15.8 FPU/g DW bagasse. Results: The first batch SSF resulted in an average productivity of 0.78 g/l/h, which is not sufficient to compete with lactic acid production processes using high-grade sugars. Addition of 1 g/l furfural to precultures can increase B. coagulans resistance towards by-products present in pretreated lignocellulose. Using furfural-containing precultures, productivity increased to 0.92 g/l/h, with a total lactic acid production of 91.7 g in a 1-l reactor containing 20% W/W DW bagasse. To increase sugar concentrations, bagasse was solubilized with a liquid fraction, obtained directly after acid pretreatment. Solubilizing the bagasse fibres with water increased the average productivity to 1.14 g/l/h, with a total lactic acid production of 84.2 g in a 1-l reactor. Addition of bagasse in two stages reduced viscosity during SSF, resulting in an average productivity in the first 23 h of 2.54 g/l/h, similar to productivities obtained in fermentations using high-grade sugars. Due to fast accumulation of lactic acid, enzyme activity was repressed during two-stage SSF, resulting in a decrease in productivity and a slightly lower total lactic acid production of 75.6 g. Conclusions: In this study, it is shown that an adequate production of lactic acid from lignocellulose was successfully accomplished by a two-stage SSF process, which combines acid-pretreated bagasse, B. coagulans precultivated in the presence of furfural as microorganism, and GC220 as enzyme cocktail. The process may be further improved by enhancing enzyme hydrolysis activities at high lactic acid concentrations.

Pasteurella multocida hyaluronan synthase (PmHAS) is a bi-functional glycosyltransferase, containing a ß1,3-glucuronyltransferase and ß1,4-N-acetylglucosaminetransferase domain. PmHAS catalyzes the elongation of hyaluronan (HA) through the sequential addition of single monosaccharides to the non-reducing end of the hyaluronan chain. Research is focused on the relation between the length of the HA oligo- saccharide and the single-step elongation ki- netics from HA4 up to HA9. It was found that the turnover number kcat increased with length to maximum values of 11 and 14 s-1 for NAc- and UA-transfer, respectively. Interestingly, the spe- cificity constant kcat/KM increased with polymer length from HA5 to HA7 to a value of 44 mM-1·s-1, indicating an oligosaccharide binding site with increasing specificity towards a heptasaccha- ride at the UA domain. The value of kcat/KM re- mained moderately constant around 8 mM-1·s-1 for HA4, HA6, and HA8, indicating a binding site with significantly lower binding specificity at the NAc domain than at the UA domain. These find- ings are further corroborated by a structural homology model of PmHAS, revealing two dis- tinct sites for binding of oligosaccharides of different sizes, one in each transferase domain. Structural alignment studies between PmHAS and glycosyltransferases of the GT-A fold showed significant similarity in the binding of the UDP-sugars and the orientation of the ac- ceptor substrate. These similarities in substrate orientation in the active site and in essential amino acid residues involved in substrate bind- ing were utilized to localize the two HA oligo- saccharide binding sites.

Background Sugars derived from lignocellulose-rich sugarcane bagasse can be used as feedstock for production of l(+)-lactic acid, a precursor for renewable bioplastics. In our research, acid-pretreated bagasse was hydrolysed with the enzyme cocktail GC220 and fermented by the moderate thermophilic bacterium Bacillus coagulans DSM2314. Saccharification and fermentation were performed simultaneously (SSF), adding acid-pretreated bagasse either in one batch or in two stages. SSF was performed at low enzyme dosages of 10.5–15.8 FPU/g DW bagasse. Results The first batch SSF resulted in an average productivity of 0.78 g/l/h, which is not sufficient to compete with lactic acid production processes using high-grade sugars. Addition of 1 g/l furfural to precultures can increase B. coagulans resistance towards by-products present in pretreated lignocellulose. Using furfural-containing precultures, productivity increased to 0.92 g/l/h, with a total lactic acid production of 91.7 g in a 1-l reactor containing 20% W/W DW bagasse. To increase sugar concentrations, bagasse was solubilized with a liquid fraction, obtained directly after acid pretreatment. Solubilizing the bagasse fibres with water increased the average productivity to 1.14 g/l/h, with a total lactic acid production of 84.2 g in a 1-l reactor. Addition of bagasse in two stages reduced viscosity during SSF, resulting in an average productivity in the first 23 h of 2.54 g/l/h, similar to productivities obtained in fermentations using high-grade sugars. Due to fast accumulation of lactic acid, enzyme activity was repressed during two-stage SSF, resulting in a decrease in productivity and a slightly lower total lactic acid production of 75.6 g. Conclusions In this study, it is shown that an adequate production of lactic acid from lignocellulose was successfully accomplished by a two-stage SSF process, which combines acid-pretreated bagasse, B. coagulans precultivated in the presence of furfural as microorganism, and GC220 as enzyme cocktail. The process may be further improved by enhancing enzyme hydrolysis activities at high lactic acid concentrations. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0646-3) contains supplementary material, which is available to authorized users.

