CN104450808A - Method for producing biodegradable polymer and biomass fuel through carbon source conversion by genetic recombination microbe - Google Patents

Abstract

The invention discloses a method for producing biodegradable polymer and biomass fuel through carbon source conversion by genetic recombination microbe, which comprises the following steps: A)a gene recombination technology is used for constructing the gene recombination microbe having glycerin utilization enzyme gene and polyhydroxyalkanoate synthetase gene; B)the gene recombination microbe is cultured in glycerin-containing environment; C)the gene recombination microbe is induced for expressing the glycerin utilization enzyme and polyhydroxyalkanoate synthetase gene, and producing the biodegradable polymer of polyhydroxyalkanoate and biomass fuel of ethanol; and D)separating to obtain polyhydroxyalkanoate and ethanol; wherein the polyhydroxyalkanoate generated in cells of the gene recombination microbe at least accounts for 30% of biomass (w/w), and the glycerin consumption can reach more than 90%(w/w).

Description

Carry out carbon source with genetically modified microorganism and transform the method for producing biodegradable polymer and raw matter fuel

Technical field

The present invention relates to a kind of method utilizing genetically modified microorganism production specific product, particularly relate to a kind of method of carrying out carbon source conversion production biodegradable polymer and raw matter fuel with genetically modified microorganism.

Background technology

Current China biodiesel is fast-developing, and the allround promotion B2 biodiesel interpolation in 2010, in view of the output of the waste products raw glycerine after manufacture biodiesel rises thereupon, cause environmental pollution; If raw glycerine refining recycled, its economic benefit is limited.Therefore, the important key that this by product of raw glycerine becomes development biodiesel is processed.With regard to world trend, all have relevant Energy policy method revision decision-making in advanced country, and majority all lists the main project of national development in.Such as by the oil-fired vehicle of use, the vehicle using alternative fuel must be replaced with specified proportion, and increase step by step when particular point in time.Under this trend, biodiesel production capacity target also will improve accordingly, and the useless raw glycerine when the time comes manufactured by huge biodiesel production capacity certainly will become the awkward situation of environmental pollution.Therefore, if microorganism can be utilized raw glycerine to be done effectively process or utilize, such as: produce the poly-biological decomposable plastics of this kind, hydroxyl alkane ester (Polyhydroxyalkanoate, PHA) with raw glycerine, so can improve the value added of biodiesel.Poly-hydroxyl alkane ester (polyhydroxyalkanoate, PHA) be a kind of microbiocidal polyester, generally mainly as the reserve substance of carbon source and energy, when microorganism faces nutritional imbalance state, PHA can be accumulated in born of the same parents with hydrophobic inclusion body in born of the same parents, exist with the form of hydrophobic particle, its content can exceed 90% of dry cell weight under certain condition.Chemically structure is said, PHA is the polymkeric substance of hydroxy fatty acid (Hydroxyalkanoic acid, HA), and general structure is as follows:

Molecular weight is generally several ten thousand to millions of, m=1 ~ 4, n=100 ~ 3000, R=C1 ~ C15 is not etc., different PHA and monomer whose have different side chain R group, and common PHA synthetics comprises on poly(hydrobutyl ester) (PHB), poly(hydroamyl ester) (PHV), the copolymer p HBV(of PHB and PHV and molecular chain has PHB and PHV simultaneously) etc. several.According to the difference of monomer composition, PHA has a series of different materialogy character such as the elastomerics expecting softness from the hard plastic that hard matter is crisp.PHA can be Material synthesis by bio-renewables, can be degradable by biologies such as bacteriums after entering nature, can, in order to substitute traditional non-degradable plastics, be the thermoplastic polymer with the characteristic such as bio-compatible and biological degradation, therefore in medical science and industrially have good applicability.

