Upcycling of Agricultural Waste Stream to High‐Molecular‐Weight Bio‐based Poly(ethylene 2,5‐furanoate)

Orange peel and sugar beet pulp contain large quantities of pectin, which can be turned via galactaric acid into furan dicarboxylic acid (FDCA) and its esters. In this work, we show the polymerisation of these FDCA esters into high-molecular-weight, 70–100 kg/mol, poly(ethylene 2,5-furanoate) (PEF). PEF is an emerging bio-based alternative for poly(ethylene terephthalate) (PET), widely used in for example packaging applications. Closing the loop, we also demonstrated and confirmed that PEF can be hydrolysed by enzymes, which are known to hydrolyse PET, back into FDCA for convenient recycling and recovery of monomers

Enhanced Triacylglycerol Production With Genetically Modified Trichosporon oleaginosus

Mitochondrial pyruvate dehydrogenase (PDH) is important in the production of lipids in oleaginous yeast, but other yeast may bypass the mitochondria (PDH bypass), converting pyruvate in the cytosol to acetaldehyde, then acetate and acetyl CoA which is further converted to lipids. Using a metabolic model based on the oleaginous yeast Yarrowia lipolytica, we found that introduction of this bypass to an oleaginous yeast should result in enhanced yield of triacylglycerol (TAG) on substrate. Trichosporon oleaginosus (formerly Cryptococcus curvatus) is an oleaginous yeast which can produce TAGs from both glucose and xylose. Based on the sequenced genome, it lacks at least one of the enzymes needed to complete the PDH bypass, acetaldehyde dehydrogenase (ALD), and may also be deficient in pyruvate decarboxylase and acetyl-CoA synthetase under production conditions. We introduced these genes to T. oleaginosus in various combinations and demonstrated that the yield of TAG on both glucose and xylose was improved, particularly at high C/N ratio. Expression of a phospholipid:diacyltransferase encoding gene in conjunction with the PDH bypass further enhanced lipid production. The yield of TAG on xylose (0.27 g/g) in the engineered strain approached the theoretical maximum yield of 0.289 g/g. Interestingly, TAG production was also enhanced compared to the control in some strains which were given only part of the bypass pathway, suggesting that these genes may contribute to alternative routes to cytoplasmic acetyl CoA. The metabolic model indicated that the improved yield of TAG on substrate in the PDH bypass was dependent on the production of NADPH by ALD. NADPH for lipid synthesis is otherwise primarily supplied by the pentose phosphate pathway (PPP). This would contribute to the greater improvement of TAG production from xylose compared to that observed from glucose when the PDH bypass was introduced, since xylose enters metabolism through the non-oxidative part of the PPP. Yield of TAG from xylose in the engineered strains (0.21–0.27 g/g) was comparable to that obtained from glucose and the highest so far reported for lipid or TAG production from xylose

Marketing of forest reproductive material: the use of microsatellites for identification of registered tree clones in Finland

According to the current legislation on the marketing of forest reproductive material, tree clones marketed in the EU must have certain traits which make them identifiable, and these traits must have been accepted and registered by an official body. Due to this obligation, there is a need for reliable, functional and practicable methods for specifying these distinctive characters.We have developed a clone identification method for European and hybrid aspens and curly birch, based on nuclear microsatellites, which can be used for determining the distinctive characters mentioned in the directive. For aspens, we have used 18 loci, of which nine were developed earlier for P. tremuloides and nine for P. nigra. For curly birch (Betula pendula var. carelica), we have used seven loci developed for B. pendula and three loci developed for B. platyphylla var. japonica. Most of the aspen clones were easily identifiable using only part of the loci. In the case of curly birch, however, two clones could not be separated from each other despite the relatively high number and polymorphism of the loci, which suggests that these two clones were actually samples from the same clone. These kinds of mistakes further emphasise the urgent need for a reliable clone identification method

Efficient Production of l-Lactic Acid from Xylose by Pichia stipitis

Microbial conversion of renewable raw materials to useful products is an important objective in industrial biotechnology. Pichia stipitis, a yeast that naturally ferments xylose, was genetically engineered for l-(+)-lactate production. We constructed a P. stipitis strain that expressed the l-lactate dehydrogenase (LDH) from Lactobacillus helveticus under the control of the P. stipitis fermentative ADH1 promoter. Xylose, glucose, or a mixture of the two sugars was used as the carbon source for lactate production. The constructed P. stipitis strain produced a higher level of lactate and a higher yield on xylose than on glucose. Lactate accumulated as the main product in xylose-containing medium, with 58 g/liter lactate produced from 100 g/liter xylose. Relatively efficient lactate production also occurred on glucose medium, with 41 g/liter lactate produced from 94 g/liter glucose. In the presence of both sugars, xylose and glucose were consumed simultaneously and converted predominantly to lactate. Lactate was produced at the expense of ethanol, whose production decreased to ∼15 to 30% of the wild-type level on xylose-containing medium and to 70 to 80% of the wild-type level on glucose-containing medium. Thus, LDH competed efficiently with the ethanol pathway for pyruvate, even though the pathway from pyruvate to ethanol was intact. Our results show, for the first time, that lactate production from xylose by a yeast species is feasible and efficient. This is encouraging for further development of yeast-based bioprocesses to produce lactate from lignocellulosic raw material