Advances in physiological activities and synthesis of β-nicotinamide mononucleotide / 生物工程学报

Nicotinamide mononucleotide (NMN) is one of the key precursors of coenzyme Ⅰ (NAD+). NMN exists widely in a variety of organisms, and β isomer is its active form. Studies have shown that β-NMN plays a key role in a variety of physiological and metabolic processes. As a potential active substance in anti-aging and improving degenerative and metabolic diseases, the application value of β-NMN has been deeply explored, and it is imminent to achieve large-scale production. Biosynthesis has become the preferred method to synthesize β-NMN because of its high stereoselectivity, mild reaction conditions, and fewer by-products. This paper reviews the physiological activity, chemical synthesis as well as biosynthesis of β-NMN, highlighting the metabolic pathways involved in biosynthesis. This review aims to explore the potential of improving the production strategy of β-NMN by using synthetic biology and provide a theoretical basis for the research of metabolic pathways as well as efficient production of β-NMN.

Advances on microbial synthesis of L-proline and trans-4-hydroxy-L-proline / 生物工程学报

L-proline (L-Pro) is the only imino acid among the 20 amino acids that constitute biological proteins, and its main hydroxylated product is trans-4-hydroxy-L-proline (T-4-Hyp). Both of them have unique biological activities and play important roles in biomedicine, food and beauty industry. With the in-depth exploration of the functions of L-Pro and T-4-Hyp, the demand for them is gradually increasing. Traditional methods of biological extraction and chemical synthesis are unable to meet the demand of "green, environmental protection and high efficiency". In recent years, synthetic biology has developed rapidly. Through the intensive analysis of the synthetic pathways of L-Pro and T-4-Hyp, microbial cell factories were constructed for large-scale production, which opened a new chapter for the green and efficient production of L-Pro and T-4-Hyp. This paper reviews the application and production methods of L-Pro and T-4-Hyp, the metabolic pathways for microbial synthesis of L-Pro and T-4-Hyp, and the engineering strategies and advances on microbial production of L-Pro and T-4-Hyp, aiming to provide a theoretical basis for the "green bio-manufacturing" of L-Pro and T-4-Hyp and promote their industrial production.

New targeted compounds-biosynthesis of phytocannabinoids / 生物工程学报

Phytocannabinoids are bioactive terpenoids that are exclusive to Cannabis sativa L. The main pharmacologically active phytocannabinoids are Δ9-tetrahydrocannabinol and cannabidiol, both target endogenous cannabinoid receptors. Δ9-tetrahydrocannabinol and cannabidiol have extensive therapeutic potential due to their participation in many physiological and pathological processes in human body by activating the endocannabinoid system. At present, Δ9-tetrahydrocannabinol, cannabidiol and their analogues or combination preparations are used to treat epilepsy, vomiting in patients with cancer chemotherapy, spasticity in multiple sclerosis and relieve neuropathic pain and pain in patients with advanced cancer. With the further exploration of the application value of Δ9-tetrahydrocannabinol and cannabidiol as well as the increasing demand for standardization of pharmaceutical preparations, it is imminent to achieve large-scale production of Δ9-tetrahydrocannabinol and cannabidiol in the pharmaceutical industry. In this article, pharmacological research progress of phytocannabinoids in recent years, biosynthetic pathways of phytocannabinoids and the mechanism of key enzymes as well as various product development strategies of cannabinoids in pharmaceutical industry are reviewed. By exploring the potential of synthetic biology as an alternative strategy for the source of phytocannabinoids, it will provide a theoretical basis for the research and development of microbial engineering for cannabinoids synthesis, and promote the large-scale production of medicinal cannabinoids.

Breeding of Hyphomicrobium denitrificans for high production of pyrroloquinoline quinone by adaptive directed domestication / 生物工程学报

Pyrroloquinoline quinone (PQQ) is widely distributed in organisms and has physiological functions such as boosting body growth, maintaining mitochondrial function, promoting synthesis of nerve growth factor and regulating free radical levels in the body. It has broad application prospects in the fields of medicine, food and cosmetics. In order to improve the PQQ production of Hyphamicrobium denitrificans FJNU-6, the high-concentration methanol was used as the antagonistic factor for laboratory adaptive domestication. The PQQ positive mutants were selected using rapid screening system by spectroscopy. After 6 rounds of adaptive domestication, about 10% mutants were acquired with a doubled yield, and over 90% positive mutation rate of each round of domestication was reached. Subsequently, the mutant strain FJNU-R8 was fermented by 5 L fermenter. Compared with the original strain, the expression of pqq and moxF gene clusters were higher at different methanol concentrations and similar to each other. Meanwhile, the methanol consumption rate and growth rate were slower than the original strain. Finally, the PQQ yield was increased by 1.42 times to 1 087.81 mg/L (143 h), indicating good industrial application potential. The adaptive domestication combined with rapid screening system described in this study can easily and rapidly obtain mutants with high yield of PQQ, which can be used as reference for high-throughput screening of other high-yield PQQ mutants of methylotrophic bacteria.

Construction of Saccharomyces cerevisiae mutant with knockout of SNF4 gene / 生物工程学报

Construction and ethanol production effects of SNF4 gene knockout in Saccharomyces cerevisiae were described in this paper. For knockout of SNF4 gene in S. cerevisiae YS2, a PCR-amplified disruption cassette was used, encoding the short flanking homologous regions to the SNF4 gene and Kan(r) as selectable marker. The SNF4 gene disruption cassette was transformed into S. cerevisiae YS2 through LiAc/SS Carrier DNA/PEG. The positive transformants were grown on G418 plates and verified by PCR. The Kan(r) marker was rescued by transforming plasmid pSH65 into positive transformants and inducing expression of Cre recombinase in galactose-containing medium. Lastly, the YS2-deltaSNF4 strain, in which SNF4 allele gene were completely knocked out, was obtained by repeating the same procedure. The result of anaerobic fermentation showed that ethanol production of the SNF4 gene knockout strain had increased by 7.57 percent as compared with the original strain YS2. The experiment indicated ethanol production could be improved significantly with the approach ofSNF4 gene knockout by Cre-LoxP system.

Expression of a pectin lyase A gene from Aspergillus niger in Pichia pastoris GS115 / 生物工程学报

In this study, the mature peptide sequence of a pectin lyase gene A was amplified from Aspergillus niger strain EIM-6 by using RT-PCR reverse transcription technique. The cloned gene was then inserted into a Pichia pastoris expression vector pPIC9k to produce the recombinant expression plasmid pPIC9K-pelA. By using electric shocks, we successfully transformed the recombinant pPIC9K-pelA into Pichia pastoris GS115. The activity of the engineered strain reached to 2.3 U/mL after induction with the final concentration of 1.5% methanol. SDS-PAGE analysis revealed that the pPIC9K-pelA transformant had an additional protein band of approximately 38 kD, which was not present in the control. There were no significant differences between the recombinant and native pectin lyase with regard to their hydrolysis activities.