The production development of PHAs starts from the 1970s age, and at that time, ICI company adopted microorganism in natural soil to produce PHAs by fermenting process.Meanwhile, Massachusetts Institute of Technology (MIT) starts to adopt through engineering approaches technology with microorganisms producing PHAs.This research and development achievement derived Metabolix company in 1992.The technical patent of ICI is then transferred the possession of and is given Zeneca company, and after this this patent is more sold and given Monsanto company.Metabolix company then merges in calendar year 2001 from Monsanto corporate buyout correlation technique patent and with being certainly fruitful.Metabolix company in 2004 with ArcherDaniels Midland (ADM) company contract, will make PHA biodegradable plastic push heavy industrialization to, by construction 50,000 tons of production capacity mass production devices will in 2007 the end of the year ~ 2008 at the beginning of operation.Adopt the production cost of Metabolix microorganism fermentation process to be about US $ 3-5/kg, but industrial volume production device is expected to make production cost lower than USD2-3/kg.The target of Metabolix company produces PHAs in innovative modifying device, and this improved route is expected to make productive expense be reduced to below USD1-2/kg again.But the Media Components required for fermentation processing procedure that cost is the highest in PHAs produces, account for 50 ~ 80% of running cost, inquire into the analysis of upstream and downstream raw materials cost, wherein carbon source and nitrogenous source cost reach material source cost more than 80%.High carbon source cost forms the obstacle of PHAs commercialization and Promote Competitive, and the raw glycerine that biodiesel factory produces if therefore can utilize, as carbon source, can be conducive to reducing PHAs and be produced into and originally reached target.

The research overview of external researcher on PHA field, generally speaking, comprise the improvement (having E.coli and Wauteria eutropha) of bacterial classification, and the mutation research of gene is (as phaP, the genes such as phaR), amplify the volume of culture of bacterial classification and utilize fermenter to produce in a large number and the technology such as industrial applications test, except the research of gene, also focusing on the upgrading of bacterial classification simultaneously.In China's Mainland, the 2008 Beijing Olympic Games also adopts Biodegradable polymer to be the emphasis of conference environmental protection demand, and its achievement in research on Biodegradable plastics of planned extensive publicity.Korea S aspect in addition, not only change at basic bacterial classification grow, the improvement of pathways metabolism has outside quite deep research, the technological achievement separately in high density fermentation volume production PHAs also has much reference value.And also have for Ralstonia eutropha microorganism in the research team of Germany, by decoding complete for whole gene order, and in phaC, phaB, phaA gene, homologous genes has been found in karyomit(e), not only there is important breakthrough to the research of R.eutropha, the mode of gene comparison and engineered gimmick also can be utilized to carry out the improvement of product at other bacterial classifications.The research team of Britain then utilizes Bacillus cereus SPV then can produce the PHA series of biologic polyester macromolecule of various ways, as: 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV) and 4-hydroxybutyrate (4HB).The research team of Switzerland emphasizes to use the gene being different from microflora to turn to grow plant to produce PHA.The U.S side, except carry out the PRODUCTION TRAITS of Biopolvester with gene recombined escherichia coli production system except, in order to make the application in downstream more extensive, the material character also for co-polymer is inquired into.Australia is then produce the gene aspect regulator control system of PHA for Pseudomonas Pseudomonas to have deep discussion, even the bacterium between belonging to together but has obvious difference in the expression of gene level.The research team of India utilizes photosynthetic bacteria to carry out PHA production, and Canadian research team then tests for the biological degradability of PHAs material.

Summary of the invention

Because the trend of biodiesel fast development, and utilize the demand of microorganism volume production PHAs, the raw glycerine that biodiesel factory produces if can utilize is as carbon source, obtain combining efficient rate utilizing raw glycerine as carbon source by the technology of gene recombination, and effectively produce PHAs further, can be conducive to reducing PHAs production cost.

Edge this, namely object of the present invention is to provide a kind ofly carries out carbon source with genetically modified microorganism and transforms the method for producing biodegradable polymer and raw matter fuel.