The enzyme system AlkBGT from Pseudomonas putida GPo1 can efficiently ω-functionalize fatty acid methyl esters. Outer membrane protein AlkL boosts this ω-functionalization. In this report, it is shown that whole cells of Escherichia coli expressing the AlkBGT system can also ω-oxidize ethyl nonanoate (NAEE). Coexpression of AlkBGT and AlkL resulted in 1.7-fold-higher ω-oxidation activity on NAEE. With this strain, initial activity on NAEE was 70 U/g (dry weight) of cells (g cdw ), 67% of the initial activity on methyl nonanoate. In time-lapse conversions with 5 mM NAEE the main product was 9-hydroxy NAEE (3.6 mM), but also 9-oxo NAEE (0.1 mM) and 9-carboxy NAEE (0.6 mM) were formed. AlkBGT also ω-oxidized ethyl, propyl, and butyl esters of fatty acids ranging from C 6 to C 10 . Increasing the length of the alkyl chain improved the ω-oxidation activity of AlkBGT on esters of C 6 and C 7 fatty acids. From these esters, application of butyl hexanoate resulted in the highest ω-oxidation activity, 82 U/g cdw . Coexpression of AlkL only had a positive effect on ω-functionalization of substrates with a total length of C 11 or longer. These findings indicate that AlkBGT(L) can be applied as a biocatalyst for ω-functionalization of ethyl, propyl, and butyl esters of medium-chain fatty acids. IMPORTANCE Fatty acid esters are promising renewable starting materials for the production of ω-hydroxy fatty acid esters (ω-HFAEs). ω-HFAEs can be used to produce sustainable polymers. Chemical conversion of the fatty acid esters to ω-HFAEs is challenging, as it generates by-products and needs harsh reaction conditions. Biocatalytic production is a promising alternative. In this study, biocatalytic conversion of fatty acid esters toward ω-HFAEs was investigated using whole cells. This was achieved with recombinant Escherichia coli cells that produce the AlkBGT enzymes. These enzymes can produce ω-HFAEs from a wide variety of fatty acid esters. Medium-chain-length acids (C 6 to C 10 ) esterified with ethanol, propanol, or butanol were applied. This is a promising production platform for polymer building blocks that uses renewable substrates and mild reaction conditions.

The tricarboxylic acid (TCA) cycle has been used for decades in the microbial production of chemicals such as citrate, L-glutamate, and succinate. Maximizing yield is key for cost-competitive production. However, for most TCA cycle products, the maximum pathway yield is lower than the theoretical maximum yield (YE). For succinate, this was solved by creating two pathways to the product, using both branches of the TCA cycle, connected by the glyoxylate shunt (GS). A similar solution cannot be applied directly for production of compounds from the oxidative branch of the TCA cycle because irreversible reactions are involved. Here, we describe how this can be overcome and what the impact is on the yield. The TCA cycle is a source of industrially important chemicals. Current pathway yields of chemicals from the TCA cycle are below their theoretical maximum (YE).YE becomes achievable by combining the oxidative and reductive part of the TCA cycle.Metabolic engineering is required to reverse some of the thermodynamically unfeasible steps.

