[Effects of manipulating lactate dehydrogenase gene on metabolism of HEK-293 and production of human adenovirus].

Reducing lactate accumulation has always been a goal of the mammalian cell biotechnology industry. When animal cells are cultured in vitro, the accumulation of lactate is mainly the combined result of two metabolic pathways. On one hand, glucose generates lactate under the function of lactate dehydrogenase A (LDHA); on the other hand, lactate can be oxidized to pyruvate by LDHB or LDHC and re-enter the TCA cycle. This study comprehensively evaluated the effects of LDH manipulation on the growth, metabolism and human adenovirus (HAdV) production of human embryonic kidney 293 (HEK-293) cells, providing a theoretical basis for engineering the lactate metabolism in mammalian cells. By knocking out ldha gene and overexpression of ldhb and ldhc genes, the metabolic efficiency of HEK-293 cells was effectively improved, and HAdV production was significantly increased. Compared with the control cell, LDH manipulation promoted cell growth, reduced the accumulation of lactate and ammonia, significantly enhanced the efficiency of substrate and energy metabolism of cells, and significantly increased the HAdV production capacity of HEK-293 cells. Among these LDH manipulation measures, ldhc gene overexpression performed the best, with the maximum cell density increased by about 38.7%. The yield of lactate to glucose and ammonia to glutamine decreased by 33.8% and 63.3%, respectively; and HAdV titer increased by at least 16 times. In addition, the ATP production rate, ATP/O2 ratio, ATP/ADP ratio and NADH content of the modified cell lines were increased to varying degrees, and the energy metabolic efficiency was significantly improved.

Regulation of the pyruvate metabolism node by monogene and polygene engineering of HEK-293 cells.

HEK-293 cells are increasingly being used in the production of human adenovirus (HAdV) vaccines. However, the production of HAdV vaccine has not met the requirements of industrial production. Recently, we investigated the effects of various regulatory genes of the pyruvate metabolism node on the substance and energy metabolism and adenovirus reproduction in HEK-293 cells. Initially, single regulatory genes, including pkm2, pdhα, pyc2, mpc3, aralar1, ldha and pdk1, were studied. We found that metabolic performance and adenovirus reproduction capacity in HEK-293 cells were improved, and maximum adenovirus titre was increased approximately 15-fold. Next, we co-overexpressed the key genes, including pkm2, pyc2 and aralar1. The PYC2-A-P-L cells that had the appropriate co-overexpression levels of three genes had the most pronounced regulatory effect. The maximum cell density and maximum specific growth rate were increased by 21% compared with that in the control. The ΔLac/ΔGlc and ΔNH3/ΔGln were decreased by 26% and 27%, respectively. The ATP production rate and the ATP/O2 ratio were increased by 110% and 20%, respectively. The level of reactive oxygen species (ROS) was reduced by 60%. The adenovirus reproductive ability of the PYC2-A-P-L cells was approximately 30-fold higher than that of the control. The results showed that proper overexpression of the aralar1, pkm2 and pyc2 genes can significantly improve the substance and energy metabolism efficiency in HEK-293 cells, maximize the metabolic balance of pyruvate, and ultimately improve HAdV reproduction. This study provides a method of regulation of pyruvate metabolism and polygenic metabolic engineering in mammalian cells cultured in vitro and suggests an effective method for efficient HAdV production.

Effects of Cordyceps militaris (L.) Fr. fermentation on the nutritional, physicochemical, functional properties and angiotensin I converting enzyme inhibitory activity of red bean (Phaseolus angularis [Willd.] W.F. Wight.) flour.

The effects of solid-state fermentation with Cordyceps militaris (L.) Fr. on the nutritional, physicochemical, and functional properties as well as angiotensin I converting enzyme (ACE) inhibitory activity of red bean (Phaseolus angularis [Willd.] W.F. Wight.) flour were determined. Fermentation increased the amount of small peptides but significantly decreased large peptides. Fermentation also increased proteins and essential amino acids (by 9.31 and 13.89%, respectively) and improved the in vitro protein digestibility (6.54%) of red beans. Moreover, fermentation increased the water holding capacity (from 2.36 to 2.59 mL/g), fat absorption capacity (from 84.65 to 114.55%), emulsion activity (from 10.96 to 52.77%), emulsion stability (from 5.43 to 53.82%), and foaming stability (from 11.95 to 20.68%). Fermented red bean flour achieved a lower least gelation concentration of 14% than that of the control (18%). In contrast to the non-fermented red bean, the fermented red bean showed ACE inhibitory activity, with IC50 value of 0.63 mg protein/mL. Overall, fermentation improved the nutritional, physicochemical, and functional properties as well as the biological activity of red bean flour. Thus, fermented red bean flour may serve as a novel nutritional and functional ingredient for applications in food design.