The present invention is that the technique means that the problem of solution known techniques adopts is to provide a kind of method of carrying out carbon source conversion production biodegradable polymer and raw matter fuel with genetically modified microorganism, and its step comprises: (A) has with gene recombination technology structure the genetically modified microorganism that glycerine utilizes ferment gene and poly-hydroxyl alkane Lipase absobed enzyme gene; (B) this genetically modified microorganism is incubated in the environment containing glycerine (such as the raw glycerine of purity more than 70%); (C) inducing this genetically modified microorganism to express this glycerine utilizes ferment and this poly-hydroxyl alkane Lipase absobed enzyme also to produce the poly-biodegradable polymer of hydroxyl alkane ester and the raw matter fuel of ethanol; (D) separation obtains this poly-hydroxyl alkane ester and ethanol; Wherein the interior poly-hydroxyl alkane ester produced of the cell of this genetically modified microorganism at least accounts for 30% (w/w) of its biomass, and its glycerine consumption can reach more than 90% (w/w).Forementioned gene recombinant microorganism is deposited in Leibniz institute DSMZ-Germany microorganism and (depositary institution address: Yin Huofen street, Braunschweig, Germany city 38124 7B, cell culture harvest center on September 12nd, 2013; Deposit number: DSM27723; Classification: Escherichia coli).

Preferably, wherein, this glycerine utilizes ferment gene to comprise aldehyde reductase (aldehyde reductase, alrd) gene and aldehyde dehydrogenase (aldehyde dehydrogenase, aldH) gene; This poly-hydroxyl alkane Lipase absobed enzyme gene is that PhaCAB gathers hydroxyl alkane Lipase absobed enzyme gene; This glycerine utilizes that ferment is gene constructed expresses plastid in first, and this first is expressed plastid and can bring out it by the first inductor and express, and this first inductor is preferably L-arabinose (L-arabinose); This glycerine utilizes ferment gene constructed in the second expression plastid, and this second is expressed plastid and can bring out it by the second inductor and express, this second inductor is preferably isopropyl-β-D-thiogalactoside(IPTG) (isopropyl-beta-D-thiogalactopyranoside, IPTG); The culture temperature scope of this genetically modified microorganism is between 25 DEG C ~ 37 DEG C, and its microbial inoculant amount is more than 3% (v/v), under can being incubated at anaerobic environment or oxygen-depleted environment; This genetically modified microorganism regulates and controls the consumption of its glycerine and the production of poly-hydroxyl alkane ester with two benches induced gene; This genetically modified microorganism is prokaryotic organism, is preferably intestinal bacteria (Escherichia coli); Or can be eukaryote.

Via the technology used in the present invention means, via the structure of double mass genetically modified microorganism, and with the gene induced control methods of two benches, comprise and carry out aldehyde reductase (aldehyde reductase (alrd)) and aldehyde dehydrogenase (aldehyde dehydrogenase (aldH)) glycerine with L-arabinose (L-arabinose) and utilize ferment to induce, and with isopropyl-β-D-thiogalactoside(IPTG) (isopropyl-beta-D-thiogalactopyranoside, IPTG) induction of PHAs NOS is carried out, when making it be incubated at oxygen-depleted environment, can utilize to exceed 90% (w/v) above glycerine and transform the poly-hydroxyl alkane ester content of production and reach 30% (w/w), the glycerine utilization ratio of genetically modified microorganism is 2 times of indigenous microorganisms.Can be used for relaxing slow global evolution alternative energy problem of environmental pollution with create its value added, by the poly-hydroxyl alkane ester of microbial metabolism glycerine synthesis and ethanol.Improve known techniques shortcoming: such as production scale does not reach can volume production stage and the problem such as fermentation raw material cost (nitrogenous source, microbiotic and inductor) is excessively high.Via the present invention, removing can using the waste raw glycerine of biodiesel factory directly as the nutrition source of culturing micro-organisms, the microorganism of carrying double mass is provided to carry out metabolism and synthesis, to releive the subjects under discussion such as raw glycerine surplus, environmental pollution, more can the substituting plastics of poly-hydroxyl alkane ester of production environment friendliness and biomass energy ethanol, can be applicable to the aspects such as biological example technical industry, foodstuff manufacturing, chemical material production, medical science, industry and chemical product manufacturing future.