By-products resulting from thermo-chemical pretreatment of lignocellulose can inhibit fermentation of lignocellulosic sugars to lactic acid. Furfural is such a by-product, which is formed during acid pretreatment of lignocellulose. pH-controlled fermentations with 1 L starting volume, containing YP medium and a mixture of lignocellulosic by-products, were inoculated with precultures of Bacillus coagulans DSM2314 to which 1 g/L furfural was added. The addition of furfural to precultures resulted in an increase in l(+)-lactic acid productivity by a factor 2 to 1.39 g/L/h, an increase in lactic acid production from 54 to 71 g and an increase in conversion yields of sugar to lactic acid from 68 to 88 % W/W in subsequent fermentations. The improved performance was not caused by furfural consumption or conversion, indicating that the cells acquired a higher tolerance towards this by-product. The improvement coincided with a significant elongation of B. coagulans cells. Via RNA-Seq analysis, an upregulation of pathways involved in the synthesis of cell wall components such as bacillosamine, peptidoglycan and spermidine was observed in elongated cells. Furthermore, the gene SigB and genes promoted by SigB, such as NhaX and YsnF, were upregulated in the presence of furfural. These genes are involved in stress responses in bacilli.

Like natural tropoelastin, polypeptides based on an elastin-like VPGXG repeat have a characteristic inverse temperature response, which leads to coacervate formation above a certain transition temperature and which could be useful for a variety of applications. The key advantage of elastin-like polypeptides (ELPs) over (tropo)elastin is a full control over this temperature response by adjustment of either the amino acid composition or the chain length, according to insights provided by extensive research. Future application of ELPs will require efficient ELP production systems, and in a previous article, we described the successful use of Pichia pastoris for secreted production of an ELP, with an overall yield of ≈ 200 mg L(-1). In this study, we investigated the influence of changed amino acid composition and chain length on the yield of secreted ELP. We have found that both parameters have a distinct impact on the overall yield, with higher yield for shorter and more hydrophilic ELPs. Because yield and transition temperature (Tt) thus appear to be positively correlated, we hypothesize that good solubility of ELP below the Tt promotes the secreted production and coacervate formation above Tt decreases it.

Pseudomonas putida KT2440-JD1 is able to cometabolize benzoate to cis, cis-muconate in the presence of glucose as growth substrate. P. putida KT2440-JD1 was unable to grow in the presence of concentrations above 50 mM benzoate or 600 mM cis, cis-muconate. The inhibitory effects of both compounds were cumulative. The maximum specific uptake rate of benzoate was higher than the specific production rate of cis, cis-muconate during growth on glucose in the presence of benzoate, indicating that a benzoate derivative accumulated in the cells, which is likely to be catechol. Catechol was shown to reduce the expression level of the ben operon, which encodes the conversion of benzoate to cis, cis-muconate. To prevent overdoses of benzoate, a pH-stat fed-batch process for the production of cis, cis-muconate from benzoate was developed, in which the addition of benzoate was coupled to the acidification of the medium. The maximum specific production rate during the pH-stat fed-batch process was 0.6 g (4.3 mmol) g dry cell weight -1 h -1, whereas 18.5 g L -1 cis, cis-muconate accumulated in the culture medium with a molar product yield of close to 100%. Proteome analysis revealed that the outer membrane protein H1 was upregulated during the pH-stat fed-batch process, whereas the expression of 10 other proteins was reduced. The identified proteins are involved in energy household, transport, translation of RNA, and motility. © 2011 American Institute of Chemical Engineers (AIChE).

