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Construction and pplication of thermostable yeast engineering strain for L-lactic acid production

A technology of yeast engineering and strains, applied to the biological field

Active Publication Date: 2019-01-25
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are also many studies on the use of yeast to ferment and produce lactic acid, but they are basically carried out at 30°C; and most of them use pure sugar as a carbon source

Method used

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  • Construction and pplication of thermostable yeast engineering strain for L-lactic acid production
  • Construction and pplication of thermostable yeast engineering strain for L-lactic acid production
  • Construction and pplication of thermostable yeast engineering strain for L-lactic acid production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1. Preparation of bacterial strains:

[0055] 1. the construction of various plasmid vectors among the present invention:

[0056] 1) Construction of gene expression plasmids pKX004, pKX005, pKX007, pKX008 of L-lactate dehydrogenase derived from Bacillus subtilis, Bacillus megaterium, Lactococcus lactis, and Rhizopus oryzae

[0057] BsLDH, BmLDH, L1LDH, and RoLDH were amplified from the genomes of Bacillus subtilis, Bacillus megaterium, Lactococcus lactis, and Rhizopus oryzae. Inserted into the EcoR I and Not I sites of the pZJ042 vector respectively, constructing plasmids pKX004, pKX005, pKX007, pKX008 ( figure 2 ).

[0058] The specific operation steps are as follows:

[0059] (1) With the genomes of the above-mentioned various bacteria as templates, the primer pair BsLDH--EcoRI-F (SEQ ID No.1) and BsLDH-NotI-R (SEQ ID No.2), BmLDH-EcoRI-F (SEQ ID No.2) ID No.3) and BmLDH-NotI-R (SEQ IDNo.4), LlLDH-EcoRI-F (SEQ ID No.5) and LlLDH-NotI-R (SEQ ID No.6), ...

Embodiment 2

[0157]Embodiment 2. The situation that engineering strains containing different genes are fermented at high temperature

[0158] This example is used to compare the effects of co-utilizing glucose and xylose to produce L-lactic acid with strains containing different genes constructed in the present invention. The results showed that among all the engineering genes, strain YKX071 had the best effect of co-utilizing glucose and xylose to produce L-lactic acid.

[0159] 1. Resuscitate all frozen strains on YPD solid medium plates: YKX013, YKX014, YKX016, YKX017, YKX012, YKX019, YKX029, YKX049, YKX055, YKX071. Incubate at 37°C for 24 hours.

[0160] 2. Single clones were picked and inoculated in 5ml YPD liquid medium. Incubate overnight at 37°C on a shaker at 250rpm.

[0161] 3. Configure 30 bottles of 30ml YPDX liquid medium in 250ml Erlenmeyer flasks. YPDX formula: 10g / L yeast extract, 20g / L bacto-peptone, 80g / L glucose, 20g / L xylose. Sterilized and ready to use.

[0162] 4...

Embodiment 3

[0165] Embodiment 3.YKX071 bacterial strain is to the utilization situation of corncob

[0166] This example is used to verify whether the final strain YKX071 can ferment and produce L-lactic acid with the corncob residue after alkali treatment as the sole carbon source.

[0167] 1. Resuscitate the frozen strain YKX071 on the YPD solid medium plate, and culture it upside down in a 37°C incubator for 24 hours.

[0168] 2. Pick a single clone and inoculate it in 50ml YPD liquid medium. Incubate overnight at 37°C on a shaker at 250rpm.

[0169] 3. Prepare 500ml of fermentation medium and put it in a 1L fermentation tank. Formula: 10g / L yeast extract, 20g / L bacto-peptone, 180g / L alkali-treated corn cob residue. Sterilized and ready to use.

[0170] 4. Transplant the strain into 500ml fermentation medium, make the initial OD600 1, add cellulase at the ratio of 15FPU / g corncob residue, and ferment at 42°C, 250rpm, and 0.5vvm oxygen flow.

[0171] 5. Take samples at intervals, a...

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Abstract

The present invention relates to construction and application of thermostable yeast engineering strain for L-lactic acid production. The invention provides an L-lactic acid production yeast engineering strain. The yeast engineering strain is Kluyveromyces marxianus engineering stain prepared by recombinant expression of Plasmodium falciparum L-lactate dehydrogenase gene PfLDH and / or Bacillus megaterium L-lactate dehydrogenase gene BmLDH, for example, the deposit number of the engineering strain is CGMCC No.16192. The invention also provides a method for constructing the engineering strain, which is used in L-lactic acid production and method for production of L-actic acid. The engineering strain of the invention can efficiently co-utilize glucose and xylose for fermentation at relatively high temperature (for example, 42 DEG C), and can rapidly produce large quantities of high optical purity L-lactic acid, can also be quickly and efficiently used as a representative of corncob lignocellulose fermentation production of L-lactic acid, is the first lignocellulosic substances can be used to produce lactic acid engineering yeast.

Description

technical field [0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to a construction method and use of an engineered thermotolerant Kluyveromyces marxianus producing L-lactic acid. The invention also relates to a construction method and application of a heat-resistant engineering yeast strain that can directly use corncob residue as a carbon source to ferment and produce L-lactic acid. Background technique [0002] As a kind of lignocellulose, about 20 million tons of corn cobs are produced in China every year. At present, a large part of them is used to produce xylooligosaccharides, and the remaining residue (CCR) mainly contains glucose and a small amount of xylo Sugar (Bai et al., 2016; Samanta et al., 2012). CCR belongs to industrial waste, which will cause environmental pollution, and the cost of obtaining it is very low. This means that finding effective ways to utilize CCR will greatly reduce costs and re...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/53C12N15/54C12N15/60C07K14/395C12P7/56C12R1/645
CPCC12N9/0006C12N9/1205C12N9/88C12N15/815C12P7/56C12Y101/01009C12Y101/01027C12Y101/01028C12Y101/01307C12Y101/05003C12Y207/01011C12Y401/01001C07K14/395Y02A50/30
Inventor 洪泂孔昕王冬梅
Owner UNIV OF SCI & TECH OF CHINA
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