Accompanying drawing explanation

Fig. 1 represents that the present invention uses the double mass strategy of genetically modified microorganism;

Fig. 2 represents that pARD23 and pARD33 and pBHB2 elemental body build schematic diagram;

Fig. 3 represents that the double mass of pARD33 and pBHB2 builds schematic diagram;

Fig. 4 represents the alcohol production amount of E.coli (dual-plasmid) in shake flask test.

Embodiment

The present invention mainly discloses a kind of method of carrying out carbon source conversion production biodegradable polymer and raw matter fuel with genetically modified microorganism, it is built by gene recombination technology has the genetically modified microorganism that glycerine utilizes ferment gene and poly-hydroxyl alkane Lipase absobed enzyme gene, this genetically modified microorganism is incubated at (such as the raw glycerine of purity more than 70%) in the environment containing glycerine, and it generates with two benches induction regulating controlling, comprise and carry out aldehyde reductase (aldehyde reductase with L-arabinose (L-arabinose), alrd) with aldehyde dehydrogenase (aldehyde dehydrogenase, aldH) glycerine utilizes ferment to induce, with isopropyl-β-D-thiogalactoside(IPTG) (isopropyl-beta-D-thiogalactopyranoside, IPTG) induction of poly-hydroxyl alkane Lipase absobed enzyme is carried out, with the raw matter fuel of the biodegradable polymer and ethanol of producing poly-hydroxyl alkane ester.Do not change the use glycerine efficiency of growing primary type microorganism be 50% (w/v) and lack PHAs NOS, thus cannot produce poly-hydroxyl alkane ester.And the present invention is by turning the genetically modified microorganism grown through gene, it is made to be incubated at oxygen-depleted environment, can utilize to exceed 90% (w/v) above glycerine and transform the poly-hydroxyl alkane ester content of production and reach 30% (w/w), the glycerine utilization ratio of genetically modified microorganism is that indigenous microorganisms reaches 2 times.

Some bacterium can accumulate PHA under field conditions (factors), comprises and carrying out with different biosynthetic pathways, the enzyme needed for it and having nothing in common with each other by matter specificity.According to the difference of route of synthesis and the product specificities of relevant enzyme, the monomer structure of PHA is also varied, wherein 3-hydroxy aliphatic acid mono includes all members of 3-hydroxy-propionic acid to 3-hydroxy-palmitic acid, also have in addition 4-, 5-, 6-hydroxy fatty acid and containing unsaturated link(age), with the 3-hydroxy fatty acid of methyl chains or other functional groups as the situation of PHA monomer.And the present invention adopts acetyl-CoA to synthesize the approach of PHA, it uses the double mass strategy of genetically modified microorganism as shown in Figure 1, after metabolism, pyruvic acid is obtained as carbon source using glycerine, acetyl-CoA is generated through glycolytic pathway, beta-keto thiolase PhaA catalysis 2 acetyl-CoA condensations form acetoacetyl-CoA, then under the effect of the Acetoacetyl-CoA reductase PhaB of NADPH dependence, be reduced to (R)-3-hydroxybutyryl A, finally aggregate into PHA by pha synthesizing enzyme PhaC.

Embodiment 1: double mass E.coli (dual-plasmid) strain construction

Double mass E.coli (dual-plasmid) bacterial strain of the present invention is with constructed by llowing group of materials and method:

Use material: bacterial strain used in the present invention and plastid list as follows, refer to as shown in table 1.