The lipase-catalyzed synthesis of cyclic ester oligomers from non-activated succinic acid (A) and 1,4-butanediol (B) in the presence of immobilized Candida antarctica lipase B was investigated. Batch and pulse fed-batch systems were implemented to increase the formation of cyclic ester products. The substrate conversions after 24 h were 86% and 95% under batch and fed-batch operation, respectively and the product of the reaction was, for both systems, a mixture of cyclic (CEOs) and linear (LEOs) ester oligomers. Fed-batch operation afforded a product containing 71% cyclic ester oligomers (CEOs) as compared with only 52% CEOs in batch operation. Cyclic ester oligomers accumulated as the reaction progressed, with the dimer CEO1 the most predominant product (i.e. 50% of the total products formed in fed-batch operation). The pulse fed-batch operation system was superior to the batch operation not only because higher substrate conversion and more CEOs were obtained, but also because it resulted in products with a higher degree of polymerization (DP up to 7). Cyclic ester oligomers are produced from the early stage of the reaction simultaneously with the linear ester oligomers by a ring-closure reaction on the active site of the enzyme, and not as a result of ring-chain equilibria

Heparosan synthase catalyzes the polymerization of heparosan (-4GlcUAβ1-4GlcNAcα1-)(n) by transferring alternatively the monosaccharide units from UDP-GlcUA and UDP-GlcNAc to an acceptor molecule. Details on the heparosan chain initiation by Pasteurella multocida heparosan synthase PmHS2 and its influence on the polymerization process have not been reported yet. By site-directed mutagenesis of PmHS2, the single action transferases PmHS2-GlcUA(+) and PmHS2-GlcNAc(+) were obtained. When incubated together in the standard polymerization conditions, the PmHS2-GlcUA(+)/PmHS2-GlcNAc(+) showed comparable polymerization properties as determined for PmHS2. We investigated the first step occurring in heparosan chain initiation by the use of the single action transferases and by studying the PmHS2 polymerization process in the presence of heparosan templates and various UDP-sugar concentrations. We observed that PmHS2 favored the initiation of the heparosan chains when incubated in the presence of an excess of UDP-GlcNAc. It resulted in a higher number of heparosan chains with a lower average molecular weight or in the synthesis of two distinct groups of heparosan chain length, in the absence or in the presence of heparosan templates, respectively. These data suggest that PmHS2 transfers GlcUA from UDP-GlcUA moiety to a UDP-GlcNAc acceptor molecule to initiate the heparosan polymerization; as a consequence, not only the UDP-sugar concentration but also the amount of each UDP-sugar is influencing the PmHS2 polymerization process. In addition, it was shown that PmHS2 hydrolyzes the UDP-sugars, UDP-GlcUA being more degraded than UDP-GlcNAc. However, PmHS2 incubated in the presence of both UDP-sugars favors the synthesis of heparosan polymers over the hydrolysis of UDP-sugars.

The synthesis of linear ester oligomers (LEOs) and cyclic ester oligomers (CEOs) from non-activated succinic acid (A) in combination with di-anhydro hexitols (B, DAH) in a toluene based medium using immobilized Candida antarctica lipase B (CAL B), was studied. The conversion is highest for isomannide and decreases in the order isomannide > isosorbide ≫ isoidide. These experimental results were corroborated by substrate-imprinted docking indicating that the hydroxyl group oriented inwards the “V”-shaped plane of the DAHs (endo-hydroxyl) is preferred over the outwards oriented hydroxyl group (exo-hydroxyl) by CAL B. The maximum conversions under optimized conditions were 88.2% and 93.7% for succinic acid and isomannide, respectively. MALDI-TOF detected products at 24 h were a mixture of cyclic (35.1%) and linear ester oligomers (64.9%). Cyclic ester oliogomers were the most abundant products during the first 8 h of reaction (32.5–48.7%), where the first cyclic of the series (CEO1) was the most predominant cyclic product (23–40%).

Elastin-like polypeptides (ELPs) are biocompatible designer polypeptides with inverse temperature transition behavior in solution. They have a wide variety of possible applications and a potential medical importance. Currently, production of ELPs is done at lab scale in Escherichia coli shake flask cultures. With a view to future large scale production, we demonstrate secreted production of ELPs in methanol-induced fed-batch cultures of Pichia pastoris and purification directly from the culture medium. The production of ELPs by P. pastoris proved to be pH dependent within the experimental pH range of pH 3 to 7, as an increasing yield was found in cultures grown at higher pH. Because ELP produced at pH 7 was partly degraded, a pH optimum for production of ELP was found at pH 6 with a yield of 255 mg of purified intact ELP per liter of cell-free medium.

