Accumulation of gene-targeted Bacillus subtilis mutations that enhance fermentative inosine production

Abstract

In order to test a possible approach to enhance fermentative inosine production by Bacillus subtilis, seven gene-targeted mutations were introduced in the laboratory standard strain168 in a stepwise fashion. The mutations were employed in order to prevent inosine 5'-monophosphate (IMP) from being consumed for AMP and GMP synthesis, to minimize inosine degradation, and to expand the intracellular IMP pool. First, the genes for adenylosuccinate synthase (purA) and IMP dehydrogenase (guaB) were inactivated. Second, two genes for purine nucleoside phosphorylase, punA and deoD, were inactivated. Third, to enhance purine nucleotide biosynthesis, the pur operon repressor PurR and the 5'-UTR of the operon, containing the guanine riboswitch, were disrupted. Finally, the -10 sequence of the pur promoter was optimized to elevate its transcription level. The resulting mutant was capable of producing 6 g/L inosine from 30 g/L glucose in culture broth without the detectable by-production of hypoxanthine. This indicates the validity of this approach for the breeding of the next generation of B. subtilis strains for industrial nucleoside production.

Endophytic bacteria fromPinellia ternata, a new source of purine alkaloids and bacterial manure

“…It should be noted that alkaloid production by these five endophytic strains was low, compared with that of 1 g/L inosine by mutant of E. coli (Matsui et al, 2001), 6 g/L inosine by B. subtilis (Asahara et al, 2010), and 20 g/L guanosine by mutants of B. subtilis (Kuninaka, 2008). The production of guanosine and inosine by Bacillus subtilis has been increased by optimizing fermentation conditions, breeding new bacterial strains, and regulating the metabolic pathway of the bacteria (Bai et al, 2003;Liu et al, 2004;Qian et al, 2003;Zhu et al, 2004).…”

Deregulation of purine pathway in Bacillus subtilis and its use in riboflavin biosynthesis

“…For improving the pur operon promoter activity, the original -10 sequence (TAAGAT) of Ppur in BS102 was mutated with frequently used -10 sequence (TATAAT) in B. subtilis [[13]] to generate mutant strain BS103 (-10*). A 75-bp region which was defined as att corresponding to the attenuator region in 5’-UTP [[12]] was also deleted, leading to mutant strains BS104 (Δ att ) based on BS102 and BS107 (-10*, Δ att ) based on BS103, respectively.…”

“…The 5’-UTR of pur operon mRNA contains a stretch so called “riboswitch”, which binds to guanine to form a specific fold to enable a terminator structure to abort the transcription prematurely [[12]]. Disruption of purR gene and the guanine-sensing riboswitch in B. subtilis increased the activity of pur operon promoter so as to enhance purine nucleotides biosynthesis [[13]].…”

“…PRPP amidotransferase, encoded by purF , catalyzes the initial reaction of purine pathway and is the key regulatory enzyme in this pathway. Expression of purF is repressed at transcriptional level by extracellular purines [[13]], and the activity of PRPP amidotransferase is rigorously regulated by feedback inhibition through the specific binding of adenine and guanine nucleotides [[5]]. As reported, the structural features of B. subtilis PRPP amidotransferase have been described, and several mutated residues (S283A, K305Q, R307Q and S347A) were successfully introduced into PRPP amidotransferase to release it from feedback regulation [[15]].…”

“…B. subtilis has a long tradition as safe and stable producer for purine nucleotides [[13],[20],[21]]. It should be noted that B. subtilis has constituted a paradigm of environmentally friendly “white” biotechnology with regard to industrial riboflavin overproduction [[22],[23]].…”