The establishment of new protein expression system using N starvation inducible promoters in Chlorella.

Chlorella is a unicellular green microalga that has been used in fields such as bioenergy production and food supplementation. In this study, two promoters of N (nitrogen) deficiency-inducible Chlorella vulgaris N Deficiency Inducible (CvNDI) genes were isolated from Chlorella vulgaris UTEX 395. These promoters were used for the production of a recombinant protein, human granulocyte-colony stimulating factor (hG-CSF) in Chlorella vulgaris UTEX 395 and Chlorella sp. ArM0029B. To efficiently secrete the hG-CSF, the protein expression vectors incorporated novel signal peptides obtained from a secretomics analysis of Chlorella spp. After a stable transformation of those vectors with a codon-optimized hG-CSF sequence, hG-CSF polypeptides were successfully produced in the spent media of the transgenic Chlorella. To our knowledge, this is the first report of recombinant protein expression using endogenous gene components of Chlorella.

Microbial and genetically engineered oils as replacements for fish oil in aquaculture feeds.

As the global population grows more of our fish and seafood are being farmed. Fish are the main dietary source of the omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, but these cannot be produced in sufficient quantities as are now required for human health. Farmed fish have traditionally been fed a diet consisting of fishmeal and fish oil, rich in n-3 LC-PUFA. However, the increase in global aquaculture production has resulted in these finite and limited marine ingredients being replaced with sustainable alternatives of terrestrial origin that are devoid of n-3 LC-PUFA. Consequently, the nutritional value of the final product has been partially compromised with EPA and DHA levels both falling. Recent calls from the salmon industry for new sources of n-3 LC-PUFA have received significant commercial interest. Thus, this review explores the technologies being applied to produce de novo n-3 LC-PUFA sources, namely microalgae and genetically engineered oilseed crops, and how they may be used in aquafeeds to ensure that farmed fish remain a healthy component of the human diet.

History and current status of development and use of viral insecticides in China.

The use of insect viruses as biological control agents started in the early 1960s in China. To date, more than 32 viruses have been used to control insect pests in agriculture, forestry, pastures, and domestic gardens in China. In 2014, 57 products from 11 viruses were authorized as commercial viral insecticides by the Ministry of Agriculture of China. Approximately 1600 tons of viral insecticidal formulations have been produced annually in recent years, accounting for about 0.2% of the total insecticide output of China. The development and use of Helicoverpa armigera nucleopolyhedrovirus, Mamestra brassicae nucleopolyhedrovirus, Spodoptera litura nucleopolyhedrovirus, and Periplaneta fuliginosa densovirus are discussed as case studies. Additionally, some baculoviruses have been genetically modified to improve their killing rate, infectivity, and ultraviolet resistance. In this context, the biosafety assessment of a genetically modified Helicoverpa armigera nucleopolyhedrovirus is discussed.

Lentinan degradation in the Lentinula edodes fruiting body during postharvest preservation is reduced by downregulation of the exo-ß-1,3-glucanase EXG2.

Lentinan from Lentinula edodes fruiting bodies (shiitake mushrooms) is a valuable ß-glucan for medical purposes based on its anticancer activity and immunomodulating activity. However, lentinan content in fruiting bodies decreases after harvesting and storage due to an increase in glucanase activity. In this study, we downregulated the expression of an exo-ß-1,3-glucanase, exg2, in L. edodes using RNA interference. In the wild-type strain, ß-1,3-glucanase activity in fruiting bodies remarkably increased after harvesting, and 41.7% of the lentinan content was lost after 4 days of preservation. The EXG2 downregulated strain showed significantly lower lentinan degrading activity (60-70% of the wild-type strain) in the fruiting bodies 2-4 days after harvesting. The lentinan content of fresh fruiting bodies was similar in the wild-type and EXG2 downregulated strains, but in the downregulated strain, only 25.4% of the lentinan was lost after 4 days, indicating that downregulation of EXG2 enables keeping the lentinan content high longer.

Metabolic engineering of Thermobifida fusca for direct aerobic bioconversion of untreated lignocellulosic biomass to 1-propanol.