We report on the biosynthesis of 65 kDa A¿B¿A triblock copolymers consisting of pH-responsive (acidic) silklike blocks and nonresponsive collagenlike blocks, and we show that at pH values where the silklike blocks become uncharged, these polymers form transparent high-modulus gels, that is, 7¿15 kPa at 8 g·L¿1, that consist of supramolecular nanotapes with a height of 2.8 nm, a width of 14 nm, and an average length of >10 ¿m. At the concentrations employed, both of these protein triblocks essentially form the same structure, irrespective of block order. The amount of product isolated from the extracellular medium is in the gram per liter range. This high yield makes various applications of this promising class of biocompatible materials possible.

Hyaluronan (HA) is a polysaccharide with high-potential medical applications, depending on the chain length and the chain length distribution. Special interest goes to homogeneous HA oligosaccharides, which can be enzymatically produced using Pasteurella multocida hyaluronan synthase (PmHAS). We have developed a sensitive, simple, and fast method, based on fluorophore-assisted carbohydrate electrophoresis (FACE), for characterization and quantification of polymerization products. A chromatographic pure fluorescent template was synthesized from HA tetrasaccharide (HA4) and 2-aminobenzoic acid. HA4-fluor and HA4 were used as template for PmHAS-mediated polymerization of nucleotide sugars. All products, fluorescent and nonfluorescent, were analyzed with gel electrophoresis and quantified using lane densitometry. Comparison of HA4- and HA4-fluor-derived polymers showed that the fluorophore did not negatively influence the PmHAS-mediated polymerization. Only even-numbered oligosaccharide products were observed using HA4-fluor or HA4 as template. The fluorophore intensity was linearly related to its concentration, and the limit of detection was determined to be 7.4 pmol per product band. With this assay, we can now differentiate oligosaccharides of size range DP2 (degree of polymerization 2) to approximately DP400, monitor the progress of polymerization reactions, and measure subtle differences in polymerization rate. Quantifying polymerization products enables us to study the influence of experimental conditions on HA synthesis.

Itaconic acid, a C5-dicarboxylic acid, is a potential biobased building block for the polymer industry. It is obtained from the citric acid cycle by decarboxylation of cis-aconitic acid. This reaction is catalyzed by CadA in the native itaconic acid producer Aspergillus terreus. Recently, another enzyme encoded by the mammalian immunoresponsive gene 1 (irg1), was found to decarboxylate cis-aconitate to itaconate in vitro. We show that heterologous expression of irg1 enabled itaconate production in Escherichia coli with production titres up to 560 mg/L.

To obtain a mannitol-producingLactococcus lactisstrain, the mannitol 1-phosphate dehydrogenase gene (mtlD) fromLactobacillus plantarumwas overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity. High-performance liquid chromatography and13C nuclear magnetic resonance analysis revealed that small amounts (mtlD-overexpressing LDH-deficient and phosphofructokinase-reduced strains, whereas resting cells of the LDH-deficient transformant converted 25% of glucose into mannitol. Moreover, the formed mannitol was not reutilized upon glucose depletion. Of the metabolic-engineering strategies investigated in this work,mtlD-overexpressing LDH-deficientL. lactisseemed to be the most promising strain for mannitol production.

The phaC1 gene from Pseudomonas oleovorans, coding for the Pha-C1 polymerase, was introduced into the potato genome. Transgenic callus and plant lines which transcribed and translated the transgene were selected and cell suspension cultures from the wild type and transgenic lines were established. The substrate for the Pha-C1 polymerase, 3-(R)-hydroxyoctanoate, was provided to the growth medium. In the transgenic lines, but not in the wild type or in transgenic cell suspension cultures without Pha-C1 expression, evidence of medium-chain-length polyhydroxyalkanoate accumulation ranging from 0.02 to 9.7 mg of polymer per gram of dry weight was observed after feeding in the growth medium the substrate. (C) 2003 Elsevier Science B.V. All rights reserved.

