Journal of Biotechnology 187 (2014) 147–153 Contents lists available at ScienceDirect Journal of Biotechnology j ourna l ho me page: www.elsevier.com/locate/jbiotec Heterologous expression of VHb can improve the yield and quality of biocontrol fungus Paecilomyces lilacinus, during submerged fermentation Shumeng Zhang a , Jieping Wang a,b , Yale Wei a , Qing Tang a , Maria Kanwal Ali a , Jin He a,∗ a State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China b Agricultural Bio-resource Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350003, PR China a r t i c l e i n f o Article history: Received 6 May 2014 Received in revised form 18 July 2014 Accepted 23 July 2014 Available online 1 August 2014 Keywords: Paecilomyces lilacinus Vitreoscilla hemoglobin (VHb) Agrobacterium tumefaciens-mediated transformation (ATMT) Fermentation, Biomass Spore production a b s t r a c t Paecilomyces lilacinus is an egg-parasitic fungus which is effective against plant-parasitic nematodes and it has been successfully commercialized for the control of many plant-parasitic nematodes. However, dur- ing the large-scale industrial fermentation process of the filamentous fungus, the dissolved oxygen supply is a limiting factor, which influences yield, product quality and production cost. To solve this problem, we intended to heterologously express VHb in P. lilacinus ACSS. After optimizing the vgb gene, we fused it with a selection marker gene nptII, a promoter PgpdA and a terminator TtrpC. The complete expression cas- sette PgpdA-nptII-vgb-TtrpC was transferred into P. lilacinus ACSS by Agrobacterium tumefaciens-mediated transformation. Consequently, we successfully screened an applicable fungus strain PNVT8 which effi- ciently expressed VHb. The submerged fermentation experiments demonstrated that the expression of VHb not only increased the production traits of P. lilacinus such as biomass and spore production, but also improved the beneficial product quality and application value, due to the secretion of more protease and chitinase. It can be speculated that the recombinant strain harboring vgb gene will have a growth advantage over the original strain under anaerobic conditions in soil and therefore will possess higher biocontrol efficiency against plant-parasitic nematodes. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Vitreoscilla hemoglobin (VHb) is a soluble homodimeric pro- tein, which is synthesized by aerobic Gram-negative bacterium Vitreoscilla (Wakabayashi et al., 1986). Its oxygen dissociation rate constant is hundred times higher than that of other globins (Zhang et al., 2007). VHb can effectively enhance cellular capacity of oxy- gen utilization, strengthen cellular respiration intensity and reduce intracellular critical oxygen concentration. Moreover, VHb can maintain constant intracellular respiratory rate and dwindle the effects of variable oxygen environment on cell growth. Therefore, for industry-scale fermentation, heterologous expression of VHb has become a versatile tool to improve cellular growth, protein synthesis, metabolite productivity and biomass (Frey et al., 2011; Geckil et al., 2001; Liao et al., 2014; Zhang et al., 2007). Plant-parasitic nematodes can cause a significant economic loss to almost all vegetables and field crops mainly in tropical and sub-tropical agricultural areas. Annual global yield loss caused by ∗ Corresponding author. E-mail addresses: hejin@mail.hzau.edu.cn, hejinhz@yahoo.com (J. He). nematodes is about 80 billion (Nicol et al., 2011). Chemical con- trol has been a widely used option for plant-parasitic nematode management. However, chemical nematicides are now being reap- praised in respect of environmental hazard and their diminished effectiveness following repeated applications. For the biocontrol of the nematodes, the egg-parasitic fungus Paecilomyces lilacinus has been widely tested and shown promising application prospects (Anastasiadis et al., 2008; Atkins et al., 2005; Kiewnick and Sikora, 2006; Li et al., 2013; Siddiqui and Futai, 2009). Moreover, some excellent strains of P. lilacinus, such as strain 251 (Anastasiadis et al., 2008; Atkins et al., 2005; Kiewnick and Sikora, 2006) and strain PL9410 (Wang et al., 2010) have been successfully commercialized as these can deteriorate eggs of nematodes (Jatala, 1986) and sur- vive for long period around rhizosphere (Hashem and Abo-Elyousr, 2011). However, the submerged fermentation of the filamentous fungus is a typically aerobic fermentation process with a long fer- mentation period (more than ten days). Therefore, it has a high demand for dissolved oxygen supply for large-scale and high-cell density industrial fermentation process, which is a crucial factor for product quality and production cost. These problems can be par- tially alleviated by improving bioreactor configuration (e.g. rational http://dx.doi.org/10.1016/j.jbiotec.2014.07.438 0168-1656/© 2014 Elsevier B.V. All rights reserved.