Engineering of a genome-reduced strain Bacillus amyloliquefaciens for enhancing surfactin production

Fang Zhang,Yufen Quan,Chao Yang,Zhiling Zhang,Weixia Gao,Xingyi Song,Kaiyue Huo,Shufang Wang

doi:10.1186/s12934-020-01485-z

Abstract

BackgroundGenome reduction and metabolic engineering have emerged as intensive research hotspots for constructing the promising functional chassis and various microbial cell factories. Surfactin, a lipopeptide-type biosurfactant with broad spectrum antibiotic activity, has wide application prospects in anticancer therapy, biocontrol and bioremediation. Bacillus amyloliquefaciens LL3, previously isolated by our lab, contains an intact srfA operon in the genome for surfactin biosynthesis.ResultsIn this study, a genome-reduced strain GR167 lacking ~ 4.18% of the B. amyloliquefaciens LL3 genome was constructed by deleting some unnecessary genomic regions. Compared with the strain NK-1 (LL3 derivative, ΔuppΔpMC1), GR167 exhibited faster growth rate, higher transformation efficiency, increased intracellular reducing power level and higher heterologous protein expression capacity. Furthermore, the chassis strain GR167 was engineered for enhanced surfactin production. Firstly, the iturin and fengycin biosynthetic gene clusters were deleted from GR167 to generate GR167ID. Subsequently, two promoters PRsuc and PRtpxi from LL3 were obtained by RNA-seq and promoter strength characterization, and then they were individually substituted for the native srfA promoter in GR167ID to generate GR167IDS and GR167IDT. The best mutant GR167IDS showed a 678-fold improvement in the transcriptional level of the srfA operon relative to GR167ID, and it produced 311.35 mg/L surfactin, with a 10.4-fold increase relative to GR167.ConclusionsThe genome-reduced strain GR167 was advantageous over the parental strain in several industrially relevant physiological traits assessed and it was highlighted as a chassis strain for further genetic modification. In future studies, further reduction of the LL3 genome can be expected to create high-performance chassis for synthetic biology applications.

Highlights

Genome reduction and metabolic engineering have emerged as intensive research hotspots for constructing the promising functional chassis and various microbial cell factories
Construction of genome‐reduced B. amyloliquefaciens strains To adapt to the adverse environmental conditions, there is a common mechanism horizontal gene transfer (HGT) among microorganisms, enabling host bacteria to acquire larger DNA segments, i.e., genomic islands (GIs), the G+C contents of which are significantly different from that of the core genome [26]
There are latent secondary metabolic biosynthesis gene clusters scattered across the LL3 genome, which may increase the metabolic burden on cells and the purification cost of target products [28]

Summary

Introduction

Genome reduction and metabolic engineering have emerged as intensive research hotspots for constructing the promising functional chassis and various microbial cell factories. Bacillus amyloliquefaciens LL3, previously isolated by our lab, contains an intact srfA operon in the genome for surfactin biosynthesis. With the development of systems and synthetic biology, numerous studies have focused on the design and construction of the optimal functional microbial chassis with reduced genomes and superior physiological characteristics [1, 2]. Moderate genome reduction can create synthetic biology chassis with optimized genomic sequences, efficient metabolic regulatory networks and superior cellular physiological characteristics [3,4,5]. Surfactin, which contains a ring-shaped heptapeptide and a β-hydroxy fatty acid chain with 13–16 carbons, is a cyclic lipopeptide (CLP) biosurfactant with broad-spectrum antibiotic activity and mainly produced by Bacillus sp.

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Results

Conclusion