The lipids of the heterotrophic microalga Crypthecodinium cohnii contain the omega-3 polyunsaturated fatty acid (PUFA) and docosahexaenoic acid (22:6) to a level of over 30%. The pathway of 22:6 synthesis in C. cohnii is unknown. The ability of C. cohnii to use 13C-labelled externally supplied precursor molecules for 22:6 biosynthesis was tested by 13C NMR analysis. Furthermore, the presence of desaturases (typical for aerobic PUFA synthesis) was studied by the addition of specific desaturase inhibitors in the growth medium. The addition of 1-(13)C acetate or 1-(13)C butyrate in the growth medium resulted in 22:6 with only the odd carbon atoms enriched. Apparently, two-carbon units were used as building blocks for 22:6 synthesis and butyrate was first split into two-carbon units prior to incorporation in 22:6. When 1-(13)C oleic acid was added to the growth medium, 1-(13)C oleic acid was incorporated into the lipids of C. cohnii but was not used as a precursor for the synthesis of 22:6. Specific desaturase inhibitors (norflurazon and propyl gallate) inhibited lipid accumulation in C. cohnii. The fatty acid profile, however, was not altered. In contrast, in the arachidonic acid-producing fungus, Mortierella alpina, these inhibitors not only decreased the lipid content but also altered the fatty acid profile. Our results can be explained by the presence of three tightly regulated separate systems for the fatty acid production by C. cohnii, namely for (1). the biosynthesis of saturated fatty acids, (2). the conversion of saturated fatty acids to monounsaturated fatty acids and (3). the de novo synthesis of 22:6 with desaturases involved.

Lactic acid bacteria display a relatively simple and well-described metabolism where the sugar source is converted mainly to lactic acid. Here we will shortly describe metabolic engineering strategies on the level of sugar metabolism, that lead to either the efficient re-routing of the lactococcal sugar metabolism to nutritional end-products other than lactic acid such as L-alanine, several low-calorie sugars and oligosaccharides or to enhancement of sugar metabolism for complete removal of (undesirable) sugars from food materials. Moreover, we will review current metabolic engineering approaches that aim at increasing the flux through complex biosynthetic pathways, leading to the production of the B-vitamins folate and riboflavin. An overview of these metabolic engineering activities can be found on the website of the Nutra Cells 5th Framework EU-project (www.nutracells.com). Finally, the impact of the developments in the area of genomics and corresponding high-throughput technologies on nutraceutical production will be discussed.

Sugarcane bagasse is an interesting feedstock for the biobased economy since a large fraction is polymerized sugars. Autohydrolysis, alkaline and acid pretreatment conditions combined with enzyme hydrolysis were used on lignocellulose rich bagasse to acquire monomeric. By-products found after pretreatment included acetic, glycolic and coumaric acid in concentrations up to 40, 21 and 2.5 g/kg dry weight bagasse respectively. Alkaline pretreated material contained up to 45 g/kg bagasse DW of sodium. Acid and autohydrolysis pretreatment results in a furan formation of 14 g/kg and 25 g/kg DW bagasse respectively. Enzyme monomerization efficiencies of pretreated solid material after 72 h were 81% for acid pretreatment, 77% for autohydrolysis and 57% for alkaline pretreatment. Solid material was washed with superheated water to decrease the amount of by-products. Washing decreased organic acid, phenol and furan concentrations in solid material by at least 60%, without a major sugar loss.