Table 1

Bacterial strain (Strains)	Explanation
		R.eutropha H16	Primary type (Wild-type)
E.coli DH5α	Primary type (Wild-type)
		E.coli BL21	Primary type (Wild-type)
E.coli XL1-BLUE	pBHB2
Plastid (Plasmids)	Explanation
		pGEM-T easy	Carrier (Cloning vector) is grown in choosing
pET-23a	High expression level loading gage body (high level expression vector)
		pBAD33	Expression vector (expression vector)
pGEM-T easy_alrd	pGEM-T easy::alrdRe
		pGEM-T easy_aldH	pGEM-T easy::aldHRe
pBHB2	pBluescript II KS::phaCABRe
		pARD23	pET-23a::alrd/aldH
pARD33	pBAD33::alrd/aldH

First Genomic DNA is obtained from R.eutropha H16, polymerase chain reaction (Polymerase chain reaction is carried out again in this, as template (Template), PCR), after aldehyde reductase aldehydereductase (alrd) is amplified (amplification) with aldehyde dehydrogenase aldehyde dehydrogenase (aldH) protein gene amplification, after DNA electrophoretic analysis, confirm that target gene fragment exactness and restriction enzyme ferment react and two gene built respectively (construction) is paramount to be copied several pET-23a and lowly copy on the expression plastid of several pBAD33, and will the poly-alkyl alkane Lipase absobed enzyme gene phaC of R.eutropha H16 be taken from, phaB and phaA (being called for short phaCAB) builds up to and expresses plastid pBHB2, finally by pARD23, pARD33 and pBHB2 three expresses plastid, transition out of the ordinary is in E.coli BL21 bacterial strain, complete restructuring elemental body E.coli bacterial strain (as shown in Figure 2).Afterwards, test elemental body restructuring E.coli bacterial strain respectively under inducing culture, confirm the mrna expression of target gene out of the ordinary, protein-active compares and the difference of glycerine utilising efficiency, after interpretation, and then obtain glycerine utilising efficiency and preferably to recombinate E.coli bacterial strain (pARD33), and this restructuring E.coli (pARD33) bacterial strain is expressed plastid carry out double mass E.coli (dual-plasmid) strain construction (as shown in Figure 3) with restructuring E.coli (pBHB2) bacterial strain two, the double mass bacterial strain built compares carrying out follow-up glycerine utilization the test experiments accumulated with PHB (poly(hydrobutyl ester)).Aforementioned double mass bacterial strain is deposited in Leibniz institute DSMZ-Germany microorganism and (depositary institution address: Yin Huofen street, Braunschweig, Germany city 38124 7B, cell culture harvest center on September 12nd, 2013; Deposit number: DSM27723; Classification: Escherichia coli).

Embodiment 2: shake flask fermentation is tested

Substratum selected by this stage is Define medium, adds the develop simultaneously envrionment conditions that arranges and affect growth of microbial cells and induced concentration of extra carbon source and nitrogenous source in instances and inquires into situation.

(1) test of primary E.coli (WT) and restructuring E.coli (dual-plasmid)

The present invention prepares the triangular pyramidal shaking flask of several 500mL, after getting 3mL activation, experimental strain is to (inoculum size is more than 3%) in erlenmeyer, wherein add the glycerine of 30g L-1 and the yeast extract of 2.0g L-1, in shaking flask, volume of culture is quantitatively 100mL.Culture condition is cultivated under being set as the environment of temperature 37 DEG C (culture temperature scope can between 25 ~ 37 DEG C) and rotating speed 200rpm.Appropriate nutrient solution is taken out in timing subsequently from shaking flask, then measures the suction brightness of nutrient solution in Optical density600nm (OD600) wavelength with point luminometer, and object is for understanding microorganism growth situation.And the analysis of glycerine utilization ratio and PHAs cumulative amount, then by high-effect liquid chromatograph (High performance liquid chromatography, HPLC) and gas chromatograph (Gas chromatography, GC) carry out analysis confirmation.

