Microorganisms and methods for reducing bacterial contamination
Inventors
Wong, Steve Good-Sung • Jansen van Vuuren, Hendrik Jurgens
Assignees
Publication Number
US-12168763-B2
Publication Date
2024-12-17
Expiration Date
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Abstract
Provided herein are compositions and methods for reducing bacterial contamination during cell culture. Such compositions and methods utilize engineered peptides or recombinant cells capable of secreting such peptides into culture medium. Also provided are methods of using the engineered peptides for inhibiting bacterial growth during culturing of cells.
Core Innovation
Provided herein are compositions and methods for reducing bacterial contamination during cell culture that utilize engineered peptides or recombinant cells capable of secreting such peptides into culture medium. The invention includes engineered persulcatusin variants (variants of SEQ ID NO: 1) and engineered pediocin PA-1 variants (variants of SEQ ID NO: 36) with one or more specified amino acid alterations, isolated polynucleotides encoding those engineered peptides, expression vectors, and recombinant yeast capable of expressing and secreting the engineered peptides. Also provided are culture media containing engineered peptides and methods of using the engineered peptides or recombinant yeast for inhibiting bacterial growth during culturing of cells, including by fermentation.
The background describes a problem in commercial fermentation of yeast where bacterial contamination, particularly by lactic acid bacteria, competes with yeast for nutrients and inhibits yeast growth by producing lactic acid and acetic acid, which can lead to reduced ethanol yields, "stuck" fermentations, and economic loss. The background further states that small molecule antibiotics can inhibit bacterial growth but there is a desire to reduce antibiotic use due to concerns of antibiotic resistance and demand for antibiotic-free distillers grains. The background also notes that pediocin PA-1 does not have antibacterial activity against all lactic acid bacteria known to contaminate commercial fermentations, creating a need to identify alternatives and broaden the spectrum of antibacterial activity against lactic acid bacteria.
The invention addresses this need by engineering persulcatusin and pediocin PA-1 variants with alterations at identified residue positions that, as described, can improve antibacterial activity against lactic acid bacteria and, in some embodiments, inhibit growth with a minimum inhibitory concentration (MIC) at least two-fold lower than the corresponding wild-type peptide under specified assay conditions. The compositions include engineered peptides fused to secretion signals, isolated polynucleotides (including SEQ ID NOS: 38-71 and 88-102), expression vectors, and recombinant yeast (including specified yeast species) that can express and secrete the engineered peptides into culture medium. The methods include culturing recombinant yeast or culturing yeast in the presence of engineered peptides to inhibit contaminating lactic acid bacteria and to support production of bioderived compounds such as ethanol and other listed compounds.
Claims Coverage
One independent claim is identified, which presents one principal inventive feature.
Polynucleotide encoding engineered persulcatusin
An isolated polynucleotide comprising a nucleotide sequence encoding an engineered persulcatusin, wherein the engineered persulcatusin comprises a variant of the amino acid sequence of SEQ ID NO: 1 with one or more amino acid substitutions at positions selected from F6, N7, G9, R13, H14, R16, I18, R20, R21, A26, L28, F29, and R38 of SEQ ID NO: 1.
The independent claim covers an isolated polynucleotide encoding an engineered persulcatusin variant of SEQ ID NO: 1 having one or more substitutions at specified positions (F6, N7, G9, R13, H14, R16, I18, R20, R21, A26, L28, F29, and R38).
Stated Advantages
Reduce bacterial contamination during cell culture by use of engineered peptides or recombinant cells capable of secreting such peptides into culture medium.
Engineered peptides demonstrate improved antibacterial activity against lactic acid bacteria, including embodiments with at least a 2-fold lower MIC than the corresponding wild-type peptide under specified assay conditions.
Provide alternatives to small molecule antibiotics and broaden the spectrum of antibacterial activity against lactic acid bacteria, addressing concerns about antibiotic resistance and demand for antibiotic-free distillers grains.
Enable inhibition of contaminating lactic acid bacteria during culturing or fermentation to support production of bioderived compounds such as ethanol and other listed bioderived compounds.
Documented Applications
Reducing bacterial contamination during cell culture by employing engineered peptides or recombinant cells capable of secreting engineered peptides into culture medium.
Inhibiting bacterial growth in a yeast culture by culturing recombinant yeast that express engineered persulcatusin or engineered pediocin PA-1, or by culturing yeast in the presence of engineered peptides; methods include co-culturing and fermentation.
Use of engineered peptides or recombinant yeast in the production of bioderived compounds, including ethanol, xylitol, n-butanol, isobutanol, isopropanol, arabitol, ethyl acetate, phenyl-ethyl alcohol, 2-methyl-butanol, 3-methyl-butanol, and combinations of carbonyl compounds, alcohols, acetals, acids, esters and nitrogen compounds found in wine, beer, or whiskey.
Providing culture media that contain engineered persulcatusin and/or engineered pediocin PA-1 (or variants or functional fragments thereof), alone or in combination with other antibacterial agents such as pediocin PA-1 or small molecule antibiotics.
Generation and use of recombinant yeast (species listed in the document) comprising isolated polynucleotides or expression vectors encoding engineered peptides for secretion into culture medium to inhibit lactic acid bacteria contamination.
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