Background Many microalgae accumulate carbohydrates simultaneously with triacylglycerol (TAG) upon nitrogen starvation, and these products compete for photosynthetic products and metabolites from the central carbon metabolism. As shown for starchless mutants of the non-oleaginous model alga Chlamydomonas reinhardtii, reduced carbohydrate synthesis can enhance TAG production. However, these mutants still have a lower TAG productivity than wild-type oleaginous microalgae. Recently, several starchless mutants of the oleaginous microalga Scenedesmus obliquus were obtained which showed improved TAG content and productivity. Results The most promising mutant, slm1, is compared in detail to wild-type S. obliquus in controlled photobioreactors. In the slm1 mutant, the maximum TAG content increased to 57¿±¿0.2% of dry weight versus 45¿±¿1% in the wild type. In the wild type, TAG and starch were accumulated simultaneously during initial nitrogen starvation, and starch was subsequently degraded and likely converted into TAG. The starchless mutant did not produce starch and the liberated photosynthetic capacity was directed towards TAG synthesis. This increased the maximum yield of TAG on light by 51%, from 0.144¿±¿0.004 in the wild type to 0.217¿±¿0.011 g TAG/mol photon in the slm1 mutant. No differences in photosynthetic efficiency between the slm1 mutant and the wild type were observed, indicating that the mutation specifically altered carbon partitioning while leaving the photosynthetic capacity unaffected. Conclusions The yield of TAG on light can be improved by 51% by using the slm1 starchless mutant of S. obliquus, and a similar improvement seems realistic for the areal productivity in outdoor cultivation. The photosynthetic performance is not negatively affected in the slm1 and the main difference with the wild type is an improved carbon partitioning towards TAG.

Background: Microalgae are a promising platform for producing neutral lipids, to be used in the application for biofuels or commodities in the feed and food industry. A very promising candidate is the oleaginous green microalga Scenedesmus obliquus, because it accumulates up to 45% w/w triacylglycerol (TAG) under nitrogen starvation. Under these conditions, starch is accumulated as well. Starch can amount up to 38% w/w under nitrogen starvation, which is a substantial part of the total carbon captured. When aiming for optimized TAG production, blocking the formation of starch could potentially increase carbon allocation towards TAG. In an attempt to increase TAG content, productivity and yield, starchless mutants of this high potential strain were generated using UV mutagenesis. Previous studies in Chlamydomonas reinhardtii have shown that blocking the starch synthesis yields higher TAG contents, although these TAG contents do not surpass those of oleaginous microalgae yet. So far no starchless mutants in oleaginous green microalgae have been isolated that result in higher TAG productivities. Results: Five starchless mutants have been isolated successfully from over 3,500 mutants. The effect of the mutation on biomass and total fatty acid (TFA) and TAG productivity under nitrogen-replete and nitrogen-depleted conditions was studied. All five starchless mutants showed a decreased or completely absent starch content. In parallel, an increased TAG accumulation rate was observed for the starchless mutants and no substantial decrease in biomass productivity was perceived. The most promising mutant showed an increase in TFA productivity of 41% at 4 days after nitrogen depletion, reached a TAG content of 49.4% (% of dry weight) and had no substantial change in biomass productivity compared to the wild type. Conclusions: The improved S. obliquus TAG production strains are the first starchless mutants in an oleaginous green microalga that show enhanced TAG content under photoautotrophic conditions. These results can pave the way towards a more feasible microalgae-driven TAG production platform. Keywords: Starchle

The solvent-tolerant bacterium Pseudomonas putida S12, which adapts its membrane lipids to the presence of toxic solvents by a cis to trans isomerization of unsaturated fatty acids, was used to study possible in vivo regiospecificity of the isomerase. Cells were supplemented with linoleic acid (C18:2delta9-cis,delta12-cis), a fatty acid that cannot be synthesized by this bacterium, but which was incorporated into membrane lipids up to an amount of 15% of total fatty acids. After addition of 1-octanol, which was used as an activator of the cis-trans isomerase, the linoleic acid was converted into the delta9-trans,delta12-cis isomer, while the delta9-cis,delta12-trans and delta9-trans,epsilon12-trans isomers were not synthesized. Thus, for the first time, regiospecific in vivo formation of novel, mixed cis/trans isomers of dienoic fatty acid chains was observed. The maximal conversion (27-36% of the chains) was obtained at 0.03-0.04% (v/v) octanol, after 2 h. The observed regiospecificity of the enzyme, which is located in the periplasmic space, could be due to penetration of the enzyme to a specific depth in the membrane as well as to specific molecular recognition of the substrate molecules.

