Overexpression of GRAS Rhizomucor miehei lipase in Yarrowia lipolytica via optimizing promoter, gene dosage and fermentation parameters.

Abstract:

:Rhizomucor miehei lipase (RML), a GRAS catalyst with wide applications, was overexpressed in Yarrowia lipolytica, also a GRAS unconventional yeast, via a combined strategy, optimization for promoter, gene dosage and fermentation process. The lipase activity of the recombinant strain was first increased from 19.5 to 26.9 U/mL via codon optimization of rml gene. Subsequently, a method was developed for constructing hybrid promoters harboring different copy number of upstream activation sequences fragment (UAS1B), and the recombinant strain Po1g/hp12d-rml 25# reached 38.9 U/mL. On this basis, expression vectors with different optimized rml gene copy numbers were constructed and introduced into Y. lipolytica Po1g. The recombinant strain Po1g/hp12d-2rml 14# carrying 12 copies of UAS1B in the upstream of pLEUmin and 2 copies of rml gene obtained the highest lipase activity of 59.6 U/mL. Moreover, in optimized shaking flask culture parameters: 5% (m/v) of d-Sorbitol, 2% (v/v) inoculation density, initial pH 7.0, and 30 mL initial culture medium, the RML activity of Po1g/hp12d-2rml 14# further reached 157 U/mL after 84-h of incubation at 28 ℃. Overall, RML activity was enhanced about 8-fold compared with the initial recombinant strain via the combined strategy, which provides a consolidated basis for the large-scale production of RML in Y. lipolytica to match urgent demand of the market.

journal_name

J Biotechnol

journal_title

Journal of biotechnology

authors

Zhou Q,Jiao L,Qiao Y,Wang Y,Xu L,Yan J,Yan Y

doi

10.1016/j.jbiotec.2019.09.004

subject

Has Abstract

pub_date

2019-12-20 00:00:00

pages

16-23

eissn

0168-1656

issn

1873-4863

pii

S0168-1656(19)30852-1

journal_volume

306

pub_type

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