Biofuel production from renewable resources can potentially address lots of social, economic and environmental issues but an efficient production method has yet to be established. Combinations of different starting materials, organisms and target fuels have been explored with the conversion of cellulose to higher alcohols (1-propanol, 1-butanol) being one potential target. In this study we demonstrate the direct conversion of untreated plant biomass to 1-propanol in aerobic growth conditions using an engineered strain of the actinobacterium, Thermobifida fusca. Based upon computational predictions, a bifunctional butyraldehyde/alcohol dehydrogenase was added to T. fusca leading to 1-propanol production during growth on glucose, cellobiose, cellulose, switchgrass and corn stover. The highest 1-propanol titer (0.48g/L) was achieved for growth on switchgrass. These results represent the first demonstration of direct conversion of untreated lignocellulosic biomass to 1-propanol in an aerobic organism and illustrate the potential utility of T. fusca as an aerobic, cellulolytic bioprocess organism.

Increasing free-energy (ATP) conservation in maltose-grown Saccharomyces cerevisiae by expression of a heterologous maltose phosphorylase.

Increasing free-energy conservation from the conversion of substrate into product is crucial for further development of many biotechnological processes. In theory, replacing the hydrolysis of disaccharides by a phosphorolytic cleavage reaction provides an opportunity to increase the ATP yield on the disaccharide. To test this concept, we first deleted the native maltose metabolism genes in Saccharomyces cerevisiae. The knockout strain showed no maltose-transport activity and a very low residual maltase activity (0.03 µmol mg protein(-1)min(-1)). Expression of a maltose phosphorylase gene from Lactobacillus sanfranciscensis and the MAL11 maltose-transporter gene resulted in relatively slow growth (µ(aerobic) 0.09 ± 0.03 h(-1)). Co-expression of Lactococcus lactis ß-phosphoglucomutase accelerated maltose utilization via this route (µ(aerobic) 0.21 ± 0.01 h(-1), µ(anaerobic) 0.10 ± 0.00 h(-1)). Replacing maltose hydrolysis with phosphorolysis increased the anaerobic biomass yield on maltose in anaerobic maltose-limited chemostat cultures by 26%, thus demonstrating the potential of phosphorolysis to improve the free-energy conservation of disaccharide metabolism in industrial microorganisms.

Stepwise increase of resveratrol biosynthesis in yeast Saccharomyces cerevisiae by metabolic engineering.

Resveratrol is a unique, natural polyphenolic compound with diverse health benefits. In the present study, we attempted to improve resveratrol biosynthesis in yeast by different methods of metabolic engineering. We first mutated and then re-synthesized tyrosine ammonia lyase (TAL) by replacing the bacteria codons with yeast-preferred codons, which increased translation and improved p-coumaric acid and resveratrol biosynthesis drastically. We then demonstrated that low-affinity, high-capacity bacterial araE transporter could enhance resveratrol accumulation, without transporting resveratrol directly. Yeast cells carrying the araE gene produced up to 2.44-fold higher resveratrol than control cells. For commercial applications, resveratrol biosynthesis was detected in sucrose medium and fresh grape juice using our engineered yeast cells. In collaboration with the Chaumette Winery of Missouri, we were able to produce resveratrol-containing white wines, with levels comparable to the resveratrol levels found in most red wines.

Engineering the robustness of Clostridium acetobutylicum by introducing glutathione biosynthetic capability.

To improve the aero- and solvent tolerance of the solvent-producing Clostridium acetobutylicum, glutathione biosynthetic capability was introduced into C. acetobutylicum DSM1731 by cloning and over-expressing the gshAB genes from Escherichia coli. Strain DSM1731(pITAB) produces glutathione, and shows a significantly improved survival upon aeration and butanol challenge, as compared with the control. In addition, strain DSM1731(pITAB) exhibited an improved butanol tolerance and an increased butanol production capability, as compared with the recombinant strains with only gshA or gshB gene. These results illustrated that introducing glutathione biosynthetic pathway, which is redundant for the metabolism of C. acetobutylicum, can increase the robustness of the host to achieve a better solvent production.