This result of implementation arranges in table 2.All obviously excellent in the expression of primary E.coli (WT) by test-results observable go out to recombinate dry bacterium heavy (DCW), the glycerine utilization ratio of E.coli (dual-plasmid), PHA content, productive rate Yp/s and ds/t, the glycerine of Alrd and the AldH constructed by display utilizes the PHAs NOS of ferment and PhaCAB, all expresses its function.

Table 2, E.coli (WT) compare with restructuring E.coli (dual-plasmid) cultivation results

Note: mark "-" is not for detect.

(2) primary E.coli (WT) and restructuring E.coli (dual-plasmid) are in the test had, nothing is induced

The present invention utilizes for inquiring into inductor (L-arabinose and IPTG) impact accumulated with PHA in glycerine for genetically modified microorganism, its test cultural method and analytical procedure are as described in the same (1), carry out the comparing difference tested, nothing adds inductor, test-results arranges in table 3.Observable goes out E.coli (dual-plasmid) in having, without in induction situation, glycerine utilization ratio is better than E.coli (WT) and PHAs cumulative amount reaches more than 29%, wherein E.coli (dual-plasmid) is under having inducing culture, its glycerine utilization ratio and PHA cumulative amount are expressed all higher than without inducing culture, therefore, the present invention's test imposes induction and cultivates.

Table 3, E.coli (WT) and E.coli (dual-plasmid) are in the result had, nothing is induced

Note: Ind. is IPTG induction; Mark "-" for not detect.

(3) E.coli (dual-plasmid) that recombinates is incubated at anaerobic environment test

The present invention for improving microbial cell density, carried out comparing from primary E.coli (WT), have induce and compare without inducing culture, different induction time compares and different culture temperature compares, above test is all cultivated under oxygen-depleted environment.This test basis previous experiments optimum, further microorganism culturing is done in anaerobic environment and distinctly test discussion, its culture condition, analytical procedure and method are as shown in aforementioned, and carry out IPTG Induced synthesis PHAs in 37 DEG C of culture environment and 9 hours, this step-by-step test result arranges in table 4.Grow without turning under the primary E.coli (WT) grown all is unfavorable for anaerobic environment with restructuring E.coli (dual-plasmid) though can obviously be observed out by result, but restructuring E.coli (dual-plasmid) can synthesize PHAs under anaerobic environment, this test cultivation results is compared to oxygen-depleted environment, and display restructuring E.coli (dual-plasmid) is incubated at oxygen consumption and anaerobic environment and expresses all excellences and reach high-cell-density cultivation then must carry out under oxygen-depleted environment in primary E.coli (WT).

Table 4, E.coli (dual-plasmid) are in the cultivation results of anaerobic environment

Embodiment 3: fermenter fermentation test

The present invention tests in the shake flask fermentation of embodiment 2 the preliminary confirmation having completed high-cell-density cultivation condition, and use define medium substratum interpolation raw glycerine to cultivate as sole carbon source, and add L-arabinose and IPTG induction start glycerine utilize ferment and PHAs NOS, provide primary force bacterial strain utilize glycerol metabolism synthesize PHAs.And embodiment 3 is tested as Simulation scale-up shake flask test result is as the extensive probability analysis consuming glycerine and PHAs volume production of assessment, its fermenter test-results arranges in table 5.In this fermenter result, though PHAs content does not reach 30% (w/w), glycerine utilizes the efficiency still reaching 94% (w/w), object according to the invention.

Table 5, E.coli (dual-plasmid) are in fermenter cultivation results

Embodiment 4: alcohol production is tested

The test of this alcohol production be utilize the shake flask fermentation of embodiment 2 to test set up the condition of high-cell-density cultivation, proved invention be can be used as and produce one of ethanol procedure, add raw glycerine using defined medium (define medium) to cultivate as sole carbon source, add L-arabinose and IPTG induction startup glycerine and utilize ferment and the effect of PHAs NOS, be incubated in the shaking flask of temperature 37 DEG C and rotating speed 200rpm and carry out microorganism culturing test in 72 hours, test-results arranges in Fig. 2.By Fig. 2 susceptible of proof, restructuring E.coli (dual-plasmid) bacterial strain used in the present invention, utilize double mass strategy not only can consume glycerol production PHAs in a large number and alcohol production content can reach 0.8g L-1, far above without changing the alcohol production content growing primary E.coli (WT) bacterial strain 0.1g L-1, its ethanol content differs 8 times.