BACKGROUND: Genome scale metabolic reconstructions are developed to efficiently engineer biocatalysts and bioprocesses based on a rational approach. However, in most reconstructions, due to the lack of appropriate measurements, experimentally determined growth parameters are simply taken from literature including other organisms, which reduces the usefulness and suitability of these models. Pseudomonas putida KT2440 is an outstanding biocatalyst given its versatile metabolism, its ability to generate sufficient energy and turnover of NADH and NAD. To apply this strain optimally in industrial production, a previously developed genome-scale metabolic model (iJP815) was experimentally assessed and streamlined to enable accurate predictions of the outcome of metabolic engineering approaches. RESULTS: To substantially improve the accuracy of the genome scale model (iJP815), continuous bioreactor cultures on a mineral medium with glucose as a sole carbon source were carried out at different dilution rates, which covered pulling analysis of the macromolecular composition of the biomass. Besides, the maximum biomass yield (on substrate) of 0.397 gDCW . gglc-1, the maintenance coefficient of 0.037 gglc . gDCW-1 . h-1 and the maximum specific growth rate of 0.59 h-1 were determined. Only the DNA fraction increased with the specific growth rate. This resulted in reliable estimation for the Growth-Associated Maintenance (GAM) of 85 mmolATP . gDCW-1 and the Non Growth-Associated Maintenance (NGAM) of 3.96 mmolATP . gDCW-1 . h-1. Both values were found significantly different from previous assignment as a consequence of a lower yield and higher maintenance coefficient than originally assumed. Contrasting already published 13C flux measurements and the improved model allowed for constraining the solution space, by eliminating futile cycles. Furthermore, the model predictions were compared with transcriptomic data at overall good consistency, which helped to identify missing links. CONCLUSIONS: By careful interpretation of growth stoichiometry and kinetics when grown in the presence of glucose, this work reports on an accurate genome scale metabolic model of Pseudomonas putida, providing a solid basis for its use in designing superior strains for biocatalysis. By consideration of substrate specific variation in stoichiometry and kinetics, it can be extended to other substrates and new mutants

Hyaluronan is a polysaccharide with multiple functions in the human body being involved in creating flexible and protective layers in tissues and in many signalling pathways during embryonic development, wound healing, inflammation, and cancer. Hyaluronan is an important component of active pharmaceutical ingredients for treatment of, for example, arthritis and osteoarthritis, and its commercial value far exceeds that of other microbial extracellular polysaccharides. Traditionally hyaluronan is extracted from animal waste which is a well-established process now. However, biotechnological synthesis of biopolymers provides a wealth of new possibilities. Therefore, genetic/metabolic engineering has been applied in the area of tailor-made hyaluronan synthesis. Another approach is the controlled artificial (in vitro) synthesis of hyaluronan by enzymes. Advantage of using microbial and enzymatic synthesis for hyaluronan production is the simpler downstream processing and a reduced risk of viral contamination. In this paper an overview of the different methods used to produce hyaluronan is presented. Emphasis is on the advancements made in the field of the synthesis of bioengineered hyaluronan.

The oleaginous yeast Cryptococcus curvatus is of industrial interest because it can accumulate triacylglycerols up to 60% of the cell dry weight. We are aiming at genetic modification of fatty acid biosynthesis for the production of tailor-made triacylglycerols in C. curvatus. As a first step in the development of a transformation and expression system a gene encoding the delta-9 fatty acid desaturase of C. curvatus (CBS 570) was cloned. The 1470 bp gene encodes a protein of 493 amino acids with a calculated molecular mass of 55 kDa. The gene shows strong similarity to previous cloned delta-9 desaturase genes from rat and Saccharomyces cerevisiae, 62 and 72%, respectively. Expression of the delta-9 desaturase gene was studied. Supplementation of the growth medium with oleic acid (C18:1(c9)) showed a strong repression (90%) on the mRNA level, while supplementation with petroselinic acid (C18:1(c6)) had no effect on the amount of mRNA.