High-Level Overproduction of Thermobifida Enzyme in Streptomyces lividans Using a Novel Expression Vector

Abstract

In this study, we constructed a novel Streptomyces-E.coli shuttle vector pZRJ362 combining the xylose isomerase promoter and amylase terminator. A gene encoding the endoglucanase Cel6A in Thermobifida fusca was amplified by PCR, cloned into Streptomyces lividans host strain using the novel expression vector and Pichia pastoris GS115 host strain using the vector pPICZα-C, respectively. Afterwards, the expression pattern and the maximum expression level were comparatively studied in both expression systems. The maximum enzyme activity of Cel6A-(His)6 secreted in S. lividans supernatant after 84-h of cultivation amounted to 5.56 U/mL, which was dramatically higher than that secreted in P. pastoris about 1.4 U/mL after 96-h of cultivation. The maximum expression level of Cel6A-(His)6 in S. lividans supernatant reached up to 173 mg/L after 84-h of cultivation. The endoglucanase activity staining SDS-PAGE showed that there were some minor proteins in S. lividans supernatant which may be the Cel6A derivant by proteolytic degradation, while there was no proteolytic product detected in supernatant of P. pastoris.

Monitoring Protein Secretion in Streptomyces Using Fluorescent Proteins

“…Beside their use as reporters, eGFP and mRFP provided high production yields. Tat-dependent eGFP secretion of >10 mg/L is higher than the amount of human tumor necrosis factor (hTNF) α and interleukin (IL) secreted in S. lividans TK24 via the Tat pathway (Vrancken et al, 2007) and Sec-dependent secretion of mRFP (∼300 mg/L or 700 mg/g DCW) is more than the Sec- dependant secretion of thermostable cellulase A (70 mg/L) (Hamed et al, 2017), xyloglucanase (100–150 mg/L) (Sianidis et al, 2006), endoglucanase from Thermobifida fusca (173 mg/L) (Li et al, 2013), phospholipase D (PLD) from Streptoverticillium cinnamoneum (118 mg/L) (Ogino et al, 2004) and two major lipoproteins (A) and (B) from Mycobacterium tuberculosis (80 and 200 mg/L, respectively) (Tremblay et al, 2002), while less than laccase from S. coelicolor A3(2) (350 mg/L) (Dube et al, 2008) and chitinase C from S. coelicolor A3(2) (1070 mg/L) (Nguyen-Thi and Doucet, 2016). The values obtained with mRFP make them amongst the highest yields obtained so far in S. lividans .…”

Designing novel cellulase systems through agent-based modeling and global sensitivity analysis

“…For example, C. thermocellum ATCC 27405, has been shown to secrete two non-cellulosome bound cellulases in addition to expressing a cellulosome. 10 Advances in recombinant DNA technology have enabled the development of efficient and scalable expression systems to produce cellulase enzymes using more traditional industrial organisms such as Escherichia coli, 11,12 Bacillus subtilis, 13 Saccharomyces cerevisiae, 14 Pichia pastoris, 15 Streptomyces lividans, 16 and species of Aspergillus and Trichoderma. 17 Cellulases originating from these organisms have a wide range of applications in the biotechnology industry.…”

Streptomycetes: Surrogate hosts for the genetic manipulation of biosynthetic gene clusters and production of natural products

“…The expression of thermophilic genes (Kieser et al, 2000; Diaz et al, 2008; Li et al, 2013a), secretory enzymes (Noda et al, 2010), superoxide dismutase (SOD) genes (Halliwell and Gutteridge, 1985; Kang et al, 2006; Kang et al, 2007; Kanth et al, 2011), and glycosyltransferases (Quiros et al, 2000; Nakazawa et al, 2011) are some of the most common examples.…”

“…In addition to exogenous secondary metabolic gene clusters, S. lividans is widely used for expression or overproduction of enzymes derived from diverse microbial and environmental sources. The expression of thermophilic genes (Kieser et al, 2000; Diaz et al, 2008; Li et al, 2013a), secretory enzymes (Noda et al, 2010), superoxide dismutase (SOD) genes (Halliwell and Gutteridge, 1985; Kang et al, 2006; Kang et al, 2007; Kanth et al, 2011), and glycosyltransferases (Quiros et al, 2000; Nakazawa et al, 2011) are some of the most common examples.…”