The present invention is by genetically modified microorganism metabolism glycerine to synthesize poly-hydroxyl alkane ester and ethanol, and the double mass E.coli utilized at present need not carry out pH regulation and control can change into ethanol and poly-alkyl alkane ester by comprising the carbon source such as glucose and glycerine.Though the present invention is for procaryotic E.coli, also can utilizes and produce addicted to methyl yeast Pichia pastoris as Eukaryotic, be not limited only to prokaryotic organism.

As seen from the above embodiment, utility value in the true tool industry of method of carrying out carbon source conversion production biodegradable polymer and raw matter fuel with genetically modified microorganism provided by the present invention, describing is only only the preferred embodiments of the present invention explanation, allly be skillful in this those skilled in the art when can do other all improvement according to above-mentioned explanation, only these changes still belong in spirit of the present invention and following defined the scope of the claims.

Claims (13)

1. carry out carbon source with genetically modified microorganism and transform a method of producing biodegradable polymer and raw matter fuel, its step comprises:

(A) with gene recombination technology structure, there is the genetically modified microorganism that glycerine utilizes ferment gene and poly-hydroxyl alkane Lipase absobed enzyme gene;

(B) this genetically modified microorganism is incubated in the environment containing glycerine;

(C) inducing this genetically modified microorganism to express this glycerine utilizes ferment and this poly-hydroxyl alkane Lipase absobed enzyme also to produce the poly-biodegradable polymer of hydroxyl alkane ester and the raw matter fuel of ethanol;

(D) separation obtains this poly-hydroxyl alkane ester and ethanol;

Wherein, the poly-hydroxyl alkane ester produced in the cell of this genetically modified microorganism at least accounts for 30% (w/w) of its biomass, and its glycerine consumption can reach more than 90% (w/w).

2. method according to claim 1, wherein, this glycerine utilizes ferment gene to comprise aldehyde reductase (aldehyde reductase, alrd) gene and aldehyde dehydrogenase gene.

3. method according to claim 1, wherein, this poly-hydroxyl alkane Lipase absobed enzyme gene is that PhaCAB gathers hydroxyl alkane Lipase absobed enzyme gene.

4. method according to claim 1, wherein, this glycerine utilizes that ferment is gene constructed expresses plastid in first, and this first is expressed plastid and can bring out it by the first inductor and express.

5. method according to claim 4, wherein, this first inductor is L-arabinose.

6. method according to claim 1, wherein, this glycerine utilizes that ferment is gene constructed expresses plastid in second, and this second is expressed plastid and can bring out it by the second inductor and express.

7. method according to claim 6, wherein this second inductor is isopropyl-β-D-thiogalactoside(IPTG).

8. method according to claim 1, wherein, the culture temperature scope of this genetically modified microorganism is between 25 DEG C ~ 37 DEG C, and its microbial inoculant amount is more than 3% (v/v).

9. method according to claim 1, wherein, this genetically modified microorganism is incubated at anaerobic environment or oxygen-depleted environment.

10. method according to claim 1, wherein, this genetically modified microorganism regulates and controls the consumption of its glycerine and the production of poly-hydroxyl alkane ester with two benches induced gene.

11. methods according to claim 1, wherein, this genetically modified microorganism is prokaryotic organism.

12. methods according to claim 1, wherein, these prokaryotic organism are intestinal bacteria.

13. methods according to claim 1, wherein, this genetically modified microorganism is eukaryote.